Transplantation of chicken egg white extract-induced rabbit PBMCs as a treatment for renal ischemia-reperfusion injury in rabbits

Guang-ping Ruan; Xiang Yao; Qing-keng Lin; Zi-an Li; Xue-min Cai; Rong-qing Pang; Xing-hua Pan

doi:10.1371/journal.pone.0244160

. 2020 Dec 28;15(12):e0244160. doi: 10.1371/journal.pone.0244160

Transplantation of chicken egg white extract-induced rabbit PBMCs as a treatment for renal ischemia-reperfusion injury in rabbits

Guang-ping Ruan ^1,^2,^3,^*, Xiang Yao ^1,^2,³, Qing-keng Lin ^1,^2,³, Zi-an Li ^1,^2,³, Xue-min Cai ^1,^2,³, Rong-qing Pang ^1,^2,³, Xing-hua Pan ^1,^2,^3,^*

Editor: Nazmul Haque⁴

PMCID: PMC7769466 PMID: 33370374

Abstract

Ischemia-reperfusion injury is an important contributor to acute kidney injury and a major factor affecting early functional recovery after kidney transplantation. We conducted this experiment to investigate the protective effect of induced multipotent stem cell transplantation on renal ischemia-reperfusion injury. Forty rabbits were divided into four groups of 10 rabbits each. Thirty rabbits were used to establish the renal ischemia-reperfusion injury model, and ten rabbits served as the model group and were not treated. Among the 30 rabbits with renal ischemia-reperfusion injury, 10 rabbits were treated with induced peripheral blood mononuclear cells (PBMCs), and 10 other rabbits were treated with noninduced PBMCs. After three weekly treatments, the serum creatinine levels, urea nitrogen levels and urine protein concentrations were quantified. The kidneys were stained with hematoxylin-eosin (HE), periodic acid-Schiff (PAS) and Masson’s trichrome and then sent for commercial metabolomic testing. The kidneys of the rabbits in the model group showed different degrees of pathological changes, and the recovery of renal function was observed in the group treated with induced cells. The results indicate that PBMCs differentiate into multipotent stem cells after induction and exert a therapeutic effect on renal ischemia-reperfusion injury.

Introduction

Clinical acute kidney injury, which has a very high incidence, might be caused many factors, and ischemia-reperfusion injury is the most common of these factors [1]. The main pathological manifestation of acute kidney injury is renal tubular epithelial damage, and the recovery of renal function depends on renal tubular epithelial cell regeneration. In the event of renal tubular necrosis, blood purification is the only method that can maintain a stable internal bodily environment due to the impairment of kidney function. The development of drugs or interventions that accelerate renal tissue repair during renal tubular epithelial self-repair processes will undoubtedly shorten the course of acute kidney injury. Early recovery of a stable internal bodily environment can reduce the complications caused by renal failure and thereby reduce mortality [2].

Renal ischemia-reperfusion injury is clinically common in the context of emergency shock recovery, kidney surgery and kidney transplantation and is thus an important factor affecting renal function [3]. The manifestations of renal ischemia-reperfusion injury include acute tubular epithelial cell injury, tubular peripheral microvascular damage, inflammation and leukocyte infiltration. Mesenchymal stem cells are effective for the treatment of ischemia-related organ dysfunction, but the exact mechanism through which they improve renal function remains unclear [4]. In this study, we investigated the protective effect of induced multipotent stem cell transplantation on renal ischemia-reperfusion injury in rabbits, and our results provide experimental evidence and ideas for clinical treatment.

Induced multipotent stem cell regeneration of renal function represents a new method for the treatment of acute tubular necrosis. In our laboratory, induced multipotent stem cells are generated by the treatment of peripheral blood mononuclear cells (PBMCs) from rabbit peripheral blood with a homemade egg white extract to reverse their differentiation [5]. This study is the first to induce PBMCs with chicken egg white extract and use the induced PBMCs for the treatment of rabbit renal ischemia-reperfusion injury. Previous studies have shown that extracts of mammalian oocytes [6] and Xenopus oocytes [7] have the potential to reprogram cells. The identification of egg extracts with the ability to maintain and enhance the survival and differentiation of cells will be widely useful in cellular biology research. Many studies have shown that animal egg extracts are able to induce the reprogramming of somatic cells [6, 8]. The chicken egg yolk is the largest egg cell, the yolk membrane comprises the cell membrane, and the egg white and eggshell, which have nutritional and protective roles, are formed by oviduct secretions. Therefore, chicken egg white extract has the capacity to induce stemness in PBMCs. In our previous research, we found that chicken egg white extract can induce the reverse differentiation of somatic cells into multipotent stem cells [5, 9].

As determined by quantitative PCR, the induction of PBMCs with the chicken egg white extract significantly increased the relative expression of the multipotency-related genes OCT4, NANOG and SOX2 and significantly decreased that of the somatic cell gene LMNA, which indicates that PBMCs differentiate into multipotent stem cells [9]. Further experiments are needed to identify the key molecules in the chicken egg white extract to further improve the induction efficiency and promote this method.

Mesenchymal stem cells with multidirectional differentiation potential have been differentiated into renal tubular epithelial cells and effectively promote the restoration of renal function in injured kidneys [4, 10–13]. In the present study, we assessed whether induced PBMCs function as mesenchymal stem cells. To this end, we divided 40 rabbits into four groups: a normal control group, a model control group, an induced cell treatment group and a noninduced cell treatment group. After successful establishment of the model, we compared the efficacies of the treatments and ultimately revealed that the induced PBMCs function as multipotent stem cells and participate in the repair of renal injury.

Materials and methods

1. Preparation of the rabbit model of renal ischemia-reperfusion injury

According to the inclusion and exclusion criteria, 40 wild-type Japanese white rabbits were numbered according to their body mass and randomly allocated to a group. The rabbits were purchased from Kunming Chushang Technology Co., Ltd., and the license number was SCXK (Dian) K2018-0001. The rabbits were anesthetized with intravenous injections of 3% sodium pentobarbital (Merck, Darmstadt, Germany) at a dose of 1 ml per kg of body weight. The abdominal skin of the rabbits was prepared, and an incision was made along the midline of the abdomen. The skin, subcutaneous tissue, and the peritoneum were separated layer by layer, and an incision was then made in the abdominal cavity. The renal pedicle was located, and the bilateral renal pedicle was rapidly clipped with a noninvasive arterial clip to restrict the blood flow for 1 hour. The artery clip was then released to restore the blood flow and allow reperfusion, which resulted in ischemia-reperfusion injury. The model was established in 30 rabbits, and the other 10 rabbits were not subjected to the operation and served as the normal control group. Among the rabbits with ischemia-reperfusion injury, 10 were used as the model control group, 10 other rabbits were injected with induced PBMCs, and the remaining 10 were injected with noninduced PBMCs. The cells were transfused on the first day after model establishment and then once a week for 3 consecutive weeks. All experimental protocols were approved by the Experimental Animal Ethics Committee of the 920th Hospital of the Joint Logistics Support Force of the People’s Liberation Army (approved number: 2019-002-01). No human subjects were included in this study, and thus, informed consent is not applicable. The DOI link of experimental steps is dx.doi.org/10.17504/protocols.io.bpyrmpv6.

2. Rabbit PBMC isolation, culture, induction and labeling

Twenty milliliters of peripheral blood was harvested from each of three rabbits, and the experiment was performed more than three times to ensure repeatability. PBMCs were isolated using a separation fluid (Haoyang Biological Products Technology Co., Ltd., Tianjin, China). After two washes, the cells were cultured in culture flasks. The cells in one flask were cultured in DMEM-F12 medium supplemented with 10% fetal bovine serum, and the cells in the other flask were cultured with 50% egg white extract in culture medium. As shown in our previous study, cells grown in 50% chicken egg white extract in culture medium will differentiate into multipotent stem cells and might be used for the treatment of diseases [9]. Thus, we used 50% chicken egg white extract-induced rabbit PBMCs as a treatment for kidney injury in the rabbit model. Three days after induction, CFSE-labeled cells were transfused into the rabbits belonging to the two treatment groups; CFSE fluoresces yellowish-green. The cells were centrifuged, and the supernatant was discarded. The cell pellet was suspended in 4 ml of PBS, and 10 μl of 10 mM CFSE (from Abcam, ab145291, Cambridge, UK) was added. The mixture was incubated at 37°C for 10 minutes, and 3 ml of medium was added to terminate the labeling reaction. The cells were centrifuged, and the supernatant was removed. The cells were suspended in saline, and each rabbit received a transfusion of 2 × 10⁶ cells. The cell survival rate was greater than 95%. The PBMCs were injected via the intravenous route. We observed that the fluorescence lasted for more than 1 month after the cells were labeled with CFSE, and because the experimental period was less than a month, the fluorescent cells could be traced.

