黄芪甲苷通过SIRT1/AP-1信号轴调控软骨细胞氧化应激的机制研究OA

Objective To explore the protective effect of astragalosideⅣ(AS-Ⅳ)on oxidative damage model induced by interleukin-1β(IL-1β)in SW1353 cells and its underlying mechanisms.Methods The SW1353 cells were treated with 100 μg/L IL-1β for 48 hours to construct an in vitro cell model of oxidative damage.The cells were randomly divided into six groups:blank control,model,AS-Ⅳ,sirtuin 1(SIRT1)-knockdown,activator protein-1(AP-1)-overexpression,and SIRT1-knockdown+AP-1-overexpression.The optimal concentration and time for IL-1β and the optimal concentration and time for AS-Ⅳ in the treatment of osteoarthritis(OA)were screened using the cell counting kit-8(CCK-8)assay.SIRT1 knockdown and AP-1 overexpression were achieved by lentiviral transfection.The contents of malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT),and glutathione peroxidase(GSH-Px)in each group of cells were detected by enzyme-linked immunosorbent assay(ELISA).The mRNA and protein expressions of AP-1,SIRT1,Bax,Bcl-2,and caspase-3 were detected by quantitative reverse transcription-polymerase chain reaction(qRT-PCR)and Western blotting,respectively.Results The contents of CAT(kU/L:7.64±2.36 vs.26.98±2.05),SOD(kU/L:25.92±1.78 vs.39.63±2.51),and GSH-Px(kU/L:176.70±8.36 vs.261.40±12.71)in the model group were significantly lower than those in the blank control group(all P<0.05),while the content of MDA was significantly higher(μmol/L:13.52±2.88 vs.6.24±0.37,P<0.05).After the addition of AS-Ⅳ,compared with the model group,the contents of CAT(kU/L:24.82±2.49 vs.7.64±2.36),SOD(kU/L:38.56±2.25 vs.25.92±1.78),and GSH-Px(kU/L:231.40±20.11 vs.176.70±8.36)in the AS-Ⅳ group were significantly increased(both P<0.05),and the content of MDA(μmol/L:7.12±1.00 vs.13.52±2.88,P<0.05)was significantly decreased.Moreover,AS-Ⅳ could effectively inhibit the apoptosis of SW1353 cells,down-regulate the protein expression of AP-1(AP-1/Tubulin:0.39±0.03 vs.1.11±0.06,P<0.05),and up-regulate the protein expression of SIRT1(SIRT1/Tubulin:1.34±0.11 vs.1.06±0.12,P<0.05).After reducing SIRT1 knockdown,AP-1 overexpression,and combined SIRT1 knockdown with AP-1 overexpression,compared with the AS-Ⅳ group,the contents of CAT(kU/L:5.32±1.22,5.93±2.11,4.13±1.93 vs.24.82±2.49),SOD(kU/L:25.19±1.18,26.02±2.04,21.02±1.56 vs.38.56±2.25),and GSH-Px(kU/L:181.40±12.71,175.50±10.40,148.00±16.64 vs.231.40±20.11)in the SIRT1 group,AP-1 group,and SIRT1+AP-1 group were significantly decreased(all P<0.05),while the MDA content increased(μmol/L:13.83±2.51,16.85±2.74,45.00±1.66 vs.7.12±1.00,all P<0.05),and the mRNA and protein expression levels of AP-1,Bax,and caspase-3 were significantly increased[AP-1 mRNA expression(2-ΔΔCt):4.93±0.25,5.30±0.57,6.10±0.18 vs.1.53±0.42,AP-1 protein expression(AP-1/Tubulin):1.11±0.04,1.18±0.10,1.60±0.09 vs.0.39±0.03;Bax mRNA expression(2-ΔΔCt):4.60±0.43,4.76±0.63,6.16±0.53 vs.1.49±0.45,Bax protein expression(Bax/Tubulin):0.80±0.11,0.92±0.06,1.48±0.10 vs.0.43±0.08;caspase-3 mRNA expression(2-ΔΔCt):7.27±0.90,7.12±1.09,9.38±0.58 vs.1.20±0.35,caspase-3 protein expression(caspase-3/Tubulin):0.75±0.11,0.79±0.06,1.02±0.11 vs.0.48±0.04,all P<0.05],while the mRNA and protein expression levels of SIRT1 and Bcl-2 were significantly decreased(SIRT1 mRNA expression(2-ΔΔCt):0.64±0.09,0.63±0.05,0.28±0.06 vs.0.86±0.05,SIRT1 protein expression(SIRT1/Tubulin):0.94±0.05,0.97±0.07,0.26±0.06 vs.1.34±0.11;Bcl-2 mRNA expression(2-ΔΔCt):0.38±0.04,0.43±0.05,0.18±0.08 vs.0.70±0.09,Bcl-2 protein expression(Bcl-2/Tubulin):0.76±0.06,0.80±0.07,0.34±0.06 vs.1.15±0.08,all P<0.05).Conclusion AS-Ⅳ can protect SW1353 cells from oxidative damage induced by IL-1β and alleviate cell apoptosis,the mechanism of its action may be related to its activation of the SIRT1/AP-1 signaling pathway.