基于lncRNA Mirg与PERK/BIP通路探究荣筋拈痛方改善软骨细胞内质网应激的作用机制

Based on lncRNA Mirg and PERK/BIP pathway, exploring the mechanism of Rongjin Niantong Fang in improving endoplasmic reticulum stress of chondrocytes

关键词:  荣筋拈痛方  骨关节炎  内质网应激  PERK/BIP通路  lncRNA Mirg  

Key words:  Rongjin Niantong Fang  osteoarthritis  endoplasmic reticulum stress  PERK/BIP pathway  lncRNA Mirg

摘要:

目的 基于lncRNA Mirg与PERK/BIP通路探讨荣筋拈痛方改善软骨细胞内质网应激的分子机制。方法 由毒胡萝卜素诱导小鼠膝关节软骨细胞构建内质网应激模型,慢病毒转染沉默lncRNA Mirg,共分为4组(sh-NC组,sh-Mirg组,TG+sh-NC组和TG+sh-Mirg组),荣筋拈痛方干预内质网应激模型软骨细胞,共分为3组(空白组,模型组,治疗组),Real-time qPCR、Western blot检测软骨细胞lncRNA Mirg基因表达水平及PERK/BIP通路相关指标PERK、BIP、ATF4、CHOP的基因表达水平和蛋白含量变化,激光共聚焦显微镜检测细胞内Ca2+浓度变化。结果 成功构建lncRNA Mirg缺失的小鼠软骨细胞(P<0.01),经毒胡萝卜素干预诱导细胞内质网应激模型,lncRNA Mirg缺失可缓解细胞内质网应激,PERK/BIP通路关键指标BIP、PERK、ATF4及CHOP的基因、蛋白表达降低(P<0.01)。荣筋拈痛方干预内质网应激软骨细胞降低了lncRNA Mirg的基因及PERK/BIP通路关键指标的蛋白含量(P<0.01),调节细胞Ca2+流动。结论 荣筋拈痛方可下调软骨细胞中lncRNA Mirg表达，调控细胞PERK/BIP通路，恢复细胞内Ca2+稳态，缓解毒胡萝卜素诱导的软骨细胞内质网应激，延缓骨关节炎软骨细胞退变。

Abstract:

OBJECTIVE Exploring the mechanism of Rongjin Niantong Fang in improving endoplasmic reticulum stress of chondrocytes based on lncRNA Mirg and PERK/BIP pathway. METHODS The model of endoplasmic reticulum stress was established by inducing mice articular chondrocytes with Thapsigarin. lncRNA Mirg was silenced by lentivirus transfection. Chondrocytese were divided into 4 groups (sh-NC group, sh-Mirg group, TG + sh-NC group and TG + sh-Mirg group), Rongjin Niantong Fang was applied to endoplasmic reticulum stress model chondrocytes, which were divided into three groups (blank group, model group, treatment group). Real-time qPCR and Western blot were used to detect the expression of lncRNA Mirg, the gene level and protein content of PERK/BIP pathway, such as PERK, BIP, ATF4 and CHOP were detected, and confocal laser microscope was used to detect the intracellular Ca2+ concentration. RESULTS We successfully constructed lncRNA Mirg-deficient chondrocytes and induced endoplasmic reticulum stress model by Thapsigarin. lncRNA Mirg deletion alleviated endoplasmic reticulum stress and reduced gene and protein expression of BIP, PERK, ATF4 and CHOP, key indicators of PERK/BIP pathway(P<0.01). Intervention of Rongjin Niantong Fang reduced the gene and protein content of lncRNA Mirg and key indicators of PERK/BIP pathway (P<0.01) in ERS chondrocytes, and regulated cellular Ca2+ flow. CONCLUSION RongjinNiantong Fang down-regulated the expression of lncRNA Mirg in chondrocytes, regulated the PERK/BIP pathway, restored the homeostasis of intracellular Ca2+, relieved the endoplasmic reticulum stress induced by toxic carotene, and delayed the degeneration of chondrocytes in osteoarthritis.