3. Identification of noninduced and induced PBMCs

3.1. Quantitative PCR-based detection of the relative expression of pluripotency-related and somatic cell genes

Three days after induction, RNA was extracted from the PBMCs, reverse transcribed, and subjected to quantitative PCR to determine the relative levels and fold changes in the expression of the pluripotency-related genes OCT4, NANOG, and SOX2 and the somatic cell gene LMNA. The primer sequences and product lengths are shown in Table 1.

Table 1. Primer sequences used in the quantitative PCR-based detection of pluripotency-related and somatic genes and lengths of the products.

Gene	Primer sequences	Product length (bp)
OCT4	F: `5’-AAGGAGAAGCTGGAGCAAACC-3’`	164
OCT4	R: `5’-CTGAACACCTTTCCAAAGAGAACCC-3’`	164
NANOG	F: `5’-TCAGCCTTCAGCAGATGCAA-3’`	150
NANOG	R: `5’-GGCACCCCTGAGTCACAC-3’`	150
SOX2	F: `5’-AACGCCTTCATGGTATGGTC-3’`	253
SOX2	R: `5’-CTCCGGGAAGCGTGTACTTA-3’`	253
LMNA	F: `5’-CTTGCTGACTTACCGCTTCC-3’`	253
LMNA	R: `5’-CAGGTCATCTCCATCCTCGT-3’`	253
GAPDH	F: `5’-CGAGACACGATGGTGAAGGT-3’`	139
GAPDH	R: `5’-TGTAGACCATGTAGTGGAGGTC-3’`	139

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3.2. Flow cytometry

Three days after induction, the PBMCs were labeled with SSEA-4-PE, OCT4-PE and NANOG-PE, and the changes in the expression of pluripotency markers were detected.

SSEA-4 is a surface-expressed antigen. The PBMCs were centrifuged, and the supernatant was discarded [9]. The pellet was suspended in 50 μl of PBS, and 20 μl of an isotype control antibody or SSEA-4-PE was added to the wells. The mixtures were incubated for 1 hour at room temperature in the dark, and the cells were then washed once with PBS, fixed with 4% paraformaldehyde in PBS, and detected using a flow cytometer (Manufacturer: Becton Dickinson; model: BD LSRFortessa; software version: BD FACSDiva Software v8.0.1, USA). All the antibodies were used in strict accordance with the manufacturer’s instructions to avoid nonspecific binding.

3.3. Immunohistochemical detection

Noninduced and induced PBMCs were subjected to immunohistochemistry analysis. Specifically, noninduced and induced PBMCs were smeared on a slide, dried, and fixed with 4% paraformaldehyde for 10 minutes. The cells were stained with an Abcam immunohistochemistry kit using primary antibodies against OCT4 and NANOG at 1:50 dilution. The procedures were performed according to the manufacturer’s instructions. DAB was used to develop the staining, and images were captured.

3.4. Western blot detection

The western blot detection of noninduced and induced PBMCs was performed as follows. Proteins were extracted from noninduced and induced PBMCs, electrophoresed, and transferred to a membrane. The membrane was blocked for 1 hour and then incubated with a 1:500 dilution of an anti-OCT4 primary antibody for 1 hour at 37°C with shaking. The membrane was subjected to three 5-minute washes with TTBS and then incubated with a 1:500 dilution of the secondary antibody for 1 hour at 37°C with shaking. The membrane was subjected to three 5-minute washes with TTBS. Enhanced chemiluminescence (ECL) was used to detect the proteins, and images were captured using a chemiluminescence imaging camera (Tanon 5200, Shanghai).

3.5. Quantitative PCR detection of relative changes in the telomere length

The relative changes in the telomere lengths in noninduced and induced PBMCs were detected as follows. Three days after induction, RNA was extracted from the PBMCs, reverse transcribed, and subjected to quantitative PCR to determine the relative changes in the telomere length. The telomere and internal control 36B4 primer sequences are shown in Table 2.

Table 2. Primer sequences for telomeres and the internal control 36B4.

Genes	Primer sequences
Telomere	Forward: `5’-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3’`
Telomere	Reverse: `5’-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3’`
36B4	Forward: `5’-CAGCAAGTGGGAAGGTGTAAATCC-3’`
36B4	Reverse: `5’-CCCATTCTATCATCAACGGGTACAA-3’`

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4. Kidney function test

After three transplantations of the labeled cells, blood samples were collected from the ear veins of rabbits in the four groups. Serum was separated and sent to a laboratory to examine the changes in the creatinine and urea nitrogen contents.

5. Changes in the urinary protein content

After three transplantations of the labeled cells, urine samples were collected from the four groups of rabbits, and the urine protein contents were measured using the BCA method.

6. Observation of labeled cells in frozen sections

After three transplantations of the labeled cells, three rabbits from each group were euthanized through the introduction of an air embolism. The renal distributions of the labeled cells in frozen kidney sections were observed. The kidney tissue collected was partly frozen to observe fluorescent cells, and the histology was partially evaluated.

7. Histopathological assessment of the kidneys

Kidney tissue was used for HE staining and immunohistochemistry analysis of the fibrosis-related factor TGF-β, Masson’s trichrome staining and PAS staining. Changes in the renal tissues from each group were observed through HE staining. The degree of renal fibrosis was detected by immunohistochemistry. The extent of fibrosis in the kidneys from each group was detected by Masson’s trichrome staining. The thickening of the renal basement membrane was detected by PAS staining. The kidney tissues of the treatment group were frozen, sectioned, incubated with the primary antibody aquaporin-1 (from Abcam, ab9566, Cambridge, UK) specific for the proximal tubules, incubated with a red fluorescence-labeled secondary antibody (from Abcam, ab150115, Cambridge, UK), and counterstained with DAPI, and the distribution of red, green, and blue fluorescence was observed under a fluorescence microscope.

8. Metabolomics analysis of the kidney

After three transplantations of the labeled cells, five rabbits from each of the four groups were euthanized via introduction of an air embolism. The kidney tissues were rapidly removed, placed in liquid nitrogen, refrozen at -80°C, and then sent to a company on dry ice for metabolomics analysis. Five biological replicates of rabbit kidney tissue samples from the four groups were analyzed, and the 20 samples were then analyzed by LC-QTOFMS. The ionization source of the LC-QTOFMS platform was electrospray ionization. Two ionization modes, positive ion mode (POS) and negative ion mode (NEG), were used for the detection of metabolites and increase the coverage rate and thus the detection rate.

9. Statistical analysis

All statistical analyses were performed using SPSS 21.0 statistical software. The measurement results are expressed as the means±standard deviations. Four-group comparisons and the above results were analyzed by one-way analysis of variance (one-way ANOVA), and p<0.05 was considered statistically significant. Post hoc comparisons between individual groups after ANOVA were needed for the assessment of significant differences between specific groups. Pairwise comparisons between groups were performed using the LSD and SNK methods.

Results

1. Successful labeling of PBMCs with CFSE

Carboxyfluorescein diacetate and succinimidyl ester (CFSE)-labeled PBMCs exhibited yellow-green fluorescence under a fluorescence microscope, as shown in Fig 1B. After counterstaining with DAPI, all the cells showed blue fluorescence, as shown in Fig 1A, and an analysis of the overlap in the fluorescence staining showed that the cells were successfully labeled, as shown in Fig 1C.

