基于PINK1/Parkin线粒体自噬通路探讨骨痹方抗膝骨关节炎的机制研究

Investigating the mechanism of Gubi Prescription against knee osteoarthritis based on PINK1/Parkin mitophagy pathway

目的观察骨痹方调控PTEN诱导假定激酶1(PINK1)/E3泛素连接酶(Parkin)线粒体自噬通路抗膝骨关节炎的机制。方法构建双荧光素酶报告基因筛选骨痹方。将软骨细胞随机分为空白组、模型组、阳性药组、骨痹方组。除空白组外其余组均以羰基-氰-对-三氟甲氧基苯腙(FCCP)诱导。CCK-8法检测软骨细胞增殖情况。RT-qPCR法检测PINK1、Parkin、微管相关蛋白1轻链3(LC3)、B细胞淋巴瘤-2(Bcl-2)、大分子B淋巴细胞瘤(Bcl-xL)、髓细胞白血病1(Mcl-1)基因表达量。将60只小鼠随机分为假手术组、模型组、阳性药组、骨痹方组,每组15只采用改良Hulth法建立小鼠膝骨关节炎模型造模成功后番红O固绿染色观察软骨组织。Western Blot检测PINK1、Parkin、LC3、Bcl-2、Bcl-xL、Mcl-1蛋白表达量。结果与空白组比较,模型组软骨细胞PINK1、LC3 mRNA表达增加,OD值及Parkin、Bcl-2、Bcl-xL、Mcl-1 mRNA表达降低(P<0.05);与模型组比较,阳性药组和骨痹方组软骨细胞PINK1、LC3 mRNA表达减少,OD值及Parkin、Bcl-2、Bcl-xL、Mcl-1 mRNA表达增加(P<0.05)。与阳性药组比较,骨痹方组软骨细胞PINK1、LC3mRNA表达降低,Parkin、Bcl-2、Bcl-xL、Mcl-1 mRNA表达升高(P<0.05)。与假手术组比较,模型组软骨组织中PINK1、LC3Ⅱ/LC3Ⅰ蛋白表达增加而Parkin、Bcl-2、Bcl-xL、Mcl-1蛋白表达减少(P<0.05);与模型组比较,阳性药组、骨痹方组PINK1、LC3Ⅱ/LC3Ⅰ蛋白表达降低,而Parkin、Bcl-2、Bcl-xL、Mcl-1蛋白表达升高(P<0.05)与阳性药组比较,骨痹方组PINK1、LC3Ⅱ/LC3Ⅰ蛋白表达降低而Parkin、Bcl-2、BclxL、Mcl-1蛋白含量升高(P<0.05)。结论骨痹方可延缓软骨退变其机制可能与抑制PINK1、LC3过度表达激活Parkin同时促进BCL2家族抗凋亡蛋白Bcl-xL、Mcl-1表达增加,阻止PINK1/Parkin复合物过度合成,有效降低过度的线粒体自噬有关。

Objective To observe the mechanism by which Gubi Prescription(GBF) regulates the PINK1/Parkin mitochondrial autophagy pathway against knee osteoarthritis(KOA).Methods Chondrocytes were randomly assigned to blank group,model group,positive drug group,and GBF group.Except for blank group,all other groups were induced with Carbonylcyanide-p-trifluoromethoxuphenylhydrazone(FCCP).The proliferation of chondrocytes was detected by CCK-8method.The gene expressions of PINK1,Parkin,microtubule-associated protein 1 light chain 3(LC3),B cell lymphoma-2(Bcl-2),macromolecular B lymphoma(Bcl-xL),and myeloid cell leukemia-1(Mcl-1) were detected by RT-qPCR.A total of 60 mice were randomly separated into 4 groups:sham operation group,model group,positive drug group,and GBF group,15 mice each.Saffron O fast green staining was uesd to observe the cartilage tissue.The protein expressions of PINK1,Parkin,LC3,Bcl-2,Bcl-xL,and Mcl-1 were detected by Western Blot.Results Compared with blank control group,the mRNA expressions of PINK1 and LC3 of model group were increased,while the OD value,mRNA expressions of Parkin,Bcl-2,BclxL,and Mcl-1 were significantly decreased(P<0.05);compared with model group,the mRNA expressions of PINK1 and LC3in positive drug group and GBF group were significantly decreased,while the OD value,Parkin,Bcl-2,Bcl-xL,and Mcl-1mRNA were significantly increased(P<0.05);compared with positive drug group,the mRNA expressions of PINK1 and LC3in GBF group were significantly decreased,while the mRNA expressions of Parkin,Bcl-2,B cl-xL,and Mcl-1 were significantly increased(P<0.05);compared with sham operation group,the protein expressions of PINK1 and LC3 Ⅱ/LC3 I in model group were significantly increased,while the protein expressions of Parkin,Bcl-2,Bcl-xL and Mcl-1 were significantly decreased(P<0.05);compared with model group,the protein expressions of PINK1 and LC3 Ⅱ/LC3 Ⅰ in positive drug group and GBF group were significantly decreased,while the protein expressions of Parkin,Bcl-2,Bcl-xL and Mcl-1 were significantly increased(P<0.05);compared with positive drug group,the protein expressions of PINK1 and LC3 Ⅱ/LC3 Ⅰ in GBF group were significantly decreased,while the protein expressions of Parkin,Bcl-2,Bcl-xL and Mcl-1 were significantly increased(P<0.05).Conclusion GBF can delay cartilage degeneration,and its mechanism may be related to inhibiting the overexpression of PINK1 and LC3,activating Parkin,promoting the increase of the expression of BCL2 family anti-apoptotic proteins Bcl-xL and Mcl-1,preventing the over-synthesis of PINK1/Parkin complex,and effectively reducing excessive mitochondrial autophagy.