Irisin ameliorates myocardial ischemia-reperfusion injury by modulating gut microbiota and intestinal permeability in rats

Qingqing Liu; Yu Zhu; Guangyao Li; Tiantian Guo; Mengtong Jin; Duan Xi; Shuai Wang; Xuezhi Liu; Shuming Guo; Hui Liu; Jiamao Fan; Ronghua Liu

doi:10.1371/journal.pone.0291022

. 2023 Sep 1;18(9):e0291022. doi: 10.1371/journal.pone.0291022

Irisin ameliorates myocardial ischemia-reperfusion injury by modulating gut microbiota and intestinal permeability in rats

Qingqing Liu ^1,^2,^‡, Yu Zhu ^1,^2,^3,^‡, Guangyao Li ^1,², Tiantian Guo ^1,², Mengtong Jin ^1,², Duan Xi ¹, Shuai Wang ¹, Xuezhi Liu ^1,^2,⁴, Shuming Guo ^1,², Hui Liu ^1,^2,⁴, Jiamao Fan ^1,^2,^5,^*, Ronghua Liu ^1,^2,^*

Editor: Pradeep Dudeja⁶

PMCID: PMC10473488 PMID: 37656700

Abstract

Recently, myocardial ischemia-reperfusion (I/R) injury was suggested associated with intestinal flora. However, irisin has demonstrated beneficial effects on myocardial I/R injury, thus increasing interest in exploring its mechanism. Therefore, whether irisin interferes in gut microbiota and gut mucosal barrier during myocardial I/R injury was investigated in the present study. Irisin was found to reduce the infiltration of inflammatory cells and fracture in myocardial tissue, myocardial enzyme levels, and the myocardial infarction (MI) area. In addition, the data showed that irisin reverses I/R-induced gut dysbiosis as indicated by the decreased abundance of Actinobacteriota and the increased abundance of Firmicutes, and maintains intestinal barrier integrity, reduces metabolic endotoxemia, and inhibits the production of proinflammatory cytokines interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α). Based on the results, irisin could be a good candidate for ameliorating myocardial I/R injury and associated diseases by alleviating gut dysbiosis, endothelial dysfunction and anti-inflammatory properties.

1. Introduction

Acute myocardial infarction (AMI) is a common cardiac emergency associated with high rates of morbidity and mortality [1]. For patients with ST-elevation MI (STEMI), thrombolytic/fibrinolytic therapy or percutaneous coronary intervention (PCI) is considered the effective reperfusion strategy that should be performed [2]. Because reperfusion can induce excessive production of reactive oxygen species, excessive inflammatory response and cell apoptosis, the process is known as myocardial ischemia-reperfusion (I/R) injury [3]. Consequently, avoiding the occurrence of myocardial I/R injury is important for the treatment of ischemic heart disease. However, an effective therapy and potential target to prevent myocardial I/R injury in patients does not yet exist.

Although the I/R mechanism is not yet clearly established, the gut microbiota appears to play an important role in the development as shown in an increasing number of studies [4–6]. Vancomycin decreased heart’s susceptibility to injury in an in vivo animal model of regional myocardial I/R by reducing the abundance of gut microbiota [7]. In patients with STEMI, the systolic function of the heart decreases, resulting in insufficient blood supply to systemic organs including the intestine, leading to dysbiosis and changes in intestinal permeability [8]. However, the increase in intestinal permeability leads to the translocation of bacterial endotoxins into the blood, which contributes to a systemic inflammatory response [9]. Therefore, we hypothesized that changes in gut microbiota and intestinal permeability were associated with the occurrence of adverse cardiovascular events after myocardial I/R injury.

Irisin is a circulating hormone that is cleaved from the precursor protein fibronectin type III domain containing 5 (FNDC5) [10]. Increasing evidence has shown that irisin has beneficial effects on cardiovascular diseases [11–13]. Furthermore, in our previous studies, irisin treatment was confirmed to modulate the mitochondrial function via the AMPK pathway, ultimately protecting the H2C9 cardiomyocytes from hypoxia and reoxygenation injury [14]. In addition, irisin showed potential to alleviate intestinal inflammation by altering the gut microbiota [15–17]. Irisin could also restore gut barrier function via the integrin αVβ5-AMPK-UCP 2 pathway [18]. Therefore, in the present study, the effects of irisin on intestinal bacteria and intestinal barrier were evaluated in the rat model of myocardial I/R injury.

2. Materials and methods

2.1 I/R rat model establishment and treatment

Adult male Wistar rats weighing 230–250 g were purchased from the Laboratory Animal Center of Shanxi Provincial People’s Hospital (Taiyuan, China). The rats were housed under a 12 h light/dark cycle at 23 ± 2°C with 40%–60% humidity, and they had free access to standard food and water. All animal experimental procedures were approved by the ethical committee of LinFen Central Hospital (Permit Number: 2021-29-1).

After 1 week of adaptation, all rats were randomly divided into three groups, namely, a sham-operated group (Sham, n = 10), a myocardial ischemia reperfusion injury group (I/R, n = 10), and an irisin group (Irisin, n = 10). The rats in the Sham and I/R groups were intraperitoneally injected with 0.2 mL of PBS, while those in the Irisin group were intraperitoneally injected with 0.2 mL of irisin (100 mg/kg, Sigma, USA, SRP8039) [19]. Treatment was continued for 7 days. The myocardial I/R injury model was induced in the Irisin and I/R groups according to the previously described procedure (S1 Fig) [20]. The rats were intraperitoneally injected with sodium pentobarbital (50 mg/kg) and intubated with a small-animal ventilator (Shanghai Yuyan Instruments Co., Ltd., China) set at a respiratory rate of 60–70 breaths per minute. The surgical area was disinfected, the left chest was opened at the third intercostal space to expose the heart, and the pericardium was separated to exteriorize the heart. The left anterior descending coronary artery was quickly ligated with 6.0 prolene suture for 30 min, after which the suture was removed for reperfusion for 120 min. Meanwhile, the rats underwent electrocardiography (ECG; Shanghai Yuyan Instruments Co., Ltd., China) with limb lead II tracing during the operation. Previous studies revealed that ischemia for 30 min would lead to significant elevation of the ST segment of ECG, and successful model establishment could be indicated by a decrease of the ST segment by at least 50% following 120 min of reperfusion (S2 Fig). Meanwhile, the sham operation included all procedures except ligation of the left anterior descending coronary artery.

2.2 Histopathological and 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) staining

The heart, colon, and ileum tissues were fixed in 4% buffered paraformaldehyde for 48 h and then embedded in paraffin. The sections (5 μm) were mounted on slides, deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol, and subjected to hematoxylin and eosin (H&E) staining. The histological score was determined as previously described [21, 22].

