荞麦花总黄酮对体内外蛋白质非酶糖基化的抑制作用(英文)

Inhibitory effects of total flavones of buckwheat flower on the non-enzymatic glycation of proteins in vivo and in vitro

背景:蛋白质非酶糖基化与糖尿病并发症相关。前期实验表明荞麦花总黄酮能改善糖耐量,此作用是否与荞麦花总黄酮抑制蛋白质的非酶糖基化有关尚不清除。目的:观察荞麦花总黄酮对蛋白质非酶糖基化终产物形成的影响。设计:观察对比实验。地点:华北煤炭医学院药理学教研室。材料:选用75只成年SD大鼠,清洁级,雌性大鼠38只,雄性大鼠37只,体质量(200±20)g,由北京中国医学科学院实验动物研究所提供(合格证号:SCXK11-00-0006)。荞麦花总黄酮自制,血糖试剂盒(北京中生北控生物科技股份有限公司),青霉素(批号031020,80万单位,购自华北制药股份有限公司),链霉素(华北制药股份有限公司,批号030920,100万单位),链脲佐菌素(Sigma公司),果糖胺试剂(南京建成生物工程研究所),牛血清白蛋白(BSA)(购自Sigma公司),其他试剂为国产分析纯。方法:实验于2004-03/2004-10在华北煤炭医学院药理学教研室完成。实验1,体外大分子糖基化终产物测定:①造模和分组:将大鼠按体质量分为3组:正常对照组15只:腹腔注射生理盐水8mL/kg。治疗组45只:大鼠禁食16h后,腹腔注射链脲佐菌素80mg/kg,8mL/kg,72h后取尾静脉血测血糖,凡空腹血糖≥15mmol/L作为糖尿病大鼠,摸球法将治疗组大鼠分为3个亚组,每组15只,分别灌胃给予0.1,0.2,0.4g/kg荞麦花总黄酮。模型组15只:仅将大鼠制作成糖尿病模型,腹腔注射链脲佐菌素80mg/kg,8mL/kg。正常对照组和模型组大鼠用等容积常用水灌胃,上述各组灌胃均为1次/d,连续12周。②空腹血糖的测定:治疗组大鼠末次给药后禁食12h,取尾静脉血,采用葡萄糖氧化酶法测其空腹血糖,其他组别大鼠在相应时间点给予相应测量作为对照。③血浆及肾组织果糖胺及大分子糖基化终产物含量检测:各组大鼠乙醚麻醉后颈动脉取血,分离血浆,同时取肾脏,制成质量分数为100g/L肾脏组织匀浆,低温离心。按试剂盒要求测定血浆及肾匀浆上清果糖胺含量,用荧光分光光度计测定血浆及肾组织匀浆上清荧光强度,观察样品的大分子糖基化终产物生成量。实验2,体外大分子糖基化终产物测定:配置葡萄糖含量为0.2mol/L、牛血清白蛋白含量20g/L的磷酸盐缓冲液,加入青霉素20万U/L,链霉素20万U/L得A液。用A液配制终浓度为0.01,0.05,0.10mg/L的荞麦花总黄酮混悬液各60mL,将不加药物完整的非酶糖基化系统、不加药物不含糖的系统、加药物不含白蛋白的系统和加药物不含糖的系统设为对照,每1样品作5个平行管,作为体外糖基化的培养液,将配好的蛋白糖基化培养液放入37℃的恒温箱内培养孵化,于第4,8,12周分别无菌取部分培养液,按上述方法测定大分子糖基化终产物的荧光值,并计算抑制率,观察荞麦花总黄酮对体外大分子糖基化终产物的抑制情况。主要观察指标:实验1中大鼠空腹血糖、血浆及肾组织果糖胺及大分子糖基化终产物含量检测结果;实验2中荞麦花总黄酮对体外大分子糖基化终产物的抑制情况。结果:实验1:纳入大鼠75只,56只造模成功并进入结果分析。模型组大鼠血糖值、血浆及肾脏果糖胺均明显高于正常对照组(t=7.572,10.186,5.794,P<0.01)。荞麦花总黄酮3个剂量治疗组血糖值均明显低于模型组(t=3.357,4.382,3.938,P<0.05～0.01),荞麦花总黄酮0.1g/kg组血浆及肾脏果糖胺与模型组相近(P>0.05),荞麦花总黄酮0.2,0.4g/kg组血浆及肾脏果糖胺均明显低于模型组(t=5.109,4.605,3.731,3.097,P<0.05～0.01)。模型组大鼠血浆及肾脏大分子糖基化终产物生成量均明显高于正常对照组(t=6.463,12.704,P>0.05),而治疗组大鼠血浆大分子糖基化终产物含量与模型对照组相近(P<0.01)。治疗组各亚组肾脏大分子糖基化终产物含量均明显低于模型组(t=9.845,12.799,12.899,P<0.01)。实验2:随时间的推移,各组大分子糖基化终产物的产生均逐渐增加,而各组荞麦花总黄酮均能不同程度的抑制大分子糖基化终产物的产生,并有明显的量效关系,呈现出剂量和时间依赖性。结论:荞麦花总黄酮能明显抑制在体内外蛋白质非酶糖基化终产物的形成。

