荣筋拈痛方调控lncRNA MGC-Mirg减轻骨关节炎软骨细胞内质网应激的作用

Effect of Rongjin Niantong Formula in reducing endoplasmic reticulum stress response via lncRNA MGC-Mirg in osteoarthritis chondrocytes

目的:探讨荣筋拈痛方调控lncRNA MGC-Mirg减轻骨关节炎(OA)软骨细胞内质网应激的作用与机制。方法:构建lncRNA MGC-Mirg沉默软骨细胞;采用荧光原位杂交实验检测25μmol/L毒胡萝卜素(T G)诱导的软骨细胞中lncRNA MGC-Mirg的分布及表达;Real-timePCR检测lncRNA MGC-Mirg沉默后的软骨细胞中EDEM1、Caspase-3 mRNA水平变化;使用第一代软骨细胞进行后续实验,设置空白组、模型组、荣筋拈痛方组,其中空白组予以含10%胎牛血清的DMEM培养;模型组先以25μmol/L TG干预4 h,再予以含10%胎牛血清的DMEM继续培养;荣筋拈痛方组先以25μmol/L TG干预4 h,再更换含300μg/mL的荣筋拈痛方继续培养12 h。采用Real-time PCR检测TG诱导的软骨细胞中lncRNA MGC-Mirg、EDEM1、PERK、ATF4、BIP、GADD153、Caspase-3基因相对表达水平;流式细胞术检测软骨细胞凋亡率。结果:lncRNA MGCMirg主要分布于软骨细胞的胞质中,与对照组比较,模型组软骨细胞中lncRNA MGC-Mirg相对拷贝数明显增多。与TG+sh-NC组比较,TG+sh-Mirg组EDEM1、Caspase-3 mRNA及蛋白表达显著降低(P<0.05)。与空白组比较,模型组软骨细胞中lncRNA MGC-Mirg、EDEM1、PERK、ATF4、BIP、GADD153、Caspase-3 mRNA相对表达水平显著增高(P<0.05);与模型组比较,荣筋拈痛方组lncRNA MGC-Mirg、EDEM1、PERK、ATF4、BIP、GADD153、Caspase-3 mRNA相对表达水平显著降低(P<0.05)。模型组软骨细胞凋亡率高于空白组和荣筋拈痛方组。结论:荣筋拈痛方可通过调控lncRNA MGC-Mirg,减轻OA软骨细胞内质网应激反应,延缓软骨细胞退变。

Objective:To investigate the effect and mechanism of Rongjin Niantong Formula(RJNTF)reduce endoplasmic reticulum stress response via lncRNA MGC-Mirg in osteoarthritis.Methods:Construction of lncRNA MGC-Mirg silenced chondrocytes;FISH was used to detect the distribution and expression of lncRNA MGC-Mirg in 25μmol/L TG induced chondrocytes.The mRNA levels of EDEM1 and Caspase-3 in lncRNA MGC-Mirg silenced chondrocytes were detected by Real-time PCR.The first generation of chondrocytes were used for subsequent experiments,including blank group,model group and RJNTF group.The blank group was cultured with DMEM containing 10%fetal bovine serum.The model group was first treated with 25μmol/L TG for 4 h,and then treated with DMEM containing 10%FBS.RJNTF group was first treated with 25μmol/L TG for 4 h,and then replaced with 300μg/mL RJNTF for 12 h.Real-time PCR was used to detect the relative expression levels of lncRNA MGC-Mirg,EDEM1,PERK,ATF4,BIP,GADD153 and Caspase-3 in TG induced chondrocytes.The apoptosis rate of chondrocytes was detected by flow cytometry.Results:lncRNA MGC-Mirg was mainly distributed in the cytoplasm of chondrocytes.Compared with the control group,the relative copy number of MGC-Mirg in chondrocytes in model group was significantly increased.Compared with TG+sh-NC group,mRNA levels and protein expression of EDEM1 and Caspase-3 in TG+sh-Mirg group were significantly decreased(P<0.05).Compared with the blank group,the relative expression level of lncRNA MGC-Mirg,EDEM1,PERK,ATF4,BIP,GADD153 and Caspase-3 in chondrocytes in model group was significantly increased(P<0.05).Compared with blank group,the mRNA relative expression levels of lncRNA MGC-Mirg,EDEM1,PERK,ATF4,BIP,GADD153,Caspase-3 were significantly decreased(P<0.05).The apoptosis rate of chondrocytes in model group was higher than that in blank group and RJNTF group.Conclusion:RJNTF can reduce endoplasmic reticulum stress response in osteoarthritis chondrocytes and delay chondrocyte degeneration by regulating lncRNA MGC-Mirg.