肝爽颗粒通过Nrf2/HO-1信号通路缓解慢性肝损伤作用及机制研究

Effect and mechanism of Ganshuang Granule alleviated chronic liver injury via Nrf2/HO-1 signal pathway

关键词:  肝爽颗粒  四氯化碳  慢性肝损伤  氧化应激

Key words:  Ganshuang Granule  CCL₄  chronic liver injury  oxidative stress

摘要:

[目的] 探讨肝爽颗粒（GSG）对四氯化碳（CCL₄）所导致的慢性肝损伤（CLI）模型小鼠的保护作用及机制。[方法] 将50只雄性BALB/c小鼠平均分为正常组、模型组以及肝爽颗粒低、中、高剂量组。除正常组外，其余各组小鼠腹腔注射20% CCL₄橄榄油溶液（2 mL/kg），每周2次，持续6周，成功建立CLI模型小鼠后，正常组与模型组每日灌胃生理盐水，GSG各给药组每日分别灌胃GSG 1、2、4 g/kg，连续给药4周。收集各组小鼠的血清和肝脏，检测血清转氨酶水平并计算肝指数，苏木精-伊红（HE）染色观察肝组织病理学特征，研究GSG对CLI模型小鼠的治疗作用。此外检测肝组织匀浆中超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、丙二醛（MDA）的含量探讨GSG对CLI小鼠氧化损伤的影响。荧光定量聚合酶链式反应（qPCR）检测肝组织中核因子E2相关因子2（Nrf2）、血红素加氧酶-1（HO-1）、谷氨酰半胱氨酸连接酶催化亚基（GCLC）、醌氧化还原酶1（NQO1）基因表达，初步探究GSG缓解CLI的作用机制。[结果] 与正常组比较，模型组小鼠血清中天门冬氨酸氨基转移酶（AST）和丙氨酸氨基转移酶（ALT）水平显著升高（P<0.05或P<0.01）；HE染色结果显示肝细胞广泛水肿、部分肝细胞坏死；同时，肝组织中MDA水平明显上升（P<0.01），SOD、GSH-Px活性显著下降（P<0.01）；肝组织中Nrf2、HO-1、GCLC、NQO1的基因表达显著下调（P<0.05或P<0.01）。与模型组比较，GSG中、高剂量组能显著降低CLI模型小鼠血清AST和ALT的水平（P<0.05或P<0.01）；GSG低、中、高剂量组能降低肝组织中MDA水平，同时升高肝组织中SOD、GSH-Px活性及Nrf2、HO-1、GCLC、NQO1基因表达（P<0.05或P<0.01）。[结论] GSG能缓解CCL₄诱导的CLI模型小鼠肝功能受损，其潜在的机制可能是通过激活Nrf2/HO-1信号通路抑制肝细胞氧化应激。

Abstract:

[Objective] This study was designed to investigate the protective and mechanism of Ganshuang Granule (GSG) on mice with chronic liver injury (CLI) caused by CCL₄.[Methods] Fifty balb/c mice were divided into five groups:normal group,CHI group,low-dose Ganshuang Granule group,medium-dose Ganshuang Granule group and high-dose Ganshuang Granule group. Mice in all groups except for the normal group were intraperitoneal injected with 20% CCL₄ olive oil solution (2 mL/kg),twice a week for 6 weeks to establish a mouse model of CLI. After the model was established,normal group and CHI group were given normal saline daily,and each dose of Ganshuang Granule group was given 1 g/kg,2 g/kg and 4 g/kg daily for four weeks. After 4 weeks intervention,blood and liver were collected. The serum transaminase level in each group was detected,and liver index was calculated. Meanwhile,pathological changes of mice liver were observed by HE staining,so as to study the therapeutic effect of GSG on mice with CLI. In addition,the contents of SOD,GSH-Px and MDA in liver homogenate were detected to study the effect of GSG on oxidative damage in mice with CLI. The Nrf2,HO-1,Gclc and NQO1 genes that related to the Nrf2/HO-1 signaling pathway in liver tissues were detected by RT-qPCR to preliminarily explore the mechanism of GSG in alleviating CLI.[Results] Compared with the normal group,serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the CLI group were significantly increased(P<0.05 or P<0.01). Hepatocytes showed extensive edema and partial nuclear fragmentation and necrosis. The activity of SOD and GSH-Px in liver homogenate decreased (P<0.01),while the level of MDA increased significantly (P<0.01). The gene expressions of Nrf2,HO-1,Gclc and NQO1 were significantly down-regulated (P<0.01,P<0.05). Compared with the CLI group,the middle and high dose groups of Ganshuang Granule could significantly reduce the serum levels of AST and ALT in mice with CLI (P<0.01,P<0.05). Each dose group of Ganshuang Granule can reduce the level of MDA in liver tissues,and increase the activity of SOD and GSH-Px,and the expression of Nrf2,HO-1,Gclc and NQO1 genes (P<0.01,P<0.05).[Conclusion] GSG could alleviate CLI caused by CCL₄,which potential mechanism may be to inhibit oxidative stress of hepatocytes by activating Nrf2/HO-1 signaling pathway.