Fig 1 — The background is black, and the cells show yellow-green fluorescence. A. DAPI shows blue fluorescence. B. CFSE-labeled PBMCs display yellow-green fluorescence. C. Overlap between blue fluorescence and yellow-green fluorescence. All the cells are labeled with yellow-green fluorescence. D. The expression of the pluripotency-related genes *NANOG*, *OCT4*, and *SOX2* was significantly increased after induction, whereas the expression of the somatic cell gene *LMNA* was decreased after induction. A statistical analysis showed that the two groups were significantly different (n = 3, p = 0.003). The difference in the somatic cell gene *LMNA* between the two groups was not statistically significant. E-M. Flow cytometry analyses of noninduced and induced PBMCs. E, F and G: OCT4-PE; H, I and J: SSEA-4-PE; and K, L and M: NANOG-PE. E, H and K: isotype control; F, I and L: noninduced PBMCs; and G, J and M: induced PBMCs. After induction, the proportion of cells positive for multipotency-related factors was significantly increased.

2. Identification of PBMCs after induction

2.1. Relative expression levels of multipotency-related and somatic cell genes in noninduced and induced PBMCs

The expression of NANOG, OCT4 and SOX2 was significantly increased in PBMCs stimulated with chicken egg white extract, and the expression of the somatic cell gene LMNA was decreased (Fig 1D), which indicated that the cells differentiated into multipotent cells. A statistical analysis showed that the two groups were significantly different (n = 3, p = 0.003). The cells were induced three times, and three biological replicates were included in the study. The difference in the somatic cell gene LMNA between the two groups was not statistically significant.

2.2. The pluripotency factor of induced PBMCs was significantly higher than that of noninduced PBMCs

Among the noninduced cells, 0.081% were positive for OCT4-PE, whereas 99.3% of the induced cells were positive for OCT4-PE. The percentages of SSEA-4-PE-positive noninduced and induced cells were 1.08% and 16.5%, respectively. Moreover, 0.495% of the noninduced cells were positive for NANOG-PE, whereas 95.8% of the induced cells were found to be NANOG-PE-positive (Fig 1E–1M). The percentages of isotype control cells positive for OCT4-PE, SSEA-4-PE and NANOG-PE were 0.546%, 0.401% and 0.249%, respectively.

2.3. Immunohistochemical analyses yielded positive results for the induced PBMCs

Immunohistochemical analyses of OCT4 and NANOG showed that induced PBMCs, but not noninduced PBMCs, expressed these markers (Fig 2A–2D). Fig 2A and 2C show uninduced PBMCs, and Fig 2B and 2D show induced PBMCs. In addition, OCT4 expression is shown in Fig 2A and 2B, and Fig 2C and 2D show the expression of NANOG.

2.4. Western blot analyses yielded positive results for induced PBMCs

According to the Western blot results, OCT4 was expressed in induced PBMCs but not in noninduced PBMCs (Fig 2E).

2.5. The relative telomere lengths were significantly longer in induced PBMCs

Based on the quantitative PCR results, the telomeres of induced PBMCs (1.83807±0.84756) were significantly longer than those of noninduced PBMCs (1±0.08307) (Fig 2F, means±standard deviations, n = 5, p = 0.013), which indicated that the cells differentiated into young stem cells. The relative telomere lengths actually increased after dedifferentiation.

3. The serum urea nitrogen and creatinine levels were decreased in the induced cell treatment group

The model control group had a urea nitrogen content of 22.1 mmol/l and a creatinine content of 452 μmol/l. After three rounds of transplantation with induced cells, the urea nitrogen content was 7 mmol/l, and the creatinine content was 74 μmol/l; these values were similar to the normal levels. The levels in the noninduced cell treatment group remained elevated (Fig 2G). A statistical analysis showed statistically significant differences among the results of the four groups (p = 0.031).

4. The urinary protein concentrations were decreased in the induced group

The urinary protein concentration in the model control group was 8.17 mg/ml. After the three treatments, the urinary protein concentration in the induced group was 4.35 mg/ml, which was similar to the normal level. However, the urinary protein content of the noninduced group remained high at 7.96 mg/ml (Fig 2H). A statistical analysis showed statistically significant differences among the results obtained from the four groups (p = 0.001).

5. Labeled cells were detected in the induced group

As shown in Fig 3A–3D, many fluorescent cells were distributed in the kidneys of the induced cell treatment group, whereas the kidney tissues from the other three groups did not display any fluorescent cells. A possible explanation is that induced cells are transported to the injured kidney to repair the damage. The distribution of fluorescently labeled induced PBMCs in frozen kidney sections suggested that these cells were involved in repairing the injured kidney.

6. The structure of the kidney of the induced group exhibited a normal phenotype

Based on the hematoxylin-eosin (HE) staining results, the structure of the kidneys of the rabbits in the model control group was damaged. After treatment with the induced cells, the renal tissue structure exhibited a normal phenotype. In contrast, the damage to the kidney tissue persisted in the noninduced cell treatment group (Fig 3E–3H). The acute tubular necrosis (ATN) score is shown in Fig 3I.

7. The IOD of the induced cell groups was significantly reduced

Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) software was used to perform an immunohistochemical analysis of the cumulative optical density (IOD). For each group, at least six 200× magnification fields were randomly selected from each section, and images were captured. We attempted to view the entire field of vision to ensure that every photograph had the same background. Image-Pro Plus 6.0 software was used to select the same brown color as a uniform standard for judging the positive staining in all the images. Each image was analyzed to determine the IOD of positive staining. The IOD was significantly increased in the model control group, whereas the IODs of the normal control and induced cell groups were reduced and significantly reduced, respectively. The IOD of the noninduced cell treatment group was not markedly reduced compared with that of the model control group (Fig 3J). A statistical analysis showed significant differences among the results of the four groups (p = 0.001).

8. Fibrosis was improved in the induced group

Substantial collagen fiber deposition was observed in the model control group, and serious fibrosis occurred. This fibrosis was improved or eliminated in the induced cell treatment group, whereas severe fibrosis was still observed in the noninduced cell-treated group (Fig 4A–4D).

Fig 4 — A. The normal control group showed no obvious collagen fiber deposition or fibrosis. B. The model control group showed a large number of collagen fibers, hyperplasia, and severe fibrosis. C. The treatment with the induced cells improved or eliminated the fibrosis observed in the model control group. D. In the noninduced cell-treated group, a large number of collagen fibers, hyperplasia, and severe fibrosis were observed. E-G. Renal PAS staining of the four groups after the three treatments. E. Significant thickening of the basement membrane was not observed in the normal control group. F. Significant thickening of the glomerular basement membrane, basilar membrane of the renal capsule, and tubular basement membrane was observed in the model control group. G. After treatment, significant thickening of the basement membrane was not observed in the induced cell treatment group. H. A thicker basement membrane was observed in the noninduced cell treatment group. I. Basement membrane thickness score (mean±standard deviation, n = 3). A statistical analysis showed significant differences among the results of the four groups (p = 0.024). * indicates p<0.05 compared with the model group. J. Renal tissue immunofluorescence results. The immunofluorescence results showed that the transplanted cells exhibited green and red fluorescence at the same time, which indicated that the transplanted cells had differentiated into tubular epithelial cells (TECs).

9. Thickening of the basement membrane was not observed in the induced group

The thicknesses of the glomerular basement membrane, basilar membrane of the renal capsule and tubulointerstitial membrane were significantly increased in the model control group. In the induced cell treatment group, the basement membrane did not display any significant thickening, whereas thicker basement membranes were still observed in the noninduced cell treatment group (Fig 4E–4H). The basement membrane thickness scores are shown in Fig 4I.

10. Transplanted cells differentiated into tubular epithelial cells

The immunofluorescence results showed that the transplanted cells exhibited green and red fluorescence at the same time, which indicated that the transplanted cells were differentiated into tubular epithelial cells (TECs) (Fig 4J).

