ABSTRACT
To investigate the effect of bone marrow mesenchymal stem cells (MSCs) on ovarian and testicular function of aging Sprague-Dawley (SD) rats induced by D-galactose (D-gal) and try to clarify the underlying functional mechanism. Adherent culture was used to isolate and purify rat MSCs. The status, proliferation and differentiation of MSCs were detected by hematoxylin-eosin staining, MTT, colony formation, flow cytometry and directional differentiation. The aging rat model was established by subcutaneous injection of D-gal, and the homing of MSCs was detected by fluorescence microscope after infusion of GFP-labeled MSCs through caudal vein. ELISA was used to detect the content of sex hormone in serum, and HE staining was used to observe the structure and morphology of testis and ovary. The isolated and purified MSCs were in good condition, and most of the cells were in G1 phase, which had strong abilities of cell proliferation, colony formation and differentiation. After GFP-labeled MSCs were infused, MSCs could be homed into the testis and ovary of rats. MSCs infusion could significantly improve the morphology of testis and ovary, increase the contents of P and E2 while decrease the contents of LH and FSH in female rats, and increase the content of testosterone in male rats (P < 0.01). It also increased the activity of superoxide dismutase (SOD) in serum of ovary and testis and significantly decreased the content of malondialdehyde (MDA). MSCs affected the content of MDA and the activity of SOD by reducing the expression of cyclin-dependent kinase inhibitor 2A (p16) and increasing proliferating cell nuclear antigen (PCNA), consequently improving the aging and injury of reproductive organs.
KEYWORDS: MSCs, D-galactose, aging SD rat model
1. Introduction
Senescence, also known as aging, is a generalized, multifaceted, complex and progressive degradation process which is affected by factors such as genetics, environmental conditions, and lifestyles with age [1]. This process is accompanied by changes in behavior, neuroendocrine, immune regulation and other physiological functions, and with the aggravation of aging, the body will gradually lose its ability to adapt to the environment and its susceptibility to diseases will be increased. At present, the researches on the mechanism of aging can be roughly divided into free radical theory, metabolism theory, telomere theory and immune system degeneration theory, but none of them can fully explain the mechanism [2–4]. Aging affects individual reproductive ability, and in female reproductive organs, ovaries play an important role in maintaining the stability of endocrine and reproductive function of the body and the aging of ovaries is much earlier than the other body tissues and organs [5]. Testes are male gonads, which not only produce sperm to maintain male reproductive function, but also secrete male hormones to regulate spermatogenesis and maintain male sexual function. As age increases or using agents in senescence animal model construction, testicular structure and function are found to be impaired or decayed [6,7].
Bone marrow mesenchymal stem cells (MSCs) are a type of mesoderm-derived stem cells that can differentiate into a variety of cell types, including osteoblasts, chondrocytes, adipocytes, and muscle cells [8]. In addition to multiple differentiation potentials, it is currently believed that MSCs can secrete a variety of trophic factors in response to injury localities, including brain-derived neurotrophic factor, nerve growth factor, vascular endothelial growth factor, basic fibroblast growth factor, etc. [9,10]. These factors can promote the differentiation and proliferation of various cells, thus delaying the aging process. In addition, MSCs have been found to promote the proliferation and metastasis of head and neck cancer and leukemia cells [11,12]. Extracellular vesicles produced by MSCs can reduce microglia-mediated neuroinflammation after cortical injury in elderly Ganges macaques [13]. MSCs have the ability to differentiate into germ cells (GC). G M etal . found that in vitro-induced GC survived, homed and formed colonies in testes [14]. Moreover, MSC-secreted factors may protect the spermatogenic dysfunction after busulfan treatment by reducing the apoptosis of spermatogenic cells and enhancing the expression of intercellular adhesion molecules [15]. Transplantation of BMMSCs can improve follicular production in mice with induced polycystic ovary syndrome [16]. Other studies have found that transplanted placenta-derived MSCs (PD-MSCs) can induce follicular development and enhance ovarian function [17]. These studies have found that MSCs can regulate testicular and ovarian function, but the effects of MSCs on testicular and ovarian aging have not been reported. Therefore, in this study, an aging rat model was constructed and injected with MSCs to explore the alleviation of impairment of ovaries and testes in rats by MSCs and clarify its mechanism.
