盐酸小檗碱通过上调SIRT1的表达发挥对多柔比星心脏毒性的保护作用

目的 通过建立多柔比星(doxorubicin,DOX)大鼠急性心脏毒性模型和大鼠心肌细胞模型来验证盐酸小檗碱(Ber)对心脏毒性的保护作用并阐明相关的作用机制。方法 采用一次性腹腔注射DOX 20 mg·kg^-1致大鼠急性心脏损伤模型。大鼠随机分为5组:空白对照(Con)组,DOX组,DOX+Ber 5、10及20 mg·kg^-1组。Ber组均采用灌胃给药,每天1次,连续10 d。Con组及DOX组均给予等容积的蒸馏水,每日1次,连续10 d。于第8天除对照组外,其余各组均i.p. DOX 20 mg·kg^-1。10 d后观察大鼠生存率的变化并将大鼠麻醉,剖开动物的胸腔,取一部分心脏组织用于组织病理学检查,一部分制成匀浆备心肌组织中谷胱甘肽过氧化物酶(GSH-PX)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)和丙二醛(MDA)的检测。另取急性分离的心肌细胞建立DOX心肌细胞氧化损伤模型,MTT法检测生存率;双氯荧光素(DCFH-DA)探针检测细胞活性氧(ROS)水平从而明确Ber对DOX所致心脏损伤是否有保护作用,并从氧化应激的角度对其机制进行探讨。在心肌组织及心肌细胞水平用Western blot检测沉默信息调节因子2同源蛋白1(SIRT1)蛋白表达。罗丹明123(Rh123)染色荧光显微镜照像测定线粒体膜电位(ΔΨ_m)、荧光染料rhod-2-AM(分子探针)测定线粒体Ca²⁺浓度([Ca²⁺]_m)以评价线粒体损伤。结果 Ber预处理能显著改善DOX引起的病理学改变并提高DOX大鼠心肌组织CAT、SOD和GSH-PX活性,降低MDA水平。Western blot结果显示Ber可上调心肌组织及心肌细胞中SIRT1蛋白表达,并且这种上调可被SIRT1的抑制剂EX527所取消。此外,Ber通过调节心肌细胞内ROS、ΔΨ_m和[Ca²⁺]_m水平,显著改善DOX诱导的心肌细胞氧化损伤和线粒体损伤。而这种保护作用可被EX527所取消。结论 Ber可减轻DOX引起的心肌氧化损伤,这种保护作用可能是通过上调SIRT1来实现的。

Berberine Hydrochloride Inhibits Doxorubicin Induced Oxidative Damage of Cardiomyocytes by Up Regulating SIRT1 Expression

OBJECTIVE To verify the protective effect of berberine(Ber) on cardiotoxicity and clarify the relevant mechanism by establishing the doxorubicin(DOX) rat acute cardiotoxicity model and the rat cardiomyocyte model. METHODS Rats with acute heart injury induced by DOX 20 mg·kg^-1 were given a single intraperitoneal injection. The rats were randomly divided into five groups: Con group, DOX group, DOX+Ber 5, 10 and 20 mg·kg^-1 group. Berberine group was given by gavage once a day for 10 days. Con group and DOX group were given equal volume of distilled water once a day for 10 d. On the 8th day, i.p. DOX 20 mg·kg^-1 was given to all groups except the control group. After 10 d, the survival rate of the rats was observed and the rats were anesthetized. The thoracic cavity of the rats was cut open. A part of the heart tissue was taken for histopathological examination, and a part was made into homogenate to test the levels of glutathione peroxidase(GSH-Px), catalase(CAT), superoxide dismutase(SOD) and malondialdehyde(MDA). In the acute isolated cardiomyocytes, the oxidative damage model of cardiomyocytes was established by DOX. The level of ROS in cardiomyocytes was measured by DCFH-DA probe to determine whether Ber has protective effect on DOX induced cardiac injury, and the mechanism was discussed from the perspective of oxidative stress. Western blot was used to detect SIRT1 protein expression in myocardium and cardiomyocytes. The mitochondrial membrane potential(ΔΨ_m) was measured by rhodamine 123(Rh123) staining fluorescence microscopy, and the mitochondrial Ca²⁺ concentration([Ca²⁺]_m) was measured by rhod-2-AM(molecular probe) to evaluate mitochondrial damage. RESULTS Ber pretreatment could significantly increase the activities of cat, SOD and GSH-Px, decrease the level of MDA, and improve the histopathological changes of DOX rats. Western blot showed that Ber could up regulate the expression of SIRT1 protein in myocardium and cardiomyocytes, and this up regulation could be cancelled by the inhibitor EX527 of SIRT1. In addition, by regulating the levels of ROS, ΔΨ_m and[Ca²⁺]_m in cardiomyocytes, Ber significantly improved the oxidative damage and mitochondrial damage induced by DOX. This protective effect can be cancelled by EX527. CONCLUSION Ber can reduce the oxidative damage of myocardium induced by DOX, and this protective effect may be achieved by up regulating SIRT1.