11. The identified substances were increased in the model group and decreased after treatment

In the negative ion mode, the two substances that displayed significantly increased levels in the model control group were 2'-deoxy-D-ribose and N-acetylglucosamine 1-phosphate (Fig 5A). The levels of both of these compounds were significantly reduced after induced cell therapy compared with the levels found in the model control group (Fig 5A). In the positive ion mode, the levels of three substances (D-pinitol, lysyl-glycine and glutamyl-asparagine) were significantly increased in the model control group (Fig 5B), and the levels of these three substances were significantly reduced after induced cell therapy compared with those found in the model control group (Fig 5B).

Fig 5 — A: The results obtained in the negative ion mode showed significantly increased levels of two substances in the model control group compared with the normal control group (mean±standard deviation, n = 5). B: In the positive ion mode, the levels of three substances were significantly increased in the model control group compared with the normal control group (mean±standard deviation, n = 5). * indicates p = 0.035 compared with the model group. In the positive ion mode, the levels of three substances were significantly decreased in the induced cell treatment group compared with the model control group (mean±standard deviation, n = 5). * indicates p = 0.041 compared with the model group.

In the negative ion mode, the levels of the two identified substances were increased in the model group and decreased after treatment, which indicated a meaningful correlation between the levels of the two substances.

In the positive ion mode, the levels of the three identified substances were increased in the model control group and decreased after treatment, which indicated meaningful correlations among the levels of these three substances.

11. Significant changes in the pyrimidine metabolism and phenylalanine, tyrosine and tryptophan biosynthesis pathways were observed

In the negative ion mode, a significant change in the pyrimidine metabolism pathway was detected between the model control group and the normal control group (Fig 6A). In addition, significant changes in this pathway were observed in the induced cell treatment group compared with the model control group (Fig 6B), which indicated that the pyrimidine metabolism pathway is a significantly altered pathway. In the positive ion mode analysis, the phenylalanine, tyrosine and tryptophan biosynthesis pathways were significantly altered in the model control group compared with the normal control group (Fig 6C), and these pathways were significantly altered in the induced cell treatment group compared with the model control group (Fig 6D), which indicated that the phenylalanine, tyrosine and tryptophan biosynthesis pathways are meaningful pathways. In Fig 6, a darker bubble color and a larger volume indicates a more significant difference. As shown by the arrows in Fig 6A and 6B, the difference in the pyrimidine metabolism pathway is significant, and as indicated by the arrows in Fig 6C and 6D, the differences in phenylalanine, tyrosine and tryptophan biosynthesis pathways are significant.

A. In the negative ion mode, the model control group displayed significant changes in the pyrimidine metabolism pathway compared with the normal control group. B. In the negative ion mode, significant changes in the pyrimidine metabolism pathway were also observed in the induced cell treatment group compared with the model control group. C. In the positive ion mode, significant changes in the phenylalanine, tyrosine and tryptophan biosynthesis pathways were detected in the model control group compared with the normal control group. D. In the positive ion mode, the phenylalanine, tyrosine and tryptophan biosynthesis pathways also exhibited significant changes in the induced cell treatment group compared with the model control group. In Fig 6, a darker bubble color and a larger volume indicate a more significant difference. As shown by the arrows in Fig 6A and 6B, the difference in the pyrimidine metabolism pathway is significant, and as indicated by the arrows in Fig 6C and 6D, the differences in the phenylalanine, tyrosine and tryptophan biosynthesis pathways are significant.

Discussion

Acute kidney injury caused by ischemia-reperfusion is a common clinical disease, and the mortality rate of patients with acute kidney injury is approximately 30% to 50% due to limited treatment measures [14]. During ischemia, the activation of enzymes induces cytoskeletal destruction, cell membrane damage, and DNA degradation, which eventually leads to cell necrosis and apoptosis [15]. Ischemia-reperfusion also activates complement proteins, cytokines, and chemokines, among other molecules, and the mechanisms underlying ischemia-reperfusion injury are of substantial importance [16]. Additionally, transplanted stem cells promote the migration and recruitment of residual renal stem cells to the injury site and induce their differentiation into TECs, but the exact mechanism is unclear [17].

The mortality of patients with ischemic-reperfusion injury is high due to the lack of an effective clinical treatment [18]. The condition requires long-term or lifelong renal replacement therapy or renal transplantation [19]. The pathophysiological mechanism of ischemia-reperfusion injury is very complicated and results from numerous interactions among inflammatory cells, vascular endothelial cells and cytokines [20]. In recent years, mesenchymal stem cells have become the focus of research on ischemia-reperfusion injury [21] and have been shown to improve ischemia-reperfusion-induced renal injury. As shown in studies using ischemia-reperfusion models, mesenchymal stem cells play an important role in regulating immunity, participating in vascular reconstruction [22] and repairing the renal microenvironment by secreting paracrine antiapoptotic factors, mitogenic factors and angiogenic factors. A recent study using stem cells of different origins revealed that induced pluripotent stem cells exhibit high potential for clinical applications. These cells can migrate to damaged sites, produce large amounts of anti-inflammatory cytokines and growth factors, and exhibit immunomodulatory properties that endow them with greater potential and more advantages than other types of stem cells [22]. We believe that the induced cells are more likely to be mesenchymal stem cells because they are more mature than induced pluripotent stem cells.

After induced pluripotent stem cell transplantation, molecules expressed on the surface of the induced pluripotent stem cells interact with T cells to regulate their biological activity. T cells can evade the immune system following damage and reduce the intensity of the immune response in the surrounding tissue, and these cells thus play a role in protecting the function of damaged tissue to some extent [23]. We successfully established a rabbit model of renal interstitial fibrosis and demonstrated that the transplantation of induced autologous stem cells can repair kidney damage within 8 weeks [24]. Stem cells provide an advantageous microenvironment for the repair of renal tubular epithelial damage [24]. In this experiment, after the administration of three consecutive weekly treatments, the serum creatinine and urea nitrogen levels in the induced cell treatment group were restored to the same level observed in the normal control group, whereas the levels in the noninduced cell treatment and model control groups were significantly elevated.

As shown by Haynesworth et al. [25], mesenchymal stem cells secrete a variety of growth factors, colony-stimulating factors, adhesion molecules, and interleukins (IL-6, IL-7, IL-8, IL-11, IL-14, and IL-15), which promote the mitosis of renal TECs and tissue repair and thereby inhibit the elevation of creatinine and urea nitrogen concentrations.

In the present study, the expression levels of the pluripotency factors SSEA-4, NANOG and OCT4 in induced PBMCs were detected by flow cytometry, and the results revealed that PBMCs dedifferentiated into multipotent cells. The quantitative PCR analysis demonstrated that the telomeres in induced PBMCs were significantly longer than those in noninduced PBMCs. Based on our evidence, PBMCs obtained from rabbit peripheral blood dedifferentiate into multipotent stem cells following treatment with chicken egg white extracts and exhibit the characteristics of mesenchymal stem cells [9], which allows their eventual participation in the repair of renal injury. Studies have shown that mammalian egg cell and Xenopus egg cell extracts can reprogram somatic cells [6, 7]. Our previous research showed that chicken egg extract can also reprogram somatic cells [5, 9]. The chicken egg is the largest egg cell, and its ability to reprogram somatic cells will thus advance cell biological research. The mammalian egg cell and Xenopus egg cell extract-induced reprogramming steps are cumbersome, and the extracts are difficult to obtain; however, chicken eggs are the largest egg cells, and a large amount of chicken egg white extract can thus be obtained. If the procedure is performed in a sterile manner, the obtained extract does not need to be filtered and sterilized. The activity of the extract can be maintained well. We repeated the induction experiment using final concentrations of chicken egg white extract of 10%, 20%, 30%, 40%, and 50% to induce the PBMCs (see S1 to S3 Figs). As the concentration of the chicken egg white extract increased, the pluripotency factor positive rate gradually increased, and the highest positive rate was obtained with a final concentration of 50%. However, a chicken egg white extract concentration higher than 50% will affect cell growth. We used protease, DNase, and RNase to lyse the protein, DNA, and RNA in egg white extract, respectively, and then conducted our induction experiment. We found that the egg white extract obtained after protein lysis no longer has the ability to reprogram cells, and the chicken protein extract obtained after DNA and RNA lysis still has the ability to reprogram cells. Therefore, these findings confirmed that the main role of the extract is played by the protein component (results not shown).