To measure the area of MI, 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) (Solarbio, China, T8170) staining was used. The left ventricle was cut transversely into six sections of the same thickness and stained with 2% TTC at 37°C for 30 min without exposure to light. After staining, normal areas of the myocardium were stained red and infarcted areas were left unstained.

2.3 TUNEL and immunofluorescence (IF) staining

The sections (5 μm) were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. A TUNEL kit (Beyotime, China, C1088) was used for TUNEL staining according to the manufacturer’s instructions. Apoptotic rate was counted as previously described [23]. The sections were submerged into Tris-ethylenediaminetetraacetic acid antigenic retrieval buffer and heated for 5 min by pressure cooker. The sections were then treated with 3% hydrogen peroxide in methanol, blocked with 5% bovine serum albumin, and incubated with anti-zonula occludens-1 (ZO-1, 1: 1000, Abcam, UK, ab221546) and anti-occludin antibodies (1: 200, Abcam, UK, ab216327) overnight at 4°C followed by incubation with Alexa Fluor 594-conjugated secondary antibodies (1: 500, Bioss, China, bs-0295G-AF594). Nuclear staining was performed using 4’,6-diamidino-2-phenylindole (DAPI, BOSTER, China, AR1176). An Olympus inverted fluorescence microscope was used to observe section staining. The images were evaluated using ImageJ software.

2.4 Serum cardiac troponin I (cTnI), creatine phosphokinase (CK), lipopolysaccharide (LPS) and Zonulin measurements

Rats were sacrificed and their blood serum centrifuged. Serum levels of cardiac troponin I (cTnI), creatine phosphokinase (CK), lipopolysaccharide (LPS) and Zonulin were determined using the cTnI ELISA kit (Solarbio, China, SEKR-0048), CK ELISA kit (Solarbio, China, BC1145), LPS ELISA kit (Signalway Antibody, USA, EK3762) and Zonulin ELISA kit (Jianglaibio, China, JL45867) according to the manufacturer’s instructions.

2.5 Western blot analysis

Colon and ileum tissues (0.5 g) were lysed on ice with 500 μL of RIPA lysis buffer for 30 min in the presence of protease and phosphatase inhibitors and then sonicated for 1 min at 60 Hz. After centrifugation at 12,000 rpm for 15 min at 4°C, the supernatant was harvested. Protein concentrations were determined with the BCA protein assay kit (Solarbio, China, PC0020) and the protein was thermally denatured at 100°C for 10 min. The protein was then isolated using sodium dodecyl sulfate polyacrylamide gels and transferred to nitrocellulose membranes. After blocking the membranes with skim milk powder, the membranes were incubated overnight at 4°C with rabbit anti-mouse primary antibodies including interleukin 1β (IL-1β, 1: 1000, Abcam, UK, ab283818), interleukin 6 (IL-6, 1: 1000, CST, USA, 12912T), tumor necrosis factor α (TNF-α, 1: 1000, CST, USA, 11948T), ZO-1 (1: 1000, Abcam, UK, ab221546); occludin (1: 1000, Abcam, UK, ab216327), and β-actin (1: 1000, CST, USA, 37000T). After washing with TBST, the membranes were incubated with goat anti-rabbit secondary antibody (1: 3000, CST, USA, 7074) at 37°C for 1 h followed by incubation with a chemiluminescent substrate for visual detection using the Tanon imaging system. ImageJ software was used to calculate the integrated optical density (IOD).

2.6 Extraction of fecal DNA and high-throughput sequencing

The cecal contents of the rats were collected in sterile tubes and stored in a refrigerator at -80°C. DNA in the cecal contents was extracted using a DNA extraction kit (Accurate, China, 37000T) and the quality of DNA extraction was determined using 0.8% agarose gel electrophoresis and quantified with a UV spectrophotometer. The 16S rRNA hypervariable region (V3-V4) was amplified with PCR using the extracted DNA as a template. The primer sequences were ACTGCATCCGCAGCGTCGA and CCTGTACGGTCTTGCATAT. The PCR products were detected using 2% agarose gel electrophoresis and purified with AXYGEN gel extraction kit. The preliminary quantification results of electrophoresis were obtained by fluorescing the PCR-amplified products using the Quant-iT PicoGreen dsDNA assay kit (Thermo Fisher Scientific, USA, P7589). Based on the quantitative results, purified amplicons were pooled in equimolar amounts and paired-end sequenced on an Illumina MiSeq PE300 platform/NovaSeq PE250 platform (Illumina, USA) according to Majorbio Bio-Pharm Technology Co. Ltd. (China) standard protocols.

2.7 Bioinformatic analysis

To minimize the effects of sequencing depth on α and β diversity measures, the number of 16S rRNA gene sequences from each sample were rarefied to 20,000, which still yielded an average Good’s coverage of 99.09%. Bioinformatic analysis of the gut microbiota was performed using the Majorbio Cloud platform (https://cloud.majorbio.com). Based on the operation taxonomic unit (OTU) information, rarefaction curves and α diversity indices, including observed OTUs, Chao1 richness, Shannon index, and Good’s coverage were calculated using Mothur v1.30.1. The similarity among the microbial communities in different samples was determined using principal component analysis (PCA), principal coordinate analysis (PCoA), and partial least squares discrimination analysis (PLS-DA) based on Bray–Curtis dissimilarity using Vegan v2.5–3 package. The PERMANOVA test was used to assess the percentage of variation explained by the treatment with its statistical significance using Vegan v2.5–3 package. The linear discriminant analysis (LDA) effect size (LEfSe; http://huttenhower.sph.harvard.edu/LEfSe) was performed to identify the significantly abundant taxa (phylum to genera) of bacteria among the different groups (LDA > 4.00, p < 0.05). Spearman correlation analyses were used to assess correlations between the 10 top genus and blood parameters, the infarct area, colon and ileum barrier function, degree of bacterial translocation, and inflammatory response.

2.8 Statistical analysis

The data are presented as the mean ± standard error of mean (SEM) of five or more independent experiments. Normal distribution was confirmed using the Shapiro–Wilk test. One-way analysis of variance (ANOVA), least significant difference (LSD), or Tamhane test were used to compare the statistical differences among multiple groups. A p-value < 0.05 was considered statistically significant. SPSS 22.0 was used for statistical analysis.

3. Results

3.1 Effects of irisin on gut microbial diversity and richness

To characterize the microbial populations in the rat gut, the bacterial populations were measured using 16S rRNA gene sequencing. The rarefaction curves of all samples indicated that the sequencing coverage was sufficient to reflect the composition of intestinal flora (S3A Fig). Rank-Abundance curves showed that species were evenly distributed (S3B Fig). In gut microbial α diversity, both richness and evenness were indicated based on the Chao, ACE, Simpson, and Shannon indices. In I/R rats, ACE index and Shannon index increased and Simpson index decreased, and irisin completely restored these effects (Fig 1A).

