基于ATF6/GRP78/CHOP信号通路探讨龟鹿二仙胶对膝骨关节炎大鼠退变软骨细胞内质网稳态的影响  

Effect of Guilu Erxian(龟鹿二仙)Gel on Endoplasmic Reticulum Homeostasis of Degenerative Chondrocytes in Rats with Knee Osteoarthritis Based on ATF6/GRP78/CHOP Signaling Pathway

作　　者：吴伟欣 郑珍萍 顾富城 杨美鑫 马文凯 王和鸣[1] 耿秋东 李楠[1,2] WU Weixin;ZHENG Zhenping;GU Fucheng;YANG Meixin;MA Wenkai;WANG Heming;GENG Qiudong;LI Nan(Fujian University of Traditional Chinese Medicine,Fuzhou 350122;Key Laboratory of Traditional Chinese Medicine for Bone Injury and Sports Rehabilitation,Ministry of Education,Fuzhou 350122)

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：《中药药理与临床》2024年第10期33-40,共8页Pharmacology and Clinics of Chinese Materia Medica

基　　金：福建省自然科学基金项目(编号:2022J01364);国家自然科学基金面上项目(编号:81973880)。

摘　　要：目的:探讨龟鹿二仙胶对毒胡萝卜素诱导的大鼠退变软骨细胞激活转录因子6(ATF6)/葡萄糖调节蛋白78(GRP78)/C/EBP同源蛋白(CHOP)信号通路的影响。方法:用大鼠膝关节提取软骨细胞进行体外培养。将软骨细胞分为空白对照组、模型对照组、10.8 g/kg 10%龟鹿二仙胶含药血清组、2μmol/L蛋白运输蛋白SX61抑制剂组。干预24 h后,以CCK-8法检测各组软骨细胞活性;免疫荧光染色检测蛋白运输蛋白SX61(SEC61)因子的表达情况;流式细胞术及TUNEL染色检测各组软骨细胞凋亡情况;Western blot及qPCR法检测软骨细胞ATF6/GRP78/CHOP、Ⅱ型胶原α1(COL2A1)、基质金属蛋白酶-13(MMP-13)蛋白及mRNA的表达。结果:与空白对照组相比,模型对照组软骨细胞活性显著下降(P<0.01),软骨细胞凋亡率及凋亡指数显著增高(P<0.01),SEC61荧光强度显著增强(P<0.01),GRP78、ATF6、CHOP、COL2A1、MMP-13蛋白及mRNA的表达明显上调(P<0.05);与模型对照组相比,10.8 g/kg 10%龟鹿二仙胶含药血清组及2μmol/L蛋白运输蛋白SX61抑制剂组软骨细胞活性显著提高(P<0.01),软骨细胞凋亡率及凋亡指数显著降低(P<0.01),SEC61荧光强度显著降低(P<0.01),GRP78、ATF6、CHOP、MMP-13蛋白及mRNA表达明显下调(P<0.05),COL2A1蛋白及mRNA表达上调(P<0.05);与10.8 g/kg 10%龟鹿二仙胶含药血清组相比,2μmol/L蛋白运输蛋白SX61抑制剂组软骨细胞活性显著提升(P<0.01),软骨细胞凋亡率及凋亡指数降低(P<0.05或P<0.01),SEC61蛋白荧光强度显著降低(P<0.01),GRP78、ATF6、CHOP、MMP-13蛋白及mRNA表达下调(P<0.05),COL2A1蛋白及mRNA表达上调(P<0.05)。结论:龟鹿二仙胶可通过ATF6/GRP78/CHOP信号通路抑制退变软骨细胞内质网应激(ERS)反应,调控细胞外基质(ECM)分解合成平衡,改善软骨细胞ER稳态,进而减少软骨细胞凋亡。Objective:To investigate the effect of Guilu Erxian(龟鹿二仙)Gel on the activation of the transcription factor 6(ATF6)/glucose regulated protein 78(GRP78)/C/EBP homologous protein(CHOP)signaling pathway in degenerative chondrocytes induced by thapsigargin(TG)in rats.Methods:Chondrocytes were extracted from rat knee joints and cultured in vitro.Chondrocytes were divided into a blank control group,a model control group,a 10%Guilu Erxian Gel group,and a 2μmol/L protein transport protein SX61(SEC61)inhibitor group.After 24 hours of intervention,the cell activity of each group was detected by CCK-8 method.The expression of SEC61 factor was detected by immunofluorescence staining.Flow cytometry and TUNEL staining were used to detect the apoptosis of chondrocytes in each group,and Western blot(WB)and real-time fluorescence-based quantitative polymerase chain reaction(qPCR)were used to detect the expression of ATF6/GRP78/CHOP,type II collagen alpha 1(COL2A1),matrix metalloproteinase-13(MMP-13)protein and mRNA in chondrocytes of each group.Results:Compared with the blank control group,the cell activity of chondrocytes in the model control group decreased significantly(P<0.01),the apoptosis rate and apoptosis index of chondrocytes increased significantly(P<0.01),SEC61 fluorescence intensity increased significantly(P<0.01),the expression of GRP78,ATF6,CHOP,COL2A1,MMP-13 protein and mRNA increased(P<0.05).Compared with the model control group,the cell activity of chondrocytes in the 10%Guilu Erxian Gel group and the 2μmol/L SEC61 inhibitor group increased significantly(P<0.01),the apoptosis rate and apoptosis index of chondrocytes decreased significantly(P<0.01),SEC61 fluorescence intensity decreased significantly(P<0.01).Moreover,the expression of GRP78,ATF6,CHOP,MMP-13 protein and mRNA decreased(P<0.05),and the expression of COL2A1 protein and mRNA increased(P<0.05).Compared with the 10%Guilu Erxian Gel group,the cell activity of chondrocytes in the 2μmol/L SEC61 inhibitor group increased significantly(P<0.01),the a

关 键 词：龟鹿二仙胶 软骨细胞 膝骨关节炎 激活转录因子6/葡萄糖调节蛋白78/C/EBP同源蛋白信号通路 

分 类 号：R285.5[医药卫生—中药学]