miR-210-5p对脓毒症小鼠心肌损伤的调控作用及其分子机制

Regulatory effect of miR-210-5p on myocardial injury in septic mice and its molecular mechanism

目的观察miR-210-5p对脓毒症小鼠心肌损伤的调控作用并分析其分子机制。方法将40只C57BL/6小鼠随机分为对照组、脓毒症组、脓毒症+miR-210-5p激动剂组、脓毒症+miR-210-5p拮抗剂组,每组10只。除对照组外均采用盲肠结扎穿孔术建立小鼠脓毒症模型,对照组小鼠采用不结扎、不穿刺的剖腹手术进行对照。脓毒症+miR-210-5p激动剂组、脓毒症+miR-210-5p拮抗剂组小鼠在建模前1 d及建模术后即刻分别经尾静脉注射miR-210-5p激动剂agomir及miR-210-5p拮抗剂antagomir,对照组和脓毒症组在相同时间经尾静脉注射等体积生理盐水。比较各组心功能指标左心室射血分数(LVEF)、左心室缩短分数(LVFS),心肌组织病理改变,血清心肌损伤标志物肌酸激酶同工酶(CK-MB)、肌钙蛋白I(cTnI),细胞凋亡率,凋亡蛋白剪切半胱氨酸天冬氨酸蛋白酶3(cleaved Caspase-3)表达,髓过氧化物酶(MPO)活性,炎症因子肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、IL-6、单核细胞趋化蛋白1(MCP-1)表达,抗氧化物超氧化物歧化酶(SOD)、谷胱甘肽(GSH)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化物丙二醛(MDA)表达及超氧化物阴离子荧光探针(DHE)荧光强度,沉默调节蛋白1(SIRT1)通路相关蛋白SIRT1、叉头框蛋白O1(FOXO1)、乙酰化FOXO1(Ac-FOXO1)、核因子κB亚基p65(NF-κB p65)、乙酰化NF-κB p65(Ac-NF-κB p65),计算Ac-FOXO1/FOXO1、Ac-NF-κB p65/NF-κB p65。结果小鼠LVEF、LVFS对照组>脓毒症+miR-210-5p激动剂组>脓毒症组>脓毒症+miR-210-5p拮抗剂组(P均<0.01)。HE染色显示,小鼠心肌组织病理损伤对照组<脓毒症+miR-210-5p激动剂组<脓毒症组<脓毒症+miR-210-5p拮抗剂组。小鼠血清CK-MB、cTnI水平对照组、脓毒症+miR-210-5p激动剂组<脓毒症组<脓毒症+miR-210-5p拮抗剂组(P均<0.05)。小鼠心肌细胞凋亡率对照组<脓毒症+miR-210-5p激动剂组<脓毒症组<脓毒症+miR-210-5p拮抗剂组,cleaved Caspase-3蛋白表达对照组、脓毒症+miR-210-5p激动剂组<脓毒症组<脓毒症+miR-210-5p拮抗剂组(P均<0.05)。小鼠心肌组织MPO活性及TNF-α、IL-1β、IL-6、MCP-1表达对照组、脓毒症+miR-210-5p激动剂组<脓毒症组<脓毒症+miR-210-5p拮抗剂组(P均<0.05)。小鼠心肌组织SOD、GSH、GSH-Px表达对照组、脓毒症+miR-210-5p激动剂组>脓毒症组>脓毒症+miR-210-5p拮抗剂组,心肌组织MDA、DHE荧光强度对照组、脓毒症+miR-210-5p激动剂组<脓毒症组<脓毒症+miR-210-5p拮抗剂组(P均<0.05)。小鼠心肌组织SIRT1蛋白表达对照组、脓毒症+miR-210-5p激动剂组>脓毒症组>脓毒症+miR-210-5p拮抗剂组,AcFOXO1/FOXO1、Ac-NF-κB p65/NF-κB p65对照组、脓毒症+miR-210-5p激动剂组<脓毒症组<脓毒症+miR-210-5p拮抗剂组(P均<0.05)。结论miR-210-5p可通过抑制心肌组织炎症反应和氧化应激减轻脓毒症诱导的小鼠心肌损伤,其分子机制可能与激活SIRT1信号通路、减轻SIRT1下游分子NF-κB p65及FOXO1乙酰化程度有关。

