Curcumin Alleviates The Senescence Of Canine Bone Marrow Mesenchymal Stem Cells During In Vitro Expansion Part 2

Please contact oscar.xiao@wecistanche.com for more information

2.5.Autophagy Inovoloes in Exerting the Protective Effect of Cur in cBMSC Senescence

To explore the effect of Cur-induced autophagy on cBMSC senescence, the autophagy level was modulated through the employment of RAP(200 nM) or 3-MA(5 mM).3-MA exerts a significant inhibitory action on autophagic activity, manifested by the significant downregulation of the mRNA expression of microtubule-associated protein 1 light chain 3(LC3), autophagy-related gene(ATG)7, ATG12, and unc51-like autophagy-activating kinase-1(ULK1); the decreased microtubule-associated protein 1 light chain 3 type Ⅱ/I (LC3-I/I) expression ratio; and the increased expression of p62 compared with the control group (Figure 5A, B). Accordingly, compared with the Cur group, a decrease in autophagic activity was observed in the 3-MA+Cur group (Figure 5A, B). In contrast, an increase in autophagic activity was observed in the RAP group, as evidenced by the upregulated mRNA expression of LC3, ATG12, and ATG7; the increased conversion of LC3-I to LC3-II; and the degradation of p62 (Figure 5A, B).

Firstly, the autophagic activity was investigated systematically. The formation of autophagic vacuoles (also termed autophagosomes)was assessed by morphological observation, and the results indicated that there were more autophagic vacuoles and LC3 dots in the Cur group and RAP group compared with the control group, while a reduced number of autophagic vacuoles and fewer LC3 dots were observed in the 3-MA and 3-MA+Cur groups (Figure 5C, E).In addition, LysoTracker staining indicated that lysosomal acidification was enhanced by RAP and Cur in cBMSCs and decreased in the 3-MA and 3-MA+Cur groups(Figure 5D). The results show that Cur and RAP exert a similar positive effect on autophagy activation and that autophagic activity is suppressed by 3-MA. cistanche penis size, However, the inhibition effects of 3-MA on autophagy can be partially rescued by the employment of Cur.

Please click here to know more

To confirm whether autophagy participates in the regulation of CB MSc senescence, we further examined senescence-associated phenotypes after promoting or suppressing autophagy through pharmacological treatment. The results showed that a reduced number of SA-β-gal-positive cells were present in the Cur and RAP groups, while an increased number of SA-β-gal-positive cells were observed in the 3-MA group compared with the control group. It is noteworthy that the number of SA-β-gal-positive cells was significantly increased in the 3-MA+Cur group compared with the Cur group (Figure). The results of an RT-qPCR analysis indicated that treatment with RAP and Cur increased the expression level of SOX-2 and Nanog and decreased the expression level of IL-6, TNF-a, p21, and p16 compared with the control group. However, the inhibition of autophagy by 3-MA upregulated the expression of p16, p21, TNF-α, and IL-6(Figure 6C-E). Furthermore, compared with the control group, the colony-forming efficiency of cBMSCs was increased in the Cur and RAPgroups, while the number and the size of CFU-F were significantly decreased in the 3-MA group. Additionally, it was shown that the enhanced effect of Cur on the colony-forming number of cBMSCs could be abolished through preconditioning with 3-MA (Figure 6B). cistanche powder This evidence indicates that RAP and Cur can ameliorate cBMSC senescence, while 3-MA aggravates cBMSC senescence and attenuates the beneficial effects exerted by Cur.

Taken together, these results suggest that the inhibition of autophagy with 3-MA accelerates cBMSCs senescence, while the activation of autophagy with Cur and RAP alleviates cBMSC senescence (Figure 6F). Notably, the protective effects exerted by Cur were attenuated by pretreatment with 3-MA(Figure 6F), suggesting that Cur-induced autophagy is a potential molecular mechanism for ameliorating cBMSC senescence.

