【作者】 张强；

【导师】 王兵；

【作者基本信息】 上海交通大学 ， 中西医结合临床， 2021， 硕士

【摘要】 目的:1.探求不同配比柴胡人参药对对NAFLD大鼠脂代谢的影响。2.探求柴胡人参药对治疗NAFLD的药效物质基础及潜在作用靶点。3.揭示柴胡人参药对的有效成分豆甾醇通过调节PPARγ—UCP-1信号通路治疗NAFLD的作用机制。4.阐释具有疏肝健脾法的柴胡人参药对治疗NAFLD的机制。方法:1.将48只Wistar大鼠随机分为正常组(C组)8只和造模组40只。C组给予普通饲料及0.9%NaCl溶液灌胃,造模组给予高脂高糖饲料及0.9%NaCl溶液灌胃,造模成功后将造模组大鼠随机分成模型组(M组)、柴胡人参1:1组(CR1:1组)、柴胡人参1:2.5组(CR1:2.5组)、柴胡人参2.5:1组(CR2.5:1组)、奥贝胆酸组(OB组),每组8只。各用药组给予高脂高糖饲料及相应药物灌胃。各组干预结束后称量大鼠体质量、肝湿重,观察病理染色,检测肝功能、血脂。2.利用TCMSP数据库获取柴胡人参药对的活性成分,结合PubChem和SwissTargetPrediction数据库预测柴胡人参药对活性成分潜在作用靶点并建立柴胡人参药对活性成分与作用靶点网络。3.从DisGeNET数据库中获取NAFLD相关靶点。Venny分析得到柴胡人参药对活性成分与NAFLD作用的交集靶点。4.针对交集靶点作PPI分析,GO功能分析与KEGG通路富集分析。综合评价得出核心靶点,建立柴胡人参药对活性成分与核心作用靶点网络。5.利用Autoduck Vina工具将柴胡人参药对活性成分与核心作用靶点进行分子对接实验,预测其潜在结合活性。6.建立NAFLD细胞模型,分别以低剂量(30μM),高剂量(60μM)的豆甾醇干预HepG2细胞进行实验。HepG2细胞分为空白对照组(C组),模型组(M组),豆甾醇低剂量组(L组)和豆甾醇高剂量组(H组)。采用油红O染色,尼罗红染色观察病理改变。检测生化指标TG。Real-time PCR检测PPARγ,CPT-2,UCP-1表达水平。Western blot 检测 PPARγ,CPT-2,UCP-1表达水平。利用免疫荧光技术检测PPARγ的蛋白荧光水平。7.利用Crisper cas9技术构建PPARγ敲除的HepG2细胞模型进行实验,PPARγ敲除的HepG2细胞分为PPARγ-/-组和PPARγ-/-+豆甾醇组。采用油红O染色,尼罗红染色观察病理改变。检测生化指标TG。Real-time PCR检测PPARγ,UCP-1表达水平。Western blot检测PPARγ,UCP-1表达水平。利用免疫荧光技术检测PPAR-/-细胞PPARγ的蛋白荧光水平。结果:1.与C组比较,M组大鼠丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、胆固醇(TC)、三酰甘油(TG)水平升高,肝脏脂肪变及炎症程度加重。与M组比较,各用药组脂代谢水平有一定程度的改善。具体而言,各用药组之间比较,CR2.5:1组的体质量、肝湿重,以及TC、ALT、AST、脂肪变及炎症评分下降较其余两组显著,配伍2.5:1时的药效作用最佳;与OB组比较,CR2.5:1组体质量水平下降更为显著,而肝湿重、TC、AST及炎症评分的改善不如奥贝胆酸组显著。2.柴胡人参药对活性成分与核心作用靶点网络显示,豆甾醇为柴胡和人参共有活性成分之一,分子对接结果预测豆甾醇和PPARγ有良好的潜在结合活性,且PPARγ的激活具有改善NAFLD的作用。3.油红O染色显示,与C组相比,M组HepG2细胞内着色脂滴沉积较多,同视野比较脂变率明显高于C组,豆甾醇处理后脂滴沉积有所改善。尼罗红染色结果显示,与C组相比,M组细胞中脂质含量较高,豆甾醇处理后有明显改善。与C组相比,M组细胞TG含量升高(P<0.01);经过豆甾醇处理后,TG含量明显下降(P<0.01)。Real-time PCR结果显示,与C组相比,M组细胞PPARγ,CPT-2,UCP-1mRNA水平明显降低(P<0.05),经过豆甾醇干预后,PPARγ,CPT-2,UCP-1mRNA 水平明显上调(P<0.05)。Western blot 结果显示,与C组相比,M组细胞PPARγ,CPT-2,UCP-1蛋白水平明显下调(P<0.05),经过豆甾醇干预后,PPARγ,UCP-1蛋白水平明显上升(P<0.05),而CPT-2蛋白水平没有明显变化(P>0.05)。免疫荧光结果显示,与C组相比,M组细胞PPARγ蛋白荧光强度明显降低(P<0.05),经过豆甾醇干预后,PPARγ蛋白荧光强度明显上升(P<0.05),PPARγ被豆甾醇激活。4.敲低HepG2细胞的PPARγ后,构建PPARγ-/-细胞系。油红O染色显示,与PPARγ-/-组相比,PPARγ-/-+豆甾醇组细胞着色脂滴沉积未见明显变化,同视野脂变率也没有明显差异。尼罗红染色结果显示,与PPARγ-/-组相比,PPARγ-/-+豆甾醇组细胞尼罗红染色中脂质含量没有明显变化。与PPARγ-/-组相比,PPARγ-/-+豆甾醇组细胞TG含量没有明显改变。Real-time PCR结果显示,与PPARγ-/-组相比,PPARγ-/-+豆甾醇组细胞PPARγ,UCP-1mRNA 水平没有明显变化(P>0.05)。Western blot 结果显示,与PPARγ-/-组相比,PPARγ-/-+豆甾醇组细胞PPARγ,UCP-1蛋白水平没有明显变化(P>0.05)。免疫荧光结果显示,与PPARγ-/-组相比,PPARγ-/-+豆甾醇组细胞PPARγ蛋白荧光强度没有明显变化(P>0.05)。结论:1.柴胡人参药对可以改善NAFLD大鼠的脂代谢,其最佳配伍比例为柴胡人参2.5:1。2.柴胡人参药对活性成分与核心靶点调控网络及分子对接技术聚焦于豆甾醇对PPARγ的调控作用。3.豆甾醇可以改善HepG2细胞中脂质蓄积,降低TG水平。敲除PPARγ后,豆甾醇对HepG2细胞脂代谢的改善作用被逆转。4.豆甾醇作用于PPARγ—UCP-1信号通路改善脂代谢可能是柴胡人参药对治疗NAFLD的机制之一。更多还原

【Abstract】 OBJECTIVE:1.Explore the effects of different compatibility ratios of Drug pair of Radix Ginseng and Radix Bupleuri on lipid metabolism in NAFLD rats.2.Explore the material basis and potential targets of herb pair of Radix Ginseng and Radix Bupleuri for the treatment of NAFLD,3.Reveal the mechanism of action of stigmasterol(the active ingredient of herb pair of Radix Ginseng and Radix Bupleuri)in the treatment of NAFLD by regulating the PPARγ一UCP-1 signaling pathway.4.Explain the molecular mechanism of herb pair of Radix Ginseng and Radix Bupleuri in the treatment of NAFLD.METHOD:1.48 Wistar rats were randomly divided into a normal group(group C)with 8 rats and a modeling group with 40 rats.Group C was given normal feed and 0.9%NaCl solution by gavage,and the modeling group was given high-fat and high-sugar feed and 0.9%NaCl solution by gavage.After the model was successfully built,the model rats were randomly divided into model group(group M),Drug pair of Radix Ginseng and Radix