基于PERK通路探讨荣筋拈痛方对软骨细胞内质网应激反应抑制作用  

Inhibitory Effect of Rongjin Niantong Decoction on Endoplasmic Reticulum Stress Response of Chondrocytes Based on PERK Pathway

作　　者：付长龙 谢新宇 何俊君 邱志伟 郑春松 叶锦霞 马德尊 FU Changlong;XIE Xinyu;HE Junjun;QIU Zhiwei;ZHENG Chunsong;YE Jinxia;MA Dezun(Academy of Integrative Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou,Fujian 350122,China;Fujian Key Laboratory of Integrative Medicine on Geriatrics,Fuzhou,Fujian 350122,China;Periodicals Agency of Fujian University of Traditional Chinese Medicine,Fuzhou,Fujian 350122,China)

机构地区：[1]福建中医药大学中西医结合研究院,福建福州350122 [2]福建省中西医结合老年性疾病重点实验室,福建福州350122 [3]福建中医药大学杂志社,福建福州350122

出　　处：《福建中医药》2022年第4期22-24,28,共4页Fujian Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(82104888);福建中医药大学高层次人才科研启动资金项目(X2019011-人才)。

摘　　要：目的基于PERK通路探讨荣筋拈痛方(RJNTD)对毒胡萝卜素(TG)诱导的软骨细胞内质网应激反应的抑制作用。方法将30只4周龄SPF级雄性C57BL/6小鼠处死后,体外分离双膝软骨细胞置于低糖DMEM培养基培养,经Ⅱ型胶原免疫细胞化学染色法鉴定后将软骨细胞分成空白组、对照组、模型组和中药组。空白组常规培养4 h,对照组常规培养4 h后更换300μg/mL RJNTD培养液培养12 h,模型组用25μmol/L TG培养液培养4 h后改为常规培养,中药组用25μmol/L TG培养液培养4 h后更换300μg/mL RJNTD培养液培养12 h。干预后采用Real-time PCR检测各组软骨细胞中miRNA-377-3p相对表达水平;Western blot检测各组软骨细胞中内质网降解增强子(EDEM1)、蛋白激酶R样内质网激酶(PERK)、转录激活子4(ATF4)、免疫球蛋白重链结合蛋白(BIP)与DNA损伤诱导基因153(GADD153)蛋白表达水平。结果对照组各指标与空白组比较均无统计学意义(P均>0.05);与空白组比较,模型组及中药组miRNA-377-3p相对表达水平显著降低(P<0.05),且中药组较模型组降低更不明显(P<0.05);与空白组比较,模型组及中药组EDEM1、PERK、ATF4、BIP、GADD153蛋白表达水平均升高(P均<0.05),且中药组上述各指标较模型组升高更不明显(P均<0.05)。结论RJNTD可通过PERK通路抑制内质网应激反应,进而发挥延缓TG诱导的软骨细胞退变作用,其潜在作用机制可能与调节miRNA-377-3p相关。Objective:To investigate the inhibitory effect of Rongjin Niantong Decoction(RJNTD)on endoplasmic reticulum stress response of chondrocytes induced by thapsigargin(TG)based on PERK pathway.Methods:Thirty 4-week-old male C57BL/6mice of SPF grade were killed.The chondrocytes of both knees were isolated in vitro and cultured in low sugar DMEM.After identified by typeⅡcollagen immunocytochemical staining,the chondrocytes were divided into blank group,control group,model group and RJNTD group.The blank group was normally cultured for 4 h;the control group was cultured in 300μg/mL RJNTD medium for12 hours after 4 hours of conventional culture;the model group was cultured with 25μmol/L TG medium for 4 hours and then changed to conventional culture;the RJNTD group was cultured with 25μmol/L TG medium for 4 hours and then changed to 300μg/mL RJNTD medium for 12 hours.The relative expression level of miRNA-377-3p in chondrocytes was detected by Real-time PCR;the expression levels of ER degradation enhancer mannosidase alpha-like 1(EDEM1),protein kinase R-like endoplasmic reticulum kinase(PERK),transcription activator 4(ATF4),immunoglobulin heavy chain binding protein(BIP)and growth arrest and DNA damage-inducible gene 153(GADD153)in chondrocytes were detected by Western blot.Results:The index in control group and blank group had no significant difference(P>0.05).Compared with the blank group,the model group and RJNTD group significantly decreased the relative expression level of miRNA-377-3p(P<0.05),increased the protein expression levels of EDEM1,PERK,ATF4,BIP and GADD153(P<0.05).Compared with the model group,the RJNTD group significantly increased the relative expression level of miRNA-377-3p(P<0.05),decreased the protein expression levels of EDEM1,PERK,ATF4,BIP and GADD153(P<0.05).Conclusion:RJNTD can inhibit endoplasmic reticulum stress response through PERK pathway,and then delay chondrocyte degeneration induced by TG.Its potential mechanism may be related to the regulation of miR-377-3p.

关 键 词：膝骨关节炎 荣筋拈痛方 软骨细胞退变 内质网应激 PERK 

分 类 号：R285.5[医药卫生—中药学]