姜黄素激活TGF-β1/Smad2通路保护关节软骨细胞损伤实验研究

Curcumin Protected Articular Chondrocyte Injury by Activating TGF-β1/Smad2 Pathway

目的探讨姜黄素对关节软骨中转化生长因子-β1/信号转导蛋白Smad(TGF-β1/Smad2)通路的调节作用,及对软骨细胞增殖、凋亡、细胞外基质(ECM)合成和降解的影响。方法将细胞分成对照组,模型组,姜黄素组,TGF-β抑制剂组和姜黄素+抑制剂组,共5组。采用细胞计数试剂盒-8(CCK-8)检测细胞活性,采用流式细胞术检测细胞凋亡,采用Western Blot检测细胞中B细胞淋巴瘤2蛋白(Bcl-2)、Bcl-2相关X蛋白(Bax)、TGF-β1、Smad2、磷酸化Smad2(p-Smad2)、基质金属蛋白酶-13(MMP-13)、Ⅱ型胶原蛋白(COL2α1)的蛋白表达,qRT-PCR检测MMP-13、COL2α1 mRNA表达。结果姜黄素对白介素1β(IL-1β)诱导构建的骨关节炎(OA)细胞模型的最佳干预量为10μmol/L。与对照组比较,模型组细胞增殖能力下降(P<0.05),凋亡率上升(P<0.05),Bcl-2、TGF-β1、p-Smad2蛋白表达均下调(P<0.05),COL2α1 mRNA和蛋白表达均下调(P<0.05),Bax蛋白、MMP-13 mRNA和蛋白表达均上调(P<0.05)。与模型组比较,姜黄素组细胞增殖能力上升(P<0.05),凋亡率下降(P<0.05),Bcl-2、TGF-β1、Smad2蛋白表达均上调(P<0.05),COL2α1 mRNA和蛋白表达均上调(P<0.05),Bax蛋白、MMP-13 mRNA和蛋白表达均下调(P<0.05),TGF-β抑制剂处理加重了模型组细胞的损伤。与TGF-β抑制剂组比较,姜黄素+抑制剂组数据变化呈相反结果(P<0.05)。结论姜黄素能改善IL-1β诱导的关节软骨细胞损伤,减轻细胞凋亡,进而抑制ECM降解,其机制可能与激活TGF-β1/Smad2通路有关。

Objective To study the effects of curcumin on regulating transforming growth factor-β1(TGF-β1)/signal transduction protein Smad pathway in articular cartilage,and its effects on chondrocyte proliferation,apoptosis,extracellular matrix(ECM)synthesis and degradation.Methods The cells were divided into five groups:control group,model group,curcumin group,inhibitor group,and curcumin+inhibitor group.Cell activity was detected with cell counting kit-8(CCK-8).Flow cytometry was used to detect apoptosis.Western Blot was used to detect expressions of B-cell lymphoma 2 protein(Bcl-2),Bcl-2 associated X protein(Bax),TGF-β1,Smad2,phosphorylated Smad2(p-Smad2),matrix metalloproteinase-13(MMP-13),type Ⅱ collagen(COL2α1)in cells.qRT-PCR was used to detect the mRNA expressions of MMP-13 and COL2α1.Results The optimal dose of curcumin for IL-1β-induced osteoarthritis cell model was 10μmol/L.Compared with control group,the proliferation ability of cells in model group decreased(P<0.05),the apoptosis rate increased(P<0.05),protein expression levels of Bcl-2,TGF-β1,and p-Smad2 were all down-regulated(P<0.05),mRNA and protein expressions of COL2α1 were down-regulated(P<0.01),Bax protein,MMP-13 mRNA and protein expressions were all up-regulated(P<0.05).Compared with model group,cell proliferation increased(P<0.05),apoptosis rate decreased(P<0.05),protein expression levels of Bcl-2,TGF-β1,and Smad2 were up-regulated(P<0.05),mRNA and protein expressions of COL2α1 were up-regulated(P<0.05),Bax protein,MMP-13 mRNA and protein expressions were all downregulated(P<0.05)in curcumin group.TGF-β1 inhibitor aggravated the injury of model cells.Compared with TGF-β inhibitor group,an opposite trend showed in curcumin+inhibitor group.Conclusion Curcumin ameliorated IL-1β-induced articular chondrocyte injury,attenuated apoptosis,and thus further inhibited ECM degradation,which was possibly associated by activating TGF-β1/Smad2 pathway.