健脾祛湿方治疗非酒精性脂肪肝大鼠的作用研究

Study on the Efficacy of Jianpi Qushi Decoction in the Treatment of Nonalcoholic Fatty Liver in Rats

[目的]阐明健脾祛湿方(Jianpi Qushi decoction,JQD)治疗大鼠非酒精性脂肪肝(nonalcoholic fatty liver disease,NAFLD)的药效和分子机制。[方法]试验通过饲喂高脂饲料建立大鼠NAFLD模型,将大鼠随机分为模型、奥利司他(32.4 mg/kg)和JQD低(0.35 g/kg)、高(0.70 g/kg)剂量组,另设对照组,每组6只。奥利司他和JQD不同剂量组大鼠灌胃给药,1 mL/只,每天1次,连续7周,对照组与模型组大鼠灌胃等剂量的生理盐水。试验结束后采集大鼠血液、肝脏组织。通过ELISA法测定大鼠血清血脂水平,主要包括总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL-C)、高密度脂蛋白(HDL-C);统计大鼠肝脏指数,检测肝脏抗氧化指标(谷胱甘肽(GSH)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、丙二醛(MDA));采用HE染色和油红O观察大鼠肝脏组织形态结构。利用药物和疾病靶点相关数据库分析JQD治疗NAFLD的活性成分和关键靶点;通过Cytoscape 3.7.2软件构建“药物-成分-靶点”网络图;通过分子对接和实时荧光定量PCR验证JQD主要活性成分和关键靶点的作用效果。[结果]与对照组相比,模型组大鼠血清中TC、TG、LDL-C和肝脏中MDA含量及肝脏指数均显著或极显著升高(P<0.05;P<0.01),血清中HDL-C含量和肝脏中GSH含量及SOD、CAT活性均显著或极显著降低(P<0.05;P<0.01)。与模型组比,JQD低、高剂量组大鼠血清中TC、TG含量和肝脏中MDA含量及肝脏指数均显著降低(P<0.05),肝脏中SOD、CAT活性和GSH含量均显著升高(P<0.05)。HE染色和油红O染色结果显示,对照组大鼠肝细胞形态正常,肝索整齐;模型组大鼠肝脏发生明显的脂肪变形,肝细胞萎缩,布满大小不一的空泡,肝索形态紊乱;JQD低、高剂量组大鼠肝脏空泡数量明显减少,肝索清晰可见。网络药理学共筛选出JQD的有效活性成分35个和NAFLD疾病靶点1890个;网络拓扑分析结果显示,JQD治疗NAFLD的核心活性成分为山柰酚、异鼠李素和槲皮素等,其核心治疗靶点为肿瘤坏死因子(TNF)、过氧化物酶体增殖物激活受体γ(PPARG)和血管内皮生长因子A(VEGFA)等。分子对接结果显示,核心活性成分和关键靶点的结合能均<―5.0 kJ/mol,相互之间具有较好结合活性。实时荧光定量PCR结果显示,与模型组相比,JQD低、高剂量组大鼠肝脏中TNF和PPARG基因表达量均极显著降低(P<0.01),JQD高剂量组VEGFA基因表达量极显著降低(P<0.01)。[结论]JQD对NAFLD显示出良好的治疗效果,其作用机制可能与降低血脂水平、提高肝脏抗氧化酶活性有关。

[Objective]The purpose of this experiment was to elucidate the pharmacological efficacy and molecular mechanism of Jianpi Qushi decoction(JQD)in the treatment of non-alcoholic fatty liver disease(NAFLD)in rats.[Method]After the rat NAFLD model was established by feeding high-fat diet,the rats were randomly divided into model,orlistat(32.4 mg/kg),JQD low(0.35 g/kg)and high(0.70 g/kg)dose groups,and control group was set up,with 6 rats in each group.Rats in orlistat and JQD different dose groups were given intragastric administration,1 mL/rat,once a day for 7 weeks,and the rats in control and model groups were given intragastric administration of equal dose of normal saline.After the experiment,the blood and liver tissues of rats were collected.Serum lipid levels of rats were determined by ELISA,including total cholesterol(TC),triglyceride(TG),low density lipoprotein(LDL-C)and high density lipoprotein(HDL-C).The liver index of rats was measured and the liver antioxidant indexes,including glutathione(GSH),superoxide dismutase(SOD),catalase(CAT)and malondialdehyde(MDA)were detected.HE staining and oil red O staining were used to observe the morphological structure of liver.The active ingredients and key targets of JQD for NAFLD were analyzed using drug and disease target database.The“drug-ingredient-target”network diagram was constructed by Cytoscape 3.7.2 software.The effects of main active ingredients of JQD and key targets were verified by molecular docking and Real-time quantitative PCR.[Result]Compared with control group,the contents of TC,TG and LDL-C in serum,MDA content in liver and liver index in model group were significantly or extremely significantly increased(P<0.05 or P<0.01),HDL-C content in serum,GSH content,SOD and CAT activities in liver were significantly or extremely significantly decreased(P<0.05 or P<0.01).Compared with model group,the contents of TC and TG in serum,MDA content in liver and liver index of rats in JQD low and high dose groups were significantly decreased(P<0.05),and the activities of SOD and CAT,and GSH content in liver were significantly increased(P<0.05).The results of HE staining and oil red O staining showed that the liver cells of control group were normal and the liver cords were neat.In the model group,the liver showed obvious adipose deformation,hepatocyte atrophy,vacuoles of different sizes,and disordered liver cord morphology.The number of liver vacuoles decreased significantly in JQD low and high dose groups,and hepatic cords were clearly visible.A total of 35 active ingredients of JQD and 1890 disease targets of NAFLD were identified by network pharmacology.The results of network topology analysis showed that the core active ingredients of JQD in the treatment of NAFLD were kaempferol,isorhamnetin and quercetin,and the core therapeutic targets were tumor necrosis factor(TNF),peroxisome proliferating-activated receptorγ(PPARG)and vascular endothelial growth factor A(VEGFA).Molecular docking results showed that the binding energies of core active ingredients and key targets were less than―5.0 kJ/mol,and they had good binding activity with each other.Real-time quantitative PCR results showed that compared with model group,the expressions of TNF and PPARG genes in liver of JQD low and high dose groups were extremely significantly decreased(P<0.01).The expression of VEGFA gene in JQD high dose group was extremely significantly decreased(P<0.01).[Conclusion]JQD had shown good therapeutic effect on NAFLD,and the mechanism might be related to reducing blood lipid level and increasing antioxidant enzyme activity in liver.