基于网络药理学探讨桂枝加葛根汤防治血脂异常的机制研究及实验验证

Research on the Mechanism of Guizhi Jiagegen Decoction in Preventing and Treating Dyslipidemia Based on Network Pharmacology and Experimental Verification

目的通过网络药理学技术预测桂枝加葛根汤防治血脂异常可能作用靶点和机制,并进行动物实验验证。方法通过中药系统药理学数据库与分析平台(TCMSP)或中药分子机制的生物信息学分析工具(BATMAN-TCM)检索葛根,桂枝,白芍,生姜,大枣,甘草的化学组分,活性成分的筛选标准以口服生物利用度(OB)>30%,化合物类药性(DL)>0.18或Score cutoff≥20。已筛选出的靶点名称应用数据库Uniprot对其标准化;同时在Genecards和OMIM两个数据库中检索血脂异常相关基因,并将上述预测靶基因合并作为疾病相关基因数据。采用Draw Venn Diagram在线程序将桂枝加葛根汤和血脂异常的靶基因取交集,获得桂枝加葛根汤和血脂异常的共同靶点。采用Cytoscape3.7.1绘制桂枝加葛根汤药物成分和血脂异常疾病靶点的网络图。在R3.6.0软件中安装程集包ggplot2、colorspace、clusterProfiler、pathview、stringi、DOSE等进行GO富集分析和KEGG富集分析。并进一步采用动物实验进行验证,选取SPF级SD雄性大鼠,运用高脂饲料喂养建立高脂血症模型,桂枝加葛根汤干预30天后检测各组大鼠TC、TG、HDL-C、LDL-C水平,qRT-PCR法对TNF信号通路上的关键分子IL-6、IL-1β、PTGS2、MAPK1、MAPK3、MAPK8进行验证。结果预测出桂枝加葛根汤可能作用靶点222个,动脉粥样硬化疾病相关基因1250个,桂枝加葛根汤和血脂异常的共同靶点119个。GO功能富集分析结果表明分子功能相关条目114个,细胞组分相关条目70个,生物过程相关的条目2143个。同时在KEGG通路富集到165条信号通路。然后对三组间大鼠的血脂水平进行比较,与正常组相比,模型组大鼠血清中HDL-C水平显著降低(P<0.01),LDL-C、TG、TC水平显著升高(P<0.01),可以证明造模成功;与模型组相比,桂枝加葛根汤组血清中HDL-C水平显著升高(P<0.05),LDL-C、TG、TC水平显著降低(P<0.01,P<0.05),说明桂枝加葛根汤可以改善大鼠血脂水平。与正常组比较,模型组肝脏组织PTGS2、MAPK1、MAPK3、MAPK8、IL-6、IL-1βmRNA表达均显著上升(P<0.01);与模型组比较,桂枝加葛根汤组肝脏组织PTGS2、MAPK1、MAPK3、MAPK8、IL-6、IL-1βmRNA表达上均显著降低(P<0.01,P<0.05)。结论桂枝加葛根汤通过多靶点、多通路治疗血脂异常,为进一步深入探讨其分子机制奠定基础,拓宽经方桂枝加葛根汤临床应用范围,为后续临床治疗血脂异常提供新的思路。

Objective To predict the possible targets and mechanisms of Guizhi Jiagegen Decoction in preventing and treating dyslipidemia through network pharmacology technology,and to verify it in animal experiments.Methods The chemical components of Radix Puerariae,Ramulus Cinnamomi,Radix Paeoniae Alba,Rhizoma Zingiberis Recens,Fructus Jujubae,and Radix Glycyrrhizae were searched through the TCM System Pharmacology Database and Analysis Platform(TCMSP)or TCM Molecular Mechanism Bioinformatics Analysis Tool(BATMAN-TCM),and based on the compounds Drug-like properties(DL)>0.18,oral bioavailability(OB)>30%,or Score cutoff≥20 were the criteria for screening active ingredients of drugs.Through the Uniprot database,standardize the names of the targets that had been screened,and establish a target data set of active pharmaceutical ingredients.At the same time,genes related to dyslipidemia were searched in the two databases of Genecards and OMIM,and the aforementioned predicted target genes were combined as disease-related gene data.The Draw Venn Diagram online program was used to cross the target genes of Guizhi Jiagegen Decoction and dyslipidemia to obtain the common target of Guizhi Jiagegen Decoction and dyslipidemia.Cytoscape 3.7.1 was used to draw a network diagram of Guizhi Jiagegen Decoction drug components and dyslipidemia disease targets.Using R3.6.0 software,install colorspace,stringi,DOSE,clusterProfiler,pathview and ggplot2 and other packages for GO enrichment analysis and KEGG enrichment analysis.It was further verified by animal experiments.SPF-grade SD male rats were selected and fed with high-fat feed to establish a hyperlipidemia model.The levels of TC,TG,HDL-C and LDL-C in each group of rats were detected after the intervention of Guizhi Jiagegen Decoction for 30 days.The qRT-PCR method verified the key molecules IL-6,IL-1β,PTGS2,MAPK1,MAPK3 and MAPK8 in the TNF signaling pathway.Results We predicted 222 targets of Guizhi Jiagegen Decoction,1250 genes related to atherosclerosis,and 119 common targets of Guizhi Jiagegen Decoction and dyslipidemia.GO analysis results showed that there were 2143 items related to biological processes,70 items related to cell components,and 114 items related to molecular functions.KEGG pathway analysis revealed 165 signal pathways.Compared with the normal group,the serum levels of TG,TC,and LDL-C in the model group increased significantly(P<0.01),and the level of HDL-C decreased significantly(P<0.01),proving that the model was successful.Compared with the model group,the levels of TG,TC and LDL-C in the Guizhi Jiagegen Decoction group decreased significantly(P<0.01,P<0.05),and the level of HDL-C increased significantly(P<0.05),indicating that Guizhi Jiagegen Decoction could improve blood lipid levels in rats.Compared with the normal group,the expression of PTGS2,MAPK1,MAPK3,MAPK8,IL-6,and IL-1βmRNA in the liver tissue of the model group increased significantly(P<0.01).Compared with the model group,the expression of PTGS2,MAPK1,MAPK3,MAPK8,IL-6 and IL-1βmRNA in the liver tissues of the Guizhi Jiagegen Decoction group decreased significantly(P<0.01,P<0.05).Conclusion Guizhi Jiagegen Decoction treats dyslipidemia through multiple targets and multiple pathways,which lays the foundation for further in-depth study of its molecular mechanism,broadens the clinical application of Guizhi Jiagegen Decoction,and provides new ideas for subsequent clinical treatment of dyslipidemia.