摘要
目的:探究柴胡提取物对非酒精性脂肪性肝病(non-alcoholic fatty liver disease, NAFLD)的影响及与含铜胺氧化酶3(amine oxidase copper-containing 3,AOC3)-磷脂酰肌醇3-激酶(phosphoinositide 3-kinase, PI3K)/蛋白激酶B(protein kinase B,AKT)信号通路的关系。方法:选择2022年5月至2023年5月于镇江市中西医结合医院确诊的69例NAFLD患者,另选择70例健康对照者,采用ELISA法检测血清中苯丙氨酸(Phe)和酪氨酸(Tyr)含量,分别采用qRT-PCR和蛋白免疫印迹法检测血清中AOC3 mRNA和蛋白相对表达。构建C57BL/6小鼠NAFLD模型,造模成功后分为对照组、模型组及柴胡提取物低剂量组(50 mg/kg)、中剂量组(75 mg/kg)、高剂量组(100 mg/kg),每组6只,柴胡提取物组每天分别给予对应柴胡提取物灌胃,对照组和模型组予以对应体积的生理盐水灌胃,4周后采用ELISA法检测血清总胆固醇(total cholesterol, TC)、三酰甘油(triglyceride, TG)、高密度脂蛋白胆固醇(high-density lipoprotein cholesterol, HDL-C)、低密度脂蛋白胆固醇(low-density lipoprotein cholesterol, LDL-C)、一氧化氮(nitric oxide, NO)、肿瘤坏死因子-α(tumor necrosis factor-alpha, TNF-α)含量及肝组织超氧化物歧化酶(superoxide dismutase, SOD)活性以及丙二醛水平,采用HE染色评价肝组织病理。通过转染siRNA敲低AOC3表达,筛选出最适siRNA2(50 nmol/L);将肝细胞分为对照组、模型组、siRNA2组(50 nmol/L)、柴胡组(100μg/mL)和联合组(50 nmol/L siRNA2+柴胡100μg/mL),采用MTT、流式细胞术及蛋白免疫印迹分别检测细胞增殖率、凋亡率以及p-PI3K、p-AKT蛋白表达。结果:NAFLD患者及模型小鼠血清中Phe、Tyr含量及AOC3 mRNA和蛋白相对表达水平分别显著高于健康者和对照组(P<0.01)。与模型组相比,柴胡中、高剂量组小鼠血清中Phe、Tyr、TC、TG、LDL-C、NO、TNF-α水平明显降低(P<0.05或P<0.01),HDL-C水平显著升高(P<0.01),肝纤维化及活性氧沉积均显著改善(P<0.01)。与siRNA2组和柴胡组相比,联合组肝细胞增殖率明显升高(P<0.01),细胞凋亡率明显降低(P<0.01),脂毒性代谢物(TG、TC)含量显著降低(P<0.01),氧化应激明显抑制(丙二醛含量降低,SOD/GSH-Px升高,P<0.01或P<0.05),肝功能显著改善(ALT、AST含量降低,P<0.01),PI3K/AKT信号通路活化(AOC3、p-PI3K、p-AKT表达下降,P<0.01)。结论:柴胡提取物可能通过调节NAFLD相关氨基酸代谢,降低过氧化物含量,阻断AOC3介导的PI3K/AKT通路过度活化,降低AOC3表达以及p-PI3K、p-AKT表达,进而改善NAFLD。
Objective:To explore the effect of Bupleurum extract on non-alcoholic fatty liver disease(NAFLD)and its relationship with amine oxidase copper-containing 3(AOC3)-phosphatidylinositol 3-KINASE(PI3K)/protein kinase B(AKT)signaling pathway.Methods:A total of 69 patients with NAFLD in Zhenjiang Hospital of Chinese Traditional and Western Medicine from May 2022 to May 2023,and another 70 healthy controls were enrolled in the study.The contents of phenylalanine(Phe)and tyrosine(Tyr)in serum were measured by ELISA.The relative expressions of AOC3 mRNA and protein in serum were detected by qRT-PCR and Western blotting,respectively.The NAFLD model of C57BL/6 mice was constructed.After modeling,they were divided into control group,model group,and low-dose group(50 mg/kg),medium-dose group(75 mg/kg),and high-dose group(100 mg/kg)of Bupleurum extract,with 6 mice in each group.The Bupleurum extract group was given the corresponding Bupleurum extract,the control group and the model group were given corresponding volumes of normal saline by gavage every day,respectively.Four weeks later,the contents of total cholesterol(TC),triglyceride(TG),and high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),nitric oxide(NO),and tumor necrosis factor-alpha(TNF-α)in serum were detected by ELISA.Additionally,superoxide