露水草调节MAPK/NF-κB通路对db/db糖尿病小鼠肝损伤修复作用研究

Study on the Repair Effect of MAPK/NF-κB Pathway Regulated by Cyanotis Arachnoides on Liver Injury in db/db mice

中文关键词:露水草  糖尿病  肝损伤  MAPK/NF-κB通路

keywords:Cyanotis  arachnoides, Diabetes, Liver  injury, MAPK/NF-κB  pathway

中文摘要:目的：研究露水草对db/db糖尿病小鼠肝损伤的修复作用,并基于丝裂原活化蛋白激酶(MAPK)/核转录因子-κB(NF-κB)通路探讨露水草的作用机制。方法：40只C57BL/KsJ-db/db糖尿病模型小鼠随机分为Model组、LSC-L组、LSC-M组、LSC-H组、Positive组,每组8只,另8只空白背景鼠作为对照组。LSC-L组、LSC-M组、LSC-H组分别按照0.15g、0.3g、0.6g/kg灌胃给予小鼠露水草提取物,Positive组按照250mg/kg灌胃给予小鼠盐酸二甲双胍,所有组别给药1次/d,连续干预8w。全自动生化分析仪测定空腹血糖(FBG)、空腹血浆胰岛素(FINS)、总胆固醇(TC)、总甘油三酯(TG)水平,酶联免疫吸附法(ELISA)试剂盒测定血清谷草转氨酶(AST)、谷丙转氨酶(ALT)、碱性磷酸酶(ALP)、γ-谷氨酰转移酶(γ-GGT)、总胆红素(TBIL)水平,测定小鼠体重和肝脏重量并计算肝脏指数,ELISA试剂盒测定肝脏肿瘤坏死因子(TNF)-??、白介素(IL)-6、IL-1β、转化生长因子(TGF)-β、超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-PX)水平,苏木精-伊红(HE)染色法检查肝脏病理变化,蛋白免疫印迹法测定肝脏p-NF-κB、NF-κB、p-p38MAPK、p38MAPK、p-ERK、ERK蛋白水平。结果：与Normal组比较,Model组FBG、FINS、TC、TG、AST、ALT、ALP、γ-GGT、TBIL、TNF-??、IL-6、IL-1β、TGF-β、MDA水平,小鼠体重、肝脏重量和肝脏指数,肝脏p-NF-κB、p-p38MAPK、p-ERK蛋白水平升高,SOD、GSH-PX降低,差异有统计学意义(P<0.05)；与Model组比较,LSC-L、LSC-M、LSC-H和Positive组FBG、FINS、TC、TG、AST、ALT、ALP、γ-GGT、TBIL、TNF-??、IL-6、IL-1β、TGF-β、MDA水平,小鼠体重、肝脏重量和肝脏指数,肝脏p-NF-κB、p-p38MAPK、p-ERK蛋白水平降低,SOD、GSH-PX升高,差异有统计学意义(P<0.05)；与LSC-L组比较,LSC-M、LSC-H组FBG、FINS、TC、TG、AST、ALT、ALP、γ-GGT、TBIL、TNF-??、IL-6、IL-1β、TGF-β、MDA水平,小鼠体重、肝脏重量,肝脏p-NF-κB、p-p38MAPK、p-ERK蛋白水平降低,SOD、GSH-PX升高,差异有统计学意义(P<0.05)；与LSC-M组比较,LSC-H组FBG、FINS、TC、TG、AST、ALT、ALP、γ-GGT、TBIL、TNF-??、IL-6、IL-1β、TGF-β、MDA水平,小鼠体重、肝脏重量,肝脏p-NF-κB、p-p38MAPK、p-ERK蛋白水平降低,SOD、GSH-PX升高,差异有统计学意义(P<0.05)。结论：露水草提取物能够调节db/db糖尿病模型小鼠糖脂代谢,抑制肝脏炎症反应及氧化应激损伤,修复肝功能,缓解db/db小鼠肝脏病理损伤,其机制可能与调节MAPK/NF-κB通路有关。

Abstract:Objective: To study the repair effect of Cyanotis arachnoides on liver injury in db/db mice and explore the mechanism of action of Cyanotis arachnoides based on the MAPK/NF-κB pathway. Methods: 40 C57BL/KsJ-db/db diabetic model mice were randomly divided into Model group, LSC-L group, LSC-M group, LSC-H group and Positive group, with 8 mice in each group. Another 8 mice with blank background were used as the control group. In the LSC-L group, LSC-M group and LSC-H group, mice were given Cyanotis arachnoides extracts by gavage at 0.15g、0.3g、0.6g/kg respectively. In the Positive group, mice were given metformin hydrochloride by gavage at 250mg/kg. All groups were administered once a day for continuous intervention for 8 weeks. The levels of fasting blood glucose (FBG), fasting plasma insulin (FINS), total cholesterol (TC), and total triglycerides (TG) were measured by an automatic biochemical analyzer. The levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ -glutamyl transferase (γ-GGT), and total bilirubin (TBIL) were determined by enzyme-linked immunosorbent assay (ELISA) kit. The body weight and liver weight of mice were measured and the liver index was calculated. ELISA kit was used to determine the levels of liver tumor necrosis factor (TNF)-??, interleukin (IL)-6, IL-1β, transforming growth factor (TGF)-β, superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-PX) Pathological changes of the liver were examined by hematoxylin-eosin (HE) staining. The protein levels of p-NF-κB, NF-κB, p-p38MAPK, p38MAPK, p-ERK and ERK in the liver were determined by Western blotting. Results: Compared with the Normal group, the levels of FBG, FINS, TC, TG, AST, ALT, ALP, γ-GGT, TBIL, TNF-??, IL-6, IL-1β, TGF-β, MDA, body weight, liver weight and liver index of mice in the Model group, the protein levels of p-NF-κB, p-p38MAPK and p-ERK in the liver were increased, while SOD and GSH-PX were decreased, and the differences were statistically significant (P<0.05). Compared with the Model group, the levels of FBG, FINS, TC, TG, AST, ALT, ALP, γ-GGT, TBIL, TNF-??, IL-6, IL-1β, TGF-β, MDA, body weight, liver weight and liver index, and the protein levels of p-NF-κB, p-p38MAPK and p-ERK in the liver of mice in the LSC-L, LSC-M, LSC-H and Positive groups were decreased, while SOD and GSH-PX were increased, and the differences were statistically significant (P<0.05). Compared with the LSC-L group, the levels of FBG, FINS, TC, TG, AST, ALT, ALP, γ -ggt, TBIL, TNF-??, IL-6, IL-1β, TGF-β, MDA, as well as the body weight, liver weight of mice, and the protein levels of p-NF-κB, p-p38MAPK and p-ERK in the liver of the LSC-M and LSC-H groups were decreased, while SOD and GSH-PX were increased, and the differences were statistically significant (P<0.05). Compared with the LSC-M group, the levels of FBG, FINS, TC, TG, AST, ALT, ALP, γ -ggt, TBIL, TNF-??, IL-6, IL-1β, TGF-β, MDA, body weight, liver weight, and the protein levels of p-NF-κB, p-p38MAPK and p-ERK in the liver of mice in the LSC-H group were decreased, while SOD and GSH-PX were increased, and the differences were statistically significant (P<0.05). Conclusion: The Cyanotis arachnoides ectracts can regulate glycolipid metabolism in db/db mice, inhibit liver inflammatory response and oxidative stress damage, repair liver function, and alleviate liver pathological damage in db/db mice. The mechanism may be related to the regulation of the MAPK/NF-κB pathway.