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-1 β-induced AMPK/mTOR/ULK1 pathway media-ted autophagy in cultured chondrocytes in vitro
吴伟欣 1郑珍萍 1顾富城 1杨美鑫 1耿秋东 1沈默金 1王和鸣 2李楠3
作者信息
- 1. 福建中医药大学中医学院,福建福州 350122
- 2. 福建中医药大学,福建福州 350122
- 3. 福建中医药大学中医学院,福建福州 350122;中医骨伤及运动康复教育部重点实验室,福建福州 350122
- 折叠
摘要
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、LC3 B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.
Abstract
Objective Investigate the effect of Guilu Erxian Jiao on the autophagy of cultured chondrocytes mediated by the IL-1β induced 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 mR-NA.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 expres-sions of ULK1,AMPK,Atg5 and LC3B were decreased(P＜0.05).Compared with model group,the chondrocyte activity in tur-tle 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 ap-optosis 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 ex-pressions of ULK1,AMPK,Atg5 and LC3B were increased(P＜0.05).Compared with CKO group,chondrocyte activity in tur-tle 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.
关键词
龟鹿二仙胶/AMPK/mTOR/ULK1信号通路/软骨细胞/自噬/C57BL/6小鼠Key words
Guilu Erxian Jiao/AMPK/mTOR/ULK1 signaling pathway/Chondrocytes/Autophagy/C57BL/6 mice引用本文复制引用
基金项目
国家自然科学基金面上项目(81973880)
福建省自然科学基金(2022J01364)
出版年
2024
NETL
NSTL
万方数据