摘要
目的甘姜苓术汤是金匮要略名方,增加丹参、小茴香二位药物形成加味方以提升疗效,本研究通过网络药理学、分子对接、动物实验证实其抗脂肪肝的影响和机制。方法通过数据库,寻找加味方和脂肪肝的共同靶点,做蛋白互作图,进行GO富集分析和KEGG通路预测。对主要化合物和主要蛋白做分子对接。动物实验中,C57BL6小鼠60%脂肪功能造模饲料造模。对照组无给药,低剂量、中剂量、高剂量加味甘姜苓术汤组采用加味甘姜苓术汤灌胃给药。原方组采用原方灌胃,阳性药组采用易善复灌胃,模型组采用同等剂量纯净水灌胃。测定肝指数,HE染色、油红O染色切片观察肝组织形态学改变,ELISA检测肝脏氧化还原水平,自动生化仪检测血脂水平。Western blot验证网络药理学和分子对接的相关通路蛋白表达水平。结果网络药理学研究发现疾病与药物成分靶点共同靶点98个。蛋白互作PPI分析证实JUN,AKT1,MAPK1,MAPK3,STAT3,HSP90AA1等为核心基因。KEGG通路预测了HIF-1、MAPK、NOD、TNF、TLR等信号通路。分子对接证实主要化合物和蛋白之间连接紧密。动物实验证实,4周给药后,加味方、原方、阳性药均减少小鼠体重,加味方中、高剂量组降低体质量的疗效高于原方组(P<0.05,P<0.001),加味方高剂量组降低体质量的疗效高于阳性药组(P<0.01)。油红O染色证实,加味方促进肝组织脂滴减少。HE染色证实,加味方改善肝细胞形态,减少了脂肪,与原方组比较,中剂量组NAS评分更低(P<0.05)。肝功能及脂质代谢指标中,与模型组比较,加味方低、中、高剂量组、原方、阳性药均有效降低肝指数、ALT、AST、TC、TG、LDL-C含量(P<0.05,P<0.01,P<0.001),提升了HDL-C含量(P<0.01,P<0.001)。与原方组比较,加味方中、高剂量组更能降低肝指数、ALT、AST、TC、TG、LDL-C(P<0.05,P<0.01,P<0.001);中、高剂量组更能升高HDL-C指标(P<0.01,P<0.001);证实加味方应用提升了护肝、抗脂质代谢紊乱疗效。加味方低、中、高剂量组能促进肝组织中的GSH水平提升(P<0.05,P<0.001),降低TNF-α、IL-6、IL-1β、MDA、ROS水平(P<0.001),发挥抗炎、调节氧化还原水平的作用。Western blot实验证实,加味方低、中、高剂量组TLR4蛋白水平下降(P<0.001)。p-P38蛋白水平下降(P<0.05,P<0.01,P<0.001),证实加味方抑制了肝组织TLR4-P38 MAPK信号通路激活。结论加味甘姜苓术汤对非酒精性脂肪肝小鼠模型具有促进恢复血脂代谢平衡,调节氧化还原水平、减轻脂肪堆积、减轻体重的作用,其效果优于甘姜苓术汤原方。TLR4-P38信号通路参与了加味甘姜苓术汤对非酒精性脂肪肝的作用机制。
Objective Ganjiang Lingzhu Decoction is a famous formula in Essentials from the Golden Cabinet.Salviae Miltiorrhizae Radix et Rhizoma and Foeniculi Fructus were added to compose the modified formula and improve the curative effect.This study aimed to explore the effect of Ganjiang Lingzhu Decoction against fatty liver and its mechanism through network pharmacology,molecular docking,and animal experiments.Methods Through databases,the common targets of the modified Ganjiang Lingzhu Decoction and fatty liver were screened out,and the protein-protein interaction(PPI)map was made.Gene Ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed.Molecular docking was performed between main compounds and proteins.In animal experiments,C57BL6 mice were modeled with 60%fat functional modeling feed.The control group was not given drugs,and the low-dose,medium dose,and high-dose modified formula groups were given modified formula by gavage.The original formula group was perfused with the original formula,the positive drug group was perfused with Essentiale,and the model group was perfused with the same dose of purified water.Liver index was measured,and liver morphological changes were observed by hematoxylin-eosin(HE)staining and oil red O staining.The liver redox levels were determined by enzyme-linked immunosorbent assay(ELISA),and the blood liquid levels were determined by automatic biochemical instrument.Western blot was used to explore the protein expression levels of pathways related to network pharmacology and molecular docking.Results Ninety-eight common targets of diseases and drug components were found in the research of network pharmacology.PPI analysis showed that JUN,protein kinase B1(AKT1),mitogen-activated protein kinase(MAPK)1,MAPK3,signal transducers and activators of transcription 3(STAT3),and heat shock protein 90 alpha family class a member 1(HSP90AA1)were hub genes.KEGG pathway enrichment analysis predicted hypoxia inducible factor-1(HIF-1),MAPK,nucleotide-binding oligomerization domain(NOD),tumor necrosis factor(TNF),Toll-like receptor(TLR),and other signaling pathways.Molecular docking demonstrated that the main compounds and proteins were closely linked.Animal experiments confirmed that after 4 weeks of administration,the modified formula,the original