Principal component analysis (PCA), principal coordinate analysis (PCoA), and partial least squares discrimination analysis (PLS-DA) were used to measure the difference between microbial communities. The aggregation of the flora in the I/R group significantly stayed away from Sham and Irisin groups, and the gut microbial community structure was similar between Sham and Irisin group (Fig 1B). Notably, the difference of the rat microbial community composition was small in the Irisin and the Sham groups.

Among 711 OTUs, 544 of the total richness were shared among all groups, and OTUs were observed between two groups or in each group (Fig 1C). In addition, irisin treatment decreased OTUs in the I/R rats.

These data indicated that irisin treatment significantly improved α and β diversity of intestinal microbiota.

3.2 Effects of irisin on the gut microbiota composition

Based on the results, gut microbiota was significantly changed. Therefore, the gut microbiota composition was compared among the three groups to identify potential probiotics or harmful bacteria of irisin intervention after I/R.

At the phylum level, I/R affected the relative abundance of Firmicutes and Actinobacteriota. Notably, irisin treatment significantly increased the relative abundance of Firmicutes and decreased the relative abundance of Actinobacteriota (Fig 2A).

Fig 2 — A. Relative abundance of the major microbial phyla; B. Relative abundance of the top 10 different families; C. Relative abundance of the top 10 different genera; D. Relative abundance of the top 10 different species. Data are expressed as means ± standard error of the mean (SEM; n = 11 in each group).*p < 0.05; **p < 0.01; ***p < 0.001.

The top 10 families were significantly affected by I/R; several families (Corynebacteriaceae, Bifidobacteriaceae, Staphylococcaceae, Aerococcaceae, Akkermansiaceae, and Carnobacteriaceae) were drastically increased and others (Peptostreptococcaceae and Monoglobaceae) decreased. However, irisin significantly increased Peptostreptococcaceae and Monoglobaceae but decreased Corynebacteriaceae, Bifidobacteriaceae, Staphylococcaceae, Aerococcaceae, Akkermansiaceae, and Carnobacteriaceae to some extent (Fig 2B).

Next, the top 10 genera were analyzed. In the I/R group, several genera (Corynebacterium, norank_f__Erysipelotrichaceae, Bifidobacterium, Staphylococcus, and Jeotgalicoccus) were significantly increased, however, others (Romboutsia, Turicibacter, and Monoglobus) were markedly decreased. In contrast, irisin treatment significantly increased Romboutsia, Turicibacter, and Monoglobus but markedly reduced Corynebacterium, norank_f__Erysipelotrichaceae, Bifidobacterium, Staphylococcus, and Jeotgalicoccus (Fig 2C).

The relative abundance of 10 species was different in I/R-induced rats, with an increase of seven species (Lactobacillus_johnsonii, uncultured_Allobaculum_sp._g__norank, Bifidobacterium_animalis, Corynebacterium_stationis, uncultured_bacterium_g__Dubosiella, Staphylococcus_lentus_g__Staphylococcus, and uncultured_bacterium_g__Jeotgalicoccus) and a decrease of three species (Romboutsia_ilealis, uncultured_bacterium_g__Turicibacter, and gut_metagenome_g__Lactobacillus). In the irisin group, an increase of three species (Romboutsia_ilealis, uncultured_bacterium_g__Turicibacter, and gut_metagenome_g__Lactobacillus) and a decrease of six species (uncultured_Allobaculum_sp._g__norank, Bifidobacterium_animalis, Corynebacterium_stationis, uncultured_bacterium_g__Dubosiella, Staphylococcus_lentus_g__Staphylococcus, and uncultured_bacterium_g__Jeotgalicoccus) was observed (Fig 2D).

The bacterial composition with significant differences among the sham, I/R, and irisin groups was analyzed using LDA-LEfSe (Fig 3). In the sham group, g__Turicibacter and s__uncultured_bacterium_g__Turicibacter played critical roles and may be considered biomarkers. In addition, p__Actinobacteriota, c__Actinobacteria, f__Bifidobacteriaceae, g__Bifidobacterium, o__Bifidobacteriales, s__Bifidobacterium_animalis, g__Dubosiella, s__uncultured_bacterium_g__Dubosiella, s__uncultured_Allobaculum_sp__g__norank, and g__norank_f__Erysipelotrichaceae had an important function and may be used as biomarkers in the I/R group, and g__Romboutsia, s__Romboutsia_ilealis, f__Peptostreptococcaceae, o__Peptostreptococcales-Tissierellales, and p__Firmicutes played a crucial part and may be considered biomarkers in the irisin group.

3.3 Effects of irisin on the gut microbiota function phenotype

The gut flora performs basic physiological functions in the host and participates in regulating body’s homeostasis and health. Therefore, BugBase was employed to predict the functional potential of bacteria. Then, from our research results, it was found that irisin treatment affected Biofilm Forming, Gram Positive, Gram Negative, Pathogenic Potential, Mobile Element Containing, Oxidative Stress Tolerant and Oxygen Utilizing (S4 Fig). Gram negative and Biofilm forming bacteria were more abundant in the I/R group. However, these effects were dramatically restored by irisin (S4 Fig). Concluding, irisin treatment improved gut microbiota function phenotype.

3.4 Irisin maintains intestinal integrity in I/R rats

The gut epithelial integrity is considered the first line of defense of the gastrointestinal tract. Intestinal dysbiosis in I/R animals may affect gut permeability and subsequently lead to release of potentially harmful bacterial metabolites into the blood [24]. In the current study, I/R dramatically increased intestinal permeability and damaged the intestinal mucosa (Figs 4 and 5) but were restored with irisin treatment.

Fig 5 — A. Hematoxylin and eosin (H&E) staining of ileum sections; B. Representative fluorescent pictures of TUNEL staining of ileum sections. The apoptotic cells were detected by TUNEL (green); C. Immunofluorescence staining of the infiltration of zonula occludens-1 (ZO-1, red) and occludin (red) in ileum tissue sections; D. Histological scores for ileum sections (n = 6); E. Apoptosis rate in ileum sections (n = 6); F. Quantification of staining intensity of ZO-1 and occludin in each group (n = 6); G. ZO-1 and occludin expression in the ileum tissue from each group were evaluated using western blotting. Data are expressed as means ± standard error of the mean (SEM). *p < 0.05; **p < 0.01; ***p < 0.001.

The colon tissue of representative rats from each group showed the I/R group exhibited inflammatory cell infiltration (Fig 4A). Notably, the ileum villi arrangement was loose and disordered was observed in the I/R group (Fig 5A). In view of the obvious pathological changes of colon and ileum in I/R rats, the level of apoptosis was assessed by TUNEL staining. Compared to the Sham group, the levels of apoptosis of colon and ileum tissues were significantly increased in the I/R group (Figs 4B and 5B). After treatment with the irisin, the colon and the ileum structure and apoptosis level were alleviated. Inflammatory cell infiltration was decreased in colon tissue (Fig 4A) and the shape of ileum villi was straight finger-like protrusions neatly and densely arranged in the irisin group (Fig 5A).