We also analyzed animal blood samples after one week of treatment. However, the difference among the four groups was not significant; thus, we only show the results obtained for the four groups after three treatments. The distribution of fluorescently labeled induced PBMCs in frozen kidney sections suggested that these cells were involved in repairing the injured kidney. HE staining showed damage to the kidney structure in the model control group. After treatment with induced cells, the kidney exhibited a normal structure, whereas damage to the kidney structure was observed in the noninduced cell treatment group. After three treatments, Masson’s trichrome staining of kidney sections from the four groups revealed substantial collagen fiber deposition and serious fibrosis in the model control group. This fibrosis was improved or eliminated by treatment with the induced cells, whereas severe fibrosis was still observed in the noninduced cell treatment group. A renal metabonomics analysis of the four groups conducted in the negative ion mode revealed significantly increased levels of 2'-deoxy-D-ribose and N-acetylglucosamine 1-phosphate in the model control group compared with the normal control group, and the levels of these substances were significantly reduced in the induced cell treatment group compared with the model control group. In the positive ion mode, the levels of five substances were significantly increased in the model control group compared with the normal control group, and the levels of three of these five substances were significantly reduced in the induced cell treatment group compared with the model control group.

The postulated main mechanisms underlying the observed repair of the kidney and the accompanying reduction in the inflammatory response are listed below. ① Induced PBMCs differentiate into TECs or fuse with surviving cells to directly promote the repair of renal tissue. ② The synergistic effects of various cytokines provide a good microenvironment for the repair of renal tissues [26]. After three transplantations of induced PBMCs, the renal function and pathological indexes returned to the normal levels, and these normal levels persisted until the end of the experiment, which suggested that the transplanted multipotent stem cells effectively promoted the repair of renal tissue structure and function.

Although the specific mechanism through which induced PBMCs promote structural and functional repair of the kidney has not yet been fully understood, the results of this study suggest that the intravenous transplantation of induced PBMCs promotes repair after acute kidney injury. These results provide a valuable reference for researchers investigating the function of multipotent stem cells in regenerating damaged kidneys and for the clinical treatment of acute and chronic kidney diseases.

In summary, induced multipotent stem cell transplantation has substantial significance for parenchymal cell repair in animal models of renal failure and other kidney diseases.

Conclusions

The treatment of PBMCs with chicken egg white extract significantly increased the expression of pluripotency-related genes and proteins, which indicated that the cells had dedifferentiated into multipotent stem cells. Thus, induced PBMCs dedifferentiate into multipotent stem cells and can potentially be used to treat kidney injury. Future research is needed to identify the key molecules in chicken egg white extract and thus further improve the induction efficiency.

Supporting information

S1 Fig

(TIF)

Click here for additional data file.^{(311.3KB, tif)}

S2 Fig

(TIF)

Click here for additional data file.^{(311KB, tif)}

S3 Fig

(PPTX)

Click here for additional data file.^{(3.9MB, pptx)}

Acknowledgments

We thank American Journal Experts for assisting with the preparation of this manuscript.

Data Availability

All relevant data are within the manuscript.

Funding Statement

This work was supported by grants from Yunnan Science and Technology Plan Project Major Science and Technology Project (2018ZF007) and the Yunnan Province Applied Basic Research Program Key Project (2018FA041, 2017FA040). National Natural Science Foundation (31970515), 920th Hospital of the PLA Joint Logistics Support Force Inhospital technology plans (2019YGB17, 2019YGA05). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0244160.r001

Decision Letter 0

Nazmul Haque

27 Aug 2020

PONE-D-20-22977

Transplantation of Chicken Egg-White Extract-Induced Rabbit PBMCs as Treatment for Renal Ischemia-Reperfusion Injury in Rabbits

PLOS ONE

Dear Dr. Ruan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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[This work was supported by grants from Yunnan Science and Technology Plan Project

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Applied Basic Research Program Key Project (2018FA041, 2017FA040). National

Natural Science Foundation (31970515), 920th Hospital of the PLA Joint Logistics

Support Force In-hospital technology plans (2019YGB17, 2019YGA05).]

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5. Review Comments to the Author

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Reviewer #1: The idea for this research is interesting and offers a numerous possibilities for future investigations such as clarification of strict molecular mechanism of beneficial induced PBMC treatment.

The basic question is how the authors decided to conduct just three procedures of induced PBMC administration. Why would not we suppose that perhaps one treatment is enough for restoration of kidney architecture and function? Why did not you analyze animal blood sample after first week of treatment? Please explain and the answers on these question incorporate in discussion. Additionally, restructure some part of discussion by comparing your results with other studies instead of simple paraphrase of already shown results.

Please, use uniform phrase ACUTE KIDNEY INJURY, not ACUTE RENAL FAILURE.

Reviewer #2: The current manuscript entitled ‘Transplantation of Chicken Egg-White Extract-Induced Rabbit PBMCs as Treatment for Renal Ischemia-Reperfusion Injury in Rabbits’ by RUAN et al. is moderately organised and represented. However the following points need to be addressed for betterment of their manuscript.

1# In the Materials and Methods section the author wrote that ‘As shown in our previous study, cells grown in 50% chicken egg-white extract culture medium will differentiate into pluripotent stem cells and may be used to treat disease.’ They need to cite their paper here.

2# To check the stemness property the author checked the expression level of Oct4, Nanog and Sox2. I wonder why they overlooked Klf4 and c-Myc detection. To characterize any induced pluripotent cell type OKSM expression is very important.

3# It is highly recommended to draw a diagram of this research where all treatment groups will be included for better understanding.

4# In the histopathological assessment, the author performed immunohistochemistry analysis of the fibrosis factor TGF-β only. Is it adequate to represent fibrosis by detecting only Tgf-b expression? The authors need to add couple of more markers.

5# In the result section, discussion under the heading of ‘Infusions of labeled PBMCs to treat the rabbit model’ is not sufficient. The author should write their findings very clearly in this section. It is highly recommended to summarize each result of every sub-category at the end of description.

6# In the result section, the author wrote ‘Statistical analysis showed that the two groups were statistically significant (n=3, p=0.003)’ regarding Figure 1D. However, I failed to find any indication of this significance in the figure!!! Furthermore, the author mentioned that the expression of the somatic cell gene LMNA was decreased. But Figure 1D does not show any difference among the two candidates.

7# For statistical analysis they mentioned n=3 in everywhere. Does it represent biological replicates or technical replicates? In case of quantitative PCR do they use biological replicates? Author need to mention it in their manuscript.

8# Author needs to change all gene symbols in Italic throughout the manuscript including Tables also.

9# The picture resolution of Figure 1E-1M is very poor. The numbers and description are very hard to read. Furthermore, authors used SSEA-4 candidate to show the status of pluripotency induction. But they did not show SSEA-4 transcript expression. It would be better if they synchronize their candidates for qPCR and Flow cytometric analysis.

10# In the methods section the author needs to add the name and company of instruments they used in this research. It is recommended to check the manuscript again and correct missing one.

11# In the Figure 2, the labelling of A-D is missing. Additionally, which one is noninduced and induced PBMCs? Authors need to be more careful to organise a manuscript.

12# In the Figure 2E, it is recommended to replace 1-5 numbering by adding text. And also mention candidates as usual.

13# In the Figure 2F, the picture resolution is very poor. Failed to read the figure text. There is no sign of statistically significance though author claim it in the manuscript.

14# In the Figure 2G-2I, I failed to read the text of it. Most probably, the statistical significance signs are also missing here.

15# The Figure 3A-3D is difficult to understand. The difference is not visible. Author needs to reedit their picture for better visualisation. Furthermore, 3A-3H figures have no identifying text!!!

16# The Figure 3I-3J is difficult to read. Most probably, the statistical significance signs are also missing here!!!

