【摘要】 目的 探讨天麻素(gastrodin,GSTD)在骨关节炎中对软骨细胞自噬的影响及其作用机制。方法通过CCK-8法检测不同浓度GSTD对软骨细胞活力的影响。使用IL-1β (10 ng/mL)刺激软骨细胞建立体外骨关节炎细胞损伤模型。将细胞分为对照组、IL-1β组、IL-1β+GSTD组。培养48 h之后通过流式细胞术检测细胞凋亡率;蛋白质印迹法检测自噬相关蛋白(LC3和P62)、AMPK和mTOR蛋白的表达;酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测细胞培养上清液中COL2A1、MMP-13、TNF-α和IL-6的分泌。使用AMPK抑制剂(化合物C)观察AMPK通路在软骨细胞自噬过程中所发挥的作用。结果 GSTD在12.5～50μmol/L范围内增加细胞活力,选择25μmol/L GSTD预处理细胞进行后续试验。与对照组比较,IL-1β刺激细胞促进软骨细胞凋亡(P<0.05),抑制自噬(P<0.05),增加炎症因子的分泌(P<0.05),抑制COL2A1和增加MMP13的表达(P<0.05)。与IL-1β组比较,GSTD预处理的细胞自噬能力增强(P<0.05),细胞凋亡减少(P<0.05),炎症因子和MMP-13的分泌减少(P<0.05),COL2A1合成增加(P<0.05),促进AMPK磷酸化和抑制mTOR的磷酸化。使用AMPK通路抑制剂(化合物C)处理细胞可以逆转GSTD对软骨细胞的保护作用,导致细胞凋亡率下调,自噬水平下调,炎症因子和MMP-13分泌增加,COL2A1合成减少(P<0.05)。结论 GSTD通过调控AMPK/mTOR信号通路促进细胞自噬,从而抑制软骨细胞的凋亡和炎症反应,促进胶原合成,对软骨细胞起保护作用。更多还原

【Abstract】 Objective To investigate the effect of gastrodin(GSTD) on the autophagy of chondrocytes in osteoarthritis and its mechanism.Methods The effect of GSTD on the chondrocyte viability was detected by CCK-8 assay.Chondrocytes were stimulated with IL-1β(10 ng/mL) to establish the osteoarthritis cell injury model in vitro.Cells were divided into 3 groups:control group,IL-1 β group,and IL-1 β+GSTD group.The apoptosis rate was detected by flow cytometry after 48 h culture.Western blotting was used to detect the expression levels of autophagy related proteins(LC3and P62),AMPK,and mTOR.The secretion levels of COL2A1,MMP-13,TNF-α,and IL-6 in cell culture supernatant were determined by enzyme-linked immunosorbent assay(ELISA).AMPK inhibitor(compound C) was used to observe the role of AMPK pathway in chondrocyte autophagy.Results GSTD treatment increased cell viability in the range of 12.5～50 μmol/L.Cells pretreated with 25 μmol/L GSTD were selected for the subsequent experiments.IL-1β stimulated cells to promote chondrocyte apoptosis(P<0.05),inhibited autophagy(P<0.05),increased the secretion of inflammatory factors(P<0.05),inhibited the expression level of COL2A1 and increased the expression level of MMP-13(P<0.05),when compared with the control group.Compared with the cells in the IL-1β group,cells in the GSTD pretreated group had increased autophagy(P <0.05),decreased apoptosis(P <0.05),decreased levels of inflammatory cytokines,decreased expression level of MMP-13 and phosphorylation of mTOR(P <0.05),and increased expression level of COL2A1(P<0.05) and phosphorylation of AMPK.Treatment of cells with AMPK pathway inhibitor(compound C) reversed the protective effect of GSTD on chondrocytes,resulting in the decrease in apoptosis ratio,autophagy level and expression level of COL2A1,and the increased in secretion of inflammatory factors and expression level of MMP-13(P<0.05).Conclusion GSTD promotes the chondrocytes autophagy through the regulation of AMPK/mTOR signaling pathway,and protects chondrocytes from apoptosis and inflammatory response and by promotion of collagen synthesis.更多还原