Because intestinal mucosa damage is closely associated with the expression of epithelial mucosal proteins and tight junction proteins, the expression of ZO-1 and occludin was examined. Irisin significantly increased ZO-1 and occludin expression compared with the I/R group in both colon and ileum tissues (Figs 4C, 4G, 5C and 5G).

These findings indicate that irisin may enhance intestinal barrier integrity in myocardial I/R injury rats.

3.5 Effects of irisin on gut inflammation

Previous studies revealed that serum LPS and Zonulin level was positively correlated with intestinal permeability [24, 25]. The data obtained confirm that myocardial I/R significantly increased intestinal permeability, leading to release of bacterial LPS and Zonulin into the blood, which was restored by irisin treatment (Fig 6A). Leaky gut was shown to produce higher levels of proinflammatory cytokines in colon or ileum tissues, including IL-1β, IL-6, and TNF-α [26]. In the present study, protein expression of these cytokines was measured using western blotting; IL-1β, IL-6, and TNF-α protein expression levels were higher in colon and ileum tissues of the I/R rats compared with the sham rats (Fig 6B and 6C). Notably, the protein expression level of these cytokines was altered by irisin treatment, resulting in the protein expression level similar to the sham rats (Fig 6B and 6C).

Fig 6 — A. Serum LPS and Zonulin concentration in each group detected using ELISA; B. Interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α) expression in the colon and ileum tissue from each group were evaluated using western blotting; C. Quantification of relative protein expression of IL-1β, IL-6 and TNF-α. Data are expressed as means ± standard error of the mean (SEM). *p < 0.05; **p < 0.01; ***p < 0.001.

These results show irisin reduces inflammation in I/R rats by reducing proinflammatory cytokines in colon and ileum tissues.

3.6 Irisin reduces myocardial injury

In previous studies, ischemia for 30 min was shown to lead to significantly elevated ST segment of ECG, which decreased by at least 50% after 120 min reperfusion indicating successful model establishment [6]. The myocardial interstitium showed inflammatory cell infiltration and marked edema accompanied by dissolution, rupture and even necrosis of myocardial fibers in I/R rats [27]. In the current study, irisin treatment significantly reduced the elevated ST segment compared with I/R (S2 Fig). TTC staining was performed to analyze the infarct area. Compared with the I/R group, treatment with irisin notably reduced myocardial I/R-induced infarction (Fig 7A and 7D). In addition, the infiltration of inflammatory cells and edema were reduced in the myocardial interstitium, and the myocardial fibers were intact and neatly arranged in the irisin group (Fig 7B). Fig 7C shows the apoptosis of cardiomyocytes was reduced in the irisin group. Cardiomyocyte necrosis can release a variety of myocardial enzymes to reflect the degree of myocardial injury. The levels of serum myocardial enzymes were increased in I/R group as shown in Fig 7E. Notably, the cTnI and CK levels were altered by irisin treatment, resulting in levels similar to sham rats.

Fig 7 — A. 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) staining of myocardium sections; B. Hematoxylin and eosin (H&E) staining of myocardium sections; C. Tunnel staining of myocardium sections; D. Quantification of the infarct area in TTC staining; E. Serum cardiac troponin I (cTnI) and creatine phosphokinase (CK) concentration in each group detected using ELISA. Data are expressed as means ± standard error of the mean (SEM). *p < 0.05; **p < 0.01; ***p < 0.001.

The data obtained confirm that irisin ameliorates myocardial I/R injury.

3.7 Gut microbiota-associated blood parameters, the myocardial infarct area, colon and ileum barrier function, degree of bacterial translocation, and inflammatory response

Based on heatmap correlation analysis, in the 10 top genus, three genus were notably associated with blood parameters, the myocardial infarct area, colon and ileum barrier function, degree of bacterial translocation, and inflammatory response (Fig 8). In colon and ileum, Turicibacter was positively correlated with tight junction protein ZO-1 and occludin expression but negatively correlated with serum cTnI and CK levels, the myocardial infarct area, HE score, apoptosis rate, inflammatory factors (IL-1β, IL-6, and TNF-α) in colon and ileum and serum LPS and Zonulin concentration (Fig 8). Thus, these bacterial species may inhibit I/R development. In addition, Bifidobacterium and norank_f__Erysipelotrichaceae negatively correlated with tight junction protein ZO-1 and occludin expression in colon and ileum but positively correlated with serum cTnI and CK levels, the myocardial infarct area, HE score, apoptosis rate, inflammatory factors (IL-1β, IL-6 and TNF-α) in colon and ileum, and serum LPS and Zonulin concentration (Fig 8). Thus, these bacterial species may induce I/R. Collectively, these findings demonstrated the three bacterial species play vital roles in myocardial I/R injury.

Fig 8 — *p < 0.05; **p < 0.01; ***p < 0.001.

4. Discussion

Evidence from a compilation of animal and human studies indicates the implications of gut microbiota and their metabolites in cardiovascular diseases are well established [28, 29]. In our previous studies, irisin was shown to protect the H2C9 cardiomyocytes from hypoxia and reoxygenation injury [14]. However, whether crosstalk exists between irisin, gut microbiota and cardioprotection remains unclear. In the present study, the data indicate that myocardial I/R injury is accompanied by intestinal microbiota imbalance. Notably, irisin treatment significantly decreased the abundance of gut microbiota. Furthermore, irisin treatment decreased gut inflammation and maintained the integrity of the intestinal barrier. To the best of our knowledge, this is the first study in which the effects of irisin on intestinal flora under myocardial I/R injury stress were investigated.