2. Materials and methods
2.1. Experimental animals and schemes
32 SPF Sprague-Dawley (SD, 6 to 8-week old) rats (16 males and 16 females) weighed 130–150 g were selected and purchased from Shanghai SLAC Laboratory Animal Co., Ltd. The animals were raised at 25°C with a humidity of 60%-70%.
MSCs from two SD rats (one male and one female) were isolated and cultured, and the rest 30 SD rats were randomly divided into the normal control group, the aging model group and the MSCs treatment group. Rats in the aging model group were daily injected with D-galactose (D-gal) 400 mg/kg subcutaneously for 4 months. After the construction of aging model, 3 × 106 MSCs were weekly infused into the tail vein for 4 weeks in the treatment group. Saline of the same volume was given to the rats in the control group and model group. The animal experiment program had been approved by the ethics committee for laboratory animals.
2.2. Isolation and culture of rat MSCs
After being executed, the SD rats were sterilized with 75% alcohol for 10 min, and the femur and tibia were separated under a sterile condition. Periosteum and residual muscles were removed, and the marrow cavity was exposed. DMEM medium was used to repeatedly rinse the marrow cavity, and rinse solution was collected, filtered with a 200-mesh steel net and centrifuged at 1000 RPM for 5 min. The supernatant was discarded. Cells were washed with serum-free Dulbecco’s Modified Eagle’s Medium (DMEM) for once (centrifugation 1000 RPM for 5 min) and were resuspended with complete DMEM containing 15% fetal bovine serum (FBS) and counted. Then 2 × 106 cells were seeded in a 25 cm2 culture bottle and cultured in an incubator at 37°C with 5% CO2 in a humidified atmosphere.
Primary cells were recorded as P0. After 48 h, half of the solution was changed, and the non-adherent cells were transferred to a new culture bottle for further cultivation. The medium was changed every 3 days, and suspended cells were discarded as far as possible. The adherent cells were preserved to passage when they grew to 80–90% in confluence. During the passage, culture supernatant was firstly discarded and cells were washed with PBS twice, and then digested with 0.25% trypsin for l-3 min. When the cells were observed to shrink slightly, digestion was stopped with complete DMEM containing 10% FBS. The cell suspension was centrifuged at 1000 RPM for 5 min and resuspended in complete DMEM containing 10% FBS after discarding the supernatant. The cells were transferred into a new culture bottle in a ratio of 1: (2–3) for passage, which was recorded as P1, and so on. The passage cells were cultured with complete DMEM and 10% FBS, and the adherent cells were discarded. Each passage was performed in a new culture bottle. Cells were purified by multiple passages.
2.3. MTT and colony formation assay
Cell proliferation was measured by MTT assay. MSCs (5 × 103 cells/100 μL) were seeded in 96-well plates of each treatment run in triplicate. Cell proliferation was evaluated with sterile MTT solution (Beyptime) after cell culture for 1, 2, 3, 4, 5, 6 and 7 days, respectively, according to the instructions. The absorbance at the wavelength of 490 nm was measured by a spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).
Cell colony-forming ability was detected by colony formation assay. 6 × 103 cells/well) were exposed to 30% formaldehyde (Thermo Fisher, USA) after culture for 14 days and 15 min later for staining with 0.1% crystal violet (Thermo Fisher, USA). The number of colonies (more than 50 cells per colony) was calculated using an optical microscope.
2.4. Fluorescence microscopy
After the cells were fixed on FN slides with 3.7% formaldehyde, the fluorescence microscopic images of the cells were captured by a NikonTE2000-E fluorescence microscope equipped with a photometric Cool SNAP HQ CCD camera.
2.5. Flow cytometry (FCM)
For detection of MSCs, the well-grown third-generation cells that had reached 80%-90% in confluence were washed with PBS for 1–2 times after trypsin digestion, and then filtered through a 300-mesh nylon net. The cell volume was adjusted to approximately 2 × 105 with PBS. The cells were used to be prepared into four samples which were added with fluorescent direct-labeled monoclonal antibodies CD34, CD45, CD29, and CD44, respectively, and stored at 37°C in the dark for 30 min. After cells were washed three times with PBS, the cell surface antigen expression was detected by FCM. PBS was used as the primary antibody, and negative control was set for comparison.
