大黄素对骨关节炎软骨细胞增殖、凋亡和氧化应激的影响

Effects of emodin on the proliferation,apoptosis and oxidative stress of chondrocytes in osteoarthritis

背景:既往研究显示,沉默交配型信息调节因子2同源蛋白1(silent mating-type information regulator 2 homolog 1,SIRT1)-雷帕霉素机械靶蛋白(mechanistic target of rapamycin,mTOR)信号在骨关节炎进展中起重要作用,大黄素对骨关节炎软骨细胞有一定的保护作用。目的:基于SIRT1-mTOR探究大黄素对骨关节炎软骨细胞增殖、凋亡和氧化应激的影响。方法:体外分离培养大鼠软骨细胞,采用10 ng/mL的白细胞介素1β诱导构建体外骨关节炎细胞模型,以CCK-8法测定经0,20,40,80,120,160μmol/L的大黄素处理后的大鼠软骨细胞活力,选出合适大黄素作用浓度。将软骨细胞随机分为对照组、模型组、大黄素低剂量组、大黄素高剂量组、SIRT1抑制剂EX527组、大黄素高剂量+EX527组,除对照组外其余各组以10 ng/mL的白细胞介素1β诱导构建体外骨关节炎模型,同时以大黄素和EX527分组处理细胞后,用CCK-8法、Edu染色法及流式细胞术分别检测各组细胞增殖与凋亡情况;用试剂盒检测各组细胞活性氧相对含量、丙二醛与抗氧化酶超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶水平;免疫印迹检测各组细胞基质降解、凋亡与SIRT1-mTOR通路相关蛋白表达。结果与结论:①与对照组相比,模型组大鼠软骨细胞活力、Edu阳性率、超氧化物歧化酶与过氧化氢酶、谷胱甘肽过氧化物酶水平、Bcl-2与SIRT1蛋白表达降低,凋亡率、活性氧相对含量、丙二醛水平、Bax与基质金属蛋白酶3、基质金属蛋白酶9蛋白表达及p-mTOR/mTOR升高(P<0.05)。与模型组相比,大黄素低、高剂量组上述各项指标呈相反变化(P<0.05);EX527组各指标水平与大黄素各组呈相反趋势(P<0.05)。②与大黄素高剂量组相比,大黄素高剂量+EX527组细胞活力、Edu阳性率、超氧化物歧化酶与过氧化氢酶、谷胱甘肽过氧化物酶水平、Bcl-2与SIRT1蛋白表达降低,凋亡率、活性氧相对含量、丙二醛水平、Bax与基质金属蛋白酶3、基质金属蛋白酶9蛋白表达及p-mTOR/mTOR升高(P<0.05)。③结论:大黄素可通过激活SIRT1-mTOR信号而抑制骨关节炎软骨细胞氧化应激,从而促进软骨细胞增殖并减轻其凋亡。

BACKGROUND:Previous studies have shown that silent mating-type information regulator 2 homolog 1(SIRT1)-mechanistic target of rapamycin(mTOR)signaling plays an important role in the progression of osteoarthritis.Emodin has a protective effect on osteoarthritic chondrocytes.OBJECTIVE:To investigate the effects of emodin on the proliferation,apoptosis and oxidative stress of osteoarthritic chondrocytes based on the SIRT1-mTOR signaling.METHODS:Rat chondrocytes were isolated and cultured in vitro.Osteoarthritic chondrocyte model in vitro was induced by 10 ng/mL interleukin-1β.Cell counting kit-8 method was used to determine the viability of rat chondrocytes treated with 0,20,40,80,120,160μmol/L emodin,and the appropriate concentration of emodin was selected.Rat chondrocytes isolated and cultured in vitro were randomly divided into control group,model group,low-dose emodin group,high-dose emodin group,EX527 group,and high-dose emodin+EX527 group.In vitro osteoarthritis model was constructed by induction of 10 ng/mL interleukin 1βin all groups except the control group.The cells in the latter four groups were correspondingly treated with emodin or/and EX527.The proliferation and apoptosis of chondrocytes in each group were detected by cell counting kit-8,Edu staining and flow cytometry respectively.The relative content of reactive oxygen species and the levels of malondialdehyde,superoxide dismutase,catalase,and glutathione peroxidase in chondrocytes of rats in each group were measured with the kit.The expression of proteins related to cell matrix degradation,apoptosis and the SIRT1-mTOR pathway-related proteins in each group were detected by western blot.RESULTS AND CONCLUSION:Compared with the control group,the survival rate of chondrocytes,the positive rate of Edu,the levels of superoxide dismutase,catalase,and glutathione peroxidase,and the expression of Bcl-2 and SIRT1 proteins in the model group were decreased,while the apoptosis rate,the relative content of reactive oxygen species,the level of malondialdehyde,the expression of Bax,matrix metalloproteinase 3,matrix metalloproteinase 9 proteins,and p-mTOR/mTOR were increased(P<0.05).Compared with the model group,the survival rate of chondrocytes,the positive rate of Edu,the levels of superoxide dismutase,catalase,and glutathione peroxidase,and the expression of Bcl-2 and SIRT1 proteins in the low-and high-dose emodin groups were increased,while the apoptosis rate,the relative content of reactive oxygen species,the level of malondialdehyde,the expression of Bax,matrix metalloproteinase 3,matrix metalloproteinase 9 proteins,and p-mTOR/mTOR were decreased(P<0.05).Compared with the low-and high-dose emodin groups,the indexes of EX527 group showed the opposite trend(P<0.05).Compared with the high-dose emodin group,the survival rate of chondrocytes,the positive rate of Edu,the levels of superoxide dismutase,catalase,and glutathione peroxidase,and the expression of Bcl-2 and SIRT1 proteins in the high-dose emodin+EX527 group were decreased,while the apoptosis rate,the relative content of reactive oxygen species,the level of malondialdehyde,the expression of Bax,matrix metalloproteinase 3,matrix metalloproteinase 9 proteins,and p-mTOR/mTOR were increased(P<0.05).To conclude,emodin can inhibit oxidative stress of osteoarthritic chondrocytes by activating the SIRT1-mTOR signaling,thereby promoting chondrocyte proliferation and reducing apoptosis.