基于PERK/eIF2α信号通路探讨龟鹿二仙胶含药血清对脂多糖诱导大鼠退变软骨细胞凋亡的作用机制  

Study on the mechanism of Guilu Erxianjiao-containing serum on lipopolysaccharideinduced apoptosis of rat degenerated chondrocytes based on PERK/eIF2a signalingpathway

作　　者：耿秋东 李晔[1] 沈默金 郑珍萍 吴伟欣 牛素生[1,2] 张燕 王和鸣[1] 李楠[1,2] GENG Qiudong;LI Ye;SHEN Mojin;ZHENG Zhenping;WU Weixin;NIU Susheng;ZHANG Yan;WANG Heming;LI Nan(Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China;Key Laboratory of Orthopedics&Traumatology of Traditional Chinese Medicine and Rehabilitation,Ministry of Education,Fuzhou 350122,China)

机构地区：[1]福建中医药大学,福州350122 [2]中医骨伤及运动康复教育部重点实验室,福州350122

出　　处：《中华中医药杂志》2023年第8期3570-3576,共7页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家自然科学基金面上项目(No.81973880);福建省自然科学基金项目(No.2022J01364)。

摘　　要：目的:观察龟鹿二仙胶含药血清对脂多糖(LPS)诱导的大鼠退变软骨细胞中蛋白激酶R样内质网激酶(PERK)/真核起始因子2α(eIF2α)信号通路相关蛋白表达的影响,探讨龟鹿二仙胶治疗KOA的作用机制。方法:制备龟鹿二仙胶含药血清及空白血清。采用4周龄SD大鼠膝关节软骨提取软骨细胞并进行体外培养,用甲苯胺蓝染色法对软骨细胞进行鉴定。采用CCK-8法筛选最佳含药血清浓度进行后续实验。用10μg/mL LPS诱导F2代软骨细胞复制退变软骨细胞模型,将细胞分为空白组、模型组、龟鹿二仙胶组、PERK抑制剂组,干预24 h后,分别采用CCK-8法检测软骨细胞活性;TUNEL法检测软骨细胞凋亡情况;RT-qPCR法检测软骨细胞PERK、eIF2α、前凋亡基因C/EBP同源蛋白(CHOP)、Bcl-2相关X蛋白(Bax)、半胱氨酸蛋白酶-3(Caspase-3)、B淋巴细胞瘤-2基因(Bcl-2)mRNA的表达;Western Blot检测软骨细胞p-PERK、p-eIF2α、CHOP、Bax、活化的Caspase-3(Cleaved-Caspase-3)、Bcl-2蛋白的表达情况。结果:与空白组比较,模型组软骨细胞活性显著降低(P<0.05),软骨细胞凋亡数增多,PERK、eIF2α、CHOP、Bax、Caspase-3 mRNA及p-PERK、p-eIF2α、CHOP、Bax、Cleaved-Caspase-3蛋白表达量显著上调(P<0.01),Bcl-2 mRNA和蛋白表达显著下调(P<0.01);与模型组比较,龟鹿二仙胶组和抑制剂组软骨细胞活性显著提升(P<0.05,P<0.01),软骨细胞凋亡数减少,PERK、eIF2α、CHOP、Bax、Caspase-3 mRNA及p-PERK、p-eIF2α、CHOP、Bax、Cleaved-Caspase-3蛋白表达量显著下调(P<0.01),Bcl-2 mRNA和蛋白表达显著上调(P<0.01)。结论:龟鹿二仙胶含药血清能显著改善大鼠退变软骨细胞活性及细胞凋亡情况,其作用机制可能是通过调控PERK/eIF2α信号通路相关蛋白的表达水平,进而发挥了软骨保护作用。Objective:To observe the effect of Guilu Erxianjiao-containing serum on the expression of PERK/eIF2αsignaling pathway-related proteins in lipopolysaccharide(LPS)-induced rat chondrocytes,and to explore the mechanism of Guilu Erxianjiao in the treatment of knee osteoarthritis(KOA).Methods:Preparation of Guilu Erxianjiao-containing serum and blank serum.A stable chondrocyte culture system was established in the knee articular cartilage of 4-week-old SD rats,and the chondrocytes were identified by toluidine blue staining.The CCK-8 method was used to screen the optimal drug-containing serum concentration for subsequent experiments.The F2 generation chondrocytes were induced to replicate the degenerated chondrocyte model with 10μg/mL LPS,and the cells were divided into blank group,model group,Guilu Erxianjiao-containing serum group,and PERK inhibitor group.After 24 hours of intervention,CCK-8 method to detect chondrocyte activity and survival rate;TUNEL method to detect chondrocyte apoptosis;RT-qPCR method to detect chondrocyte PERK,eIF2α,CHOP,Bax,Caspase-3,Bcl-2 mRNA expression;Western Blot to detect chondrocyte Expression of p-PERK,p-eIF2α,CHOP,Bax,Cleaved-Caspase-3,Bcl-2 proteins.Results:Compared with the blank group,the activity and survival rate of chondrocytes in the model group were significantly decreased(P<0.05),the number of chondrocytes apoptotic was increased,PERK,eIF2α,CHOP,Bax,Caspase-3 mRNA and the expressions of p-PERK,p-eIF2α,CHOP,Bax and Cleaved-Caspase-3 proteins were significantly upregulated(P<0.01),and the expressions of Bcl-2 mRNA and protein were significantly down-regulated(P<0.01);compared with the model group,the Guilu Erxianjiao-containing serum group and inhibitor group the chondrocyte activity and cell survival rate were significantly improved(P<0.05,P<0.01),the number of chondrocytes apoptotic was decreased,and the expression of PERK,eIF2α,CHOP,Bax,Caspase-3 mRNA and p-PERK,p-eIF2α,CHOP,Bax,Cleaved-Caspase-3 protein were significantly downregulated(P<0.01),and the expression of B

关 键 词：龟鹿二仙胶 膝骨关节炎 软骨细胞 凋亡 内质网应激 

分 类 号：R285.5[医药卫生—中药学]