3. Discussion

Organisms continuously repair injured tissues and retard senescence-related processes owing to the distinctively functional characteristics of the MSCs that extensively reside within various tissues and organs [15]. Unfortunately, age and disease are key factors for MSC senescence in vivo, and internal and external differences in cellular environments accelerate MSC senescence in vitro culture, both of which negatively affect their capacity for immunosuppression, differentiation, and migration, ultimately reducing the efficacy of self-repair and transplantation in MSCs [7,14,49-51]. cistanche salsa extract Oja and colleagues indicated that human BMSCs ceased proliferation at the fifth to the ninth passage of clinical-grade cultures and exhibited typical senescence phenotypes, such as a hypertrophic and flat morphology, the activation of cell cycle kinase inhibitors p16 and p21, a decreased proliferation rate, and enhanced activity of SA-β-gal [52]. Evidently, in vitro expansion inevitably engenders the premature appearance of senescence in MSCs, which are considered to be an important model system for in vitro cellular aging research [42, A4, A5]. Our present study found that cBMSCs before the 3rd passage displayed a uniform morphology but had been observed to undergo a series of changes in terms of cellular morphology, physiology, and gene expression after the 6th passage(Figures 2 and 7), consistent with premature senescence.

Cistanche can anti-aging

An increasing amount of evidence indicates that pharmacological stimulation is a promising approach for rescuing MSCs from senescence [53,54]. With this in mind, a number of natural and synthetic compounds have been investigated extensively in order to determine their anti-inflammatory, antioxidative, and anti-senescence potential in vivo and in vitro [15,55]. As a naturally occurring phenolic compound, Cur has aroused great attention due to its beneficial effects on MSC biology [36,37,56,57]. However, the complex effects of Cur exposure on MSCs should be carefully considered before the implementation of different biomedical research. Yang and colleagues reported that high concentrations of Cur (50 and 100 uM) could induce acute toxic effects in human BMSCs in vitro, while continuous exposure (7 d)to 10uM of Cur inhibits human BMSC proliferation and induces cell apoptosis [58]. Interestingly, another study indicated that treatment with Cur(<20 uM)for 5 days ameliorates H, O,-induced oxidative stress in human ADSCs[56]. Additionally, Cur preconditioning （1 μM and5uM） for 24 or 48h can help to maintain cellular viability and improve the lifespan of rat ADSCs[36,57. Our results demonstrated that Cur(1 uM and 10 μM） was able to maintain the viability of cBMSCs and alleviate cBMSC senescence after exposure for 24 h, while the colony-forming efficiency of cBMSCs was significantly decreased at a dose of 10 μM（Figure 3）. Therefore， the beneficial effects of Cur （10 uMD may be attributed to short-term stimulation, and it can impair the proliferation potential of cBMSCs in the long term.

Recently, the biological activities of Cur have been extensively reported on various in vitro or in vivo models, particularly regarding the modulation of the biological characteristics of MSCs. The anti-aging activity of Cur has been discussed frequently, and Cur has been found to exhibit beneficial effects on aging and age-related diseases at the organismal and cellular levels. However, the mechanism underlying its regulation is complicated due to the different doses and forms of Cur, as well as the mechanism of aging [19]. As a major intracellular mechanism of molecule degradation and organelle turnover, autophagy plays a major role in protecting MSCs against stress conditions and maintaining cellular homeostasis. The modulation of autophagy is considered to be a novel strategy for the amelioration of MSC functions [59]. According to data gathered from substantial studies, the promotion or suppression of autophagy by Cur in various cell models exerts satisfactory cytoprotective effects [38-40]. cistanche stem Our data showed that increased autophagic activity was observed after exposure to Cur, as identified by the upregulation of autophagy-related genes (LC3, ULK1, Atg7, and Atg12), generation of LC3-II, increase in the number of autophagic vacuoles and acidic vesicular organelles, and a significant decrease in the p62 protein level (Figure 4). Additionally, the lysosome is regarded as an indispensable organelle for autophagy, while the dysregulation of lysosomal pH and alteration of vacuolar H+-ATPase (v-ATPase) activity were observed in the process of MSC senescence, thus promoting lysosomal acidification and autophagy and contributing to delaying MSC senescence [60]. Yan and colleagues indicated that Cur can activate the lysosome function of mouse embryonic fibroblasts (MEFs) and induce autophagy, which serves as a crucial survival signal [38]. Similarly, we observed that Cur treatment enhances lysosomal acidification in cBMSCs (Figure 4), suggesting that Cur may be involved in activating lysosome function, which is indispensable for enhancing autophagic activity.

