黄金胶囊通过铁死亡对非酒精性脂肪肝病大鼠的防治作用研究

Research on Effect of HuangJin Capsule on prevention and treatment of rats with non-alcoholic fatty liver disease by ferroptosis

关键词:  黄金胶囊  非酒精性脂肪肝病  铁死亡  炎症反应

Key words:  Huang Jin capsule  non-alcoholic fatty liver disease  ferroptosis  inflammatory response

摘要:

目的：探讨黄金胶囊通过铁死亡对NAFLD大鼠炎症反应和肝功能的影响。方法：48只SPF级雄性SD大鼠随机分为空白对照组、模型组、黄金胶囊高、中、低剂量和阳性对照组,空白对照组以普通饲料喂养,其他各组饲喂高脂饲料制备NAFLD大鼠模型。造模8周后黄金胶囊高、中、低剂量组灌胃不同剂量黄金胶囊混悬液,空白对照组与模型组给予等体积生理盐水灌胃,阳性对照组予去铁胺0.1g/kg腹腔注射,连续给药4周。4周后HE染色观察肝脏病理变化；ELISA检测血清TNF-α、IL-6、IL-8、IL-1β含量；全自动生化分析仪检测血清TC、TG、ALT和AST水平；免疫组化观察大鼠肝脏组织形态及蛋白表达；Western blot及RT-qPCR检测FTH1、Nrf2、Keap1、HO-1、NQO-1蛋白和mRNA表达。结果：HE结果显示模型组肝细胞索结构破坏,出血,少量纤维化,脂肪空泡较多,肝细胞部分坏死、溶解,大量炎性细胞浸润；阳性对照组仅存在少量肝细胞坏死和少量炎性细胞；黄金胶囊高剂量组和中剂量组脂肪变性、炎性细胞浸润和肝细胞损伤也明显减轻,低剂量组仍存在较多炎性细胞,肝细胞损伤和出血仍较明显但脂肪变性较模型组减轻。与空白对照组相比,模型组大鼠血清IL-8、IL-6、TNF-α、IL-1β含量和TC、TG、ALT、AST水平明显升高,肝组织中FTH1、HO-1、Nrf2、Keap-1、NQO1表达明显下降(P＜0.01)；与模型组相比,黄金胶囊各剂量组和阳性对照组IL-8、IL-6、TNF-α、IL-1β含量和TC、TG、ALT、AST水平明显降低,FTH1、HO-1、Nrf2、Keap-1、NQO1表达明显升高(P＜0.05),其中高剂量组和阳性对照组变化更为显著(P＜0.01)。结论：黄金胶囊可能通过抑制铁死亡减轻炎症反应,降低血清TC、TG水平,改善NAFLD肝功能和肝脏病理损害,对于NAFLD具有潜在的治疗作用。

Abstract:

Objective: To investigate the effect of HuangJin Capsule on inflammation and liver function of NAFLD rats through ferroptosis. Methods: 48 SPF male SD rats were randomly divided into normal control group, model group, high-dose, medium-dose and low-dose of HuangJin capsule and positive control group. Normal control group was fed ordinary diet, and the other groups were fed high-fat diet to prepare NAFLD rat model. After 8 weeks of modeling, the high-dose, medium-dose and low-dose of Huang Jin capsule groups were given different doses of Huang Jin capsule suspension by intragastric administration, the normal control group and the model group were given equal volume of normal saline intragastric administration, and the positive control group was given deferoxamine (0.1g/kg) intraperitoneal injection for continuous 4 weeks. The pathological changes of liver were observed by HE staining after 4 weeks. TNF-α, IL-6, IL-8 and IL-1β of serum were determined by ELISA. TC, TG, ALT and AST levels of serum were detected by automatic biochemical analyzer. The morphology and protein expression of rat liver were observed by immunohistochemistry. The protein and mRNA expressions of FTH1, Nrf2, Keap1, HO-1 and NQO-1 were detected by Western blot and RT-qPCR. Results: HE results showed that in the model group, the structure of hepatocyte cord was damaged, bleeding, a little fibrosis, fat vacuoles were more, partial necrosis and dissolution of hepatocytes, and a large number of inflammatory cells were infiltrated. In the positive control group, there was only a small amount of hepatocyte necrosis and inflammatory cells. Steatosis, inflammatory cell infiltration and hepatocyte damage were also significantly reduced in the high-dose and medium-dose of Huang Jin capsule groups, while there were still more inflammatory cells in the low-dose group, hepatocyte damage and bleeding were still obvious but steatosis was less than in the model group. Compared with normal control group, the contents of IL-8, IL-6, TNF-α and IL-1β in serum and the levels of TC, TG, ALT and AST in model group were significantly increased, while the expressions of FTH1, HO-1, Nrf2, Keap-1 and NQO1 in liver tissue were significantly decreased (P < 0.01). Compared with model group, the contents of IL-8, IL-6, TNF-α and IL-1β and the levels of TC, TG, ALT and AST of Huang Jin capsule groups and positive control group were significantly decreased, while the expressions of FTH1, HO-1, Nrf2, Keap-1 and NQO1 were significantly increased (P < 0.05). The changes of high dose group and positive control group were more significant (P < 0.01). Conclusion: Huang Jin capsule may alleviate inflammation by inhibiting ferroptosis, reduce TC and TG levels in serum, improve liver function and liver pathological damage of NAFLD, and has potential therapeutic effect on NAFLD.