磷酸肌酸钠通过miR-25-3p/肌浆网Ca^2+泵-2a信号通路改善多柔比星诱导大鼠心肌损伤

Sodium creatine phosphate ameliorates doxorubicin-induced myocardial injury in rats via miR-25-3p/SERCA2a pathway

目的探讨磷酸肌酸钠(CP)对多柔比星(DOX)所致大鼠心肌损伤的保护机制.方法①体内实验:雄性SD大鼠,随机分为正常对照组、DOX组和CP+DOX联合组,均隔天给药1次,持续26 d.DOX组前3次ip给予生理盐水5 mL·kg^-1;之后ip给予DOX 2 mg·kg^-1,共7次.CP+DOX组,前3次ip给予CP 200 mg·kg^-1;之后隔天ip给予CP 200 mg·kg^-1,30 min后再ip给予DOX 2 mg·kg^-1,共7次;之后继续隔天ip给予CP 200 mg·kg^-1,共3次.停药1周后进行指标检测.②体外实验DOX 1μmol·L^-1单独或与N-乙酰-L-半胱氨酸(NAC)0.5 mmol·L^-1或与CP 1 mmol·L^-1共同处理H9C2心肌细胞24 h.HE染色观察大鼠心肌组织病理变化;Masson染色检测大鼠心肌组织胶原纤维沉积;TUNEL染色检测大鼠心肌组织细胞凋亡;荧光定量PCR分析大鼠心肌组织和心肌细胞H9C2中miR-25-3p mRNA表达;CCK8法检测心肌细胞H9C2存活率;Western印迹法检测大鼠心肌组织和H9C2心肌细胞中超氧化物歧化酶2(SOD2)、肌浆网Ca^2+泵-2a(SERCA2a)和活化的胱天蛋白酶3蛋白表达.结果①体内实验:与正常对照组相比,DOX组心肌组织中肌纤维排列紊乱,胶原纤维累积增加,细胞凋亡增加明显,miR-25-3p mRNA和活化的胱天蛋白酶3蛋白表达水平上升(P<0.05),而SOD2和SERCA2a蛋白表达水平下降(P<0.05);与DOX组比较,CP+DOX联合组心肌组织肌纤维排列较为整齐,胶原纤维累积减轻,凋亡细胞明显减少(P<0.05),miR-25-3p mRNA和活化的胱天蛋白酶3蛋白表达水平降低,而SOD2和SERCA2a蛋白表达水平明显增加(P<0.05).②体外实验:DOX诱导心肌细胞H9C2内miR-25-3p mRNA和活化的胱天蛋白酶3蛋白表达呈时间依赖性上调(r=0.9681,r=0.66,P<0.05),而SERCA2a蛋白表达水平在DOX处理3 h后达到峰值,随后逐渐下降至低于细胞对照组.与细胞对照组比较,DOX组存活率显著降低(P<0.05);与DOX组比较,CP+DOX组细胞存活率显著升高(P<0.05);与DOX组相比,CP+DOX组H9C2细胞miR-25-3p mRNA和活化的胱天蛋白酶3蛋白表达水平下调(P<0.05),SOD2和SERCA2a蛋白表达明显增强(P<0.05).结论CP减轻氧化应激,抑制miR-25-3p mRNA表达,促进SERCA2a mRNA和蛋白表达,缓解DOX心肌毒性.

OBJECTIVE To investigate the protective mechanism of sodium creatine phosphate(CP)for doxorubicin(DOX)-induced myocardial injury in rats.METHODS①In vivo,male SD rats were randomly divided into three groups and drugs were ip given once every other day for 26 d.The normal control group was ip injected with normal saline.In the DOX group,rats were ip pre-injected with normal saline for 3 times,and then with DOX 2 mg·kg^-1 7 times.In the CP+DOX group,rats were ip injected with CP 200 mg·kg^-1 3 times,and then with DOX 2 mg·kg^-1 plus CP 200 mg·kg^-1 7 times,finally with CP 200 mg·kg-13 times.②In vitro,H9C2 cells were pretreated with CP 1 mmol·L^-1 or Nacetyl-L-cystine(NAC)0.5 mmol·L^-1 for 1 h before being treated with DOX 1μmol·L^-1 for 24 h.In the myocardial tissues of rats,the morphological changes were detected with HE staining,collagen accu⁃mulation with masson staining and apoptosis cells with TUNEL staining.The miR-25-3p mRNA was detected with qRT-PCR,the protein expressions of superoxide dismutase 2(SOD2),sarcoplasmic retic⁃ulum Ca^2+pump-2a(SERCA2a)and cleaved-caspase 3 in the myocardial tissues of rats or H9C2 cells by Western blotting,and H9C2 cell viability by CCK8 kit,respectively.RESULTS①In vivo,compared with the normal control group,the myocardial fibers were disordered,collagen accumulation and apop⁃tosis cells of myocardial tissues of those rats increased in the DOX group,the expressions of miR-25-3p mRNA and cleaved-caspase 3 protein were up-regulated(P<0.05),while SOD2 and SERCA2a protein decreased(P<0.05).Compared with the DOX group,the myocardial fibers were slightly disordered,collagen accumulation and apoptosis cells of myocardial tissues were decreased(P<0.05)in the CP+DOX group,the expressions of miR-25-3p mRNA and cleaved-caspase 3 protein were decreased(P<0.05),while SOD2 and SERCA2a protein significantly increased(P<0.05).②In vitro,the expressions of miR-25-3p and cleaved-caspase 3 protein were increased in a time-dependent manner(r=0.9681,r=0.66,P<0.05),while SERCA2a protein increased to the peak at 3 h,but subsequently decreased to a lower level than that of the cell control group at 24 h in the DOX group.Compared with the cell control group,DOX decreased cell viability(P<0.05),but compared with the DOX group,H9C2 cell viability was signif⁃icantly increased in the CP+DOX group(P<0.05).Compared with the DOX group,the expressions of miR-25-3p mRNA and cleaved-caspase 3 protein decreased,while SOD2 and SERCA2a protein increased in the CP+DOX group.CONCLUSION CP relieves DOX-induced myocardial injury by allevi⁃ating oxidative stress,suppressing miR-25-3p mRNA and increasing SERCA2a protein expression in rats.