dismutase(SOD)activity and malondialdehyde(MDA)levels in liver tissues were measured.Hepatic pathology was evaluated by hematoxylin-eosin(HE)staining.The siRNA2(50 nmol/L)was screened out by knocking down the expression of AOC3 by siRNA transfection.Hepatocytes were divided into control group,model group,siRNA2 group(50 nmol/L),Bupleurum group(100μg/mL),and combined group(50 nmol/L siRNA2+Bupleurum 100μg/mL).The cell proliferation rate,apoptosis rate and the protein expressions of p-PI3K and p-AKT were detected by MTT,flow cytometry and Western blotting,respectively.Results:The contents of Phe and Tyr in the serum of patients with NAFLD and model mice,as well as the relative expression levels of AOC3 mRNA and protein,were significantly higher than those of healthy individuals and the control group,respectively(P<0.01).Compared with the model group,the levels of Phe,Tyr,TC,TG,LDL-C,NO and TNF-αin the serum of mice in the medium and high-dose Bupleurum groups were significantly decreased(P<0.05 or P<0.01),the level of HDL-C was significantly increased(P<0.01),and liver fibrosis and reactive oxygen species accumulation were significantly attenuated(P<0.01).Compared with the siRNA2 group and the Chaihu group,the proliferation rate of hepatocytes in the combined group was significantly increased(P<0.01),the apoptosis rate was greatly decreased(P<0.01),the contents of lipotoxic metabolites(TG,TC)were significantly decreased(P<0.01),and oxidative stress was significantly inhibited(decreased content of MDA,increased SOD/GSH-Px,P<0.01 or P<0.05),liver function was significantly improved(decreased contents of ALT and AST,P<0.01),and the PI3K/AKT signaling pathway was activated(the expressions of AOC3,p-PI3K,and p-AKT decreased,P<0.01).Conclusion:Bupleurum extract may attenuate NAFLD by regulating the metabolism of amino acids related to NAFLD,reducing the content of peroxides,blocking the excessive activation of the PI3K/AKT pathway mediated by AOC3,reducing the expression of AOC3 and phosphorylating the expression of PI3K/AKT.
作者
宗斌
李冉
钱元霞
侯义林
季大伟
尉广飞
ZONG Bin;LI Ran;QIAN Yuanxia;HOU Yilin;JI Dawei;WEI Guangfei(Department of Rehabilitation Medicine,Zhenjiang Hospital of Chinese Traditional and Western Medicine,Zhenjiang Jiangsu 212002;School of Pharmacy,Jiangsu University,Zhenjiang Jiangsu 212013;Department of Pharmacy,Zhenjiang Hospital of Chinese Traditional and Western Medicine,Zhenjiang Jiangsu 212002;Department of Gastroenterology,Zhenjiang Hospital of Chinese Traditional and Western Medicine,Zhenjiang Jiangsu 212002;Taizhou Institute of Drug Control,Taizhou Jiangsu 225316,China)
出处
《江苏大学学报(医学版)》
2025年第3期197-211,共15页
Journal of Jiangsu University:Medicine Edition
基金
2022年度江苏省卫健委老年健康课题项目(LKM2022082)。