We performed 16S rRNA gene sequencing on the gut microbiota. In this study, significant microbiota changes in cecal contents in both I/R and irisin groups were observed. The results were compatible with a recent study in which α diversity reportedly significantly increased in I/R rats [30]. β diversity revealed significant separation of the community compositions between the sham and I/R groups, further confirming the possible close relationship between myocardial I/R injury and microbiota. Notably, the distribution of microbiota was not obvious in I/R rats, which may be due to the difference in the degree of acute stress. Furthermore, irisin prevented acute intestinal stress, thus, creating a microbiota similar to the sham group. Zhou et al. [8] suggested that increased richness and distinct structure of microbiome may be caused by the transportation of intestinal bacteria into the blood of I/R rats. In the present study, the relative abundance of gut bacteria significantly changed at the phylum, family, genera, and species level. LEfSe results were used to analyze the potential pathogenic bacteria such as p__Actinobacteriota, c__Actinobacteria, f__Bifidobacteriaceae, g__Bifidobacterium, o__Bifidobacteriales, s__Bifidobacterium_animalis, g__Dubosiella, s__uncultured_bacterium_g__Dubosiella, s__uncultured_Allobaculum_sp__g__norank, g__norank_f__Erysipelotrichaceae in the I/R rat intestine. Bifidobacteriaceae is a common probiotic that maintains gut microbiota balance [31]. However, the present study results are contradictory. If the relative abundance of probiotics is too high, the balance of bacterial flora may be disrupted and potentially become pathogenic bacteria. Data supporting the role of probiotics in other conditions are often conflicting [32]. A possible explanation for these results is that different gut environments may affect the abundance and composition of gut microbiota. Furthermore, g__Romboutsia, s__Romboutsia_ilealis, f__Peptostreptococcaceae, o__Peptostreptococcales-Tissierellales, and p__Firmicutes played a crucial part and could be considered a potential probiotic in the irisin group. Romboutsia species, such as Romboutsia ilealis [33] and Peptostreptococcaceae [34], could utilize glucose and carbohydrates to generate short-chain fatty acids to promote intestinal barrier integrity. Most Firmicutes are probiotics, except Lactobacillus, and other beneficial bacteria can produce butyrate [35]. In particular, butyrate is important for maintaining health by regulating the immune system and preserving the epithelial barrier [13, 36]. In summary, the findings showed the improvement of myocardial I/R injury due to irisin is closely associated with the changes in intestinal flora.

MI causes ischemic stress such as intestinal hypoperfusion, loss of tight junction protein occludin, intestinal mucosal damage, and increased intestinal permeability [37]. Tight junctions are important in the intestinal mucosal barrier and located at the top of the intestinal epithelium and consist of a number of proteins including ZO-1 and occludin of adjacent intestinal cells [38]. Zonulin is currently the only physiological regulator of intestinal permeability [39]. Upon stimulation by potentially harmful bacteria, Zonulin is released in large quantities into the intestinal lumen, where it binds to the Zonulin receptor. It can down-regulate the expression of tight junction protein, destroy the integrity of tight junction complex, and increase intestinal permeability [40]. In the current study, the structure of the colon and ileum was destroyed, the apoptosis of intestinal epithelial cells was significantly increased, the expression of ZO-1 and occludin was decreased and serum Zonulin levels were increased in the I/R rats. These results indicate that myocardial I/R can increase intestinal permeability in rats, however, irisin treatment can reverse this effect. Irisin possibly increases the abundance of beneficial bacteria in the intestine to maintain intestinal barrier integrity. Gut barrier breakdown leads to bacteria and endotoxin (LPS) translocation into the systemic circulation [41]. Then, low levels of LPS in the blood may activate TLR4 signaling in various cells, causing systemic and targeted inflammation [42–44]. In the present study, serum LPS concentration and the protein expression of inflammatory factors in colon and ileum tissues were determined. The results indicate that irisin inhibited the transfer of LPS from the intestine to the systemic circulation and caused overexpression of inflammatory factors IL-1β, IL-6, and TNF-α in I/R rats. Therefore, the beneficial effects induced by irisin treatment may be attributed to specific changes in the gut microbiota and maintenance of intestinal barrier integrity.

Furthermore, our data indicated that Turicibacter was obviously and negatively correlated with serum cTnI and CK levels, the myocardial infarct area, HE score, apoptosis rate, inflammatory factors (IL-1β, IL-6, and TNF-α) in colon and ileum and serum LPS and Zonulin concentration. Tang et al. found that the gut microbiota was a crucial element necessary for optimal cardiac repair after myocardial infarction (MI). Gut microbiota-derived short-chain fatty acids (SCFAs) alleviate the inflammatory microenvironment after myocardial infarction by regulating the immune system [45]. SCFAs could not only maintain intestinal barrier function, but also regulate immune response to inhibit inflammation [46]. Turicibacter is classified as beneficial bacteria that promote the production of SCFAs [47]. Turicibacter was significantly increased in the Irisin group. In this case, our experiments provided unequivocal evidence for protective role of irisin in myocardial I/R injury and irisin or probiotics supplementation may be an alternative or adjunct therapy for cardiovascular diseases treatment.

The results of this study indicated that intestinal microbiota is involved in irisin alleviating myocardial I/R injury. The effects of gut microbiota and intestinal barrier are likely a part of the mechanism underlying the irisin treatment process. However, the pathway through which irisin affects intestinal microbiota and its effects on metabolites need to be further investigated.

5. Conclusion

Myocardial I/R promotes alterations in gut microbiota that may enhance intestinal permeability and LPS translocation. However, irisin supplementation reversed the changes in gut microbiota, restored intestinal barrier structure, reduced LPS translocation, and decreased the inflammatory response, thus, exerting cardioprotective effects. In conclusion, irisin treatment can be an important tool for preventing and treating patients with myocardial I/R and intestinal flora is the mechanism affecting myocardial I/R injury (Fig 9). The results can be used to develop new prevention and diagnostic strategies to promote the health of patients with myocardial I/R.

Fig 9 — Irisin supplementation restored the intestinal flora and intestinal barrier to inhibit bacteria and lipopolysaccharide (LPS) translocation as well as inhibit the inflammatory response and apoptosis of cardiomyocytes, thus, exerting cardioprotective effects.

Supporting information

S1 Fig. Experimental design.

Timeline of the experimental process of this research treatment and ischemia-reperfusion (I/R) injury induction in rats. (i. p.: intraperitoneal injection).

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S2 Fig. The effect of irisin on the electrocardiogram waveform was evaluated in I/R rats.

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S3 Fig. Sample sequencing evaluation.

(A) Rarefaction curve indicating that the amount of sequencing reads per sample has reached saturation. (B) Rank-Abundance curves were shown.

(TIF)

Click here for additional data file.^{(977.2KB, tif)}

S4 Fig. The gut microbiota function phenotype characterization of different groups based on BugBase analysis.

The relative abundance of Biofilm Forming, Gram Positive, Gram Negative, Pathogenic Potential, Mobile Element Containing, Oxidative Stress Tolerant and Oxygen Utilizing were shown.

(TIF)

Click here for additional data file.^{(1.9MB, tif)}

Abbreviations

I/R: ischemia reperfusion
AMI: acute myocardial infarction
STEMI: ST elevation myocardial infarction
PCI: percutaneous coronary intervention
SPF: specific pathogen-fre
ECG: electrocardiogram
cTnI: Cardiac Troponin I
CK: Creatine phosphokinase
LPS: lipopolysaccharide
H&E: hematoxylin and eosin
IF: immunofluorescence
ZO-1: zonula occludens-1
DAPI: 4’ 6- diamidino- 2- phenylindol
IL-1β: interleukin-1-beta
IL-6: interleukin-6
TNF-α: tumour necrosis factor-alpha
PCA: Principal component analysis
PCoA: principal coordinate analysis
PLS-DA: partial least squares discrimination analysis
LDA: linear discriminant analysis
LEfSe: linear discriminant analysis effect size
ANOVA: One-way analysis of variance

Data Availability

The data that support the findings of this study are openly available in the BioSample database at http://www.ncbi.nlm.nih.gov/bioproject/940075, reference number [PRJNA940075].

