摘要
[目的]传统中医即以灵芝为重要配伍组份用于内分泌、代谢紊乱相关疾病的治疗。本文以蛋白酪氨酸磷酸酶1B(PTP1B)为靶点从灵芝子实体中筛选具有有效降血糖效果的PTP1B抑制剂,并对该抑制剂进行理化性质及降血糖机理的研究。
[方法]利用醇提、水提和碱提等手段结合醇沉、凝胶柱层析等技术从灵芝子实体中筛选有效降血糖组分；利用高效液相色谱,红外光谱,核磁共振光谱对提取物进行纯度和组成表征；利用分析单糖组成、氨基酸组成、多糖含量、蛋白含量等,建立这些参数与对PTP1B抑制能力的相关性,并研究对PTP1B抑制的动力学；利用链脲霉素(STZ)诱导2型糖尿病小鼠及大鼠,db/db基因型2型糖尿病小鼠对有效降糖提取物进行药效学研究,并与现用临床一线西药二甲双胍及罗格列酮相对照;同时进行了有效提取物的初步急性安全性试验；利用western blotting技术,免疫沉淀技术分析STZ致糖尿病大鼠及基因糖尿病小鼠给药前后肝和骨骼肌组织中PTP1B表达水平和活性的变化及胰岛素受体p亚基(IRβ)磷酸化水平的变化,从而分析有效降糖提取物在体内的降血糖机理；另外,用酶标仪、荧光光谱等研究提取物对a-葡萄糖苷酶、a-淀粉酶、自由基等抑制能力和动力学；此外,用试剂盒分析与糖尿病有关的代谢性疾病的体内血液生化指标。
[结果]从灵芝中筛选出7种对PTP1B有抑制效果的组分,其中抑制最有效的组分IC50=5.12±0.05μm/mL,将该组分命名为FYGL;对FYGL组成分析表明,FYGL为分子量2.6×105的蛋白多糖,其中蛋白与多糖含量比为17：77。多糖部分主要由葡聚糖组成,蛋白部分含有19种常见氨基酸；对PTP1B抑制动力学分析表明,FYGL对PTP1B的抑制呈竞争性抑制,即与PTP1B的底物竞争相同的活性位点。动物药效学试验证明,FYGL能显著降低STZ致2型糖尿病模型小鼠及大鼠的血糖,并呈剂量和时间依赖性,且与二甲双胍及罗格列酮相当；同样,db/db基因型糖尿病小鼠药效学实验证明,FYGL亦显著降低小鼠血糖,且与临床一线西药相当；初步急性安全性试验表明,小鼠半致死量LD50=6g/kg,安全性很高；药理学试验证明,对于STZ致糖尿病大鼠,FYGL能降低骨骼肌PTP1B的表达水平和活性,进而调控IRp亚基的磷酸化水平；对于db/db基因型糖尿病小鼠,FYGL能显著降低肝脏和骨骼肌PTP1B的表达水平,降低骨骼肌PTP1B的活性,增加肝脏和骨骼肌IRp亚基的磷酸化水平。此外,STZ致糖尿病大鼠试验表明,FYGL不仅能显著降低血糖,而且能增加血清胰岛素浓度,降低胰岛素抵抗指数,增加胰岛素分泌指数,提示FYGL可能有修复胰岛细胞的功能；FYGL亦能降低db/db小鼠胰岛素抵抗指数,但不能促进胰岛素的分泌。另外,FYGL对a-葡萄糖苷酶、α-淀粉酶、自由基等亦有抑制能力,对α-葡萄糖苷酶亦呈竞争性抑制机理,提示FYGL可能具有多靶点降糖作用功能。同时,FYGL能有效改善STZ致2型糖尿病大鼠及db/db小鼠中与代谢紊乱综合症相关的血脂生化指标,如自由脂肪酸(FFA)、甘油三脂(TG)、总胆固醇(TC),低密度脂蛋白胆固醇(LDL-C)和高密度脂蛋白胆固醇(HDL-C)。
[结论]FYGL有显著降低血糖能力,与临床一线西药二甲双胍及罗格列酮相当,且安全性高,其降血糖机理是通过降低体内肝脏和骨骼肌PTP1B表达水平,降低骨骼肌PTP1B活性,进而调控胰岛素受体p亚基的磷酸化水平,同时降低胰岛素抵抗指数。另外,FYGL可能具有多靶点降糖作用功能,同时改善与糖尿病代谢紊乱综合症相关的血脂生化指标。FYGL是一种新型胰岛素增敏剂,有望成为糖尿病治疗的候选新药。
Objectives:
Ganoderma lucidum has traditionally been used for the treatment of endocrine-related diseases, metabolic disorders and diabetes. This work aimed to study the antihyperglycemic mechanism in vivo of a Ganoderma Lucidum extraction, FYGL, screened from the fruiting bodies of Ganoderma Lucidum (Fr.) Karst.