BACKGROUND： Non-enzymatic glycation of proteins is involved in the complications of diabetes mellitus. Previous experiments have demonstrated that total flavones of buckwheat flower （TFBF） could improye carbohydrate tolerance. However, it is little known whether TFBF inhibit the non-enzymatic glycation of proteins. OBJECTIVE： To investigate the influences of TFBF on the non-enzymatic advanced glycation end products （AGEs） of proteins in vivo and in vitro. DESIGN： Completely randomized controlled trial. SETTING： Department of Pharmacology, North China Coal Medical College. MATERIALS： Totally 75 adult SD rats , of clean grade, weighing （200±20） g, including 38 female rats and 37 male rats, were provided by the institute of experimental animals, Chinese Academy of Medical Sciences （Certification No.SCXK11-00-0006）. TFBF was extracted by our laboratory from flowers of buckwheat. The blood glucose kit was purchased from Beijing Biosino Biotechnology Company Ltd. Penicillin （Batch No. 031020, 8×10^5 U） and streptomycin （Batch No. 030920, 1×10^6 U） were purchased from North China Pharmaceutical Company. Streptozotocin and BSA were purchased from Sigma Company. Fructosamine kit was purchased from Nanjing Jiancheng Bioengineering Institute, and the other chemicals were analytical pure produced domestically. METHODS： This experiment was carried out in the Department of Pharmacology, North China Coal Medical College from March to October 2004.In the first experiment, in vivo macromolecular AGEs was measured： （1） Modeling and grouping： Rats were divided into 3 groups according to body mass： Normal control group （n=15）, the rats were treated with 8 mL/kg normal saline intraperitoneally. Streptozotocin-treated group （n=45）, the rats were fasted for 16 hours and then treated with 80 mg/kg streptozotocin of 8 mL/kg intraperitoneally. Twenty-two hours later, the blood of all rats was harvested from vena caudalis to measure the level of blood sugar. Those with fasting blood glucose ≥15 mmol/L were acted as diabetic rats. Streptozotocin-treated group were divided into 3 subgroups, 15 rats in each subgroups. Each rat was given intragastric administration of 0.1, 0.2 and 0.4 g/kg TFBF. Model group （n=15）： Rats were only treated with 80 mg/kg streptozotocin of 8 mL/kg . The rats in normal control group and model group were given the same volume of salt water. The administration was once a day for 12 weeks successively. （2）Measurement of fasting blood glucose： After the last administration, the rats of streptozotocin-treated group were fasted for 12 hours and the blood was harvested from vena caudalis. The fasting blood glucose was measured by glucose oxidase method. （3）The levels of blood plasma and nephridial tissue fructosamine and maeromolecular AGEs were measured： The rats of each group were anesthetized with ethyl ether on the second day following the last administration. Blood was chosen from carotid artery, and plasma was separated. Kidneys were taken at the same time, prepared into 100 g/L tissue homogenate and centrifuged at low temperature. The levels of fructosamine in plasma and the supernatant fluid of kidney homogenate were measured according to the instructions of the kit. AGEs in plasma and renal tissue were determined by fluorospectrophotometer. The products of macromolecular AGEs were calculated, in the second experiment, in vitro macromolecular AGEs were measured as below： 0.01, 0.05, 0.10 mg/L TFBF of 6 mL respectively was prepared with solution A （0.2 mol/L glucose, 2×10^6 U/L penicillin, 2×10^6 U/L streptomycin , PBS containing 20 g/L bovine serum albumin）. Control groups were set： （1） without TFBF, （2） without TFBF and glucose, （3） without BSA, （4） without glucose. Five parallels of each sample were sterilized by filtration and incubated in the attemperator at 37℃. The fluorescence of AGEs （F） in the culture was determined at the 4^th, 8^th and 12^th weeks. Inhibition ratio （IR） was calculated and the inhibition of TFBF on AGEs was observed. MAIN OUTCOME MEASURES： In the first experiment, the levels of fasting blood glucose, fructosamine in kidney and plasma, and AGEs were measured. In the second experiment, the inhibition of TFBF on AGEs in vitro was measured. RESULTS： In the first experiment, 75 rats were involved, and 56 successful rats entered the stage of result analysis. The levels of blood glucose, froctosamine in kidney and plasma of rats in the model group were significantly higher than those of rats in the normal control group （t =7.572, 10.186, 5.794,P 〈 0.01 ）. The level of blood glucose of rats in the 3 subgroups was significantly lower than that of rats in the model group （t =3.357,4.382,3.938,P 〈 0.05-0.01 ）; The levels of fructosamine in kidney and plasma of rats in the 0.2 and 0.4 g/kg TFBF groups were significantly lower than those in the model group （t=5.109, 4.605, 3.731,3.097, P 〈 0.05-0.01 ）. The levels of AGEs in plasma and kidney of rats in the model group were significantly higher than those in the normal control group （t=6.463, 12.704,P 〈 0.01 ）, while the levels of AGEs in plasma of rats in the streptozotocin-treated group were similar to those in the model control group （P 〉 0.05）, and those in kidney of rats in the streptozotocintreated subgroups were significantly lower than those in the model group （t=9.845, 12.799, 12.899,P 〈 0.01）. In the second experiment, the level of macromolecular AGEs of each group was gradually increased with time. TFBF could inhibit the formation of macromolecular AGEs in dose- and time-dependent manner. CONCLUSION： TFBF ohviously inhibited the formation of AGEs of proteins in vivo and in vitro.