微RNA-375-5p对心力衰竭大鼠的保护作用及机制

Protective effect and mechanism of microRNA-375-5p on heart failure rats

目的探讨微RNA(miR)-375-5p对心力衰竭(HF)大鼠的保护作用及相关机制。方法将50只雄性Sprague Dawley大鼠随机分为假手术组、HF组、miR-NC组、miR-375-5p组、miR-375-5p+Compound C(CC)组,每组10只。HF组、miR-NC组、miR-375-5p组、miR-375-5p+CC组大鼠腹腔注射阿霉素溶液制备HF模型,假手术组大鼠腹腔注射等量的NaCl溶液。造模结束后次日,miR-375-5p组大鼠经尾静脉注射miR-375-5p mimics 100μL,miR-NC组大鼠经尾静脉注射miR-NC mimics 100μL,假手术组和HF组大鼠经尾静脉注射等量生理盐水,miR-375-5p+CC组大鼠经尾静脉注射miR-375-5p mimics 100μL和腺苷酸活化蛋白激酶(AMPK)抑制剂CC(0.2 mg·kg^(-1));各组大鼠均每日给药1次,连续给药4周。使用彩色多普勒超声仪检测各组大鼠心功能指标,反转录聚合酶链反应检测各组大鼠心肌组织中miR-375-5p表达,酶联免疫吸附试验检测各组大鼠血清中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6、IL-1β、丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)水平,苏木精-伊红染色观察各组大鼠心肌组织病理学变化,末端脱氧核苷酸转移酶介导的末端标记法检测各组大鼠心肌细胞凋亡情况,蛋白质印迹检测各组大鼠心肌组织中磷酸化腺苷酸活化蛋白激酶(p-AMPK)、AMPK、沉默信息调节因子3(SIRT3)蛋白的相对表达量。结果与假手术组比较,HF组、miR-NC组和miR-375-5p组大鼠的左心室射血分数(LVEF)、左心室短轴缩短率(LVFS)、血清中SOD活性、GSH水平及心肌组织中p-AMPK/AMPK、miR-375-5p表达量和SIRT3蛋白相对表达量显著降低,左心室舒张末期内径(LVEDD)、左心室收缩末期内径(LVESD)、心肌细胞凋亡率及血清中TNF-α、IL-6、IL-1β、MDA水平显著升高(P<0.05)。miR-NC组与HF组大鼠LVEF、LVFS、LVEDD、LVESD、心肌细胞凋亡率、血清中TNF-α、IL-6、IL-1β、MDA、GSH水平、SOD活性、心肌组织中p-AMPK/AMPK、miR-375-5p表达量及SIRT3蛋白相对表达量比较差异无统计学意义(P>0.05)。与HF组相比,miR-375-5p组大鼠LVEF、LVFS、血清中SOD活性、GSH水平、心肌组织中p-AMPK/AMPK及miR-375-5p表达量和SIRT3蛋白相对表达量显著升高,LVEDD、LVESD、心肌细胞凋亡率及血清中TNF-α、IL-6、IL-1β、MDA水平显著降低(P<0.05)。与miR-375-5p组相比,miR-375-5p+CC组大鼠LVEF、LVFS、血清中SOD活性、GSH水平及心肌组织中p-AMPK/AMPK、miR-375-5p表达量和SIRT3蛋白相对表达量显著降低,LVEDD、LVESD、心肌细胞凋亡率及血清中TNF-α、IL-6、IL-1β、MDA水平显著升高(P<0.05)。假手术组大鼠心肌细胞规律排列,细胞核明显且无炎症细胞浸润;HF组大鼠心肌细胞形态发生明显改变,排列紊乱,细胞间隙变大,染色变浅,且出现心肌纤维化,有大量的炎症细胞浸润,HF组和miR-NC组大鼠心肌组织病理学变化无明显差异;与HF组相比,miR-375-5p组大鼠心肌细胞排列明显有序,细胞坏死程度和范围明显减少;miR-375-5p+CC组与HF组大鼠心肌组织病理学变化相似。结论miR-375-5p能抑制HF大鼠心肌细胞凋亡,进而对HF大鼠发挥保护作用,其机制可能与激活AMPK/SIRT3信号通路有关。

