黄连解毒汤减轻脓毒症大鼠心功能障碍机制研究

Study on the mechanism of Huanglian Jiedu Decoction in alleviating sepsis induced myocardial dysfunction in rats

目的探讨黄连解毒汤减轻脓毒症大鼠心功能障碍(SIMD)的作用机制。方法60只大鼠中随机抽取7只作为假手术组,其余大鼠建立SIMD模型,分为核富集丰富转录本1(NEAT1)沉默组、空载组、模型组、阳性对照组、黄连解毒汤组,每组7只。造模28 d时进行干预,假手术组、模型组予等体积生理盐水灌胃,阳性对照组予氯沙坦钾5 mg·kg^(-1)灌胃,NEAT1沉默组、空载组、黄连解毒汤组予12 g·kg^(-1)黄连解毒汤灌胃。HE染色观察大鼠心肌组织病理学变化,RNA FISH法检测长链非编码RNA-核富集丰富转录本1(lncRNA-NEAT1)亚细胞定位,ELISA法检测炎症因子表达,RT-PCR法检测lncRNA-NEAT1、微小核糖核酸-132-3p(miR-132-3p)、解偶联蛋白2(UCP2)表达,Western blot法检测细胞增殖、凋亡相关蛋白表达。结果给药后,黄连解毒汤组大鼠心肌纤维排列紊乱减轻,心肌细胞肿胀、坏死及炎症细胞浸润现象较模型组明显改善。lncRNA-NEAT1激发紫红色,在心肌细胞细胞核、细胞质中均有分布。与模型组比较,阳性对照组、黄连解毒汤组、空载组、NEAT1沉默组肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)水平、lncRNA-NEAT1 mRNA相对表达量、Bax、Caspase-3、Caspase-9表达均降低(P<0.05),NEAT1沉默组最低;miR-132-3p、UCP2 mRNA相对表达量、Ki67表达均升高(P<0.05),NEAT1沉默组最高。结论黄连解毒汤减轻脓毒症大鼠心功能障碍的机制可能是下调lncRNA-NEAT1、竞争性结合miR-132-3p后解除对UCP2抑制,促进UCP2表达后进一步抑制炎症反应。

Objective To explore the mechanism of Huanglian Jiedu Decoction in alleviating sepsis induced myocardial dysfunction(SIMD)in rats.Methods From 60 SPF grade SD male rats,20 rats were randomly selected and divided into long noncoding RNA nuclear-enriched abundant transcript 1(NEAT1)silencing group and the no-load group.From the remaining 40 rats,7 rats were selected as the sham operation group,and the remaining 33 rats were used to construct the sepsis model.In this experiment,27 rats were successfully modeled and randomly divided into the model group,the positive control group,and the Huanglian Jiedu Decoction group,with 9 rats in each group.At 28 days of modeling,the intervention was carried out,the sham operation group and the model group were given equal volumes of normal saline by gavage,the positive control group was given losartan potassium of 5 mg·kg^(-1) by gavage,and the NEAT1 silencing group,the no-load group and Huanglian Jiedu Decoction group were given Huanglian Jiedu Decoction of 12 mg·kg^(-1) by gavage.Myocardial histopathological changes were observed by HE staining.Long noncoding RNA nuclear-enriched abundant transcript 1(lncRNANEAT1)subcellular localization was detected by RNA FISH.The expression of inflammatory factors was detected by ELISA.The expression of lncRNA-NEAT1,microRNAs-132-3p(miR-132-3p)and uncoupling protein 2(UCP2)was detected by RT-PCR.Western blot assay was used to detect the expression of apoptosis and proliferation related proteins.Results After administration,compared with the model group,the disorder of myocardialfiber arrangement in Huanglian Jiedu Decoction group was relieved,and the swelling and necrosis of cardiomyocytes,and inflammatory cell infiltration were significantly improved.IncRNA-NEAT1 stimulated purplish red and was distributed in both the nucleus and cytoplasm of cardiomyocytes.Compared with the model group,microRNAs-132-3p levels of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β),the relative expression of lncRNANEAT1 mRNA,and the expression of Bax,Caspase-3 and Caspase-9 in the positive control group,Huanglian Jiedu Decoction group,the no-load group and NEAT1 silencing group were decreased(P<0.05),with NEAT1 silencing group being the lowest;The relative expression levels of miR-132-3p and UCP2 mRNA and the expression of Ki67 were all increased(P<0.05),with NEAT1 silencing group being the highest.Conclusion The mechanism which Huanglian Jiedu Decoction alleviates SIMD in rats may be that after downregulating lncRNA-NEAT1 and competitively binding to miR-132-3p,the inhibition of UCP2 is relieved,and after promoting UCP2 expression,the inflammatory response is further inhibited.