麦冬皂苷D调控IGF-1/mTOR/PGE2通路对脂多糖所致骨关节炎软骨细胞损伤的保护作用

Ophiopogonin D protects lipopolysaccharide-induced osteoarthritis chondrocytes injury by regulating IGF-1/mTOR/PGE2 pathway

目的探讨麦冬皂苷D对脂多糖(LPS)所致骨关节炎软骨细胞损伤的保护作用及其机制。方法提取10只SPF级SD大鼠关节软骨的正常软骨细胞,进行体外培养,将培养后的软骨细胞随机分为正常对照组、模型组及麦冬皂苷D低、中、高剂量组。除正常对照组外,其余各组均用16 mol/L的LPS诱导骨关节炎模型。造模成功后,麦冬皂苷D低、中、高剂量组分别加入5、10、20 mol/L麦冬皂苷D,而正常对照组和模型组均加入等量0.9%氯化钠注射液。CCK-8检测各组细胞的活性,ELISA法检测各组细胞肿瘤坏死因子(TNF)-、白介素(IL)-1、IL-6、胰岛素样生长因子(IGF)-1、前列腺素E2(PGE2)浓度,Hoechst 33342染色法观察各组细胞核的形态,Western blotting检测各组细胞的Bcl-2、Bax、Caspase-3、Caspase-9及mTOR的蛋白表达。结果麦冬皂苷D可显著改善LPS所致骨关节炎软骨细胞的活性(均P<0.05),同时能抑制LPS所致骨关节炎软骨细胞的TNF-、IL-1、IL-6和PGE2的表达(均P<0.05),促进IGF-1的表达(P<0.05);麦冬皂苷D可改善LPS所致骨关节炎软骨细胞的凋亡,抑制LPS所致骨关节炎软骨细胞的Bax、Caspase-3、Caspase-9及mT OR的表达(均P<0.05),促进Bcl-2的表达(P<0.05)。结论麦冬皂苷D可能通过IGF-1/mT OR/PGE2信号通路上调软骨细胞IGF-1的表达,下调PGE2和mTOR的表达,从而保护骨关节炎软骨细胞。

Objective To study the protective effect of ophiopogonin D on the osteoarthritis chondrocytes injury induced by lipopolysaccharide(LPS)and its mechanism.Methods Normal chondrocytes were extracted from ten SPF grade SD rat articular cartilage and cultured in vitro.The cultured chondrocytes were randomly divided into five groups:normal control group,model group,low dose group,middle dose group and high dose group of saponin D,with 5,10,20μmol/L of ophiopogonin D,respectively.Except for the normal control group,the other groups of cells were induced osteoarthritis model with 16μmol/L LPS.After successful modeling,5,10,20μmol/L of ophiopogonin D were added to the low,middle and high dose groups,while the normal control group and the model group were added with the same amount of normal saline.The activity of each group of cells was detected by CCK-8.The concentrations of tumor necrosis factor-a(TNF-α),interleukin(IL-1β),interleukin(IL-6),Insulin-like growth factor-1(IGF-1)and prostaglandin(PGE2)in each group were detected by ELISA.The morphology of the nucleus of each group was observed by Hoechst 33342.The protein expression of Bcl-2,Bax,Caspase-3,Caspase-9 and mTOR in each group was detected by Western blotting.Results Ophiopogonin D could improve the activity of osteochondral chondrocytes induced by LPS significantly(P<0.01).Ophiopogonin D could significantly inhibit the expression of TNF-α,IL-1β,IL-6 and PGE2(all P<0.05)and promote the expression of IGF-1(P<0.05)in osteoarthritic chondrocytes induced by LPS.Ophiopogonin D could improve the apoptosis of osteoarthritic chondrocytes induced by LPS.Ophiopogonin D could significantly inhibited the expression of Bax,Caspase-3,Caspase-9 and mTOR(allP<0.05)and promoted the expression of Bcl-2(P<0.05)in osteoarthritic chondrocytes induced by LPS.Conclusions Ophiopogonin D may up-regulate the expression of IGF-1 and down-regulate the expression of PGE2 and mTOR in osteoarthritic chondrocytes through IGF-1/mTOR/PGE2 signaling pathway to protect osteoarthritis chondrocytes..