Curcumin Inhibits Aging Of Adipose-derived Mesenchymal Stem Cells And Improves The Therapeutic Effect Of Osteoarthritis In Mice

Objective:Osteoarthritis(OA)is a common senile degenerative joint disease with various treatment methods.Among them,intra-articular injection of mesenchymal stem cells(MSCs)is one of the most potential treatments,and clinical experiments have been carried out in Peking University Third Hospital.However,ex vivo subculture of MSCs is prone to senescence and greatly affects the efficacy.Curcumin(CCM)is the main active ingredient in turmeric,which has good antioxidant capacity and can reduce cellular senescence.The aim of this study was to investigate the therapeutic effect of CCM-pretreated adipose-derived mesenchymal stem cells(ADSCs)in a mouse OA model and to explore its mechanism of action.Methods:In vitro experiments:Adipose was isolated from the groin of C57BL/6 mice at 4–6 weeks,and ADSCs were extracted and primary cultured and identified by cell immunofluorescence.Tert-butylhydroperoxide(TBHP)was used to induce ADSCs and construct a cell senescence model.Cell counting kit(CCK-8)was used to detect cell proliferation ability and drug toxicity.β-galactosidase staining was used to observe the proportion of senescence-positive cells.Cell immunofluorescence was used to detect senescence and autophagy-related gene expression levels morphologically.Western blot experiments were performed to detect markers associated with aging and autophagy.In vivo experiment:32 C57BL/6 mice aged 10 weeks(about 20 g)were randomly divided into 4 groups of 8 mice each.That is,the control group,OA group,ADSCs group,and ADSCs+CCM group.The control group only underwent arthrotomy,and the other three groups were used to construct OA mouse models,that is,the medial meniscus instability model(DMM),which were treated with intra-articular injection after 4 and 6weeks of modeling,the control and OA groups were injected with 8μl sterile PBS,the ADSCs group was injected with 1×10~5 ADSCs dissolved in 8μl PBS,and the ADSCs+CCM group was injected with 1×10~5 CCM-pretreated ADSCs dissolved in 8μl PBS.After 8 weeks of behavioral testing,the mice were sacrificed and the affected knee joints were taken for Micro CT examination,HE,safranin fast green staining and immunohistochemical experiments.Results:In vitro experiments:Immunofluorescence experiments showed that CD44 and CD90 were positively expressed in ADSCs.CCK8 showed that CCM from 0μM to 15μM was not toxic to ADSCs,and CCM from 5μM showed the best cell proliferation ability.As the concentration of TBHP added increased,the degree of cellular senescence also increased.To assess the therapeutic effect of CCM on TBHP-induced ADSCs,ADSCs were pre-protected with different concentrations of CCM for 2 h,followed by incubation with TBHP for 24 h.CCK8 showed that 5μM CCM showed a good protective effect.By observing the proportion ofβ-galactosidase positive cells in each group,it was known that the proportion ofβ-galactosidase positive cells in the TBHP group was significantly higher than that in the blank group.After CCM pretreatment,the proportion ofβ-galactosidase positive cells decreased significantly despite the addition of TBHP.The protein expression levels of P16 and P21 genes in TBHP group were higher than those in blank group and CCM pretreatment group,and the fluorescence intensity of P21gene was also stronger.Numerous studies have shown that autophagy plays an important role in cellular senescence,and the protein levels of autophagy-related markers(LC3B,Beclin1)and cellular immunofluorescence results of LC3B gene showed that LC3B and Beclin1 gene levels were significantly lower in the TBHP group than in the CCM pretreatment and blank groups.LC3B gene also showed lower fluorescence intensity.However,when autophagy inhibitor(3-MA)was added,no significant difference was observed in the protein expression levels of LC3B and Beclin1 genes between TBHP and CCM pretreatment groups.In vivo experiment:The results of rotarod test showed that the residence time of OA mice on rotarod was lower than that of treatment group and control group,and the residence time of ADSCs+CCM mice was longer than that of ADSCs group.The results of safranin fast green staining and HE staining showed that cartilage wear,synovial hyperplasia,inflammatory infiltration were severe and pannus exposure was observed in the OA group,but these phenomena showed different degrees of improvement in the treatment group.OARSI and synovial scores showed more intuitive differences between groups,with ADSCs+CCM having lower scores than ADSCs and significantly lower scores than OA.Micro CT results also showed less subchondral bone damage in the ADSCs+CCM group.COL2A and MMP13 immunohistochemical experiments showed that COL2A expression was decreased and MMP13-positive cell rate was increased in the OA group,on the contrary,COL2A expression was restored and MMP13-positive cell rate was also significantly decreased in the treatment group.Conclusion:CCM inhibits the senescence of ADSCs by increasing autophagy in vitro.In vivo experiments have demonstrated that CCM-pretreated ADSCs are more effective in the treatment of OA,providing a new method for MSCs in the treatment of OA.