姜黄素激活转化生长因子-β_(1)/Smad2通路保护关节软骨细胞损伤的机制研究

Mechanism of TGF-β_(1)/Smad2 pathway activated by curcumin to protect articular chondrocyte injury

目的 研究姜黄素对关节软骨细胞损伤的保护作用,以及对转化生长因子(TGF)-β_(1)/Smad2通路的影响。方法 选取6周龄SD雌性大鼠,脱颈椎处死后分离获取膝关节软骨细胞,甲苯胺蓝染色进行细胞鉴定。将细胞分为空白对照组、模型组以及姜黄素干预组(低、中、高剂量),其中模型组以及姜黄素干预组用5μg/L脂多糖预处理1 h,干预组(低、中、高剂量)加入含5、15、50μmol/L姜黄素的培养基,空白对照组、模型组加入不含姜黄素的培养基。培养24 h,采用Hoechst 33258染色法观察细胞凋亡情况,采用蛋白免疫印迹法检测细胞凋亡蛋白(Bcl-2、Bax、caspase-3、caspase-9)、软骨细胞外基质蛋白(Aggrecan、COLL2)以及TGF-β_(1)/Smad信号通路相关蛋白(TGF-β_(1)、Smad2/3、Smad7)表达水平,采用实时荧光定量聚合酶链反应(PCR)法检测TGF-β_(1)/Smad2信号通路基因表达情况。结果 模型组凋亡小体减少,细胞核固缩深染,姜黄素干预组随着浓度增加,凋亡小体较模型组呈逐渐降低趋势。5组细胞凋亡蛋白(Bcl-2、Bax、caspase-3、caspase-9)表达水平差异有统计学意义(F=30.382,17.362,39.284,18.855;P均<0.05),其中姜黄素干预组Bax、caspase-3、caspase-9高于空白对照组,低于模型组,而干预组Bcl-2低于空白对照组,高于模型组;5组细胞软骨细胞外基质蛋白(Aggrecan、COLL2)表达水平差异有统计学意义(F=26.605,40.699;P均<0.05),其中姜黄素干预组Aggrecan、COLL2高于空白对照组,模型组低于空白对照组,细胞外基质蛋白以及凋亡蛋白表达水平呈剂量依赖(P<0.05)。5组细胞TGF-β_(1)/Smad信号蛋白、基因(TGF-β_(1)、Smad2/3、Smad7)表达水平差异有统计学意义(F=27.667,13.684,26.029,61.032,25.877,35.934;P均<0.05),其中姜黄素干预组TGF-β_(1)、Smad2/3高于空白对照组、模型组,而Smad7低于空白对照组、模型组,TGF-β_(1)/Smad信号通路蛋白、基因表达水平呈剂量依赖(P<0.05)。结论 姜黄素可改善脂多糖诱导的软骨细胞炎性损伤,抑制细胞凋亡,促进细胞外基质合成,其作用机制可能与激活TGF-β_(1)/Smad2通路有关。

Objective To study the protective effect of curcumin on articular chondrocyte injury and the effect on TGF-β_(1)/Smad2 pathway.Methods The 6-week-old SD female rats were selected and sacrificed to iso-lated and obtained knee chondrocytes.And chondrocytes were identified by toluidine blue staining.The cells were divided into blank control group,model group and curcumin intervention group(low,medium and high dose).The model group and curcumin intervention group were pretreated with 5μg/L lipopolysaccharide for 1h,the interven-tion group(low,medium and high dose)was added with medium containing 5,15 and 50μmol/L curcumin,and the blank control group and model group were added with medium without curcumin.After culturing for 24 hours,Hoechst 33258 staining was used to observe the cell apoptosis,and Western blotting was used to detect cellular collagen(Bcl-2,Bax,caspase-3,caspase-9),protective extracellular matrix proteins(Aggrecan,COLL2)and the ex-pression levels of TGF-β_(1)/Smad signaling pathway related proteins(TGF-β_(1),Smad2/3,Smad7),and real-time fluo-rescence quantitative PCR was used to detect the gene expression of TGF-β_(1)/Smad2 signaling pathway.Results In the model group,the number of apoptotic bodies were decreased,and the cell nuclei were condensed and deeply stained.With the concentration of curcumin increased,the number of apoptotic bodies in the curcumin in-tervention group were gradually decreased compared with the model group.The expression levels of apoptotic pro-teins(Bcl-2,Bax,caspase-3,caspase-9)in the 5 groups were significantly differences(F=30.382,17.362,39.284,18.855;all P<0.05).The expression levels of Bax,caspase-3,caspase-9 in the curcumin intervention group were higher than those in the blank control group,but lower than those in the model group.Bcl-2 in the intervention group was lower than that in the blank control group but higher than that in the model group.There were signifi-cant differences in the expression levels of chondrocyte extracellular matrix protein(Aggrecan and COLL2)in the 5 groups(F=26.605,40.699;all P<0.05).The expression levels of Aggrecan and COLL2 in the curcumin interven-tion group were higher than those in the blank control group,and those in the model group were lower than those in the blank control group.The expression levels of extracellular matrix protein and apoptosis protein were dose-dependent(P<0.05).There were statistically significant differences in the expression levels of TGF-β_(1)/Smad signal-ing proteins and genes(TGF-β_(1),Smad2/3,Smad7)in the 5 groups(F=27.667,13.684,26.029,61.032,25.877,35.934;all P<0.05),among which,the levels of TGF-β_(1) and Smad2/3 in curcumin intervention group were higher than those in blank control group and model group,while the levels of Smad7 were lower than those in blank con-trol group and model group.The protein and gene expression levels of TGF-β_(1)/Smad signaling pathway were dose-dependent(P<0.05).Conclusion Curcumin can improve the inflammatory injury of chondrocytes induced by lipopolysaccharide,inhibit cell apoptosis,and promote extracellular matrix synthesis,the mechanism of which may be related to the activation of TGF-β_(1)/Smad2 pathway.