Autophagy is predominantly a cytoprotective mechanism, and an increasing amount of evidence has indicated that the anti-aging properties of natural and synthetic compounds are correlated with autophagy modulation [29,54,61,62]. However, the effects of autophagy modulation on MSC senescence and corresponding mechanisms have not yet been fully evaluated and explored. Initial reports have indicated that autophagy is a predominantly cytoprotective mechanism and that an increased level of autophagy can delay cellular senescence by reducing the accumulation of toxic metabolites and restoring the function of organelles [63]. Interestingly, recent investigations have also shown that increased numbers of autophagic vacuoles and autophagy-related proteins (LC3-II, ATG7, and ATG12)were observed during MSC senescence, while the inhibition of autophagy with bafilomycin A1 and 3-MA was shown to reduce the percentage of SA-β-gal-positive cells and the expression of p16 and p21 [35]. To further elucidate the relationship between Cur-induced autophagy and its effects on cBMSCsenescence, autophagy was modulated by pretreatment with rapamycin or 3-MA. Obviously, autophagic activity was found to be attenuated by 3-MA, whereas RAP and Cur (1 uM) were shown to significantly enhance autophagy (Figure 5). Consistent with previous reports [5], our findings also demonstrate that the inhibition of autophagy by 3-MA accelerated cellular senescence in cBMSCs.Almost consistent effects on the activation of autophagy were exerted by Cur(1uMand RAP while analogous cytoprotective effects in cBMSCs were displayed. Accordingly, when autophagy was inhibited by 3-MA, the protective effects exerted by Cur were decreased (Figure ), suggesting that Cur-induced autophagy is a potential molecular mechanism for ameliorating cBMSC senescence (Figure 7).

Under physiological conditions, autophagy occurs at a basal level in all eukaryotic cells to maintain cellular homeostasis. However, various stress conditions can lead to abnormal autophagy, which has an influence on cell fate unless autophagy is restored to an optimal level [41,44,64]. Our evidence confirmed that Cur-induced autophagy exhibits beneficial effects on the regulation of cBMSC senescence. In this scenario, diverse stress-producing stimuli and extracellular settings should be carefully considered before Cur treatment, and it would be interesting to investigate whether Cur-induced autophagy can selectively improve the function of MSCs at a predetermined dose and duration. Additionally, a nanotechnology-based curcumin delivery system has exhibited better aqueous-phase solubility and bioavailability levels [65,66]; it could be a promising tool by which to delay and counteract MSC senescence. The answer to the question of whether autophagy is the primary underlying mechanism in delaying MSC senescence still requires more details that will be provided by future research.

4. Materials and Methods

4.1.Animals

Bone marrows samples were collected from 6 healthy adult female Chinese rural dogs (12-month-old). All studies were approved by the Faculty Animal Care and Use Committee of Sichuan Agricultural University (approval no.2020-0608)and conducted in accordance with the ethical standards of the animal protection laws of the People's Republic of China.

4.2. Preparation of Curcumin Solution

Cur(HPLC≥98%, CAS number:458-37-7; Solar Science& Technology Co., Ltd., Beijing, China) was dissolved in DMSO to a stock concentration of 20 mmol/L. filtered through a 0.22 μm organic microporous filter membrane and stored at -80°C. Different Cur solutions were prepared in a medium for in vitro study.

4.3. Cell Culture and Expansion

cBMSCs were obtained from bone marrow. The cells were cultured in a complete medium consisting of low-glucose Dulbecco's Modified Eagle Medium (LG-DMEM, Gibco Grand Island, NY, USA), 10% fetal bovine serum(FBS, TransGen Biotech Co., Ltd., Beijing, China), and 1%penicillin/streptomycin. At an 80-90% confluence, the adherent cells were released with Trypsin Digestion Solution (Beyotime Biotechnology Co., Ltd., Shanghai, China) and further expanded at a ratio of 1:2-1:3 [67].

4.4. Cell Growth Curie

To determine the proliferative ability of cBMSCs in passages (P)3,6, and 9, the cells were seeded in three 48-well plates (2500 cells/well). After 48 h incubation, the cells were released with Trypsin Digestion Solution and counted with a hemocytometer. The cell counting procedure was repeated every 48 h and sustained for 14 d.

