基于Nrf2/HO-1信号通路调控的恰玛古多糖抗多柔比星心肌毒性的机制研究

Mechanistic studies on the anti-DOX cardiotoxicity of polysaccharides of Brassica rapa L. based on the regulation of Nrf2/HO-1 signaling pathway

本研究主要探讨了恰玛古多糖(polysaccharides of Brassica rapa L.,BRPs)对多柔比星(doxorubicin,DOX)心脏毒性的作用及相关机制,研究选用H9c2细胞作为研究对象,采用Cell counting kit-8(CCK-8)法检测BRPs对DOX致H9c2细胞损伤的影响;将H9c2细胞分成对照组、模型组和药物组(0.5~3 mg·mL^(-1)),对照组于正常条件下培养,其余各组在处理后进行1μmol·L^(-1) DOX诱导24 h。通过流式细胞仪检测各组细胞凋亡;分别测定各组细胞乳酸脱氢酶(lactate dehydrogenase,LDH)、细胞内超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)的含量;检测细胞内活性氧(ROS)和线粒体膜电位(mitochondrial membrane potential,MMP);Western blot检测凋亡及转录因子NF-E2相关因子(NF-E2-related factor 2,Nrf2)/血红素加氧酶-1(heme oxygenase-1,HO-1)通路相关蛋白表达。与对照组相比,DOX致H9c2细胞损伤的细胞活力下降、凋亡率增加,LDH、MDA水平升高,SOD活性降低,ROS水平显著增多,MMP显著下降;B淋巴细胞瘤-2(B cell lymphoma-2,Bcl-2)蛋白水平降低,Bcl-2关联X蛋白(Bcl associated X protein,Bax)水平显著升高;且模型组Nrf-2、HO-1、醌氧化还原酶1(quinone oxidoreductase 1,NQO1)蛋白表达水平降低,Kelch样环氧氯丙烷相关蛋白1(Kelch-like ECH-associated protein 1,Keap1)和磷酸化p38丝裂原活化蛋白激酶蛋白表达水平显著升高。与模型组相比,BRPs各组在0.5~3 mg·mL^(-1)对DOX致H9c2细胞损伤具有保护作用,降低细胞凋亡及LDH、MDA和细胞内ROS水平,增强SOD活性,升高MMP;同时,BRPs可上调凋亡相关Bcl-2,并下调Bax水平;而且,BRPs可使Nrf2、HO-1、NQO1蛋白表达水平升高,Keap1和磷酸化p38丝裂原活化蛋白激酶水平降低。本研究认为BRPs能够保护H9c2细胞,抑制细胞凋亡,可能与其调节Nrf2/HO-1通路拮抗氧化应激有关。

To investigate the role of chamagogic polysaccharides(polysaccharides of Brassica rapa L.,BRPs)against doxorubicin(DOX)cardiotoxicity and related mechanisms,H9c2 cells were selected for the study,and the effects of BRPs on DOX induced damage in H9c2 cells were detected by cell counting kit-8(CCK-8);H9c2 cells were divided into the control group,the model group,and the drug group(0.5-3 mg·mL^(-1));the control group was cultured under normal conditions,and the remaining groups were induced for 24 h by 1μmol·L^(-1) DOX after treatment.Apoptosis was detected by flow cytometry;the levels of lactate dehydrogenase(LDH),superoxide dismutase(SOD)and malondialdehyde(MDA)were measured in each group;intracellular reactive oxygen species(ROS)and mitochondrial membrane potential(MMP)were detected.Western blot was used to detect the expression of proteins related to the apoptosis and transcription factor NF-E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)pathway.Compared with the control group,DOX-induced H9c2 cell injury was characterized by decreased cell viability,increased apoptosis,elevated LDH and MDA levels,decreased SOD activity,significantly increased ROS levels,and significantly decreased MMP;the level of B cell lymphoma-2(Bcl-2)protein decreased,and the level of Bcl-2 associated X protein(Bax)increased significantly;In the model group,the expression levels of Nrf-2,HO-1,quinone oxidoreductase 1(NQO1)were reduced,and the expression levels of Kelch-like ECH-associated protein 1(Keap1)and phosphorylated p38 mitogen-activated protein kinase were significantly increased,Moreover,BRPs(0.5-3 mg·mL^(-1))increased the protein expression levels of Nrf2,HO-1,and NQO1,and decreased the levels of Keap1 and phosphorylated p38 mitogen-activated protein kinase.In summary,the ability of BRPs to protect H9c2 cells and inhibit apoptosis may be related to their regulation of the Nrf2/HO-1 pathway to antagonize oxidative stress.