17# In the Figure 4, authors are asked to label A-H, to add statistical significance sign in I and to add text for magnification bar in J. Furthermore, the author wrote ‘Immunofluorescence results showed that the transplanted cells had green fluorescence and red fluorescence at the same time, indicating that the transplanted cells were differentiated into tubular epithelial cells.’ But it is very hard to find any red fluorescence in Figure 4J. Need to use arrow for any specific presentation.

18# In Figure 5, the author needs to add line bars among their comparable candidates in the figure. It is hardly detectable among which the statistical analysis was done.

19# In Figure 6, the author needs to edit all texts. Nothing is visible here. It is very tough to comment more than that….!!!

20# The author wrote in summary ‘the results of this study suggest that the intravenous transplantation of induced PBMCs promotes the repair of acute kidney injury’. However, they did not check any expression of marker genes. They need to add couple of marker gene expressions, if possible.

21# Authors need to submit their metabolomics data in any open access platform.

Reviewer #3: The manuscript, “Transplantation of Chicken Egg-White Extract-Induced Rabbit PBMCs as Treatment for Renal Ischemia-Reperfusion Injury in Rabbits” does not present any novel scientific concept. The study was not presented properly, the figures were not labelled and there are number of unclear images presented without paying due attention.

• Induction of PBMCs by chicken egg white extract is an old concept. Number of studies have been conducted two-three decades ago. Heterogeneous nature of the chicken egg white components and allergenic substances could affect the outcome.

• Authors need to explain the results of the previous studies with egg white extract on inducing cells in the introduction.

• Chicken egg white contains different growth factors and enzymes. Which component of chicken egg white are you interested in and expecting to induce cells?

• The research gap addressed by the study is not well defined.

• How many rabbits were used for harvesting peripheral blood? Please explain the procedure and mention how the animal well-being was maintained.

• What is the rationale of using 50% egg white extract medium? How was it formulated? Was the protocol standardized? If so, please cite the methodology.

• Instead of formulating egg white extract medium, authors could have used purified egg white lysozymes as done in other studies to avoid interferences with host biology.

• Methodology is not sub-sectioned well. Please put a title instead of using sentences and explain the methodology thereafter.

• Results section also needs proper headings rather than using sentences

• In the discussion, the results of the egg white extract induction mechanism were not discussed.

• Please mention other methods that have been used for induction, and compare the results of those methods with egg white extract method.

• Figures are not labelled. Therefore, referring to the figures is very difficult.

• The axes of the graphs are not labelled properly.

• There are number of unclear images presented without paying due attention.

• The manuscript needs revision for English language.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PLoS One. 2020 Dec 28;15(12):e0244160. doi: 10.1371/journal.pone.0244160.r002

Author response to Decision Letter 0

20 Sep 2020

Dear Editor,

Thank you very much for your letter and advice. We have revised the paper and would like to resubmit it for your consideration. We have addressed the reviewers’ comments. This manuscript has been edited by American Journal Experts.

We hope that our revised manuscript is acceptable for publication, and we look forward to hearing from you at your earliest convenience.

Best wishes,

Guang-ping Ruan

Attachment

Submitted filename: 2-Response to Reviewers.doc

Click here for additional data file.^{(88KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0244160.r003

Decision Letter 1

Nazmul Haque

8 Oct 2020

PONE-D-20-22977R1

Transplantation of Chicken Egg-White Extract-Induced Rabbit PBMCs as a Treatment for Renal Ischemia-Reperfusion Injury in Rabbits

PLOS ONE

Dear Dr. Ruan,

Please submit your revised manuscript by 06 November 2020. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Nazmul Haque

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #3: No

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: No

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #3: Although authors have revised the manuscript up to a certain degree, there are inadequately addressed concerns as mentioned below.

1. As a response to the reviewer's comments, authors have mentioned that the induction effect of the chicken egg white extract obtained according to their methodology is "stable", with our giving any supportive evidence. How do the authors justify this?

2. Authors have added only a single sentence on chicken egg white extract as the previous literature. There should be a more extensive literature review in the introduction in relation to this.

3. What is the protein in chicken egg white extract that you consider as the active factor?

4. Research gap needs to be logically stated in the introduction.

5. How was the protocol on 50% egg white extract medium standardised?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

PLoS One. 2020 Dec 28;15(12):e0244160. doi: 10.1371/journal.pone.0244160.r004

Author response to Decision Letter 1

21 Oct 2020

PONE-D-20-22977R1

Transplantation of Chicken Egg-White Extract-Induced Rabbit PBMCs as a Treatment for Renal Ischemia-Reperfusion Injury in Rabbits

PLOS ONE

Dear Dr. Ruan,

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Nazmul Haque

Academic Editor

PLOS ONE

Dear Editor,

We hope that our revised manuscript is acceptable for publication, and we look forward to hearing from you at your earliest convenience.

Best wishes,

Guang-ping Ruan

We would like to express our sincere thanks to the reviewers for their constructive and positive comments.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

________________________________________

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #3: No

________________________________________

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

________________________________________

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

________________________________________

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: No

________________________________________

6. Review Comments to the Author

Reviewer #1: (No Response)

Responses to Reviewer 3

Reviewer #3: Although authors have revised the manuscript up to a certain degree, there are inadequately addressed concerns as mentioned below.

We repeated the induction experiment and performed flow cytometry, and the results showed that our induction experiment was stable. The result is as follows.

As the concentration of the egg white extract increases, the positive rate gradually increases, and the positive rate is the highest at a final concentration of 50%.

2. Authors have added only a single sentence on chicken egg white extract as the previous literature. There should be a more extensive literature review in the introduction in relation to this.

We have made a more extensive literature review in the introduction in relation to this. “In our laboratory, induced multipotent stem cells are generated by treating peripheral blood mononuclear cells (PBMCs) from rabbit peripheral blood with a homemade egg-white extract to reverse differentiation[5]. This is the first report to use chicken egg-white extract to induce PBMCs and treat rabbit renal ischemia-reperfusion injury. Studies have shown that extracts of mammalian oocytes[6] and Xenopus oocytes[7] have the potential to reprogram cells. The identification of egg extracts with the ability to maintain and enhance the survival and differentiation of cells will be widely useful in cellular biology research. Many studies have reported that animal egg extracts are able to induce the reprogramming of somatic cells[6, 8]. The chicken egg yolk is the largest egg cell, where the yolk membrane comprises the cell membrane, and the egg white and eggshell, which have nutritional and protective roles, are formed by oviduct secretions. Therefore, chicken egg white extract has the capacity to induce stemness in PBMCs.”

3. What is the protein in chicken egg white extract that you consider as the active factor?

Because we have done the following experiments, using protease, DNase, and RNase to respectively lyse the protein, DNA, and RNA in the egg white extract, and then conduct our induction experiment, and found that the egg white extract after protein lysis no longer has the ability to reprogram cells, and the chicken protein extract after DNA and RNA lysis still has the ability to reprogram cells. So we proved that the main role of the extract is the protein component.

4. Research gap needs to be logically stated in the introduction.

We have added a sentence in the introduction: “Further experiments need to find the key molecules in the chicken egg white extract in order to further improve the induction efficiency and promote this method.”

5. How was the protocol on 50% egg white extract medium standardised?

“We repeated the induction experiment, using 10%, 20%, 30%, 40%, 50% final concentration of chicken egg-white extract to induce cells. As the concentration of the chicken egg-white extract increased, the pluripotency factor positive rate gradually increased, and at the final concentration of 50% the positive rate is the highest. But if the concentration of chicken egg-white extract exceeds 50%, cell growth will be affected. Thus, we used 50% chicken egg-white extract-induced rabbit PBMCs as a treatment for kidney injury in the rabbit model.” The result is shown below.

________________________________________

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

________________________________________

In compliance with data protection regulations, you may request that we remove your personal registration details at any time. (Remove my information/details). Please contact the publication office if you have any questions.

