miR-145-5p对多柔比星诱导小鼠心肌损伤的调控作用及其机制

Improvement of miR-145-5p on mice with doxorubicin-induced myocardial injury and the mechanism

目的 观察miR-145-5p对多柔比星(DOX)诱导小鼠心肌损伤的影响,并探讨其潜在的分子机制。方法将50只C57BL/6小鼠随机分为Control组(腹腔注射生理盐水)、DOX组(腹腔注射DOX 15 mg/kg)、DOX+NC组(腹腔注射DOX+miRNA NC)、DOX+inhibitor组(腹腔注射DOX+miR-145-5p inhibitor)和DOX+mimic组(腹腔注射DOX+miR-145-5p mimic),每组10只。比较五组注射DOX后14 d的心功能[左心室射血分数(LVEF)和左心室短轴缩短率(LVFS)],心肌组织病理改变,血清乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)水平,心肌组织细胞凋亡率及裂解半胱天冬酶3(cleaved Caspase-3)蛋白表达,心肌组织miR-145-5p、沉默调节蛋白5(sirt5)mRNA表达(TargetScan软件和双荧光素酶报告基因实验结果显示sirt5是miR-145-5p的靶基因)。将40只C57BL/6小鼠随机分为Control1组(腹腔注射生理盐水)、DOX1组(腹腔注射DOX 15 mg/kg)、DOX+sirt5组(腹腔注射DOX 15 mg/kg+尾静脉注射sirt5慢病毒质粒)及DOX+共处理组(腹腔注射DOX 15 mg/kg+尾静脉注射sirt5慢病毒质粒及miR-145-5p mimic),每组10只。比较四组分组处理后14 d心肌组织二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽(GSH)表达和超氧化物阴离子荧光探针(DHE)荧光强度,心肌组织核因子E2相关因子2(Nrf2)、Kelch样ECH关联蛋白1(keap1)、超氧化物歧化酶1(SOD1)、血红素加氧酶1(HO-1)、醌氧化还原酶1(NQO-1)蛋白表达。结果 五组小鼠LVEF、LVFS及心肌组织sirt5 mRNA相对表达量:DOX+inhibitor组、Control组>DOX组、DOX+NC组>DOX+mimic组;血清LDH、CK-MB水平及心肌组织细胞凋亡率、cleaved Caspase-3蛋白、miR-145-5p相对表达量:DOX+inhibitor组、Control组DOX+sirt5组、Control1组(P均<0.05)。结论 miR-145-5p参与了DOX诱导的小鼠心肌损伤,其机制可能与靶向负调控sirt5表达,并进一步抑制sirt5下游通路分子Nrf2、SOD1、HO-1和NQO-1表达,从而促进心肌氧化应激和心肌细胞凋亡有关。

Objective To investigate the effect of miR-145-5p on mice with doxorubicin(DOX)-induced myocardial injury and the potential molecular mechanism.Methods Fifty C57BL/6 mice were randomly divided into the Control group(intraperitoneal injection of normal saline),DOX group(intraperitoneal injection of DOX 15 mg/kg),DOX+NC group(intraperitoneal injection of DOX+miRNA NC),DOX+inhibitor group(intraperitoneal injection of DOX+mi R-145-5p inhibitor)and DOX+mimic group(intraperitoneal injection of DOX+miR-145-5p mimic),with 10 mice in each group.Cardiac function[left ventricular ejection fraction(LVEF)and left ventricular short-axis shortening rate(LVFS)],myocardial histological changes,serum lactate dehydrogenase(LDH)and creatine kinase isoenzyme(CK-MB)levels were compared among the five groups at 14 days after DOX injection.Apoptosis rate and cleaved Caspase-3 protein expression were assessed.The miR-145-5p and sirt5 m RNA expression levels were detected(Target Scan software and dual luciferase reporter assay results showed that sirt5 was the target gene of mi R-145-5p).Forty C57BL/6 mice were randomly divided into the Control1 group(intraperitoneal injection of normal saline),DOX1 group(intraperitoneal injection of DOX 15 mg/kg),DOX+sirt5 group(intraperitoneal injection of DOX 15 mg/kg+tail vein injection of sirt5 lentiviral plasmid),and DOX+co-treatment group(intraperitoneal injection of DOX 15 mg/kg+tail vein injection of sirt5 lentiviral plasmid and mi R-145-5p mimic),with 10 mice in each group.The levels of dialdehyde(MDA),superoxide dismutase(SOD)and glutathione(GSH)and the fluorescence intensity of dihydroethidium(DHE)were compared among the four groups at 14 days after treatment.Expression levels of nuclear factor E2-related factor 2(Nrf2),Kelch-like ECH-associated protein 1(keap1),superoxide dismutase 1(SOD1),heme oxygenase 1(HO-1),and quinone oxidoreductase 1(NQO-1)were detected in the myocardial tissues.Results Comparisons of LVEF and LVFS values and the expression of sirt5 m RNA among the five groups were as follows:DOX+inhibitor group,Control group>DOX group,DOX+NC group>DOX+mimic group.Comparisons of serum LDH and CK-MB levels,apoptosis rate,cleaved Caspase-3 protein expression and mi R-145-5p expression among the five groups were as follows:DOX+inhibitor group,Control groupDOX+sirt5 group,Control1 group(all P<0.05).Conclusion The mi R-145-5p can alleviate DOX-induced myocardial injury,and the mechanism may be related to negatively regulating sirt5 and further inhibiting the expression levels of sirt5 downstream molecules(Nrf2,SOD1,HO-1 and NQO-1),thereby reducing myocardial oxidative stress response.