黄芪糖蛋白干扰lncRNA GAS5/miR-21/TLR4信号轴抑制细胞焦亡改善佐剂性关节炎大鼠心肌损伤

Huangqi Glycoprotein improves myocardial injury in rats with adjuvant arthritis by interfering with lncRNA GAS5/miR-21/TLR4 signaling axis to inhibit pyroptosis

目的基于长链非编码RNA生长停滞特异性转录本5/微小RNA 21/Toll样受体4(lncRNA GAS5/miR-21/TLR4)信号轴探究黄芪糖蛋白(HQGP)对佐剂性关节炎(AA)大鼠心肌损伤的作用及机制。方法将SD大鼠随机分为正常对照组、模型组、甲氨蝶呤(MTX)组、HQGP组,每组6只。复制AA模型,第19天开始给药,连续4周。检测各组AA大鼠足趾肿胀度、关节炎指数(AI),HE染色观察大鼠心肌组织病理变化,透射电镜观察心肌组织超微结构的变化及焦亡小体情况,ELISA检测血清白细胞介素6(IL-6)、IL-18、IL-1β和肿瘤坏死因子α(TNF-α)水平,乳酸脱氢酶(LDH)试剂盒检测心肌组织LDH释放量,实时荧光定量PCR检测心肌组织核因子κB p65(NF-κB p65)、胱天蛋白酶1(caspase-1)、含pyrin结构域核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、消皮素D(GSDMD)的mRNA以及lncRNA GAS5/miR-21/TLR4信号轴分子的表达,Western blot法检测心肌组织TLR4、NF-κB p65、caspase-1、NLRP3和GSDMD蛋白水平。结果与正常对照组相比,模型组大鼠足趾肿胀度、AI显著升高;大鼠心肌纤维溶解、断裂,肌节结构模糊,心肌纤维排列紊乱,组织间有炎性细胞浸润,线粒体嵴明显断裂稀疏,焦亡小体数量增加;血清促炎细胞因子IL-6、IL-18、IL-1β和TNF-α表达显著升高;心肌组织中GAS5表达显著降低,miR-21表达显著升高,LDH释放量、TLR4、NF-κB p65、caspase-1、NLRP3、GSDMD的mRNA和蛋白表达水平显著升高。与模型组比较,HQGP组大鼠足趾肿胀度、AI和心肌组织病理状态明显改善;血清IL-6、IL-18、IL-1β和TNF-α表达显著降低;心肌组织中GAS5表达显著升高,miR-21表达显著降低,LDH释放量、TLR4、NF-κB p65、caspase-1、NLRP3的mRNA和蛋白表达水平显著降低,GSDMD的mRNA表达降低且蛋白水平显著降低。结论HQGP通过上调lncRNA GAS5抑制miR-21/TLR4信号传导,抑制细胞焦亡,减少促炎细胞因子表达,改善AA大鼠心肌损伤。

Objective To investigate the effect and mechanism of Huangqi glycoprotein(HQGP)on myocardial injury in rats with adjuvant arthritis(AA)based on the long non-coding RNA growth arrest-specific transcript 5/microRNA-21/Toll-like receptor 4(lncRNA GAS5/miR-21/TLR4)signal axis.Methods SD rats were randomized into normal control,model,methotrexate(MTX)and HQGP groups,with 6 rats in each group.The AA rat model was established,and the drug was administered on the 19th day after the model was established,for 4 consecutive weeks.The degree of toe swelling and the arthritis index(AI)of AA rats in each group were measured.The pathological changes of rat myocardial tissue were observed by HE staining,and the changes in the ultrastructure of the myocardial tissue and the situation of pyroptotic vesicles were observed by transmission electron microscope.The levels of serum interleukin 6(IL-6),IL-18,IL-1βand tumor necrosis factorα(TNF-α)were detected by ELISA.The release of lactate dehydrogenase(LDH)in myocardial tissue was detected by kit.Real-time quantitative PCR was used to detect the mRNA expression of nuclear factor-kappa B p65(NF-κB p65),cysteine aspartate specific proteinase 1(caspase-1),nucleotide binding oligomerization domain like receptor family pyrin domain containing 3(NLRP3),gasdermin D(GSDMD),and molecules in the lncRNA GAS5/miR-21/TLR4 signaling axis in the myocardial tissue.The protein levels of TLR4,NF-κB p65,caspase-1,NLRP3 and GSDMD in myocardial tissue were detected by Western blot.Results Compared with the normal control group,the toe swelling and AI of the model group rats were significantly increased.The myocardial fibers of rats dissolved and broken,the structure of sarcomere was blurred,the arrangement of myocardial fibers was disordered,there was inflammatory cell infiltration between tissues,the mitochondrial cristae were significantly broken and sparse,and the number of pyroptotic vesicles increased.The expression of serum pro-inflammatory cytokines IL-6,IL-18,IL-1βand TNF-αincreased significantly.In myocardial tissue,the expression of GAS5 reduced significantly,the expression of miR-21 increased significantly,the release of LDH,and the mRNA and protein levels of TLR4,NF-κB p65,caspase-1,NLRP3 and GSDMD increased significantly.Compared with the model group,the toe swelling,AI,and the pathological status of myocardial tissue in the HQGP group rats were improved significantly.The expressions of serum IL-6,IL-18,IL-1βand TNF-αreduced significantly.In myocardial tissue,the expression of GAS5 increased significantly,the expression of miR-21 decreased significantly,the release of LDH,the mRNA and protein levels of TLR4,NF-κB p65,caspase-1 and NLRP3 decreased significantly,and the mRNA expression of GSDMD reduced while the protein level significantly reduced.Conclusion HQGP improves myocardial injury in AA rats by inhibiting miR-21/TLR4 signalling through up-regulation of lncRNA GAS5,inhibiting pyroptosis,and reducing pro-inflammatory cytokine expression.