For cell cycle assessment, MSCs in the growth stage were added with 3 mL PBS and then digested with 1 mL trypsin for 1–5 min after liquid removal. The cell suspension was prepared by adding 5 mL PBS and transferred to a 15 mL centrifuge tube for centrifugation at 1500 RPM for 5 min, after which the supernatant was discarded. Cells were gently beat upon into cell suspension with 500 μL PBS, and fixed with 2 mL 95% ice-cold ethanol at 20°C for 30 min. The cells were sequentially centrifuged at 1500 RPM for 5 min. After supernatant removal, the cells were resuspended again with 5 mL PBS and centrifuged at 1500 RPM for 5 min, with the supernatant discarded at the end. The cells were dyed by 800 μL propidium iodide at room temperature in dark for 30 min. Finally, the cell cycle was detected by FCM.
2.6. Directed induction of MSCs
Directed induction and identification of lipoblasts: cells were seeded into 6-well plates at a density of 2 × 104 cells/cm2, and added with 1 μmol/L dexamethasone, 10 mg/L insulin, 0.2 mmol/L Indomethacin, H-DMEM with 10% FBS for induction for about 14 days. Oil Red O staining was used for cell identification.
Directed induction and identification of osteoblasts: cells were inoculated into 6-well plates at a density of 2 × 104 cells/cm2, and cultured in the osteoblast induction medium (H-DMEM containing 10% FBS, 1.0 × 108 mol/L dexamethasone, 2.0 × 104 mol/L ascorbate, 7.0 × 103 mol/L β-sodium glycerophosphate). The solution was changed every 3 days and observation was made once a day for about 20 days. Then alizarin red staining was conducted to observe osteoblasts.
2.7. Cell transfection
According to the reference instructions, lentivirus vector labeled with green fluorescent protein (GFP) was transfected into MSCs cells by Lipofectamine® 3000 (Invitrogen Inc., USA), after which the cells were cultured for 96 h and then treated with purinomycin for 4 weeks for cell screening.
2.8. Tissue microscopy analysis
The left ovarian and left testicular tissue blocks from each group were fixed in 10% formalin and paraffin embedded. 5-μm-thick slices were stained by HE and the changes of ovarian tissues and testicular tissues were observed under a 100-fold field of view to count the number of corpus luteum, primary follicles and seminiferous epithelium.
2.9. Immunohistochemistry (IHC)
Conventional dewaxing hydration: xylene immersion (10 min×2), anhydrous ethanol washing (5 min×2), 95% ethanol washing (5 min×2), 75% ethanol washing (5 min×1) and DdH2O flushing (5 min×1). After repaired with 0.1 M sodium citrate-hydrochloric acid buffer solution (pH 6.0) in the microwave oven (750 W for 3-min boiling, 90 W for 11.5 min), the tissue sections were naturally cooled to room temperature. After being washed with PBS/T (5 min×3), sections were cultured with 3% H2O2 at room temperature for 10 min. Then, sections were washed again with PBS/T (5 min×3) and blocked with 10% goat serum at room temperature for 10 min. The sections were incubated overnight at 4°C in the diluted primary antibodies of anti-GFP and anti-Ki67. After being washed with PBS/T (5 min×3), the sections were cultured with DAKO Real Envision anti rabbit at room temperature for 30 min. Thereafter, PBS/T was used to wash the sections (5 min×3) and diaminobezidin (DAB) was used to stain sections at room temperature for 5 min, which was observed under a microscope. The sections were washed with ddH2O (5 min×3), counterstained with hematoxylin, differentiated by 1% acid alcohol and returned blue by using running water. Finally, dehydration, clearing, neutral balsam mounting and microscopy were performed.
2.10. ELISA double antibody sandwich method
Estradiol (E2), progesterone (P), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T) in serum of rats were determined by ELISA double antibody sandwich method. The corresponding antibodies were purchased from Abcam, China.
2.11. Malondialdehyde (MDA) and superoxide dismutase (SOD) detection
After the end of the experiment, ovarian and testicular tissues of the rats were collected, and the content of serum MDA and activity of SOD in each group were measured by thiobarbituric acid and xanthine oxidation methods, respectively.