Bupleuri 1:1 group(CR1:1 group),Drug pair of Radix Ginseng and Radix Bupleuri 1:2.5 group(CR1:2.5 group),Drug pair of Radix Ginseng and Radix Bupleuri 2.5:1 group(CR2.5:1 group),obeticholic acid group(OB group),8 rats in each group.Each medication group was given high-fat and high-sugar feed and corresponding drugs by gavage.After the intervention of each group,the rats were weighed,Liver weight,observe pathological staining,detect liver function and blood lipids.2.Use the TCMSP database to obtain the active ingredients of herb pair of Radix Ginseng and Radix Bupleuri,combine PubChem and SwissTargetPrediction database to predict the potential targets of the active ingredients of herb pair of Radix Ginseng and Radix Bupleuri,and establish a network of active ingredients of herb pair of Radix Ginseng and Radix Bupleuri and target.3.Obtain NAFLD related targets from the DisGeNET database.Through Venny analysis,the intersection target of herb pair of Radix Ginseng and Radix Bupleuri on active ingredients and NAFLD was obtained.4.PPI analysis,GO function analysis and KEGG pathway enrichment analysis are carried out for the intersection target.The key targets were obtained through comprehensive evaluation,and the network of active ingredients and key action targets of herb pair of Radix Ginseng and Radix Bupleuri was established.5.In order to predict the potential binding activity of herb pair of Radix Ginseng and Radix Bupleuri to active ingredients and key targets,molecular docking experiments were carried out using Autoduck Vina tools.6.The NAFLD cell model was established,and experiments were performed with low-dose(30μM)and high-dose(60μM)stigmasterol intervention in HepG2 cells.HepG2 cells were divided into blank control group(group C),model group(group M),lowdose stigmasterol group(group L)and high-dose stigmasterol group(group H).Oil red O staining and Nile red staining were used to observe the pathological changes.Detect biochemical indicators TG.Real-time PCR was used to detect the expression levels of PPARγ,CPT-2 and UCP-1.Western blot was used to detect the expression levels of PPARγ,CPT-2 and UCP-1.Use immunofluorescence technology to detect the protein fluorescence level of PPARγ.7.The Crisper cas9 technology was used to construct a HepG2 cell model of PPARγ knockout.HepG2 cells knocked out of PPARγwere divided into PPARγ-/-group and PPARγ-/-+ stigmasterol group.Oil red O staining and Nile red staining were used to observe the pathological changes.Detect biochemical indicators TG.Real-time PCR was used to detect the expression levels of PPARγ and UCP-1.Western blot was used to detect the expression levels of PPARγ and UCP-1.RESULT:1.Compared with group C,alanine aminotransferase(ALT),aspartate aminotransferase(AST),cholesterol(TC),triacylglycerol(TG)increased,liver steatosis and inflammation aggravated in group M of rats.Compared with group M,the lipid metabolism of each medication group was improved to a certain extent.Comparison between medication groups,the body weight,liver weight,TC,ALT,AST,steatosis and inflammation scores of the CR 2.5:1 group decreased significantly compared with the other two groups.The drug effect is the best when the compatibility is 2.5:1;compared with the OB group,the body mass level of CR 2.5:1 group decreased significantly,while the liver weight,TC,AST and inflammation score was not as significant as that of OB group.2.The network of active ingredients and core target points of herb pair of Radix Ginseng and Radix Bupleuri shows that stigmasterol is one of the common active ingredients of herb pair of