结合网络药理学和分子对接技术分析绞股蓝皂苷在肥胖小鼠中的微生态调节机制

Elucidation of microecological regulatory mechanism of gypenosides in obese mice through integrated network pharmacology and molecular docking techniques

目的采用网络药理学和分子对接技术并结合体内实验探讨绞股蓝皂苷(Gyp)对高脂饮食小鼠肠道黏膜菌群及体重的影响,探析Gyp干预肥胖小鼠的微生态机制。方法18只SPF级小鼠按照随机数表法分为6只正常组(C组)和12只造模组,造模组给与高脂饲料喂养,C组给与普通饲料喂养,持续12周。造模结束后,按照随机数表法将造模组分为6只模型组(M组)和6只Gyp组。Gyp组小鼠每日灌胃Gyp溶液,300 g/kg,2次/天,连续4周,同期灌胃C组和M组等量无菌水。收集小鼠小肠黏膜,利用16S rRNA高通量测序技术分析小鼠小肠黏膜菌群特征。同时结合网络药理学和分子对接技术预测绞股蓝治疗肥胖的潜在机制。结果网络药理学结果显示,绞股蓝可以调节90个与肥胖生成相关的靶点。这些靶点的PPI网络分析、GO和KEGG富集分析结果主要与代谢和炎症反应相关。分子对接验证结果显示,gypenosideⅩⅩⅤⅢ_qt作为绞股蓝的主要活性成分,与核心靶点蛋白TP53、TNF、AKT1的结合具有较强的亲和力。体重变化率结果显示,Gyp干预后,与M组相比,Gyp组小鼠体重增长趋缓。测序结果显示,造模改变了小肠黏膜菌群的α多样性和β多样性;Gyp干预后,与M组相比,Gyp组弧菌属相对丰度升高(t=6.662,P<0.001),uncultured bacterium f Lachnospiraceae相对丰度下降(t=2.850,P=0.017)。LEfSe分析结果显示,葡萄球菌属和弧菌属为Gyp组的优势菌属。相关性分析结果显示,uncultured bacterium f Muribaculaceae(r=0.733,P=0.020)、乳杆菌属(r=0.746,P=0.017)、韦荣球菌属(r=0.709,P=0.027)、脱硫弧菌属(r=0.758,P=0.015)、Candidatus Saccharimonas(r=0.721,P=0.023)、Lachnospiraceae NK4A136 group(r=0.794,P=0.008)、Ruminococcaceae UCG 014(r=0.733,P=0.020)、uncultured bacterium f Lachnospiraceae(r=0.830,P=0.005)与小鼠体重变化率呈显著正相关;葡萄球菌属(r=-0.721,P=0.023)和弧菌属(r=-0.649,P=0.049)与Gyp干预后的小鼠体重变化率呈显著负相关。结论绞股蓝通过多靶点、多通路作用于肥胖,其中TP53、AKT1、TNF、JUN为核心靶点。Gyp改变了肠道菌群的群落结构,增加了黏膜菌群的丰富度和多样性,减缓了肥胖小鼠体重的增长。

Objective To explore the effects of gypenosides(Gyp)on the intestinal mucosal microbiota and body weight of high-fat diet-induced obese mice using network pharmacology and molecular docking technology combined with in vivo experiments,and the microecological mechanism involved in the effect of Gyp on obese mice.Methods Eighteen SPFgrade mice were randomly allocated into a control group(Group C,n=6)and a model group(n=12).The model group was fed a high-fat diet for 12 weeks,while the Group C received a standard diet.After the modeling,the obese mice were identified using a weight threshold exceeding 20%above the mean weight of the Group C.The model group was then randomly divided into a model group(Group M,n=6)and a Gyp group(n=6).The mice in the Gyp group received daily intragastric administration of Gyp solution(300 mg/kg,b.i.d.)for 4 consecutive weeks,while the Group C and Group M received equivalent volumes of sterile water intragastrically.Intestinal mucosa samples were collected and analyzed using 16S rRNA high-throughput sequencing to assess the characteristics of intestinal microbiota.Network pharmacology and molecular docking techniques were employed to predict the potential mechanisms of Gynostemma pentaphyllum(Thunb.)Makino in treating obesity.Results Network pharmacology analysis showed that Gynostemma pentaphyllum controlled 90 targets implicated in the development of obesity;these 90 targets were primarily associated with metabolism and the inflammatory response according to protein–protein interaction(PPI)network analysis and Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses.The molecular docking validation results demonstrated that as the principal active component of Gynostemma pentaphyllum,gypenosideⅩⅩⅤⅢ_qt exhibited strong binding affinity with the core target proteins TP53,TNF and AKT1.The body weight change rate indicated that following Gyp intervention,the mice in the Gyp group exhibited a decelerated increase in body weight compared to the Group M.The sequencing results revealed that modeling altered theα-diversity andβ-diversity of intestinal mucosal microbiota.After Gyp intervention,compared to the Group M,the relative abundance of Vibrio(t=6.662,P<0.001)increased in the Group Gyp,while the relative abundance of uncultured bacterium f Lachnospiraceae(t=2.850,P=0.017)decreased.LEfSe analysis revealed enrichment of Staphylococcus and Vibrio.Correlation analysis results revealed that the relative abundances of uncultured_bacterium_f_Muribaculaceae(r=0.733,P=0.020),Lactobacillus(r=0.746,P=0.017),Veillonella(r=0.709,P=0.027),Desulfovibrio(r=0.758,P=0.015),Candidatus Saccharimonas(r=0.721,P=0.023),Lachnospiraceae NK4A136 group(r=0.794,P=0.008),Ruminococcaceae UCG 014(r=0.733,P=0.020),uncultured bacterium f Lachnospiraceae(r=0.830,P=0.005)and the rate of mouse body weight change.Conversely,significant negative correlations were observed between the relative abundances of Staphylococcus(r=−0.721,P=0.023)and Vibrio(r=−0.649,P=0.049)and the rate of mouse body weight change.Conclusion Gynostemma pentaphyllum acts on obesity through multiple targets and pathways,with TP53,AKT1,TNF and JUN identified as key core targets.Gyp alters the community structure of intestinal microbiota,increasing the richness and diversity of mucosal microbiota slowing the weight gain in obese mice.