2.12. Quantitative real-time (qRT) PCR
Total RNA was extracted from tissues using Trizol (Invitrogen) in accordance with the manufacturer’s protocol. cDNA was synthesized using a reverse transcription system kit (Invitrogen). qRT-PCR was performed on ABI 7900HT (Applied Biosystems, USA). Quantitative PCR was performed using miScript SYBR Green PCR Kit (Qiagen, Germany) under the following thermal cycling conditions: Pre-denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 2 min, 95°C for 5 s and 60°C for 30 s. The expression was normalized to β-Actin. The relative expression of the target gene in the control group and the experimental group were compared using 2−ΔΔCt method. The primers used in the experiment were listed in Supplement Table 1.
2.13. Western blot
Total proteins were extracted by RIPA lysis buffer (BB-3209, BestBio Science, Shanghai, China). The proteins were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membrane at a constant voltage of 80 V. After being blocked with skimmed milk powder for 1 h, the membrane was incubated with diluted primary antibodies including anti-PCNA, anti-P21, anti-p16 and anti-GAPDH (Abcam, Cambridge, UK) at 4°C overnight. Subsequently, the membrane was washed with TBST buffer solution (5 min×3), and cultured with secondary antibody horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG H&L (Abcam, Cambridge, UK) at room temperature for 1 h. Finally, the protein samples were subjected to an enhanced chemiluminescence detection system (Bio-Rad lab, Hercules, CA, USA).
2.14. Statistical analysis
All the data were processed by SPSS 22.0 statistical software, and exhibited as mean ± standard deviation. The comparison between two groups was analyzed by t-test, and the comparison among multiple groups was analyzed by one-way analysis of variance. P < 0.05 indicated the difference was statistically significant.
3. Results
3.1. Isolation, culture and identification of rat MSCs
MSCs cultured by adherent method could be partially adherent after 24 h. After 48–72 h, the number of adherent cells was increased significantly, and cells were gradually extended into spindle shape and polygon shape, protruded cytoplasmic protrusions of varying lengths and thickness with 1–4 nucleus centered and large proportion of nucleoplasm. Small colonies could be seen within 3–4 d. The non-adherent cells were removed when changing the solution. In primary culture, cells grew adherent to the wall in a scattered and clonal way. After 8–14 d, the adjacent colonies fused with more than 80% in confluence and overlapping between the colonies, and no contact inhibition occurred. The sub-culture cells began to adhere to the wall at 2–4 h, and were completely adherent within 24 h. The morphology was similar to that of the primary cells, but the proliferation rate was significantly increased, and the MSCs grew evenly after passage. When MSCs were passed to the third generation, the cells were found to be star-shaped and spindle shaped under a light microscope (Figure 1(a)). After HE staining, the nucleus was round and bluish, which was located in the center of the cell, and the cytoplasm was hypertrophy with clear cell membranes (Figure 1(b)). After six passages, there was no significant change in cell morphology, indicating that MSCs had been further purified. The results of MTT showed that the cell number was increased significantly from the 3rd day after inoculation, and cell proliferation reached a peak on the 5th day and kept at a steady state subsequently (Figure 1(c)). Colony formation assay suggested that when P3 generation cells were inoculated into the culture plate with a low density, scattered colonies could be formed after 1 week, and the CFE-F on the 14th day was (22.6 ± 2.4) (Figure 1(d)). MSCs induction experiment on lipoblasts found that after the induction solution was added for 72 h, small lipid droplets began to appear in the cells, which were mainly concentrated around the nucleus. As the induction time prolonged, the lipid droplets gradually accumulated into large lipid bubbles and dark orange-red lipid droplets could be seen after Oil Red O Staining (Figure 1(e)). MSCs induction experiment on osteoblasts found that on the 21st day of induction, alizarin red-positive calcified nodules were observed. As the induction time prolonged, the calcified nodules increased significantly, and the positive reaction was more obvious on the 28th day (Figure 1(f)). The results of FCM discovered that G1 phase cells accounted for 81.6% and S+ G0 + M phase cells accounted for 19%, indicating that most cells were in G1 phase (DNA pre-synthetic phase) with a strong proliferation ability (Figure 1(g)). The number of CD29- and CD44-positive cells reached 99.94% and 99.84%, respectively. The number of CD34- and CD45-negative cells were 93.72% and 97.08%, respectively (Figure 1(h)), indicating that MSCs were the main third-generation adherent cells.
Figure 1.