Radix Ginseng and Radix Bupleuri.The results of molecular docking predict that stigmasterol and PPARγ have potential binding activity,and the activation of PPARγ can improve NAFLD.3.Oil red O staining showed that compared with group C,there were more pigmented lipid droplets in HepG2 cells in group M,and the rate of lipid change was significantly higher than that in group C compared with the visual field.The lipid droplet deposition was improved after stigmasterol treatment.The results of Nile red staining showed that compared with group C,the lipid content in the cells of group M was higher,and the stigmasterol treatment was significantly improved.Compared with group C,the TG content of cells in group M increased(P<0.01);after stigmasterol treatment,the TG content decreased significantly(P<0.01).Real-time PCR results showed that compared with group C,the levels of PPARγ,CPT-2 and UCP-1 mRNA in group M were significantly reduced(P<0.05).After stigmasterol treatment,PPARγ,CPT-2,and UCP-1 mRNA levels were up-regulated.(P<0.05).Western blot results showed that compared with group C,the protein levels of PPARγ,CPT-2,and UCP-1 in group M were significantly down-regulated(P<0.05).After stigmasterol intervention,the protein levels of PPARγ and UCP-1 were significantly increased(P<0.05),while the level of CPT-2 protein did not change significantly(P>0.05).Immunofluorescence results showed that compared with group C,the fluorescence intensity of PPARγ protein in group M decreased significantly(P<0.05).After stigmasterol intervention,the fluorescence intensity of PPARγ protein increased significantly(P<0.05),and PPARγ was activated by stigmasterol.4.After knocking down the PPARγ of HepG2 cells,the PPARγ-/-cell line was constructed.Oil red O staining showed that compared with the PPARγ-/-group,the PPARγ-/-+stigmasterol group showed no significant changes in the deposition of colored lipid droplets in the cells,and there was no significant difference in the rate of lipid change in the same visual field.The results of Nile Red staining showed that compared with the PPARγ-/-group,the lipid content in the Nile Red staining of the PPARγ-/-+ stigmasterol group did not change significantly.Compared with the PPARγ-/-group,the PPARγ-/-+stigmasterol group had no significant changes in TG content.Real-time PCR results showed that compared with the PPARγ-/-group,the PPARγ and UCP-1mRNA levels of the PPARγ/-+stigmasterol group did not change significantly(P>0.05).Western blot results showed that compared with the PPARγ-/-group,the PPARγ and UCP-1 protein levels in the PPARγ-/-+stigmasterol group did not change significantly(P>0.05).The immunofluorescence results showed that compared with the PPARγ/-group,the PPARγ protein fluorescence intensity of the PPARγ-/-+stigmasterol group did not change significantly(P>0.05).CONCLUSION:1.Drug pair of Radix Ginseng and Radix Bupleuri can improve the glucose and lipid metabolism of NAFLD rats,and the best compatibility ratio of Radix Bupleuri to Radix Ginseng is 2.5:1.2.Through network pharmacology and molecular docking technology,the mechanism of herb pair of Radix Ginseng and Radix Bupleuri in the treatment of NAFLD focuses on the effect of stigmasterol on PPARγ.3.Stigmasterol improves lipid accumulation in HepG2 cells and reduces TG levels.After knocking out PPARγ,the effect of stigmasterol on lipid metabolism of HepG2 cells was reversed.4.Stigmasterol improves lipid metabolism through the PPARγ—UCP-1 signaling pathway,which may be one of the mechanisms of herb pair of Radix Ginseng and Radix Bupleuri in the treatment of NAFLD.更多还原