基于Keap1-Nrf2/ARE信号通路探讨五参汤对阿霉素所致心脏毒性的保护作用

Exploring the protective effect of Wushen Decoction on adriamycin induced cardiac toxicity based on Keap1-Nrf2/ARE signaling pathway

关键词:  五参汤  阿霉素  Keap1-Nrf2/ARE信号通路  心脏毒性

Key words:  Wushen Decoction  adriamycin  Keap1-Nrf2/ARE signaling pathway  cardiac toxicity

摘要:

[目的] 基于Kelch样环氧氯丙烷相关蛋白-1（Keap1）-核因子E2相关因子2（Nrf2）/抗氧化反应元件（ARE）信号通路探讨五参汤（WSD）对阿霉素（DOX）所致心脏毒性的保护作用。[方法] 建立DOX诱导的大鼠模型（DOX组），将大鼠分为对照组（Control组）、DOX组、辅酶Q10组（Q10组，灌胃30 mg/kg辅酶Q10）、五参汤低剂量组（WSD-L组，灌胃12.2 g/kg五参汤）、五参汤高剂量组（WSD-H组，灌胃24.4 g/kg五参汤）、五参汤高剂量+Nrf2抑制剂组（WSD-H+ML-385组，灌胃24.4 g/kg五参汤+腹腔注射30 mg/kg的Nrf2抑制剂）；分别检测各组大鼠左室缩短率（FS）、左心室舒张末期内径（LVIDd）、左心室射血分数（LVEF）、左心室收缩末期内径（LVIDs）超声指标，检测血清cTnI心肌肌钙蛋白I（cTnI）、肌酸激酶同工酶（CK-MB）、乳酸脱氢酶（LDH）及心肌组织超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、活性氧（ROS）水平，苏木素-伊红（HE）染色观察心肌组织病理变化；TUNEL染色法检测心肌细胞凋亡，蛋白免疫印迹法（Western blot）检测心肌组织Keap1、Nrf2、血红素加氧酶-1（HO-1）蛋白表达。[结果] 与Control组比较，DOX组出现明显心肌细胞坏死与炎性细胞浸润，心肌组织线粒体肿胀，嵴减少或消失，细胞核核膜皱缩，膜内出现基质溶解，LVEF、FS、心肌组织SOD、GSH-Px水平及Nrf2、HO-1表达显著降低，细胞凋亡率、LVIDs、LVIDd、血清CK-MB、LDH、cTnI水平、心肌组织ROS水平及Keap1蛋白表达显著升高（P<0.05）；与DOX组比较，Q10组、WSD-L组、WSD-H组心肌组织病理损伤减轻，LVEF、FS、心肌组织SOD、GSH-Px水平及Nrf2、HO-1表达显著升高，细胞凋亡率、LVIDs、LVIDd、血清CK-MB、LDH、cTnI水平、心肌组织ROS水平及Keap1蛋白表达显著降低（P<0.05）；与WSD-H组比较，WSD-H+ML-385组心肌组织病理损伤加重，LVEF、FS、心肌组织SOD、GSH-Px水平及Nrf2、HO-1表达显著降低，细胞凋亡率、LVIDs、LVIDd、血清CK-MB、LDH、cTnI水平、心肌组织ROS水平及Keap1蛋白表达显著升高（P<0.05）。[结论] 五参汤调节Keap1-Nrf2/ARE信号通路改善阿霉素诱导的大鼠心脏毒性。

Abstract:

[Objective] Based on the Keap1-Nrf2/antioxidant response element（ARE） signaling pathway，investigation of the protective effect of Wushen Decoction on doxorubicin（DOX）-induced cardiotoxicity. [Methods] A DOX induced rat model（DOX group） was established，and the rats were assigned into control group，DOX group，Coenzyme Q10 group（Q10 group，orally administered 30 mg/kg Coenzyme Q10），Wushen Decoction low-dose group（WSD-L group，orally administered 12.2 g/kg Wushen decoction），Wushen Decoction high-dose group（WSD-H group，orally administered 24.4 g/kg Wushen decoction），and Wushen Decoction high-dose+Nrf2 inhibitor group（WSD-H+ML-385 group，orally administered 24.4 g/kg Wushen decoction+intraperitoneal injection of 30 mg/kg Nrf2 inhibitor）. The left ventricular shortening rate（FS），left ventricular end diastolic diameter（LVIDd），and left ventricular ejection fraction（LVEF），left ventricular end systolic diameter（LVIDs） ultrasound indicators were measured. Serum cardiac troponin I（cTnI），creatine kinase isoenzyme（CK-MB），lactate dehydrogenase（LDH），and myocardial tissue superoxide dismutase（SOD），glutathione peroxidase（GSH-Px），and reactive oxygen species（ROS） were detected. Hematoxylin-eosin（HE） staining was performed to observe pathological changes in myocardial tissue. TUNEL staining was performed to measure myocardial cell apoptosis. Western blot was used to measure the Keap1，Nrf2，and heme oxygenase-1（HO-1） proteins in myocardial tissue. [Results] For the control group，the DOX group showed obvious myocardial cell necrosis and inflammatory cell infiltration，mitochondrial swelling in myocardial tissue，reduced or absent cristae，nuclear membrane shrinkage，and matrix dissolution within the membrane. The LVEF，FS，and the myocardial tissue SOD，GSH-Px，Nrf2，and HO-1 were clearly reduced，while the apoptosis rate，LVIDs，LVIDd，serum CK-MB，LDH，cTnI，myocardial tissue ROS，and Keap1 protein were clearly increased（P<0.05）. For the DOX group，the Q10 group，WSD-L group，and WSD-H group showed reduced pathological damage to myocardial tissue. The LVEF，FS，and the myocardial tissue SOD，GSH-Px，Nrf2，and HO-1 were obviously increased，while the apoptosis rate，LVIDs，LVIDd，serum CK-MB，LDH，cTnI，myocardial tissue ROS，and Keap1 protein were obviously reduced（P<0.05）. For the WSD-H group，the myocardial tissue pathological damage in the WSD-H+ML-385 group worsened. The LVEF，FS，and the myocardial tissue SOD，GSH-Px，Nrf2，and HO-1 were clearly reduced，while the apoptosis rate，LVIDs，LVIDd，serum CK-MB，LDH，cTnI，myocardial tissue ROS，and Keap1 protein were obviously increased（P<0.05）. [Conclusion] Wushen Decoction regulates the Keap1-Nrf2/ARE signaling pathway and improves adriamycin-induced cardiac toxicity in rats.