Isolation, culture and identification of rat MSCs.
a: The morphology of MSCs was examined under a light microscope (100×); b: HE staining of MSCs (100×); c: Growth curves of MSCs; d: The colony ability of MSC (40×)s; e: Oil red O staining after lipoblast induction (200×); f: Alizarin red staining after osteoblast induction (200×); g: The cell cycle of MSCs; h: Surface markers of MSCs (Data of 001: CD29; The Data of 004: CD34; The Data of 007: CD44; The Data of 010: CD45)
3.2. MSCs can delay the aging of reproductive organs in SD aging rats
Three days after MSCs infusion, testicular and ovarian tissues were removed for HE section preparation, and the fluorescent cells were observed under a fluorescence microscope (Figure 2(a-b)). Besides, fluorescence could be detected in frozen tissue sections on day 7 and day 14 (Figure S1). Meanwhile, IHC was used to detect the localization of GPF in testicular and ovarian tissues (400 x), and it was observed that GPF was positively expressed in testicular and ovarian tissues of the MSC treatment group, indicating that MSC had been successfully introduced into the testicular and ovarian tissues of rats (Figure 2(c-d)). In addition, IHC also detected the expression of proliferation-related protein Ki67 in testicular and ovarian tissues of each group. The results exhibited that the expression of Ki67 in testicular and ovarian tissues of rats in the model group was significantly lower than that in the normal group and the MSC treatment group, suggesting that after treatment with MSC, the cell proliferation capacity in the testes or ovaries of rats was enhanced (Figure 2(e-f)). Moreover, optical microscope analysis of the testicular tissues in each group found that there were various levels of spermatogenic cells and supporting cells neatly arranged in the basal membrane of the normal group, and there were more spermatogenic cell layers, more mesenchymal cells in seminiferous epithelium. The morphology and structure of testicular spermatogenic tubules in the model group were significantly changed. The arrangement of spermatogenic cells in the basal membrane was basically normal, but there were fewer layers of spermatogenic cells and mesenchymal cells, sparse cells, and less obvious intracellular secretions. The morphology and structure of testicular tubules were significantly recovered and the layer number of sperm cells was increased in the treatment group relative to those in the model group, while sperm cells remained relatively sparse with mature sperms and mesenchymal cells increased compared with control group. Additionally, the proportion of the shrinking tube was higher in the treatment group in comparison with the model group (Figure 2(g)). The percentage of different atrophy degrees of the seminiferous epithelium under a light microscope (100 x) are listed in Table 1. A light microscopy was used to observe the ovarian tissues in each treatment group, and it was examined that there were multiple corpus luteum and follicles at different stages of development in ovarian tissues in the normal group, with multilayer granulosa cells in the follicles. In the model group, the number of follicles and corpus luteum was decreased, and follicular development was blocked with fewer mature follicles. In the treatment group, multiple corpus luteum and follicles were observed, with multilayer granular cells in the follicles (Figure 2(h)). The number of follicles and corpus luteum in the ovaries under a light microscope (100 x) is exhibited in Table 2. The contents of serum P and E2 of female rats in the treatment group were higher compared with those in the model group while the contents of LH and FSH were lower (P < 0.01). The content of T of male rats was increased in the treatment group compared with that in the model group (P < 0.01). (Table 3).
Figure 2.
MSCs delay the senescence of sexual organs in SD rats.
a, b: Distribution of MSCs in testes and ovaries under a fluorescence microscope (100×); c, d: The expression of GFP in testes (c) and ovaries (d) after MSCs treatment were detected by IHC (200×); e, f: The expression of Ki67 in testes (e) and ovaries (f) of rats in each group were detected by IHC (200×); g, h: Observation of morphology changes of testicular tissues (g) and ovarian tissues (h) under a light microscope (200×);
Table 1.
Percentage of seminiferous epithelium with different atrophy degrees under a light microscope (×100, – ±S, n = 5).
| Group | Normal seminiferous tube | Partial atrophy tube | Complete atrophy tube |
|---|---|---|---|
| Control group | 87.10 ± 1.30 | 7.65 ± 0.93 | 4.9.1 ± 0.82 |
| Model group | 76.63 ± 1.80* | 15.02 ± 1.56* | 8.35 ± 0.84* |
| Treatment group | 84.78 ± 1.74Δ | 9.83 ± 0.91Δ | 5.16 ± 0.73Δ |
| F value | 56.853 | 52.179 | 28.988 |
| P value | 0.000 | 0.000 | 0.000 |
Vs the control group, * P < 0.05
Vs the model group, Δ P < 0.05
Table 2.
