NOX通路参与干细胞联合ARB预防阿霉素心肌毒性作用机制探讨

Doxorubicin (DOX) are widely used for the treatment of tumors. However, their uses may be limited by progressive cardiotoxicity. Pathomechanism of cardiomyopathy is related to the presence of reactive oxygen species(ROS). Prevention against cardiological adverse effects is very important because of the limited options to reverse them. We previously found that DOX could upregulated Angiotensin II (Ang-II) expression, while repeated intravenous bone marrow mesenchymal stem cells (MSCs) transplantation could improve cardiac function and attenuate collagen remodeling via downregulating Ang-II in DOX treated rats. Valsartan is effective to alleviate various cardiac damages by antagonizing increased Ang-II. Present study proposal is aiming to test the efficacy of combined MSCs and valsartan on preventing and treating DOX-induced cardiac toxicity. Through observing in vitro and in vivo Ang-II-Nox-ROS pathway changes post DOX use in the absence and presence of MSCs and valsartan, we aimed to defining if Ang-II-Nox-ROS pathway might be a novel pathway responsible for DOX-induced cardiac toxicity, and to present evidence of the beneficial effects of combined MSCs and valsartan on preventing and treating DOX-induced cardiac toxicity in an effort to stimulating the future clinical use of this therapy strategy to prevent and alleviate DOX-induced cardiac toxicity.

阿霉素是目前最为有效抗肿瘤药物之一，但剂量依赖性的心脏毒性限制其临床应用，目前认为蒽环类药物鳌合铁离子后触发活性氧簇（reactive oxygen species , ROS）大量释放，诱导氧化应激导致心肌损伤。我们前期研究发现阿霉素上调血管紧张素II(Angiotensin II，AngⅡ)表达，而骨髓间充质干细胞（mesenchymal stem cells,MSCs）可以下调AngⅡ表达、修复并减轻阿霉素诱导的心肌损伤，ARB可用来阻断AngⅡ心血管损伤效应，设想在阿霉素给药同时联合给予MSCs和缬沙坦，分别从两个治疗靶点减轻和阻断阿霉素导致的心肌损伤，本课题拟体内外检测阿霉素处理大鼠心肌或心肌细胞的AngⅡ-Nox-ROS通路变化，试图阐明AngⅡ-Nox-ROS通路是诱发阿霉素心肌毒性又一可能的机制，此外，探讨MSCs与ARB联合预防阿霉素心肌损害的可行性并为临床提供实验依据。

阿霉素（DOX）是目前最为有效抗肿瘤药物之一，但剂量依赖的心脏毒性限制其临床应用，机制未完全明了。基于前期发现，提出假设AngII/NOX/ROS/MAPK信号通路可能参与DOX诱导心肌损伤；验证MSCs联合AT1受体拮抗剂缬沙坦阻断上述通路减少ROS生成和释放起到心脏保护作用，探讨阿霉素心肌损伤切实可行的防护方案；研究分为体外与体内实验，体外实验：检测DOX处理后，H9c2细胞ROS形成、AT1R、NOX2、NOX4及caspase-3蛋白表达，随后分别在Val、NADPH氧化酶抑制剂(DPI)、NOX2和NOX4基因敲除和过表达下，分别与MSCs共培养，观察DOX作用24～48h后H9c2细胞caspase-9和MAPK信号转导通路凋亡蛋白表达；采用MTT法测定Val、DOX和Val+DOX作用下H9c2和MDA-MB-231乳腺癌细胞存活率。结果：DOX可促进H9c2细胞ROS生成，上调AT1R、NOX2、NOX4、caspase-3、caspase-9和MAPK信号通路（p-p38、p-JNK、p-ERK）蛋白表达；这些效应可被Val、DPI、NOX2-siRNA和NOX4-siRNA减弱。同时，过表达NOX2和NOX4显著增加DOX诱导的ROS形成，进一步上调MAPK信号通路的凋亡蛋白表达，与单用DOX相比，Val干预后H9c2细胞ROS生成减少，NOX2、NOX4和MAPK通路凋亡蛋白表达下调。同时，Val干预不影响DOX处理的MDA-MB-231乳腺癌细胞存活率，但显著提高DOX处理的H9c2心肌细胞存活率；此外，体外实验验证预防性给予Val能否减轻DOX诱导的心肌损伤。将8周龄雄性SD大鼠随机分为对照组、DOX组和DOX+Val组，造模10周，进行超声心动图及心肌病理学检查、检测心肌组织中NOX1、NOX2和NOX4的mRNA和蛋白表达，结果发现预防性Val干预心功能、病理损害明显改善，胶原体积分数明显降低，心肌NOX2、NOX4的mRNA表达明显下调，炎症及细胞因子表达降低；科学意义：证实AT1R/NOX/ROS/MAPK信号通路参与阿霉素诱导的心脏损伤；无论预防性给予缬沙坦还是MSCs联合缬沙坦都能够减轻心肌损伤，后者有更好的保护作用，为预防阿霉素心肌损伤提供切实可行的方案；NOX2 和 NOX4有可能作为防治阿霉素诱导心肌损伤新的干预靶点。