Objective To explore the protective effect and related mechanism of microRNA(miR)-375-5p on heart failure(HF)rats.Methods Fifty male Sprague Dawley rats were randomly divided into the sham operation group,HF group,miR-NC group,miR-375-5p group and miR-375-5p+Compound C(CC)group,with 10 rats in each group.The rats in the HF group,miR-NC group,miR-375-5p group and miR-375-5p+CC group were intraperitoneally injected with adriamycin solution to prepare HF models,and the rats in the sham operation group were intraperitoneally injected with an equal amount of NaCl solution.On the next day after modeling,the rats in the miR-375-5p group were intravenously injected with 100μL of miR-375-5p mimics via tail vein;the rats in the miR-NC group were intravenously injected with 100μL of miR-NC mimics via tail vein;the rats in the sham operation group and HF group were intravenously injected with an equal volume of normal saline via tail vein;the rats in the miR-375-5p+CC group were intravenously injected with 100μL of miR-375-5p mimics and adenosine monophosphate-activated protein kinase(AMPK)inhibitor CC(0.2 mg·kg^(-1))via tail vein;and the rats in each group were administered once daily for 4 consecutive weeks.The cardiac function parameters of rats in each group were detected by color Doppler ultrasound;the expression levels of miR-375-5p in myocardial tissues of rats in each group were detected by reverse transcription polymerase chain reaction;the levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,IL-1β,malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione(GSH)in serum of rats in each group were measured by enzyme-linked immunosorbent assay;the pathological changes in myocardial tissues of rats in each group were observed by hematoxylin-eosin staining;the myocardial cell apoptosis of rats in each group was detected by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling assay;and the relative expression levels of phosphorylated-AMPK(p-AMPK),AMPK and sirtuin 3(SIRT3)proteins in myocardial tissues of rats in each group were measured by Western blot.Results Compared with the sham operation group,the left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),serum SOD activity,GSH level,p-AMPK/AMPK,miR-375-5p expression level,and relative expression level of SIRT3 protein in myocardial tissues of rats in the HF group,miR-NC group and miR-375-5p group significantly decreased,while the left ventricular end-diastolic dimension(LVEDD),left ventricular end-systolic dimension(LVESD),myocardial cell apoptosis rate,and serum levels of TNF-α,IL-6,IL-1βand MDA significantly increased(P<0.05).There was no significant differences in LVEF,LVFS,LVEDD,LVESD,myocardial cell apoptosis rate,serum levels of TNF-α,IL-6,IL-1β,MDA and GSH,SOD activity,p-AMPK/AMPK,miR-375-5p expression level,and relative expression level of SIRT3 protein in myocardial tissues of rats between the miR-NC group and the HF group(P>0.05).Compared with the HF group,LVEF,LVFS,serum SOD activity,GSH level,p-AMPK/AMPK,miR-375-5p expression level,and relative expression level of SIRT3 protein in myocardial tissues of rats in the miR-375-5p group significantly increased,while the LVEDD,LVESD,myocardial cell apoptosis rate,and serum levels of TNF-α,IL-6,IL-1βand MDA significantly decreased(P<0.05).Compared with the miR-375-5p group,LVEF,LVFS,serum SOD activity,GSH level,p-AMPK/AMPK,miR-375-5p expression levels,and relative expression level of SIRT3 protein in myocardial tissues of rats in the miR-375-5p+CC group significantly decreased,while the LVEDD,LVESD,myocardial cell apoptosis rate,and serum levels of TNF-α,IL-6,IL-1βand MDA significantly increased(P<0.05).Myocardial cells of rats in the sham operation group were regularly arranged,with obvious nuclei and no inflammatory cell infiltration;myocardial cells of rats in the HF group were altered morphologically and arranged irregularly,with enlarged cell gaps,lighter staining,myocardial fibrosis,and a large number of inflammatory cell infiltration.There was no significant difference in the pathological changes of myocardial tissues between the HF group and the miR-NC group.Compared with the HF group,myocardial cells of rats in the miR-375-5p group were arranged orderly,and the degree and extent of necrocytosis were reduced.The pathological changes in myocardial cells of rats were similar between the miR-375-5p+CC group and the HF group.Conclusion miR-375-5p can inhibit the apoptosis of myocardial cells in rats with HF,which may be related to its activation of the AMPK/SIRT3 signaling pathway.