Number of follicles and corpus luteum in ovaries under a light microscope (×100, – ±S, n = 5).
| Group | follicles | Corpus luteum |
|---|---|---|
| Control group | 22.07 ± 1.82 | 10.00 ± 1.22 |
| Model group | 12.00 ± 0.91* | 5.13 ± 0.69* |
| Treatment group | 18.73 ± 1.04Δ | 8.00 ± 0.53Δ |
| F value | 75.697 | 39.793 |
| P value | 0.000 | 0.000 |
Vs the control group, * P < 0.05
Vs the model group, Δ P < 0.05
Table 3.
Effect of MSCs on sex hormones in rat serum (×100, – ±S, n = 5).
| Group | E2 (pmol/L) | P (pmol/L) | FSH (IU/L) | LH (IU/L) | T (nmol/L) |
|---|---|---|---|---|---|
| Control group | 247.70 ± 10.43 | 26.18 ± 3.59 | 1.40 ± 0.34 | 3.68 ± 0.323.28 ± 0.34 | |
| Model group | 120.35 ± 10.91* | 13.03 ± 1.88* | 3.64 ± 0.60* | 6.68 ± 0.43*1.46 ± 0.23* | |
| Treatment group | 209.63 ± 10.59Δ | 22.45 ± 1.86Δ | 1.88 ± 0.47Δ | 4.32 ± 0.56Δ | 2.46 ± 0.23Δ |
| F value | 188.401 | 34.462 | 30.452 | 61.795 | 55.88 |
| P value | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 |
Vs the control group, * P < 0.05
Vs the model group, Δ P < 0.05
3.3. MSCs may delay the senescence of rats by regulating the expressions of p16 and PCNA
Compared with the control group, SOD activity in ovaries and testes in the model group was significantly decreased (P < 0.05), while the MDA content was greatly increased (P < 0.05). The inverse results could be observed in the treatment group (P < 0.05, Table 4). The results of qRT-PCR displayed that compared with the normal group, the expression level of p16 mRNA in ovarian tissues of the model group was significantly increased, and that of PCNA mRNA was significantly reduced (P < 0.05). Compared with the model group, the treatment group had significantly lower p16 mRNA expression level and significantly higher PCNA mRNA expression level (P < 0.05). The results of ovarian tissues were consistent with the testicular tissue test results (Figure 3(a)). Besides, western blot was used to detect the protein expressions of PCNA, p16 and P21 in the testes or ovaries of rats in each group, which was similar to the results of qRT-PCR (Figure 3(b)). Compared with the aging model group, the expression of PCNA protein in the testicular or ovarian tissues of the MSC treatment group was significantly increased, while the expression of p16 was significantly decreased. P21 is also one of the genes related to aging, which is closely associated with the p16-cylinD/CDK-RB pathway. Therefore, we also detected its expression in the testes or ovaries of each group, and found that compared with the model group, the protein expression of P21 in the treatment group was significantly reduced.
Table 4.
Effect of MSCs on SOD activity and MDA content in ovaries and testes of rats (×100, – ±S, n = 5).
| Group | n | Ovaries SOD (U/mL) | Ovaries MDA (nmol/mL) | Testes SOD (U/mL) | Testes MDA (nmol/mL) |
|---|---|---|---|---|---|
| Control group | 5 | 75.10 ± 2.80 | 10.04 ± 0.61 | 90.13 ± 1.92 | 13.40 ± 1.08 |
| Model group | 5 | 43.08 ± 2.10* | 24.27 ± 1.08* | 62.73 ± 1.68* | 37.60 ± 1.70* |
| Treatment group | 5 | 69.07 ± 1.81Δ | 11.58 ± 0.70Δ | 85.32 ± 1.78Δ | 16.32 ± 1.41Δ |
| F value | 279.281 | 451.312 | 331.345 | 431.792 | |
| P value | 0.000 | 0.000 | 0.000 | 0.000 |
Vs the control group, * P < 0.05
Vs the model group, Δ P < 0.05
Figure 3.
The contents of p16, p21 and PCNA in testes and ovaries.
a: The mRNA expressions of p16 and PCNA in testes and ovaries were detected by qRT-PCR; b: The protein expressions of p16, p21 and PCNA in testes and ovaries were detected by western blot.
