基于网络药理学研究金龙补肾合剂防治代谢综合征的作用机制

Study on the mechanism of Jinlong Bushen Mixture against metabolic syndrome based on network pharmacology

目的结合网络药理学和实验验证探讨金龙补肾合剂防治高糖高脂饮食诱导代谢综合征大鼠脂质代谢紊乱的作用及机制。方法检索TCMSP和相关文献获得金龙补肾合剂中枸杞子、金樱子、龙眼肉、大枣、绞股蓝、罗汉果、母丁香药物的活性成分和作用靶点,检索GeneCards在线数据库获得代谢综合征相关靶点,利用Venny 2.1.0获得金龙补肾合剂和代谢综合征的交集靶点,采用STRING在线数据库构建交集靶点PPI网络。使用Cytoscape 3.9.0中Cyto NCA插件筛选度值前50位的核心靶点。利用DAVID Bioinformatics Resources 6.8在线分析平台进行GO功能富集与KEGG通路富集分析。采用高糖高盐高脂饲料喂养20周建立代谢综合征大鼠模型。将造模成功大鼠按随机数字表法分为模型组、阳性对照组和金龙补肾合剂组,另设空白对照组,每组8只。金龙补肾合剂组灌胃金龙补肾合剂1.8 ml/kg,阳性对照组灌胃二甲双胍溶液90 mg/kg,空白组和模型组灌胃等体积生理盐水,1次/d,连续干预4周。观察大鼠体重、腹围及空腹血糖变化,检测大鼠血脂水平,采用HE染色观察肝组织病理变化,采用ELISA法测定肝组织中三磷酸腺苷(ATP)、IL-1β、IL-6水平,采用qRT-PCR检测肝组织肝激酶B1(LKB1)、AMPK、Akt1、肉碱棕榈酰转移酶1A(CPT1A)、下调乳酸脱氢酶A(LDH-A)mRNA水平,采用Western blot测定肝组织p-LKB1、LKB1、p-AMPK、AMPK、p-Akt1、Akt1蛋白表达。结果获得金龙补肾合剂主要活性成分141个,841个作用靶点,代谢综合征靶点18763个,得到药物和疾病交集靶点820个。KEGG通路富集分析得到173条通路,主要涉及PI3K-Akt、HIF-1信号通路等。实验结果显示,金龙补肾合剂组大鼠体重、空腹血糖及血脂水平降低,大鼠肝脏糖脂质代谢紊乱改善;血清ATP水平升高(P<0.05),IL-1β、IL-6水平降低(P<0.05);肝组织LKB1、AMPK、Akt1、CPT1A mRNA水平升高(P<0.05),LDH-A mRNA水平降低(P<0.05),p-LKB1/LKB1、p-AMPK/AMPK及p-Akt1/Akt1比值升高(P<0.05)。结论金龙补肾合剂可通过多靶点、多通路改善代谢综合征脂质代谢紊乱,其机制可能通过调节LKB1/AMPK/Akt信号通路发挥作用。

Objective:To investigate the effects and mechanism of Jinlong Bushen Mixture against lipid metabolism disorders induced by high-sugar and high-fat diet in rats with metabolic syndrome by combining network pharmacology and experimental validation.Methods:TCMSP and related literature were retrieved to obtain the active components and targets of wolfberry,golden cherry,longan meat,jujube,gynostemma,rosmarinus officinalis,and motherwort in Jinlong Bushen Mixture,and GeneCards online database was retrieved to obtain metabolic syndrome-related targets.Venny 2.1.0 was used to obtain the intersection targets of Jinlong Bushen Mixture and metabolic syndrome,and STRING online STRING online database was used to construct the PPI network of intersecting targets.Core targets in the top 50 degree values were screened using the Cyto NCA plugin in Cytoscape 3.9.0.The DAVID Bioinformatics Resources 6.8 online analysis platform was used for GO functional enrichment and KEGG pathway enrichment analysis.Metabolic syndrome rat model was established using high sugar,high salt,and high fat feed for 20 weeks.The successfully modeling rats were divided into model groups,positive control group and Jinlong Bushen Mixture group according to random number table,and a blank control group was also set up,with 8 rats in each group.Jinlong Bushen Mixture group was gavaged with Jinlong Bushen Mixture 1.8 ml/kg,a positive control group was gavaged with Metformin 90 mg/kg,and the blank and model groups were gavaged with an equal volume of saline,1 time/d,for 4 weeks.The changes in body weight,abdominal circumference,and fasting blood glucose in rats were observed.The blood lipid level in rats was detected.The pathological changes of liver tissues were detected by HE staining.The levels of ATP,IL-1β,and IL-6 in liver tissues were determined by ELISA.The mRNA expression of liver kinase B1(LKB1),AMPK,Akt1,carnitine palmitoyltransferase 1A(CPT1A),and downregulated lactate dehydrogenase A(LDH-A)in liver tissue were detected by qRT-PCR.Western blot was used to determine the expression of p-LKB1,LKB1,p-AMPK,AMPK,p-Akt1,and Akt1 in liver tissue were detected by Western blotting.Results:A total of 141 main active components,841 active targets,18763 metabolic syndrome targets,and 820 drug-disease intersection targets of Jinlong Bushen Mixture were obtained.The KEGG pathway enrichment analysis yielded 173 entries,including mainly the PI3K-Akt,and HIF-1 signalling pathway.The experimental results showed that the weight,fasting blood glucose,and lipid levels of rats in the Jinlong Bushen mixture group decreased,and the disorder of liver glucose and lipid metabolism in rats improved;the levels of ATP,IL-1βand IL-6 decreased(P<0.05);The mRNA expression of LKB1,AMPK,Akt1,and CPT1A in liver tissue increased(P<0.05),while LDH-A mRNA expression decreased(P<0.05).The p-LKB1/LKB1,p-AMPK/AMPK,and p-Akt1/Akt1 ratio increased(P<0.05).Conclusion:Jinlong Bushen Mixture may restore lipid metabolism disorders in the metabolic syndrome through multiple targets and pathways.Its mechanism may exert pharmacological effects through the LKB1/AMPK/Akt signaling pathway.