Attachment

Submitted filename: 3-Response to Reviewers.doc

Click here for additional data file.^{(8.4MB, doc)}

PLoS One. doi: 10.1371/journal.pone.0244160.r005

Decision Letter 2

Nazmul Haque

26 Oct 2020

PONE-D-20-22977R2

Transplantation of Chicken Egg-White Extract-Induced Rabbit PBMCs as a Treatment for Renal Ischemia-Reperfusion Injury in Rabbits

PLOS ONE

Dear Dr. Ruan,

Please provide proper consideration to the Editor's comments and revise the manuscript carefully.

Please submit your revised manuscript by 23rd November 2020. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Nazmul Haque

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

1. Please remove the following sentences from the Materials and Methods and add in the discussion appropriately with proper citation. Mention the name of the cell type on which the experiment was conducted.

2. In answer to the question 3 you have written the following sentences:

"Because we have done the following experiments, using protease, DNase, and RNase to respectively lyse the protein, DNA, and RNA in the egg white extract, and then conduct our induction experiment, and found that the egg white extract after protein lysis no longer has the ability to reprogram cells, and the chicken protein extract after DNA and RNA lysis still has the ability to reprogram cells. So we proved that the main role of the extract is the protein component.”

Please add these sentences in the discussion appropriately with proper citation.

[Note: HTML markup is below. Please do not edit.]

PLoS One. 2020 Dec 28;15(12):e0244160. doi: 10.1371/journal.pone.0244160.r006

Author response to Decision Letter 2

26 Oct 2020

PONE-D-20-22977R2

Transplantation of Chicken Egg-White Extract-Induced Rabbit PBMCs as a Treatment for Renal Ischemia-Reperfusion Injury in Rabbits

PLOS ONE

Dear Dr. Ruan,

Please provide proper consideration to the Editor's comments and revise the manuscript carefully.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Nazmul Haque

Academic Editor

PLOS ONE

Dear Editor,

We hope that our revised manuscript is acceptable for publication, and we look forward to hearing from you at your earliest convenience.

Best wishes,

Guang-ping Ruan

We would like to express our sincere thanks to the reviewers for their constructive and positive comments.

Additional Editor Comments (if provided):

We have removed the following sentences from the Materials and Methods and add in the discussion appropriately with proper citation. The name of the cell type on which the experiment was conducted was PBMCs. “We repeated the induction experiment, using 10%, 20%, 30%, 40%, 50% final concentration of chicken egg-white extract to induce PBMCs (see supplementary material). As the concentration of the chicken egg-white extract increased, the pluripotency factor positive rate gradually increased, and at the final concentration of 50% the positive rate is the highest. But if the concentration of chicken egg-white extract exceeds 50%, cell growth will be affected.”

2. In answer to the question 3 you have written the following sentences:

Please add these sentences in the discussion appropriately with proper citation.

We have added these sentences in the discussion appropriately. “Because we have done the following experiments, using protease, DNase, and RNase to respectively lyse the protein, DNA, and RNA in the egg white extract, and then conduct our induction experiment, and found that the egg white extract after protein lysis no longer has the ability to reprogram cells, and the chicken protein extract after DNA and RNA lysis still has the ability to reprogram cells. So we proved that the main role of the extract is the protein component (results are not displayed).”

[Note: HTML markup is below. Please do not edit.]

________________________________________

Attachment

Submitted filename: 4-Response to Reviewers.doc

Click here for additional data file.^{(36KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0244160.r007

Decision Letter 3

Nazmul Haque

16 Nov 2020

PONE-D-20-22977R3

Transplantation of Chicken Egg-White Extract-Induced Rabbit PBMCs as a Treatment for Renal Ischemia-Reperfusion Injury in Rabbits

PLOS ONE

Dear Dr. Ruan,

I appreciate your effort in addressing most of the issues raised by the reviewers. However, during this revision I would like to request you to provide proper consideration to the reviewers comments. Specially

1. Please revise the subtitles in the 'results' section and convert them into statements.

2. Please revise the figures too, to make them more communicative.

3. Provide proper explanation regarding data deposition.

Please submit your revised manuscript by 13 December 2020. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Nazmul Haque

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: No

Reviewer #3: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: No

Reviewer #3: (No Response)

**********

6. Review Comments to the Author

Reviewer #2: I would like to thank the editor for providing another opportunity to review the revised version of this current manuscript. I expected that there would be a significant change in the revised manuscript. However, it was not so. The following points are major issues to me –

The weakest part of this manuscript is the arrangement of their results. In the result section, they used 11 sub-headings to represent their results. Do any of the sub-headings represent a result? They used the experiment names only. Interpretation of result shows the merit of any research. The current format of this manuscript is very hard to understand for general readers like me.

Another drawback is the arrangement of figures. It seems to me that the authors are very reluctant to rearrange their figures. The texts of several figures are very small and faint. If I can not read it then how come I comment? The author answered that the figure will be clearer if the picture will open in illustration software. However, they can easily increase the size of their texts for better viewing. I am very sorry to say that the current formats of their Figures are not up to the mark. In Figure 3A-3D, it is very hard to detect the color. The author can easily make it visible by adjusting brightness of the picture. Anyway, it is author’s choice. But the point is that they must need to provide improve figures to think forward.

Data deposition is another major issue to me. The author was asked to deposit the metabolomics data to any open resource. They reply that they will deposit it. But when? After acceptance? They need to provide the accession number in this manuscript. However, the authors mentioned that all relevant data are within the manuscript, which is not true. This activity is against the PLoS guideline.

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

PLoS One. 2020 Dec 28;15(12):e0244160. doi: 10.1371/journal.pone.0244160.r008

Author response to Decision Letter 3

24 Nov 2020

PONE-D-20-22977R3

Transplantation of Chicken Egg-White Extract-Induced Rabbit PBMCs as a Treatment for Renal Ischemia-Reperfusion Injury in Rabbits

PLOS ONE

Dear Dr. Ruan,

1. Please revise the subtitles in the 'results' section and convert them into statements.

We have revised the titles of the subsections in the 'Results' section and converted them into statements.

2. Please revise the figures too, to make them more communicative.

We have revised the figures to better convey the information.

3. Provide proper explanation regarding data deposition.

We have deposited the data in protocols.io to enhance the reproducibility of our results. The DOI link is dx.doi.org/10.17504/protocols.io.bpyrmpv6.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We have uploaded our figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool and ensured that the figures meet the PLOS requirements.

We have deposited the data in protocols.io to enhance the reproducibility of our results. The DOI link is dx.doi.org/10.17504/protocols.io.bpyrmpv6.

We look forward to receiving your revised manuscript.

Kind regards,

Nazmul Haque

Academic Editor

PLOS ONE

Dear Editor,

Thank you very much for your letter and advice. We have revised the paper and would like to resubmit it for your consideration. We have addressed the reviewers’ comments, and the manuscript has been edited by American Journal Experts.

We hope that our revised manuscript is acceptable for publication and look forward to hearing from you at your earliest convenience.

Best wishes,

Guang-ping Ruan

We would like to express our sincere appreciation to the reviewers for their constructive and positive comments.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

________________________________________

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Partly

Reviewer #3: Yes

________________________________________

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

________________________________________

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: No

Reviewer #3: (No Response)

________________________________________

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: No

Reviewer #3: (No Response)

The manuscript has been edited by American Journal Experts again.

________________________________________

6. Review Comments to the Author

Responses to Reviewer 2

We have revised the description and interpretation of the results to show the merits of our research. The current format of this manuscript allows its easy comprehension by general readers.

We have adjusted the brightness of Figs 3A-3D and uploaded Figs 3A-3D and Figs 1E-1M separately. We have uploaded our figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool and ensured that the figures meet the PLOS requirements.

We have deposited the data in protocols.io to enhance the reproducibility of our results. The DOI link is dx.doi.org/10.17504/protocols.io.bpyrmpv6.

Reviewer #3: (No Response)

________________________________________

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

We have uploaded our figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool and ensured that the figures meet the PLOS requirements.