4. Discussion
Aging is a universal natural law in organisms. It is a normal process that occurs when the body grows and matures with age. The aging problem that the body will eventually face is irreversible and no one can avoid it [18]. MSCs are expected to be widely used in the repair and reconstruction of tissues and organs, treatment of degenerative diseases and anti-aging treatment due to their self-renewal and multidirectional differentiation potential [19–22]. Relevant studies have demonstrated that MSCs can function in vivo by homing to injured tissues to participate in differentiation under the action of chemokines [23], or by activating dormant stem cells through paracrine factors [24]. As a tumor suppressor gene, p16 plays an important role in regulating G1 phase of cell cycle. It regulates cell cycle through the p16-cylinD/CDK-RB pathway, affecting cell life and telomere length [25]. In recent years, studies have reported that when cells are senescent, the increased mRNA and protein expression levels of p16 gene maintain the irreversible growth stasis of senescent cells, so it is believed that p16 is a key regulatory gene of the cell life cycle [26,27]. PCNA is a 36KD acidic protein in the nucleus, which functions in regulating cell cycle. In addition, PCNA also acts as a cofactor of DNA polymerase to help it locate, participate in DNA synthesis, repair, and promote DNA replication. DNA replication in cells mostly indicates cell proliferation, so the expression of PCNA can reflect the proliferation status of cells. Based on the above results, we speculated that the mechanism of MSCs delaying aging may be mediated by p16 and PCNA.
In this study, MSCs were firstly isolated from SD rats and purified. The cell viability and proliferation ability of MSCs were measured by MTT and colony formation assays. FCM was used to detect the cell cycle and surface markers and identified that the isolated cells were MSCs which were with good cell morphology and proliferation ability. The D-gal induced aging rat model has been widely used in the pharmacodynamics study of anti-aging drugs, which has a variety of aging characteristics such as shortened life span, learning and memory impairment, and decreased immunity [28]. A large number of studies have shown that long-term massive injection of D-gal solution can produce excessive reactive oxygen species (ROS), reduce the activity of antioxidant enzymes in various organs, form more superoxide anions and various oxidation products, and cause cell damage, resulting in the functional decline of multiple organs and systems in the body [29]. Then the SD aging rat model was established by subcutaneous injection of D-gal. After the successful establishment of the aging model, GFP-labeled MSCs were reinfused into the aging rats through the tail vein. The homing of MSCs to testicular and ovarian tissues was observed by fluorescence microscopy. Meanwhile MSCs could significantly improve the morphology and structure of testicular and ovarian tissues in aging rats, as observed by a light microscopy. Additionally, the contents of P and E2 were increased in female rats while those of LH and FSH were decreased, and the content of T was increased in male rats. The above results indicated that MSCs could be homed to testicular and ovarian tissues and they were effective in delaying aging of rats. Finally, qRT-PCR was applied to detect the mRNA expression levels of p16 and PCNA in testicular and ovarian tissues, respectively. The results displayed that MSCs could significantly reduce the expression of p16 and enhance the expression of PCNA in both testicular and ovarian tissues. Moreover, we tested the contents of MDA and SOD in ovaries and testes, and found that SOD content in tissues was significantly increased while MDA content was reduced after MSCs infusion. Based on the above results, MSCs can affect the content of MDA and activity of SOD, and improve the aging of reproductive organs by reducing p16 expression and increasing PCNA expression.
Supplementary Material
Funding Statement
This study was supported by Startup Fund for scientific research, Fujian Medical University (Grant number: 2016QH110), Joint Funds for the innovation of science and technology, Fujian province(Grant number: 2017Y9068) and High-level hospital foster grants from Fujian Provincial Hospital, Fujian province, China(Grant number:2019HSJJ01)
Authors’ contributions
All authors contributed to data analysis, drafting and revising the article, gave final approval of the version to be published, and agreed to be accountable for all aspects of the work.
Availability of data and materials
The data used to support the findings of this study are included within the article. The data and materials in the current study are available from the corresponding author on reasonable request.
Disclosure statement
The authors declare no conflicts of interest.
Supplementary material
The supplemental data for this article can be accessed here.
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Data Availability Statement
The data used to support the findings of this study are included within the article. The data and materials in the current study are available from the corresponding author on reasonable request.