Funding Statement

This work was supported by the Scientific research project of Shanxi Provincial Health Commission (2022122) and the Linfen City key research and development plan (2108).

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PLoS One. doi: 10.1371/journal.pone.0291022.r001

Decision Letter 0

Pradeep Dudeja

2 May 2023

PONE-D-23-09095Irisin ameliorates myocardial ischemia-reperfusion injury by modulating gut microbiota and intestinal permeability in ratsPLOS ONE

Dear Dr. Fan,

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Several major concerns were raised by the reviewers. It was felt that while the manuscript has merit, the barrier integrity and tight junction protein data needs to be further strengthened by additional experiments suggested. Also, the presentation and the language of the manuscript needs significant improvement as suggested by the reviewers. Please address each concern of the reviewer in a point by point manner.

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Reviewer #2: Yes

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Reviewer #1: The manuscript presents interesting data and shows the beneficial effects of Irisin in terms of reversing the I/R induced gut dysbiosis, its role in maintaining barrier integrity and its inhibitory effects on production of pro-inflammatory cytokines. Although the experiments were well designed and conducted but certain concerns were noted which significantly reduce the enthusiasm. The following concerns outlined below need to be addressed to improve the overall impact of the work.

Major Concerns:

1. Authors should provide detailed flowchart or diagrammatic representation of rat model establishment and treatment in section 2.1. It is not clear whether the I/R injury was induced in the Irisin treated group or not.

2. In section 2.3 (line 98), authors have mentioned that the sections were submerged into antigen retrieval buffer and heated for 5 min at 121°C in an autoclave. Please elaborate how this heating procedure was done in an autoclave?

3. In section 3.1, PCA, PCoA and PLS-DA plots should be elaborated in more detail.

4. In section 3.4, the results of TUNEL staining should be explained in a detailed manner. The legends for figure 4 should be more comprehensively written.

5. In section 3.4, the expression of ZO-1 and occludin is examined by immunostaining only. Authors should analyze the expression of tight junction proteins at RNA as well as protein levels by real time PCR and western blotting.

6. Authors should show higher magnification (100x) for occluding and ZO1 as the expression of both the proteins does not look like punctated as reported by other studies and recalculate the relative fluorescence intensity for the same. The number of images used for calculating the relative fluorescence intensity should be mentioned in the figure legend.

7. In figure 4 (A, B and C), Figure 5 (A, B and C) and Figure 7 (B and C) scale bars are not clearly visible on the images. The legends for these figures should be written in a more detailed and clear manner.

8. In section 3.6 and 3.7, the results of TTC staining and heat map of correlation analysis should be explained in more detail. Authors should provide references of other studies that have shown the correlation of bacterial species having impact on inhibition or development of I/R or other clinical conditions for supporting their results.

9. In lines 281- 282, authors have mentioned that significant microbiota changes in cecal contents in both I/R and irisin groups were observed. Please provide more details in section 2.6, whether these changes were analyzed in fecal or cecal content.

10. The authors should follow the same format for references, as there are multiple discrepancies in references. DOI for first 22 references are not mentioned in the manuscript.

11. The discussion section of the manuscript should emphasize more on highlighting the key findings of the study in a more extensive manner as well as providing more references for supporting the results.

Minor Concerns:

1. The manuscript needs to be read by a native English speaker as there are number of grammatical errors and punctuation mistakes. The manuscript should be proofread carefully.

2. In line 138, abbreviation of operation taxonomic unit (OTU) is written incorrectly as OUT.

3. In lines 184 and 188, repetition of “Staphylococcus” is observed.

4. Authors should provide the details about the working dilutions of antibodies, catalogue numbers of all the antibodies and kits used in the study.

5. The image quality for S3 Fig. should be improved.

6. The legends for figures 4 and 5 should be written in alphabetical order.

Reviewer #2: The manuscript "Irisin ameliorates myocardial ischemia-reperfusion injury by modulating gut microbiota and intestinal permeability in rats" was well written. The authors designed the appropriate experiments and classically presented the data. Much research documented Irisin and its importance in health diseases well. The authors utilized the available evidence and linked how irisin ameliorated myocardial I/R-mediated gut bacterial dysbiosis and barrier integrity. However, the authors need minor corrections and rewrite some of the result presentations and may need some additional experiments, which will help the manuscript strong for publication so the readers can understand clearly.

1. In the methodology section:

2.1 I/R rat model establishment and treatment. Line 74. The authors mentioned that based on reference 12, they made I/R injuries to the rats. Unfortunately, that reference did not explain the methodologies of creating myocardial I/R injury in rats. The authors requested to refer to the appropriate validated methodology reference.

2. 2.8 Statistical analysis: Line 149. The authors mentioned the data are presented as the mean±standard deviation (SD) in the results. However, none of the results and the figures are displayed with SD instead of Standard Error mean. The authors are requested to change SD to SEM in the statistical analysis section to avoid confusing readers.

3. In the Result section:

From lines 183 to 207: The authors explained that bacterial strains are differentially expressed in the I/R model and reverse in Irisin-treated models. However, the authors repeated the bacterial names, which were hard to read and understand, and reviewed the results. The authors are requested to simplify the presentation in an easily understandable manner.

4. Figure legend section:

Lines 494-512: Figure 4, Figure 5, and Figure 6: The authors did not explain the subsections' (A-F) self-explanatory information in the legends alphabetically. Comparing what is shown in the figures and the figure legends is hard. The authors are requested either change the subsections alphabetically based on the figure legends or figures accordingly.

5. Additional experiments may need to more strongly validate the Irisin mediated reversal of barrier integrity findings. The authors used only LPS levels for blood parameters to validate the barrier dysfunction. The authors are requested to consider to do to measure Zonulin and FITC-Dextran levels in the blood in both experimental models, which will strengthen the manuscript.

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2023 Sep 1;18(9):e0291022. doi: 10.1371/journal.pone.0291022.r002

Author response to Decision Letter 0

31 Jul 2023

Response: Thanks for your suggestion. We have added the detailed flowchart of rat model establishment and treatment in Supporting Information in the revised manuscript.

The detailed flowchart are shown as follows.

S1 Fig. Experimental design. Timeline of the experimental process of this research treatment and ischemia-reperfusion (I/R) injury induction in rats. (i. p.: intraperitoneal injection)

Response: Thank you, the mistake has been corrected. We have revised the sentence as “The sections were submerged into Tris-ethylenediaminetetraacetic acid antigenic retrieval buffer and heated for 5 min by pressure cooker.” in the revised manuscript and highlighted it in red colour.