________________________________________

Attachment

Submitted filename: 6-Response to Reviewers.doc

Click here for additional data file.^{(49.5KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0244160.r009

Decision Letter 4

Nazmul Haque

27 Nov 2020

PONE-D-20-22977R4

Transplantation of Chicken Egg White Extract-Induced Rabbit PBMCs as a Treatment for Renal Ischemia-Reperfusion Injury in Rabbits

PLOS ONE

Dear Dr. Ruan,

Specifically, please revise the subheadings of the results section according to the suggestions given below in the Additional Editor Comments (if provided) section.

Please submit your revised manuscript by 11 December 2020. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Nazmul Haque

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The authors have addressed almost all the issues. However, the following issues needed to be resolved before further consideration of this manuscript for publication.

1. Please revise the subheadings of the results section carefully. Few subheading are too long to consider as subheadings. Please revise the subheadings following the examples given below:

"TGF-β immunohistochemical analyses showed that the IOD of the induced cell groups was significantly reduced." change to "The IOD of the induced cell groups was significantly reduced:"

"Masson’s trichrome staining showed that fibrosis was improved in the induced group" change to "Fibrosis was improved in the induced group:"

"Renal PAS staining showed that the basement membrane did not display significant thickening in the induced group." change to "Thickening of the basement membrane was not observed in the induced group:"

"The results from renal tubular epithelial cell immunofluorescence showed that the transplanted cells were differentiated into tubular epithelial cells." change to "Transplanted cells differentiated into tubular epithelial cells:"

"The analysis of renal metabolomics pathways showed that the pyrimidine metabolism and phenylalanine, tyrosine and tryptophan biosynthesis pathways were significant." change to "Significant changes in the pyrimidine metabolism and phenylalanine, tyrosine and tryptophan biosynthesis pathways were observed"

[Note: HTML markup is below. Please do not edit.]

PLoS One. 2020 Dec 28;15(12):e0244160. doi: 10.1371/journal.pone.0244160.r010

Author response to Decision Letter 4

29 Nov 2020

PONE-D-20-22977R4

Transplantation of Chicken Egg White Extract-Induced Rabbit PBMCs as a Treatment for Renal Ischemia-Reperfusion Injury in Rabbits

PLOS ONE

Dear Dr. Ruan,

Specifically, please revise the subheadings of the results section according to the suggestions given below in the Additional Editor Comments (if provided) section.

We have revised the subheadings of the results section according to the suggestions given below in the Additional Editor Comments (if provided) section.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We have deposited our laboratory protocols in protocols.io to enhance the reproducibility of our results. The DOI link is dx.doi.org/10.17504/protocols.io.bpyrmpv6.

We look forward to receiving your revised manuscript.

Kind regards,

Nazmul Haque

Academic Editor

PLOS ONE

Dear Editor,

We hope that our revised manuscript is acceptable for publication and look forward to hearing from you at your earliest convenience.

Best wishes,

Guang-ping Ruan

Additional Editor Comments (if provided):

The authors have addressed almost all the issues. However, the following issues needed to be resolved before further consideration of this manuscript for publication.

1. Please revise the subheadings of the results section carefully. Few subheading are too long to consider as subheadings. Please revise the subheadings following the examples given below:

We have revised the subheadings of the results section carefully. We have revised the subheadings following the examples given below.

"TGF-β immunohistochemical analyses showed that the IOD of the induced cell groups was significantly reduced." change to "The IOD of the induced cell groups was significantly reduced:"

"Masson’s trichrome staining showed that fibrosis was improved in the induced group" change to "Fibrosis was improved in the induced group:"

[Note: HTML markup is below. Please do not edit.]

We have uploaded our figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool and ensured that the figures meet the PLOS requirements.

________________________________________

Attachment

Submitted filename: 7-Response to Reviewers.doc

Click here for additional data file.^{(37KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0244160.r011

Decision Letter 5

Nazmul Haque

4 Dec 2020

Transplantation of Chicken Egg White Extract-Induced Rabbit PBMCs as a Treatment for Renal Ischemia-Reperfusion Injury in Rabbits

PONE-D-20-22977R5

Dear Dr. Ruan,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Nazmul Haque

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0244160.r012

Acceptance letter

Nazmul Haque

9 Dec 2020

PONE-D-20-22977R5

Transplantation of Chicken Egg White Extract-Induced Rabbit PBMCs as a Treatment for Renal Ischemia-Reperfusion Injury in Rabbits

Dear Dr. Ruan:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Nazmul Haque

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig

(TIF)

Click here for additional data file.^{(311.3KB, tif)}

S2 Fig

(TIF)

Click here for additional data file.^{(311KB, tif)}

S3 Fig

(PPTX)

Click here for additional data file.^{(3.9MB, pptx)}

Attachment

Submitted filename: 2-Response to Reviewers.doc

Click here for additional data file.^{(88KB, doc)}

Attachment

Submitted filename: 3-Response to Reviewers.doc

Click here for additional data file.^{(8.4MB, doc)}

Attachment

Submitted filename: 4-Response to Reviewers.doc

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Data Availability Statement

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PERMALINK

Transplantation of chicken egg white extract-induced rabbit PBMCs as a treatment for renal ischemia-reperfusion injury in rabbits

Guang-ping Ruan

Xiang Yao

Qing-keng Lin

Zi-an Li

Xue-min Cai

Rong-qing Pang

Xing-hua Pan

Roles

Abstract

Introduction

Materials and methods

1. Preparation of the rabbit model of renal ischemia-reperfusion injury

2. Rabbit PBMC isolation, culture, induction and labeling

3. Identification of noninduced and induced PBMCs

3.1. Quantitative PCR-based detection of the relative expression of pluripotency-related and somatic cell genes

Table 1. Primer sequences used in the quantitative PCR-based detection of pluripotency-related and somatic genes and lengths of the products.

3.2. Flow cytometry

3.3. Immunohistochemical detection

3.4. Western blot detection

3.5. Quantitative PCR detection of relative changes in the telomere length

Table 2. Primer sequences for telomeres and the internal control 36B4.

4. Kidney function test

5. Changes in the urinary protein content

6. Observation of labeled cells in frozen sections

7. Histopathological assessment of the kidneys

8. Metabolomics analysis of the kidney

9. Statistical analysis

Results

1. Successful labeling of PBMCs with CFSE

Fig 1. CFSE-labeled PBMCs display yellow-green fluorescence.

2. Identification of PBMCs after induction

2.1. Relative expression levels of multipotency-related and somatic cell genes in noninduced and induced PBMCs

2.2. The pluripotency factor of induced PBMCs was significantly higher than that of noninduced PBMCs

2.3. Immunohistochemical analyses yielded positive results for the induced PBMCs

Fig 2. Immunohistochemical analysis of noninduced and induced PBMCs.

2.4. Western blot analyses yielded positive results for induced PBMCs

2.5. The relative telomere lengths were significantly longer in induced PBMCs

3. The serum urea nitrogen and creatinine levels were decreased in the induced cell treatment group

4. The urinary protein concentrations were decreased in the induced group

5. Labeled cells were detected in the induced group

Fig 3. Observations of labeled cells in frozen kidney sections.

6. The structure of the kidney of the induced group exhibited a normal phenotype

7. The IOD of the induced cell groups was significantly reduced

8. Fibrosis was improved in the induced group

Fig 4. Masson’s trichrome staining of kidney sections from the four groups after the three treatments.

9. Thickening of the basement membrane was not observed in the induced group

10. Transplanted cells differentiated into tubular epithelial cells

11. The identified substances were increased in the model group and decreased after treatment

Fig 5. Results of the metabolomics analysis.

11. Significant changes in the pyrimidine metabolism and phenylalanine, tyrosine and tryptophan biosynthesis pathways were observed

Fig 6. Analysis of metabolic pathways in the four groups after treatment.

Discussion

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Nazmul Haque

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Author response to Decision Letter 0

Decision Letter 1

Nazmul Haque

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Author response to Decision Letter 1

Decision Letter 2

Nazmul Haque

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Author response to Decision Letter 2

Decision Letter 3

Nazmul Haque

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Author response to Decision Letter 3

Decision Letter 4

Nazmul Haque

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Author response to Decision Letter 4