Methods:
PTP1B inhibitors were screened from the fruiting bodies of G. lucidum. The chemical components of the PTP1B inhibitor were characterized by the Infrared (IR) and Nuclear Magnetic Resonance (NMR) spectrum for the functional groups, anion-exchange chromatography for the monosaccharide contents, and the amino acid analyzer for the amino acid residues. The db/db mice and streptozotocin (STZ)-induced type2diabetic mellitus (T2DM) model mice and rats were treated with FYGL as well as metformin and rosiglitazone. The levels of plasma glucose and insulin were measured, and the expression and activity of the protein tyrosine phosphatase1B (PTP1B) and the tyrosine phosphorylation level of the insulin receptor (IR) β-subunit in the livers and skeletal muscles of the T2DM rats and db/db mice were analyzed by immunoprecipitation and Western blotting methods. In addition, the inhibity ability to a-glucosidase, a-amylase and free radical of FYGL was also investigated in vitro. At last, the levels of free fatty acid and serum lipid profile including triglyceride, total cholesterol, low density lipoprotein-cholesterol and high density lipoprotein-cholesterol were measured using commercial kits for those trailed rats.
Results:
A PTP1B activity inhibitor was screened from the fruiting bodies of G. lucidum. The inhibitor, named FYGL, has an efficient PTP1B inhibitory potency with IC50value of5.12±0.05μg/mL in a competitive inhibition kinetics mechanism. It was demonstrated that FYGL is a water soluble proteoglycan with protein to polysaccharide ratio of17:77, and a viscosity-average molecular weight of2.6×105.
Treatment for the STZ-induced type2diabetes mice, rats and db/db mice with a dose of150,120and225mg/kg FYGL for4weeks,30days and4weeks significantly decreased the fasting plasma glucose level by40.7%(p<0.01),31.8%(p<0.01) and29.1%(p<0.01), respectively, compared to the diabetic control group without drug treatment. The decrease in the plasma glucose concentration for the FYGL treatment group is comparable with that for the250mg/kg metformin and3mg/kg rosiglitazone treatment group. The FYGL does lead to the dose-and time-dependent decrease in the fasting plasma glucose. FYGL could decrease the insulin insistant of T2DM rats and db/db mice and promote the insulin release of T2DM rats. During the toxicity trial, the toxicity of FYGL was preliminarily evaluated as LD50=6g/kg with95%confidence limits of4.8-7.4g/kg.
FYGL decreased the PTP1B expression and activity in the skeletal muscles of T2DM rats, but not in the livers of T2DM rats, while decreased the PTP1B expression in the liver and skeletal muscles and decreased the PTP1B activity in the skeletal muscles of db/db mice, consequently, increased the tyrosine phosphorylation level of the IR β-subunit (p<0.05) in the skeletal muscles of T2DM rats and the tyrosine phosphorylation level of the IR p-subunit in the liver and skeletal muscles of db/db mice.
In addition, FYGL can inhibite the activity of a-glucosidase, a-amylase and free radical in vitro, which suggesting that FYGL has the multiple target point function.
At last, FYGL significantly decreased the levels of free fatty acid, triglyceride, total cholesterol and low density lipoprotein-cholesterol as well as increased the level of high density lipoprotein-cholesterol, which are the important plasma biochemistry indexes.
Conclusions:
FYGL decreases the plasma glucose level based on the mechanism of the inhibiting the PTP1B expression and activity, consequently, mediating the tyrosine phosphorylation level of the IR β-subunit; meanwhile it decrease the serum insulin insistant and maybe recover the pancreatic β-cells function. As those results, FYGL can also control the plasma biochemistry indexes relative to the type2diabetes with metabolic disorders. Therefore, FYGL is promising to be used as a drug candidate for the type2diabetes and their metabolic disorders.
引文
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