The sections (5 μm) were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. The Pressure cooker containing the tris-ethylenediaminetetraacetic acid antigenic retrieval buffer was heated to a boil on an electromagnetic stove. The sections were placed in antigen repair solution and capped for heat treatment, which ended 5 minutes after air injection from the pressure cooker valve. Then, the sections were naturally cooled. In general, high-pressure instruments can be heated to a temperature of about 120°C.

3. In section 3.1, PCA, PCoA and PLS-DA plots should be elaborated in more detail.

Response: Thanks for your suggestion. We have revised the “PCA, PCoA, and PLS-DA were used to measure the degree of difference between microbial communities. After treatment with irisin, aggregation of flora was not observed in the I/R rats (Fig 1B).” as “Principal component analysis (PCA), principal coordinate analysis (PCoA), and partial least squares discrimination analysis (PLS-DA) were used to measure the difference between microbial communities. The aggregation of the flora in the I/R group significantly stayed away from Sham and Irisin groups, and the gut microbial community structure was similar between Sham and Irisin group (Fig 1B).”

4.In section 3.4, the results of TUNEL staining should be explained in a detailed manner.

Response: Thanks for your suggestion. We have added more details in section 3.4 and highlighted it in red colour. The legends for figure 4 have been comprehensively written.

Response: Thanks for your suggestion. According to your suggestion, we have supplemented the some experimental results in Fig 4 and Fig 5 in the revised manuscript. We found that there is no statistical difference in the expression of ZO-1 and occludin genes in the colon and ileum by real time PCR. Nevertheless, protein expression of ZO-1 and occludin was increased in the Irisin group by immunofluorescence and Western Blotting. We analyzed that irisin might enhance the expression of ZO-1 and occludin proteins through acting on transcription or translation processes.

Response: Thanks for your suggestion. Our team is relatively mature in the detection of ZO-1 and occludin protein expression and does not show punctate distribution [1-2]. The expression of ZO-1 and occludin proteins showed a zonal distribution. Figures of ZO-1 and occludin protein expression in this manuscript showed a magnification of 100×. Scale bars were added clearly on the images. Meanwhile, the number of images used for calculating the relative fluorescence intensity have be mentioned in the revised manuscript.

[1] Liu Q, Yang H, Kang X, et al. A Synbiotic Ameliorates Con A-Induced Autoimmune Hepatitis in Mice through Modulation of Gut Microbiota and Immune Imbalance. Mol Nutr Food Res. 2023 Apr;67(7):e2200428. doi: 10.1002/mnfr.202200428.

[2] Liu Q, Tian H, Kang Y, et al. Probiotics alleviate autoimmune hepatitis in mice through modulation of gut microbiota and intestinal permeability. J Nutr Biochem. 2021 Dec;98:108863. doi: 10.1016/j.jnutbio.2021.108863.

Response: Thanks for your suggestion. Scale bars have been added clearly on the images.

Response: Thanks for your suggestion. We have added more details in Discussion and highlighted it in red colour.

We have performed a correlation analysis between TTC staining results and the 10 top genus (Fig. 8). Our data indicated that Turicibacter was obviously and negatively correlated with serum cTnI and CK levels, the myocardial infarct area, HE score, apoptosis rate, inflammatory factors (IL-1β, IL-6, and TNF-α) in colon and ileum and serum LPS and Zonulin concentration. Tang et al. found that the gut microbiota is a crucial element necessary for optimal cardiac repair after myocardial infarction (MI). Gut microbiota-derived short-chain fatty acids (SCFAs) alleviate the inflammatory microenvironment after myocardial infarction by regulating the immune system [1]. SCFAs could not only maintain intestinal barrier function, but also regulate immune response to inhibit inflammation [2]. Turicibacter is classified as beneficial bacteria that promote the production of SCFAs [3]. Turicibacter was significantly increased in the Irisin group. In this case, our experiments provided unequivocal evidence for protective role of irisin in myocardial I/R injury and irisin or probiotics supplementation may be an alternative or adjunct therapy for cardiovascular diseases treatment.

[1]Tang TWH, Chen HC, Chen CY, et al. Loss of Gut Microbiota Alters Immune System Composition and Cripples Postinfarction Cardiac Repair. Circulation. 2019 Jan 29;139(5):647-659. doi: 10.1161/CIRCULATIONAHA.

[2]Parada Venegas D, De la Fuente MK, Landskron G, et al. Short Chain Fatty Acids (SCFAs)-Mediated Gut Epithelial and Immune Regulation and Its Relevance for Inflammatory Bowel Diseases. Front Immunol. 2019 Mar 11;10:277. doi: 10.3389/fimmu.2019.00277.

[3]Liu X, Zhang Y, Li W, et al. Fucoidan Ameliorated Dextran Sulfate Sodium-Induced Ulcerative Colitis by Modulating Gut Microbiota and Bile Acid Metabolism. J Agric Food Chem. 2022 Nov 30;70(47):14864-14876. doi: 10.1021/acs.jafc.2c06417.

Fig 8. The relationship among blood parameters, the infarct area,colon and ileum barrier function, degree of bacterial translocation and inflammatory response, and the 10 top genus estimated using Spearman correlation analysis. *p < 0.05; **p < 0.01; ***p < 0.001

Response: Thanks for your suggestion. We have added more details in section 2.6 and highlighted it in red colour.

10. The authors should follow the same form at for references, as there are multiple discrepancies in references. DOI for first 22 references are not mentioned in the manuscript.

Response: We have adapted the format of the references to be consistent in the revised manuscript.

Response: Thanks for your suggestion. We have added more details in Discussion and highlighted it in red colour.

Minor Concerns:

1. The manuscript needs to be read by a native English speaker as there are number of grammatical errors and punctuation mistakes. The manuscript should be proofread carefully.

Response: Thanks for your suggestion. Before the first submission, we had finished polishing the language. We again thoroughly checked for the usage of the English language, typos and syntax and grammar errors carefully, and all the changes have been highlighted in red.

2. In line 138, abbreviation of operation taxonomic unit (OTU) is written incorrectly as OUT.

Response: Thank you for your suggestion, OUT has been revised in the revised manuscript.

3. In lines 184 and 188, repetition of “Staphylococcus” is observed.

Response: Thank you for your suggestion, repeated words have been deleted in the revised manuscript.

4. Authors should provide the details about the working dilutions of antibodies, catalogue numbers of all the antibodies and kits used in the study.

Response: Thanks for your suggestion. We have added the details about the working dilutions of antibodies, catalogue numbers of all the antibodies and kits in red colour.

5. The image quality for S3 Fig. should be improved.S3

Response: Thanks for your suggestion. We have revised S3 Fig in the revised manuscript, and show it as follows.

