龟鹿二仙胶对IL-1β诱导的AMPK/mTOR/ULK1通路介导的体外培养软骨细胞自噬的影响

Effect of Guilu Erxian Jiao on IL-1β-induced AMPK/mTOR/ULK1 pathway mediated autophagy in cultured chondrocytes in vitro

目的探讨龟鹿二仙胶对IL-1β诱导的AMPK/mTOR/ULK1通路介导的体外培养软骨细胞自噬的影响。方法采用酶消法体外培养野生型小鼠和mTOR-KO小鼠软骨细胞。将软骨细胞分为空白组、模型组、龟鹿组、CKO组和龟鹿+CKO组。干预24h后,采用CCK-8法检测各组软骨细胞活性;TUNEL法检测各组软骨细胞凋亡情;激光共聚焦显微镜观察各组软骨细胞LC3B蛋白表达;Western blotting和qRT-PCR检测mTOR、ULK1、AMPK、LC3B、ATG5、PARP-1蛋白及mRNA的表达。结果与空白组比较,模型组软骨细胞活性显著下降(P<0.01),凋亡率显著升高(P<0.01),LC3B光强度显著降低(P<0.01),mTOR、PARP1蛋白及mRNA表达升高(P<0.05),ULK1、AMPK、Atg5、LC3B蛋白及mRNA表达降低(P<0.05);与模型组相比,龟鹿组软骨细胞活性显著提升(P<0.01),凋亡率显著下降(P<0.01),LC3B光强度显著升高(P<0.01),ULK1、AMPK、Atg5、LC3B蛋白及mRNA表达升高(P<0.05),mTOR、PARP1蛋白及mRNA表达降低(P<0.05);与龟鹿组相比,CKO组及龟鹿+CKO组软骨细胞活性显著提升(P<0.01),凋亡率显著下降(P<0.01),LC3B光强度显著升高(P<0.01,P<0.05),mTOR、PARP1蛋白及mRNA表达降低(P<0.05),ULK1、AMPK、Atg5、LC3B蛋白及mRNA表达升高(P<0.05);与CKO组相比,龟鹿+CKO组软骨细胞活性显著提升(P<0.01),凋亡率显著下降(P<0.05),LC3B光强度显著升高(P<0.05),ULK1、AMPK、Atg5、LC3B蛋白及mRNA表达升高(P<0.05),PARP1蛋白及mRNA表达降低(P<0.05),mTOR蛋白及mRNA表达降低,差异无统计学意义(P>0.05)。结论龟鹿二仙胶可通过激活AMPK/mTOR/ULK1通路发生自噬,促进退变软骨细胞的增殖,进而减少软骨细胞凋亡。

Objective Investigate the effect of Guilu Erxian Jiao on the autophagy of cultured chondrocytes mediated by the IL-1βinduced AMPK/mTOR/ULK1 pathway.Methods Wild type mouse and mTOR-KO mouse chondrocytes were cultured in vitro by enzyme elimination method.Mouse chondrocytes were divided into blank group,model group,Guilu group,CKO group and Guilu+CKO group.After 24h of intervention,chondrocyte activity was detected by CCK-8 method and apoptosis was detected by TUNEL method;The expression of LC3B protein in chondrocytes of each group was observed by laser confocal microscope;Western blotting and qRT-PCR were used to detect the expression of mTOR,ULK1,AMPK,LC3B,ATG5,PARP-1 protein and mRNA.Results Compared with blank group,the chondrocyte activity in model group was significantly decreased(P<0.05),and the apoptosis rate of chondrocytes was significantly increased(P<0.01);LC3B light intensity decreased significantly(P<0.01);the protein and mRNA expressions of mTOR and PARP1 were increased(P<0.05),and the protein and mRNA expressions of ULK1,AMPK,Atg5 and LC3B were decreased(P<0.05).Compared with model group,the chondrocyte activity in turtle deer group was significantly increased(P<0.05),and the apoptosis rate of chondrocytes was significantly decreased(P<0.01);LC3B light intensity was significantly increased(P<0.01);the protein and mRNA expressions of ULK1,AMPK,Atg5,LC3B were increased(P<0.05),and the protein and mRNA expressions of mTOR and PARP1 were decreased(P<0.05).Compared with turtle and deer group,the chondrocyte activity in CKO group was significantly increased(P<0.05),and the apoptosis rate of chondrocytes was significantly decreased(P<0.01);LC3B light intensity was significantly increased(P<0.01,P<0.05);the protein and mRNA expressions of mTOR and PARP1 were decreased(P<0.05),and the protein and mRNA expressions of ULK1,AMPK,Atg5 and LC3B were increased(P<0.05).Compared with CKO group,chondrocyte activity in turtle deer+CKO group was significantly increased(P<0.01),and the apoptosis rate of chondrocytes was significantly decreased(P<0.05);LC3B light intensity was significantly increased(P<0.05);protein and mRNA expressions of ULK1,AMPK,Atg5,LC3B were increased(P<0.05),protein and mRNA expressions of PARP1 were decreased(P<0.05),and protein and mRNA expressions of mTOR were decreased.There was no significant difference(P>0.05).Conclusion Guilu Erxian Jiao can induce autophagy by activating AMPK/mTOR/ULK1 pathway,promote the proliferation of degenerative chondrocytes,and then reduce the apoptosis of chondrocytes.