Objective To observe the regulatory effect of miR-210-5p on myocardial injury in septic mice and to explore its molecular mechanism.Methods Forty C57BL/6 mice were randomly divided into the control group,sepsis group,sepsis+miR-210-5p agomir group,and sepsis+miR-210-5p antagomir group,with 10 mice in each group.Sepsis model was established by cecal ligation and puncture,and mice in control group were performed laparotomy without cecal ligation and puncture.Mice in the sepsis+miR-210-5p agomir group and sepsis+miR-210-5p antagomir group were injected with miR-210-5p agomir and miR-210-5p antagomir via tail vein 1 day before modeling and immediately after modeling,respectively.Mice in the control group and sepsis group were injected with the same volume of normal saline through the tail vein at the same time.Left ventricular ejection fraction(LVEF),left ventricular shortening fraction(LVFS),myocardial histopathologic changes,creatine kinase isoenzyme(CK-MB)and troponin I(cTnI)levels,apoptosis,apoptotic protein(cleaved Caspase-3)expression,myeloperoxidase(MPO)activity,inflammatory cytokines[tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-6,and monocyte chemotactic protein 1(MCP-1)]expression levels were compared.The expression levels of superoxide dismutase(SOD),glutathione(GSH),glutathione peroxidase(GSH-px)and peroxide malondialdehyde(MDA),and the fluorescence intensity of superoxide anion fluorescence probe(DHE)were analyzed.The expression levels of silent information regulator 1(SIRT1),forkhead box protein O1(FOXO1),acetylated FOXO1(Ac-FOXO1),nuclear factorκB subunit p65(NF-κB p65),acetylated NF-κB p65(Ac-NF-κB p65)were determined,and the ratios of Ac-FOXO1/FOXO1 and Ac-NF-κB p65/NF-κB p65 were calculated.Results LVEF and LVFS in rats were as follows control group>sepsis+miR-210-5p agomir group>sepsis group>sepsis+miR-210-5p antagomir group(P<0.05).HE staining showed that the pathological injury of myocardial tissues were in the following order:control group<sepsis+miR-210-5p agomir group<sepsis group<sepsis+miR-210-5p antagomir group.Serum CKMB and cTnI levels were as follows:control group,sepsis+miR-210-5p agomir group<sepsis group<sepsis+miR-210-5p antagomir group(P<0.05).The apoptosis was as follows:control group<sepsis+miR-210-5p agomir group<sepsis group<sepsis+miR-210-5p antagomir group.The cleaved Caspase-3 protein expression was in the following order:control group,sepsis+miR-210-5p agomir group<sepsis group<sepsis+miR-210-5p antagomir group(all P<0.05).MPO activity and expression levels of TNF-α,IL-1β,IL-6 and MCP-1 were in the following order:control group,sepsis+miR-210-5p agomir group<sepsis group<sepsis+miR-210-5p antagomir group(all P<0.05).The expression levels of SOD,GSH,GSH-Px were as follows:control group,sepsis+miR-210-5p agomir group>sepsis>sepsis+miR-210-5p antagomir group.MDA content,DHE fluorescence intensity were in the following order:control group,sepsis+miR-210-5p agomir groupsepsis>sepsis+miR-210-5p antagomir group.Ac-FOXO1/FOXO1,AcNF-κB p65/NF-κB p65 were as follows:control group,sepsis+miR-210-5p agomir group<sepsis+miR-210-5p antagomir group(all P<0.05).Conclusion MiR-210-5p can reduce myocardial injury induced by sepsis in mice by inhibiting myocardial inflammation and oxidative stress,and its molecular mechanism may be related to activating SIRT1 signaling pathway and reducing the acetylation of NF-κB p65 and FOXO1.