4.5.Detection of Immunophenotype of cBMSCs by Flow Cytometry

In the 3rd passage, cBMSCs were washed with PBS and trypsinized. The cells (3×105 cells/mL) were re-suspended in the staining buffer, and the cell suspensions （100 μL）were incubated with FITC， PE， or APC fluorescent-labeled monoclonal antibodies against the surface antigens CD45, CD34, and ITGB1(eBioscience, San Diego, CA, USA)and nonfluorescent-labeled CD31, CD90, and CD105(Biosynthesis biotechnology Co.Ltd. Beijing, China)for 15 min at 4°C. The cells were washed with PBS and incubated with FITC-conjugated goat anti-rabbit IgG for 15 min at 4°C. The surface antigens were detected by flow cytometry (FACS Calibur, Becton Dickinson, San Jose, CA, USA). Data analysis was carried out with the CytExpert software.

4.6. In Vitro Differentiation Assay

cBMSCs were plated at a density of 5×104 cells/mL in 6-well plates. At 70-80%confluence, the complete medium was replaced with an osteogenic or adipogenic differentiation induction medium and changed every 3 days (Cvagen, Suzhou, China). Calcium deposition was detected by Alizarin Red S staining (Solarbio, Beijing, China) after 3 weeks of osteogenic induction and lipid droplet accumulation was observed using Oil Red O staining (Solarbio, Beijing, China) after 2 weeks of adipogenic induction.

4.7.Effect of Cur on Cellular Viability

The cellular viability of cBMSCs was determined using the CCK-8kit (Vazyme Biotech Co., Ltd., Nanjing, China). cistanche tubulosa benefits and side effects cBMSCs were pre-cultured in a 96-well plate for 24 h.cBMSCs were treated with Cur at different concentrations(0.1, 0.5, 1, 5, and 10 umol/L) for 12h, 24h,48h, and 72 h. Cells were treated with 0.1%of DMSO, which was used as a control After incubating with 10 μL of CCK-8 solution per well for 2 h， the optical density was measured by a microplate reader at 450 nm (Thermo Scientific, Waltham, MA, USA). The relative cell viability was calculated in accordance with the manufacturer's instructions.

4.8. Colony Formation Assay

The self-renewal efficiency of cBMSCs was detected using a colony-forming unit-fibroblast （CFU-F） assay. cBMSCs were seeded （3× 10² cells/well） in 6-well plates. After two weeks of culture, the cells were fixed with 4% paraformaldehyde for 30 min and observed under an inverted microscope(LX73, Olympus Corporation, Tokyo, Japan) after staining with1% crystal violet for 10 min. For the CFU-F, more than 50 cells were counted. The CFU-F efficiency was calculated as follows:

CFU-F efficiency = number of CFU-F/number of seed (300 cells)[68].

4.9. Beta-Galactosidase Staining Assay

The activity of senescence-associated β-galactosidase (SA-β-gal) in cBMSCs was estimated using the SA-β-gal staining kit (Beyotime Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer's instructions. After staining, the cells were examined under an inverted microscope. Positively stained cells were counted to assess the cellular senescence.

4.10.Reverse Transcription Real-Time Quantitative PCR(RT-qPCR)

The total RNA was extracted from the cell pellets using the Trizol reagent method. cDNA was synthesized using the PrimeScriptTM RT reagent kit with the gDNA Eraser (Takara, Shiga, Japan). PCR primers (Table 2) besides GAPDH in reference to previous studies [67] were designed using the Primer Express software (Applied Biosystems, Foster City, CA, USA) based on cDNA sequences. The qPCR was performed using TB Green PCR Mix(Takara, Shiga, Japan) on the CFX96 Touch Real-time PCR Detection System (Bio-Rad, Richmond, CA, USA). The reaction conditions were as follows:95°C for 30 s and then 39 cycles of95°C for 5s and 60°C for 30 s. A melting curve analysis was performed starting at 95°Cfor10s, then ranging from 65 to95°C, increasing by 0.5°Cevery cycle. GAPDH was used as an internal control to normalize all the data and the relative expression was calculated through the comparative Cycle Threshold (Ct) method.

4.11. Tracking of Lusosomal UIsing LysoTracker

Lyso-Tracker Red (Beyotime Biotechnology Co., Ltd., Shanghai, China)was used to track lysosomes, which can exhibit an increased fluorescence intensity upon lysosomal acidification. We seeded cBMSCs in 12-well plates and treated them with Cur for 24 h, then the cells were treated with Lyso-Tracker(60 nM) and Hoechst 33,342 (2 ug/mL) for 20 min. The fluorescence was observed using an inverted fluorescence microscope after washing with PBS.

