地菍总黄酮抗2型糖尿病作用机制的网络药理学研究及实验验证

Mechanism of Melastoma dodecandrum flavonoids in treatment of type 2 diabetes mellitus on network pharmacology and validation

目的 利用网络药理学方法研究地菍总黄酮抗2型糖尿病的作用机制,通过动物实验对靶点进行验证。方法 通过文献检索收集地菍总黄酮的化学成分,并在Swiss Target Prediction平台进行筛选;依据Swiss Target Prediction、GeneCards、TTD、DrugBank数据库预测和筛选地菍总黄酮成分治疗2型糖尿病的作用靶点;由Cytoscape3.7.1构建“成分–疾病–靶点”网络;String数据库构建靶点蛋白相互作用(PPI)网络;R语言对靶点基因进行基因本体(GO)富集分析及京都基因和基因组百科全书(KEGG)通路富集分析。以高脂饮食诱导和ip链脲佐菌素(STZ)方法构建2型糖尿病模型大鼠,将2型糖尿病模型造模成功的40只大鼠随机分成模型组,二甲双胍组(0.2 g/kg)组,地菍总黄酮(0.34、0.45、0.60 g/kg)组,每组8只,继续提供高脂饲料喂养,另设对照组。各组每天ig相应药物,1次/d,对照组和模型组ig 0.5%CMC-Na溶液,连续给药5周。记录大鼠体质量、饮水量和摄食量,检测大鼠血糖及血脂的相关指标,运用q PCR法检测地菍总黄酮对2型糖尿病大鼠肝组织中磷脂酰肌醇-3-激酶(PI3K)、蛋白激酶B(Akt)、腺苷酸活化蛋白激酶(AMPK)作用靶点的调控作用。Western blotting法检测各组大鼠肝脏中Akt、AMPK、固醇调节元件结合蛋白-1C(SREBP-1C)的蛋白相对表达量。结果 从地菍总黄酮成分中筛选得到槲皮素、芹菜素、柚皮素等8种活性成分,作用于98个2型糖尿病靶点,GO功能与KEGG富集分析表明,地菍总黄酮抗糖尿病主要通过调节PI3K/Akt信号通路、AMPK相关信号通路等来发挥抗2型糖尿病的作用。动物实验证实,地菍总黄酮可降低2型糖尿病大鼠的空腹血糖(FBG),抑制血脂升高,上调2型糖尿病大鼠肝脏PI3K、Akt、AMPK m RNA表达,显著增加Akt、AMPK蛋白表达含量,降低SREBP-1C蛋白表达含量(P<0.01、0.05),对糖、脂代谢紊乱均具有改善作用,具有良好的抗2型糖尿病作用。结论 验证了地菍总黄酮多成分、多靶点、多途径治疗2型糖尿病的特点,其抗糖尿病的作用机制可能与调节PI3K、Akt、AMPK、SREBP-1C靶点的表达水平,从而改善糖脂代谢紊乱有关,为后续更进一步研究作用机制提供参考。

Objective To study the mechanism of anti type 2 diabetes by Melastoma dodecandrum flavonoid by using the method of network pharmacology,and to verify the related targets by animal experiments.Methods To collect the chemical constituents of M.dodecandrum flavonoids through literature search,and screen the target of M.dodecandrum in treatment of type 2 diabetes based on the Swiss Target Prediction,GeneCards,TTD,and DrugBank databases platform.To construct the“active ingredients-type 2 diabetes-potential target”by Cytoscape 3.7.1 software.The target protein interaction network was constructed through the String platform,GO enrichment analysis and KEGG metabolic pathway were analyzed through the R language analysis platform.To construct type 2diabetes mellitus model rats high-fat diet induction and ip administered with streptozotocin method.40 rats successfully modeled type 2 diabetes mellitus model were randomly divided into model group,metformin group(0.2 g/kg)group,and M.dodecandrum flavonoids(0.34,0.45,0.60 g/kg)groups,with 8 rats in each group.High-fat diet was continued and a control group was set up.Each group was given the corresponding drug intragaically once daily,the control group and model group were given 0.5%CMC-Na solution intragaically for 5 weeks.The body mass,water intake,and food intake of rats were recorded,and the related indexes of blood glucose and blood lipid of rats were detected.qPCR was used to detect the regulatory effects of M.dodecandrum flavonoids flavones on the targets of PI3K,Akt,and AMPK in liver tissue of type 2 diabetic rats.The protein expressions levels of Akt,AMPK,and SREBP-1c in liver tissues of rats in each group were detected by Western blotting.Results 8 Active ingredients such as quercetin,apigenin,naringin were screened from M.dodecandrum flavonoids and acted on 98 type 2 diabetes targets.GO function and KEGG enrichment analysis showed that M.dodecandrum flavonoids play an anti-diabetic role in type 2 diabetes mainly by regulating PI3K/Akt signaling pathway and AMPK related signaling pathway.Animal experiments confirmed that M.dodecandrum flavonoids can decrease FBG,inhibit the increase of blood lipid,up-regulate the mRNA expression of PI3K,Akt,and AMPK in liver of type 2 diabetic rats,and significantly increase the protein expression levels of Akt and AMPK,reduce the expression of SREBP-1C protein(P<0.01,0.05),can improve the metabolic disorders of sugar and lipid,and has a good anti-type 2 diabetes effect.Conclusion This study verified the characteristics of M.dodecandrum flavonoids in treatment of type 2 diabetes with multi-component,multi-target and multi-pathway.The anti-diabetic mechanism of of M.dodecandrum flavonoids may be related to the regulation of the expression levels of PI3K,Akt,AMPK,and SREBP-1C targets,thereby improving the disorder of glucose and lipid metabolism,providing a reference for further research on the mechanism of action.