促红细胞生成素衍生肽腹腔注射对小鼠阿基霉素诱发的心脏毒性作用防治效果及其机制

Preventive effect and mechanism of helix B surface peptid on doxorubicin-induced cardiotoxicity in mice

目的探讨促红细胞生成素衍生肽(HBSP)对阿霉素(DOX)诱发小鼠心脏毒性的预防作用及机制。方法40只C57BL/6小鼠随机分为HBSP+LY294002组、HBSP组、DOX组、Control组,HBSP+LY294002组腹腔注射DOX(15 mg/kg),同时腹腔注射30μg/(kg·d)的HBSP和20 mg/(kg·d)的LY294002,连续7 d;HBSP组一次性腹腔注射DOX(15 mg/kg),同时腹腔注射30μg/(kg·d)的HBSP,连续7 d;DOX组一次性腹腔注射DOX(15 mg/kg);Control组腹腔注射生理盐水。腹腔注射7 d后,采用生理记录仪测量左室压力上升最大速率(+dp/dtmax)和左室压力下降最大速率(–dp/dtmax)、酶联免疫吸附法检测血清中肌钙蛋白T(cTnT);采用HE染色法观察心心肌组织结构;采用Western blotting法检测心肌组织中细胞色素C(Cytochrome C)、裂解的半胱氨酸天冬氨酸蛋白酶3(Cleaved Caspase-3)、磷酸化磷脂酰肌醇-3激酶(p-PI3K)、磷酸化蛋白激酶B(p-Akt)、微管蛋白1轻链3B(LC3B)、自噬相关蛋白1(Beclin1)、溶酶体关联膜蛋白2(LAMP2)和核孔蛋白62(p62)。结果与Control组相比,DOX组+dp/dt、–dp/dt值降低,血清中cTnT含量增加(P均<0.05);心肌纤维排列紊乱并出现断裂和胞质空泡化;心肌组织Cytochrome C和Cleaved Caspase-3蛋白的相对表达量增加(P均<0.05),LC3BII/LC3BI、Beclin1、LAMP2、p62蛋白的相对表达量增加(P均<0.05),而p-PI3K、p-Akt蛋白的相对表达量降低(P均<0.05)。与DOX组相比,HBSP组dp/dt、–dp/dt值增加(P均<0.05),血清cTnT水平降低(P<0.05);心肌纤维排列改善,空泡化减少;心肌组织中Cytochrome C和Cleaved Caspase-3蛋白的相对表达量减少(P均<0.05),LC3BII/LC3BI、Beclin1、LAMP2、p62蛋白的相对表达量减少(P均<0.05),p-PI3K、p-Akt蛋白的相对表达量增加(P均<0.05)。与HBSP组比较,HBSP+LY294002组+dp/dt、–dp/dt值降低(P均<0.05),血清中cTnT含量增加(P<0.05);心肌纤维紊乱、断裂和胞质空泡化;心肌组织Cytochrome C、Cleaved Caspase-3蛋白的相对表达量增加(P均<0.05),LC3BII/LC3BI、Beclin1、LAMP2、p62蛋白的相对表达量增加(P均<0.05),而p-PI3K、p-Akt蛋白的相对表达量降低(P均<0.05)。结论HBSP可减轻阿霉素诱发的小鼠心脏毒性,其作用机制可能是通过抑制PI3K/Akt通路、从而减轻心肌细胞自噬和凋亡。

Objective To investigate the preventive effect and mechanism of helix B surface peptide(HBSP)on the doxorubicin(DOX)-induced cardiotoxicity in mice.Methods Forty C57 BL/6 mice were randomly divided into four groups(n=10):HBSP+LY294002 group,HBSP group,DOX group,and Control group.The mice in the HBSP+LY294002 group were intraperitoneally injected with 30μg/(kg·d)HBSP and 20 mg/(kg·d)LY294002 after 15 mg/kg DOX injection;mice in the HBSP group were intraperitoneally injected with 30μg/(kg·d)HBSP after 15 mg/kg DOX injection;mice in the DOX group(DOX)were intraperitoneally injected with 15 mg/kg DOX injection;mice in the Control group were intraperitoneally injected with normal saline.Seven days after intraperitoneal injection,we used a physiological recorder to measure the maximal rate of rise of left ventricular pressure(+dp/dtmax)and the maximum rate of left ventricular pressure drop(–dp/dtmax),and enzyme-linked immunosorbent assay to detect serum troponin T(cTnT);cardiac structure and function were detected by ventricular intubation and HE staining;Cytochrome c,Cleaved caspase-3,phosphorylated phosphatidylinositol-3 kinase(p-PI3 K),phosphorylated protein kinase B(p-Akt),tubulin 1 light chain-3 B(LC3 B),autophagy-associated protein 1(Beclin1),lysosome-associated membrane protein 2(LAMP2),and nucleoporin62(p62)were detected by Western blotting.Results Compared with the Control group,the cardiac function was impaired,the values of+dp/dt and-dp/dt decreased(both P<0.01),the arrangement of myocardial fibers was disordered,and the content of cTnT in serum increased(P<0.01),the expression of Cytochrome c and Cleaved caspase-3 increased(P<0.01),the ratio of LC3 BII/LC3 BI,Beclin1,and the expression of LAMP2 and p62 also increased(all P<0.01),but the expression levels of p-PI3 K and p-Akt decreased in the DOX group(all P<0.01).Compared with the DOX group,the cardiac function and structure were improved,the+dp/dt and-dp/dt values increased(both P<0.01),the arrangement of myocardial fibers was improved,the vacuolation decreased,the serum cTnT level decreased(P<0.01),the expression levels of Cytochrome c and Cleaved caspase-3 decreased(both P<0.01),the expression levels of LC3 BII/LC3 BI,Beclin1,LAMP2 and p62 decreased(all P<0.01),and the expression levels of p-PI3 K and p-Akt increased in the DOX+HBSP group(both P<0.01).Compared with the HBSP group,the cardiac function was impaired,the values of+dp/dt and-dp/dt decreased(both P<0.01),the arrangement of myocardial fibers was disordered,and the content of cTnT in serum increased(all P<0.01),the expression levels of Cytochrome c and Cleaved caspase-3 increased(both P<0.01),the ratio of LC3 BII/LC3 BI,Beclin1,and the expression levels of LAMP2 and p62 also increased(both P<0.01),while the expression levels of p-PI3 K and p-Akt decreased in the HBSP+LY294002 group(both P<0.01).Conclusion HBSP reduces doxorubicin-induced cardiotoxicity by alleviating autophagy and apoptosis through PI3 K/Akt pathway.