| Objective: BPA(bisphenol A)is an environmental endocrine-disrupting compound with a structure similar to that of estrogen that exhibits estrogenic or estrogenic antagonistic effects by binding to classical estrogen receptors α,β,and non-classical estrogen receptors,with significant effects on bone.Numerous studies have shown that BPA can cause damage to cartilage,but the exact mechanism of action needs further investigation.It was found that mi R-449 a could inhibit the expression of SIRT1,which plays an extremely important role in the signaling pathways of body metabolism,cell cycle and apoptosis.However,whether BPA causes damage to chondrocytes through mi R-449 a and SIRT1 has not been reported.Therefore,we selected SW1353 chondrocytes for in vitro culture to investigate the effects of BPA on chondrocyte proliferation,apoptosis and senescence and the regulatory roles of mi R-449 and SIRT1.This study provides new clues to elucidate the mechanism of cartilage damage caused by BPA.Methods: SW1353 cells were cultured in L-15 complete culture medium containing 10%fetal bovine serum,100 mg/L penicillin and 100 mg/L streptomycin.The effect of BPA on the survival rate of SW1353 cells was detected by CCK8 method;proteoglycan synthesis was detected by Alcian Blue staining;Western blot was used to detect cartilage extracellular matrix synthesis-related proteins(Collagen II,Collagen Ⅹ),apoptosis-related proteins(Bax,Bcl-2,Caspase3),senescence marker factors(p53,p21,p16 and BAG1),cell cycle-related proteins(Cyclin D1,CDK4 and CDK6)and SIRT1;flow cytometry was used to detect apoptosis and cell cycle;q PCR was used to detect mi R-449 a,SIRT1,cell cycle-related factors(Cyclin D1,CDK4 and CDK6),senescence markers(p53,p21,p16 and BAG1)and senescence-related proteins.and BAG1)and senescence-associated secretory phenotype SASP(IL-1α,TGF-β,IL-1β and MMP13)expression levels.dual luciferase reporter gene assay was performed to detect the direct target m RNA of mi RNA.mi R-449 a expression was upregulated by resveratrol,inhibitor of mi R-449 a,and the expression levels of the above factors were detected.expression levels.Results: 1.Effect of BPA on the survival rate of SW1353 cells: 5 μM BPA significantly decreased the cell survival rate(P<0.05),and 50 μM BPA decreased the survival rate to46%.2.Effect of BPA on the apoptosis of SW1353 cells: Flow cytometry detected the apoptosis rate and found that BPA significantly increased the apoptosis rate of SW1353cells(P<0.05);compared with the control group,the expression of pro-apoptotic The expression of caspase3,a marker protein of apoptosis,was significantly increased(P<0.05)and the expression of Bcl-2,an anti-apoptotic protein,was significantly decreased(P<0.05)compared with the control group;3.Effect of BPA on the synthesis of extracellular matrix in SW1353 cells: the effect of BPA on the synthesis of proteoglycans in SW1353 cells was detected by alcian blue staining,and the intensity of alcian blue staining was found to be weaker than that of the control group,and Alcian blue was dissolved with guanidine hydrochloride for Quantitative analysis showed that the proteoglycan production in the 10 μM BPA group was significantly lower than that in the control group(P<0.05);BPA treatment significantly inhibited the expression of cartilage extracellular matrix synthesis markers such as Collagen II and Collagen Ⅹ(P<0.05).4.Effects of BPA on senescence in SW1353 cells:β-galactosidase activity,a marker of senescence,was significantly increased(P<0.05);elevated expression of senescence marker p53,p21,p16 m RNA and protein,decreased expression of anti-aging factor BAG1 m RNA and protein(P<0.05);significantly increased expression of senescence-related secretory phenotype(P<0.05);5.Effect of BPA on SW1353 cell cycle:cell cycle was blocked in G0/G1 phase,cycle-related factors Cyclin D1,CDK4,CDK6 m RNA and protein expression were significantly reduced(P<0.05).6.BPA induced senescence in SW1353 cells through SIRT1: compared with the control group,SIRT1 protein expression level was significantly decreased after BPA treatment(P<0.05),and SIRT1 was elevated in the RES group,and compared with the BPA group,the BPA+RES group significantly increased SIRT1 protein levels compared to the BPA group.The rate of β-galactosidase positive cells was significantly increased in the BPA group compared with the control group(P<0.05),and there was no significant change in the RES group;the rate of positive cells was decreased in the BPA+RES group compared with the BPA group,and the difference was statistically significant(P<0.05).Compared with the control group,the expression of senescence-related factors m RNA and protein was significantly increased in the BPA group(P<0.05),and the expression of anti-aging factor BAG1 m RNA and protein was significantly decreased in the BPA+RES group(P<0.05);compared with the BPA group,the expression of senescence-related factors was decreased and the expression of BAG1 was significantly increased in the BPA+RES group.Compared with the control group,cells in G0/G1 phase were significantly increased in the BPA group(P<0.05);compared with the BPA group,the ratio of cells in G0/G1 phase was significantly decreased in the BPA+RES group,and the difference was statistically significant(P<0.05);the expression levels of cycle marker protein and m RNA were significantly decreased in the BPA group(P<0.05);compared with the BPA group,the BPA+RES group The expression levels of cycle marker protein and m RNA increased in the BPA group compared with the BPA group,and the difference was statistically significant(P<0.05);SASP expression was significantly higher in the BPA group compared with the control group and significantly lower in the BPA+RES group compared with the BPA group(P<0.05).7.BPA induced SW1353 cells through the inhibitory effect of mi R-449 a on SIRT1 Senescence: BPA significantly up-regulated mi R-449 a expression,and mi RDB database was used to predict the potential binding sites of mi R-449 a to SIRT1;dual luciferase reporter gene assay verified the targeting inhibitory effect of mi R-449 a on SIRT1;the m RNA and protein expression levels of SIRT1 were significantly increased in the mi R-449 a inhibitor group compared with the NC group.Compared with the negative control group,the inhibitor group significantly decreased the expression levels of senescence marker β-galactosidase activity and senescence marker factors(P<0.05)and significantly increased the expression levels of cycle-related factors and senescence-related secretory phenotypes(P<0.05),while the BPA group significantly increased the expression levels of β-galactosidase activity and senescence marker factors(P<0.05)and significantly decreased the expression levels of expression levels of cycle-related factors and senescence-related secretory phenotypes(P<0.05);compared with the BPA group,the expression levels of β-galactosidase activity,senescence marker factors and senescence-related secretory phenotypes were significantly lower(P<0.05)and the expression levels of cycle-related factors were significantly higher(P<0.05)in the BPA+inhibitor group.Conclusion: 1.BPA inhibits SW1353 extracellular matrix anabolism.2.BPA induces apoptosis in SW1353 cells.3.BPA accelerates SW1353 cell senescence through the inhibitory effect of mi R-449 a on SIRT1. |
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