花青素通过诱导细胞自噬信号通路减轻H2O2对大鼠软骨细胞损伤的研究

Study on anthocyanins attenuated H2O2-induced apoptosis in rat chondrocytes through autophagy signaling pathway

软骨细胞凋亡与骨关节炎的发生发展密切相关,而花青素具有抗炎和抗凋亡的作用,故本研究探讨了花青素活化自噬信号通路在抑制H2O2诱导的大鼠软骨细胞凋亡中的作用.采用CCK8法检测0、100μmol/L、200μmol/L、400μmol/L、600μmol/L、800μmol/L、1000μmol/L的H2O2对大鼠软骨细胞存活率的影响.将大鼠软骨细胞分为空白对照组(细胞未经任何处理)、H2O2组(400μmol/L H2O2处理大鼠软骨细胞6 h)和花青素+H2O2组(300μg/mL花青素处理24 h后,400μmol/L H2O2处理大鼠软骨细胞6 h),利用CCK8、Annexin V-FITC/PI双染色法、real-time PCR及western blot分别检测了细胞生存率、凋亡率、凋亡相关蛋白Bax和Bcl-2和自噬相关蛋白Beclin-1和p62基因和蛋白水平的表达.结果表明,随着H2O2浓度的增加,细胞的生存率逐渐降低(P<0.05;与空白对照组比较,H2O2组细胞活性明显降低,凋亡比例升高,在mRNA和蛋白水平上,Bax和p62表达升高,而Bcl-2和Beclin-1表达下降;与H2O2组比较,花青素+H2O2组细胞存活率相对升高,凋亡比例下降,在mRNA和蛋白水平上,Bax和p62表达相对下降,而Bcl-2和Beclin-1表达相对升高.花青素通过激活自噬信号通路,从而可减轻H2O2诱导的大鼠软骨细胞损伤.

Chondrocyte apoptosis is closely related to the development and progression of osteoarthritis.Anthocyanins have anti-inflammatory and anti-apoptotic effects.This study aimed to investigate the anthocyanin-activate autophagy in inhibiting H2O2-induced cell apoptosis.The effects of different concentrations(0,100μmol/L,200μmol/L,400μmol/L,600μmol/L,800μmol/L and 1000μmol/L)of H2O2 on the survival rate of rat chondrocytes were detected by CCK8 assay.Rat chondrocytes were divided into blank control group(cell without any treatment),H2O2 group(treated with 400μmol/L H2O2 on rat chondrocytes for 6 h)and anthocyanin+H2O2 group(treated with 300μmol/L anthocyanins on rat chondrocytes for 24 h,then treated with 400μmol/L H2O2 for 6 h).CCK8,flow cytometry,real-time PCR and western blot were used to detect cell viability,apoptosis and expressions of Bax,Bcl-2,Beclin-1 and p62 mRNA and protein,respectively.With the increase of H2O2 concentration,the survival rate of cells decreased gradually(P<0.05;compared with the blank control group,the H2O2 group was significantly decreased,the proportion of apoptosis increased,and the expression of Bax and p62 on the mRNA and protein levels were significantly increased.The expression of Bcl-2 and Beclin-1 were decreased,compared with H2O2 group,the cell survival rate of Anthocyanins+H2O2 group were increased,the apoptosis rate was decreased,and the expression of Bax and p62 decreased on the mRNA and protein levels were decreased.The expression of Bcl-2 and Beclin-1 was relatively increased.Anthocyanins can attenuate H2O2-induced rat chondrocyte injury by activating autophagy signaling pathway.