EGCG对顺铂所致大鼠肝损伤的保护效果评价

Evaluation of the protective effect of EGCG on cisplatin-induced liver injury in rats

关键词:  肝损伤  EGCG  顺铂

Key words:  liver injury  EGCG  cisplatin

摘要:

目的 建立顺铂(CIS)所致大鼠肝损伤模型,评价不同剂量的表没食子儿茶素没食子酸酯(EGCG)对CIS所致大鼠肝损伤的保护效果并探讨其可能机制。方法 42只Wistar大鼠随机分为7组,分别为对照组(Control)、顺铂组(CIS)、低剂量组(20 mg/kg EGCG+CIS)、中剂量组(40 mg/kg EGCG+CIS)、高剂量组(80 mg/kg EGCG+CIS)、EGCG对照组(40 mg/kg EGCG)、抑制剂组(40 mg/kg EGCG+CIS+ML385),各组大鼠连续灌胃相应剂量药物28 d,第26 d除Control组外均腹腔注射CIS(7 mg/kg)构建肝损伤模型,同时抑制剂组在第26 d腹腔注射ML385(30 mg/kg),第29 d收集大鼠血清及肝组织。首先采用生化仪测定血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)水平,然后HE染色法观察肝脏组织病变,初步评价EGCG对CIS所致大鼠肝损伤的保护效果,筛选出最佳剂量；其次,采用电镜观察肝组织超微结构变化；TUNEL检测肝组织细胞凋亡情况；试剂盒测定肝组织中超氧化物歧化酶(SOD)、谷胱甘肽(GSH)、丙二醛(MDA)含量；Western Blot检测凋亡相关蛋白(Caspase-3、Bcl-2、Bax)及Keap1/Nrf2信号通路相关蛋白表达情况。结果 与Control组相比,CIS组大鼠体重显著下降(P<0.05),血清ALT、AST水平极显著升高(P<0.01)；病理组织学观察到肝索排列不规则、大量炎性细胞浸润,肝损伤严重。与CIS组相比,中剂量组大鼠体重显著上升(P<0.05),肝功能指标极显著下降(P<0.01),肝索结构清晰、炎性细胞浸润等情况得到缓解,低、高剂量保护效果不明显。因此选用中剂量组(40 mg/kg EGCG)进行后续试验。透射电镜结果显示,预处理EGCG可明显改善线粒体肿胀程度；TUNEL结果显示,预处理EGCG降低肝组织细胞凋亡率。试剂盒结果显示,与CIS组相比,中剂量EGCG组肝组织MDA含量显著降低(P<0.05),SOD、GSH活性极显著升高(P<0.01)；Western Blot结果显示EGCG可以上调大鼠肝组织中Nrf2、Bcl-2、NQO1、HO-1蛋白表达,下调Bax、Keap1、Caspase-3蛋白表达。最后给予Nrf2信号通路抑制剂ML385后,EGCG的保护效果被抵消。结论 预处理EGCG(40 mg/kg)可通过Keap1/Nrf2信号通路改善肝组织氧化应激和细胞凋亡以缓解CIS诱导的大鼠肝损伤。

Abstract:

ABSTRACT: OBJECTIVE To evaluate the protective effect of different doses of epigallocatechin-3-gallate (EGCG) on cisplatin-induced liver injury in rats and explore its possible mechanism. METHODS Forty-two Wistar rats were randomly divided into 7 groups. The cells were divided into Control group, cisplatin group (CIS), low dose group (20 mg/kg EGCG+CIS), middle dose group (40 mg/kg EGCG+CIS), high dose group (80 mg/kg EGCG+CIS) and EGCG control group (40 mg/kg EGCG+CIS) EGCG) and inhibitor group (40 mg/kg EGCG+CIS+ML385). On the 26th day, except for the Control group, all rats were intraperitoneally injected with CIS (7 mg/kg) to establish a liver injury model. At the same time, the inhibitor group was intraperitoneally injected with ML385 (30 mg/kg) on the 26th day, and the serum and liver tissues were collected on the 29th day. First, the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by biochemical analyzer, and then the liver tissue lesions were observed by HE staining to preliminarily evaluate the protective effect of EGCG on liver injury induced by CIS in rats, and the optimal dose was screened. Secondly, the ultrastructure of liver tissue was observed by electron microscope. TUNEL was used to detect the apoptosis of liver cells. The contents of superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA) in liver tissue were determined by kits. Western Blot was used to detect the expression of apoptosis-related proteins (Caspase-3, Bcl-2, Bax) and Keap1/Nrf2 signaling pathway-related proteins. RESULTS Compared with the Control group, the body weight of the CIS group rats decreased significantly (P<0.05), serum ALT and AST levels were significantly increased (P<0.01); Histopathological examination showed irregular arrangement of hepatic cords, infiltration of a large number of inflammatory cells, and severe liver injury. Compared with the CIS group, the body weight of the rats in the medium dose group increased significantly (P<0.05), liver function indexes significantly decreased (P<0.01), the structure of hepatic cords was clear and inflammatory cell infiltration was relieved. The protective effect of low and high doses was not obvious. Therefore, the medium dose group (40 mg/kg EGCG) was selected for the subsequent experiment. Transmission electron microscopy results showed that pretreatment of EGCG significantly improved mitochondrial swelling. TUNEL results showed that pretreatment of EGCG reduced the apoptosis rate of liver tissue cells. The results of the kit showed that compared with the CIS group, the MDA content of liver tissue in the medium dose EGCG group was significantly decreased (P<0.05), and the activities of SOD and GSH were extremely significantly increased (P<0.01); Western Blot results showed that EGCG could up-regulate the protein expressions of Nrf2, Bcl-2, NQO1 and HO-1, and down-regulate the protein expressions of Bax, Keap1 and Caspase-3 in rat liver tissues. The protective effect of EGCG was abolished by the administration of ML385, an inhibitor of Nrf2 signaling pathway. CONCLUSION Pretreatment of EGCG (40 mg/kg) can alleviate CIS-induced liver injury in rats by improving oxidative stress and apoptosis in liver tissue through Keap1/Nrf2 signaling pathway. KEY WORDS: liver injury; EGCG; cisplatin