目的:为了研究Clean-DM1(C-DM1)的作用,玄参的多草药配方,黄芪,苍术,和丹参,高脂饮食(HFD)诱导的糖尿病小鼠。
方法:通过网络药理学分析获得C-DM1提取物的活性成分和分子机制信息。通过高效液相色谱-质谱(HPLC-MS)分析对C-DM1提取物中的主要化合物进行质量控制。对于体内研究,小鼠通过HFD诱导糖尿病12周。正常组(Nor)中的小鼠用常规饮食维持并用盐水通过管饲法处理。将HFD模型小鼠随机分为3组,包括一个HFD糖尿病模型组,C-DM1提取物给药组(C-DM1,500mg/kg),和二甲双胍给药组(Met,500mg/kg),每组8只小鼠。食物摄入量,体重(BW),每周记录空腹血糖(FBG)水平,共4周。治疗4周后,丙氨酸氨基转移酶(ALT),天冬氨酸转氨酶(AST),血糖,低密度脂蛋白胆固醇(LDL-C)使用自动临床化学分析仪测定,以及用于评估胰岛素抵抗(HOMA-IR)水平和口服葡萄糖耐量试验(OGTT)的稳态模型。采用苏木精-伊红染色观察肝、胰腺组织病理学改变。Westernblot检测肝、胰腺组织中胰岛素受体底物(IRS)/磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)和腺苷5'-单磷酸活化蛋白激酶(AMPK)的表达。
结果:HPLC-MS鉴定出二氢异丹参酮,二氢异丹参酮I,cryptotanshinone,harpagoside,C-DM1提取物中的苍术苷A。C-DM1提取物的给药显著降低体重,卡路里摄入量,糖尿病小鼠血糖和胰岛素水平(P<0.05或P<0.01)。C-DM1提取物的给药改善了糖尿病小鼠的糖耐量受损和胰岛素抵抗,并显着降低了LDL-C水平,ALT和AST(P<0.01)。C-DM1提取物抑制糖尿病小鼠脂肪肝组织病理学改变和胰岛增生。C-DM1提取物显著增加IRS的磷酸化,AKT,AMPK和PI3K在胰腺和肝脏组织中的表达(P<0.05或P<0.01),与网络药理学分析结果一致。
结论:C-DM1提取物通过调节胰腺和肝脏组织中IRS/PI3K/AKT和AMPK的表达改善了长期HFD诱导小鼠的糖尿病症状,提示C-DM1制剂可能有助于预防T2DM的进展。
OBJECTIVE: To investigate the effects of Clean-DM1 (C-DM1), a polyherbal formulation of Radix Scrophulariae, Radix Astragali, Rhizoma Atractylodis, and Radix Salviae Miltiorrhizae, on high-fat diet (HFD)-induced diabetes mice.
METHODS: The information about active components of C-DM1 extract and molecular mechanism was obtained from network pharmacology analysis. Main compounds of C-DM1 extract by high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis were conducted for quality control. For in vivo study, mice were induced diabetes by HFD for 12 weeks. The mice in the normal group (Nor) were maintained with a regular diet and treated with saline by gavage. The HFD model mice were randomly divided into 3 groups, including a HFD diabetic model group, a C-DM1 extract-administered group (C-DM1, 500 mg/kg), and metformin-administered groups (Met, 500 mg/kg), 8 mice in each group. Food intake, body weight (BW), and fasting blood glucose (FBG) levels were recorded weekly for 4 weeks. After 4 weeks of treatment, alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood glucose, low-density lipoprotein cholesterol (LDL-C) were determined using an automated clinical chemistry analyzer, and homeostatic model for assessing insulin resistance (HOMA-IR) levels and oral glucose tolerance test (OGTT) were detected. The histopathological changes of liver and pancreatic tissues were observed by hematoxylin-eosin staining. Insulin receptor substrate (IRS)/phosphatidylinositol 3 kinase (PI3K)/ protein kinase B (AKT) and adenosine 5\'-monophosphate-activated protein kinase (AMPK) expressions in liver and pancreas tissues were detected by Western blot analysis.
RESULTS: HPLC-MS identified dihydroisotanshinone, dihydroisotanshinone I, cryptotanshinone, harpagoside, and atractyloside A in C-DM1 extract. The administration of C-DM1 extract significantly decreased body weight, calorie intake, and the levels of blood glucose and insulin in the diabetic mice (P<0.05 or P<0.01). The C-DM1 extract administration improved the impaired glucose tolerance and insulin resistance in the diabetic mice and significantly decreased the levels of LDL-C, ALT and AST (P<0.01). The C-DM1 extract inhibited the histopathological changes of fatty liver and hyperplasia of pancreatic islets in the diabetic mice. The C-DM1 extract significantly increased the phosphorylation of IRS, AKT, and AMPK and the expression of PI3K in pancreas and liver tissues (P<0.05 or P<0.01), which was consistent with the analysis results of network pharmacology.
CONCLUSIONS: C-DM1 extract improved diabetes symptoms in long-term HFD-induced mice by regulation of IRS/PI3K/AKT and AMPK expressions in pancreas and liver tissues, suggesting that C-DM1 formulation may help prevent the progression of T2DM.