4.12. Immunofluorescence

The cBMSCs (2×104 cells/slide) were seeded onto slides and fixed with4% paraformaldehyde for 30 min. After co-incubation with 0.5% Triton X-100 for 5 min (Solarbio, Beijing, China), the sections were immersed in the blocking solution for 30 min. The enclosed liquid was removed, and cells were incubated with anti-LC3B antibodies (1:1000, Abcam, Cambridge, MA, USA)overnight at 4°C, then incubated with fluorochrome-conjugated secondary antibodies(Abcam, Cambridge, MA, USA) for 50 min at 37℃C. Finally, the cells were counterstained with DAPI (Beyotime Biotechnology, Shanghai, China) and monitored under a confocal microscope.

4.13.Western Blotting Analysis

The cell samples were lysed with the tissue and cell lysate (Solarbio, Beijing, China)containing protease inhibitor after washing with ice-cold PBS. The cell lysates containing 15 ugs of protein per sample were loaded into sodium-dodecyl sulfate-polyacrylamide (SDS-PA)(Solarbio, Beijing, China) gels and separated by electrophoresis. After transferring the proteins onto the polyvinylidene fluoride (PVDF) membrane, the latter was blocked nonspecifically with 5% nonfat dry milk (Solarbio, Beijing, China) for 1 h at room temperature. The membranes were incubated overnight with the primary antibodies anti-LC3B (1:2000, Abcam, Cambridge, MA, USA), anti-p62/SQSTM1(1:4000, Novus Biologicals, Littleton, NH, USA), and anti-β-actin (1:1000, Abcam, Cambridge, MA, USA) at 4°C, and the blots were washed with TBST(Solarbio, Beijing, China) prior to incubating them with secondary antibody (1:2000, Abcam, Cambridge, MA, USA) at 37 °C for 1 h. Subsequently, the membranes were developed by exposure to chemiluminescence reagents (Millipore, Billerica, MA, USA)and visualized with ChemiDocTM Imaging Systems(Tanon-5200, Shanghai, China). The band density was quantified using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA)for each group and normalized with β-actin.

4.14. Transmission Electron Microscopy (TEM)

The cell pellet was digested and collected into a 1.5mL centrifuge tube, then fixed with 2.5% glutaraldehyde(Solarbio, Beijing, China)for 2 h at room temperature. The samples were post-fixed with 1% osmium tetroxide for 1 h after washing with PBS, then we increased the dehydration in a stepwise manner in solutions of acetone and embedded them in812 epoxy resin(Beijing Zhongjingkeyi Technology Co., Ltd., Bejing, China). Subsequently, 50 nm sections were obtained from the ultra-microtome (EM UC7, Leica Microsystems Co., Ltd., Heidelberg, Germany). The sections were stained with uranyl acetate (Zhongjingkevi, Bejing, China)for 10-15 min and lead citrate (Zhongjingkei, Beijing, China)for 2 min. All specimens were viewed on a TEM (EM-1400PLUS, JEOL, Akishima, Tokyo, Japan).

4.15. Statistical Analysis

The results were obtained from three independent experiments and all data were shown as means ± standard deviations (SD). Statistical values were analyzed using the IBM SPSs Statistics 25 and illustrated using GraphPad Prism 9.0(GraphPad Software, San Diego, CA, USA). Statistically significant differences were determined using performed using a one-way analysis of variance (ANOVA)and the Student's t-test. p values<0.05 were considered to be significant differences.

5. Conclusions

Our findings shed light on the relationship between Cur, cBMSC senescence, and autophagy. The data from our study suggests that Cur can alleviate the senescence state of cBMSCs while activating autophagy and promoting lysosomal acidification. Moreover, further evidence demonstrated that Cur-induced autophagy is a potential mechanism for ameliorating cBMSC senescence. Cur could be a promising activator and conservator for improving the function of MSCs. In our opinion, the positive effects of Cur on aging cannot be neglected. Future studies should focus on the effect of the regulation of Cur on MSC fate to enhance the therapeutic potential of MSCs in various diseases, such as tissue damage and degenerative and inflammatory diseases.

This article is extracted from Int. J. Mol. Sci. 2021, 22, 11356. https://doi.org/10.3390/ijms222111356 https://www.mdpi.com/journal/ijms