摘要
本论文运用体外模型对一种从水翁(Cleistocalyx operculatus (Roxb.) Merr. et Perry)的干燥花蕾中提取纯化的化合物,2',4'-二羟基-6’-甲氧基-3',5'-二甲基查耳酮(DMC),进行了一系列降血糖的功效及机制研究。研究主要从以下几个方面着手：DMC对小肠内碳水化合物消化酶活性的影响；DMC对小肠上皮细胞吸收葡萄糖的影响；DMC对葡萄糖毒性损伤的胰岛p细胞分泌胰岛素的影响及机制；DMC对胰岛素敏感器官利用葡萄糖的影响,包括对骨骼肌细胞L6和脂肪细胞3T3-L1摄取葡萄糖的影响,对肝脏HepG2细胞合成肝糖原的影响；以及对3T3-L1脂肪细胞分化和产生脂滴的影响和机制。
胰腺α-淀粉酶和小肠α-葡萄糖苷酶是小肠内主要的碳水化合物消化酶,研究结果表明,DMC具有强烈的抑制胰腺α-淀粉酶(0.033Units/mL)的作用,其方式为非竞争性抑制,IC50为43μM；但是其对小肠a-葡萄糖苷酶(0.004Units/mg蛋白)并没有显著的抑制作用(<20%)。葡萄糖在小肠内的吸收是高血糖产生过程中至关重要的一步,研究表明,DMC在Caco-2单层细胞模型上显著抑制葡萄糖的转运(P<0.05)。在模拟的空腹状态下,其抑制效果具有剂量依赖性,2.5、10、和40μMDMC分别抑制了葡萄糖转运64%、40%、和18%(vs.对照组)；而在模拟的饱腹状态下则没有明显的剂量关系,DMC浓度为2.5、10、和40μM时对葡萄糖转运的抑制率分别为42%、48%、和52%(vs.对照组)。
胰岛素是体内唯一降血糖的激素,胰岛受损会导致其产生的胰岛素大幅下降。胰岛长期处于高葡萄糖状态(葡萄糖毒性状态)会导致损伤。在33mM葡萄糖浓度下孵育48h,小鼠胰岛p细胞RIN-5F的细胞活力显著降低,胰岛素分泌功能受损；DMC干预葡萄糖毒性状态的RIN-5F细胞,葡萄糖刺激的胰岛素分泌会随剂量的增加而增多,其在2μM和20μM时分别提高胰岛素分泌量1.94和4.38ng/mL(提高了63%和143%,P<0.05,vs.对照组),而各种浓度DMC处理组与其相对应的对照相比较,细胞的活力并没有受到影响,因此推断,DMC是通过提高单个胰岛β细胞的胰岛素分泌能力来提高胰岛素生成的,但DMC对基础胰岛素的分泌则没有影响；结果显示,DMC提高葡萄糖毒性损伤的RIN-5F细胞分泌胰岛素,可能是通过模拟GLP-1的作用以及提高GLP-1R的表达,从而提高胰岛素合成通路上的各个基因靶点(PDX-1、PRE-INS、GLUT2、和GCK)的表达。另一种可能是DMC通过抑制葡萄糖毒性状态引起的iNOS和MCP-1的表达,从而抑制一氧化氮(NO)的生成和其对胰岛细胞功能的损伤。
改善胰岛素抵抗状态,对治疗高血糖也是大有裨益。研究表明,10μM DMC能促进3T3-L1细胞对葡萄糖的摄取,与对照组相比,提高了78%(P<0.05),而20μM DMC则没有表现出对葡萄糖摄取的差异性。20μM DMC对骨骼肌L6细胞摄取葡萄糖和对肝脏HepG2细胞合成肝糖原都没有显著影响。高浓度DMC (10μM和μM)显著降低3T3-L1细胞的脂肪合成,分别降低了55%和20%(P<0.01vs.分化对照组)；低浓度DMC(2.5μM)则增加3T3-L1细胞的脂滴产生,增加了39%(P<0.01vs.分化对照组)；而5μM DMC则对脂肪合成没有显著影响。通过运用定量RT-PCR监测基因水平表达和Western blot对蛋白水平表达的验证,我们推测DMC对3T3-L1细胞脂肪合成的影响是通过调控PPAR-γ和C/EBP-α的来表达实现的。这些结果显示了DMC用于治疗高血糖的潜在可能性。
2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), a compound isolated and purified from the dried flower buds of Cleistocalyx operculatus (Roxb.) Merr. et Perry (Myrtaceae), was investigated for its glucose control benefits and corresponding mechanism of action using in-vitro methods. The study was carried on from the perspective of intestinal digestive enzymes, glucose transport, insulinotropic against glucotoxicity, glucose uptake/glycogen synthesis in insulin sensitive tissue cells, and3T3-L1pre-adipocyte differentiation.
For the effect on intestinal digestive enzymes, DMC showed strong non-competitive (IC50of43μM) inhibition of pancreatic α-amylase; it was, however, ineffective against intestinal a-glucosidase. As to glucose absorption, DMC exhibited remarkable glucose transport inhibition effects in both simulated fasting and fed states in Caco-2cell monolayers (P<0.05), while the inhibition of DMC was dose dependent in simulated fasting status, and no dose effect in simulated fed status.
When exposed to high glucose at the cytotoxicity level for48h, RIN-5F β-cells experienced a significant viability loss and impaired insulin secretion function. Whereas, co-treating with DMC can protect β-cells against glucotoxicity-induced decrease in glucose-stimulated insulin secretion in a dose-dependent manner without affecting basal insulin secretion. It was demonstrated that DMC increased insulin secretion against glucotoxicity by simulating the effect of GLP-1and enhancing the expression of GLP-1R, followed by activating the signal pathway of PDX-1, PRE-INS, and GLUT2-GCK. Another mechanism was that DMC avoided the pancreatic islets dysfunction of cellular damage by suppressing the production of nitric oxide (NO) by iNOS, and the expression of MCP-1. The results indicated the potential application of DMC in the intervention against glucotoxicity-induced hyperglycaemia.
DMC (10μM) treatment remarkably promoted glucose uptake in differentiated3T3-L1adipocytes(P<0.05vs.control group), and at concentration of20μM, DMC did not show-any effect in adipocytes; while, the glucose uptake in L6myoblasts and glycogen synthesis in HepG2hepatocytes were not affected by the treatment. DMC had paradoxical effect on lipid accumulation in3T3-L1cells compared with differentiation control. High concentration DMC (10and20μM) markedly diminished lipid accumulation; however, low concentration of DMC (2.5μM) enhanced lipid storage in3T3-L1cells (P<0.01vs. differentiation control group), and5μM DMC did not impose significant effect. It was demonstrated that the effect of DMC in lipid accumulation was controlled by the expression of PPAR-y. Such effects highlight therapeutic potential of DMC in the management of hyperglycemia.
引文
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