formula,and the positive drug all reduced the body weight of mice.The effects of the modified formula on reducing body weight in the medium and high-dose groups were higher than that in the original formula group(P<0.05,P<0.001),and the effect of the modified formula on reducing body weight in the high-dose group was higher than that in the positive drug group(P<0.01).Oil red O staining indicated that the modified formula promoted the reduction of lipid droplets in the liver tissue.HE staining showed that the modified formula improved the morphology of hepatocytes and reduced fat.As compared with the original formula group,the NAFLD Activity Score(NAS)of the medium-dose group was lower(P<0.05).In the indexes of liver function and lipid metabolism,compared with the model group,the low,medium,and high-dose modified formula groups,the original formula group,and the positive drug group all effectively reduced the liver indexes and the content of alanine aminotransferase(ALT),aspartate aminotransferase(AST),total cholesterol(TC),triglyceride(TG),and low-density lipoprotein cholesterol(LDL-C)(P<0.05,P<0.01,P<0.001),and increased the content of high-density lipoprotein cholesterol(HDL-C)(P<0.01,P<0.001).Compared with the original formula group,the medium and high-dose modified formula groups decreased the liver indexes,ALT,AST,TC,TG,and LDL-C(P<0.05,P<0.01,P<0.001),and increased the HDL-C index(P<0.01,P<0.001).It was confirmed that the application of the modified formula improved the efficacy of liver protection and anti-lipid metabolism disorder.The low,medium,and high-dose modified formula groups promoted the level of glutathione(GSH)in the liver tissue(P<0.05,P<0.001)and reduced the levels of TNF-α,interleukin(IL)-6,IL-1β,malondialdehyde(MDA),and reactive oxygen species(ROS)(P<0.001),which played the role of anti-inflammation and regulating redox level.Western blot suggested that the levels of TLR4 proteins in the low,medium,and high-dose modified formula groups decreased(P<0.001).The level of p-P38 protein decreased(P<0.05,P<0.01,P<0.001),which indicated that the modified formula inhibited the activation of TLR4-P38 MAPK signaling pathway in the liver tissue.Conclusion The modified Ganjiang Lingzhu Decoction can promote the recovery of blood lipid metabolic balance,regulate redox level,and reduce fat accumulation and weight loss in the mouse model of nonalcoholic fatty liver,and its effect is better than the original formula.TLR4-P38 signaling pathway is involved in the mechanism of modified Ganjiang Lingzhu Decoction in nonalcoholic fatty liver.
作者
徐永妘
张磊
张宇
高司成
卓蕴慧
XU Yong-yun;ZHANG Lei;ZHANG Yu;GAO Si-cheng;ZHUO Yun-hui(Department of Internal Medicine,Municipal Hopital of Traditional Chinese Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 200071;Department of Internal medicine,Shuguang Hospital Affiliated to Shanghai University of Taditional Chinese Medicine,Shanghai 201203;General Ward of Community Health Service Center in Ouyang Road Street of Hongkou District,Shanghai 200081;Depatment of Hepatology,Mumieipal Hospital of Traditional Chinese Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 200071;Department of Hepatology,Shuguang Hospital Afliated to Shanghai Univesity of Traditional Chinese Medicine,Shanghai 201203)
出处
《世界中西医结合杂志》
2023年第1期74-84,共11页
World Journal of Integrated Traditional and Western Medicine
基金
上海市卫健委项目(2020JQ005)。
关键词
非酒精性脂肪肝
甘姜苓术汤
血脂
炎症
氧化还原
Nonalcoholic Fatty Liver
Ganjiang Lingzhu Decoction
Blood Lipids
Inflammation
Redox