S3 Fig. The effect of irisin on the electrocardiogram waveform was evaluated in I/R rats.

6. The legends for figures 4 and 5 should be written in alphabetical order.

Response: Thanks for your suggestion. The legend for figures 4 and 5 have been corrected in the revised manuscript.

1. In the methodology section:

Response: Thanks for your suggestion. We have added the appropriate validated methodology reference in the revised manuscript. “The myocardial I/R injury model was induced in the Irisin and I/R groups according to the previously described procedure (Supplementary S1 Fig.) [20].”

[20] Zhao W, Wu Y, Ye F, et al. Tetrandrine Ameliorates Myocardial Ischemia Reperfusion Injury through miR-202-5p/TRPV2. Biomed Res Int. 2021 Mar 8;2021:8870674. DOI: 10.1155/2021/8870674.

Response: Thanks for your suggestion. We have been revised in the revised manuscript.

3. In the Result section:

Response: Thanks for your suggestion. In Section 3.2, we described the sequencing results of gut microbiota to show the effect of irisin on the composition of gut microbiota at the phylum, family, genus and species levels. Bacterial names were obtained through the standard database https://www.arb-silva.de/ and could not be stated simply or changed.

4. Figure legend section:

Response: Thanks for your suggestion. The legend for Figure 4, Figure 5, and Figure 6 have been corrected in the revised manuscript. In figure 4 (A, B and C), Figure 5 (A, B and C) and Figure 7 (B and C) scale bars have been added clearly on the images.

Response: Thank you for your guidance and making the article more complete. The results of Zonulin levels in the blood have been supplemented in the revised manuscript (Fig 6A). In general, rats are gavaged with FITC dextran for 4 hours before collecting peripheral blood for testing intestinal permeability. In acute animal models, animals have a short survival time, so the FITC-Dextran assay may not be appropriate.

Attachment

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PLoS One. doi: 10.1371/journal.pone.0291022.r003

Decision Letter 1

Pradeep Dudeja

21 Aug 2023

Irisin ameliorates myocardial ischemia-reperfusion injury by modulating gut microbiota and intestinal permeability in rats

PONE-D-23-09095R1

Dear Dr. Fan,

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.

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Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #2: All comments have been addressed

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have provided a nicely detailed and thorough response to the comments from the previous review and have addressed all my concerns.

However, there are following 2 minor mistakes that can be taken care of during proof reading:

1. In line 198 and 199, repetition of "Staphylococcus" is observed.

2. Authors should follow the same format (either uppercase or lowercase) for mentioning "DOI". As in majority of references DOI is written in uppercase but in some reference its mentioned in lowercase (Ref 24, 25, 40, 41, 46, 47 and 48).

Reviewer #2: I appreciated the authors response of each comments raised by the reviewers. This manuscript now look scientifically flawless.

**********

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 #2: No

**********

PLoS One. doi: 10.1371/journal.pone.0291022.r004

Acceptance letter

Pradeep Dudeja

25 Aug 2023

PONE-D-23-09095R1

Irisin ameliorates myocardial ischemia-reperfusion injury by modulating gut microbiota and intestinal permeability in rats

Dear Dr. Fan:

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. Pradeep Dudeja

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. Experimental design.

Timeline of the experimental process of this research treatment and ischemia-reperfusion (I/R) injury induction in rats. (i. p.: intraperitoneal injection).

(TIF)

Click here for additional data file.^{(577.1KB, tif)}

S2 Fig. The effect of irisin on the electrocardiogram waveform was evaluated in I/R rats.

(TIF)

Click here for additional data file.^{(724.7KB, tif)}

S3 Fig. Sample sequencing evaluation.

(A) Rarefaction curve indicating that the amount of sequencing reads per sample has reached saturation. (B) Rank-Abundance curves were shown.

(TIF)

Click here for additional data file.^{(977.2KB, tif)}

S4 Fig. The gut microbiota function phenotype characterization of different groups based on BugBase analysis.

The relative abundance of Biofilm Forming, Gram Positive, Gram Negative, Pathogenic Potential, Mobile Element Containing, Oxidative Stress Tolerant and Oxygen Utilizing were shown.

(TIF)

Click here for additional data file.^{(1.9MB, tif)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(951.3KB, docx)}

Data Availability Statement

The data that support the findings of this study are openly available in the BioSample database at http://www.ncbi.nlm.nih.gov/bioproject/940075, reference number [PRJNA940075].

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PERMALINK

Irisin ameliorates myocardial ischemia-reperfusion injury by modulating gut microbiota and intestinal permeability in rats

Qingqing Liu

Yu Zhu

Guangyao Li

Tiantian Guo

Mengtong Jin

Duan Xi

Shuai Wang

Xuezhi Liu

Shuming Guo

Hui Liu

Jiamao Fan

Ronghua Liu

Roles

Abstract

1. Introduction

2. Materials and methods

2.1 I/R rat model establishment and treatment

2.2 Histopathological and 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) staining

2.3 TUNEL and immunofluorescence (IF) staining

2.4 Serum cardiac troponin I (cTnI), creatine phosphokinase (CK), lipopolysaccharide (LPS) and Zonulin measurements

2.5 Western blot analysis

2.6 Extraction of fecal DNA and high-throughput sequencing

2.7 Bioinformatic analysis

2.8 Statistical analysis

3. Results

3.1 Effects of irisin on gut microbial diversity and richness

Fig 1. Effects of irisin on gut microbial diversity and richness.

3.2 Effects of irisin on the gut microbiota composition

Fig 2. Effects of irisin treatment on the gut microbiota composition.

Fig 3. Irisin alters gut microbiota biomarkers in ischemia-reperfusion (I/R) rats.

3.3 Effects of irisin on the gut microbiota function phenotype

3.4 Irisin maintains intestinal integrity in I/R rats

Fig 4. Irisin maintains intestinal integrity in ischemia-reperfusion (I/R) rats.

Fig 5. Irisin maintains intestinal integrity in I/R rats.

3.5 Effects of irisin on gut inflammation

Fig 6. Effects of irisin on gut inflammation.

3.6 Irisin reduces myocardial injury

Fig 7. Protective effects of irisin on myocardium in ischemia-reperfusion (I/R) rats.

3.7 Gut microbiota-associated blood parameters, the myocardial infarct area, colon and ileum barrier function, degree of bacterial translocation, and inflammatory response

Fig 8. The relationship among blood parameters, the infarct area, colon and ileum barrier function, degree of bacterial translocation and inflammatory response, and the 10 top genus estimated using Spearman correlation analysis.

4. Discussion

5. Conclusion

Fig 9. Protective mechanisms of irisin against myocardial ischemia-reperfusion (I/R) injury.

Supporting information

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Pradeep Dudeja

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Author response to Decision Letter 0

Decision Letter 1

Pradeep Dudeja

Roles

Acceptance letter

Pradeep Dudeja

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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