黄芪、大黄复方制剂对培养大鼠系膜细胞增殖和细胞外基质分泌的影响(英文)

Effect of compound preparation of huangqi and dahuang on proliferation and secretion of extracellular matrix in mesangial cells of cultured rats

背景:糖尿病肾病是糖尿病最严重的血管并发症之一。中药制剂黄芪、大黄组成复方在临床用于防治糖尿病肾病多年,具有确切的保护糖尿病患者肾脏的作用,但其作用机制需观察探讨。目的:观察以黄芪、大黄复方组成的中药制剂肾康丸对高糖培养大鼠系膜细胞增殖及细胞外基质分泌的影响。设计:随机对照观察。单位:南方医科大学珠江医院中西医结合肾病中心和南方医科大学南方医院药局。材料:血清药理学实验于2005-04在南方医科大学实验动物研究中心完成。细胞培养实验于2005-04/07在南方医科大学细胞培养室完成。选择健康雄性Wistar大鼠16只,体质量190～220g。方法:16只大鼠随机分为4组,正常血清组、开搏通组、肾康丸大小剂量组(肾康丸主要由黄芪、大黄、水蛭、芡实、玉米须等组成,由南方医科大学珠江医院药剂科生产,制剂号20031214)。①开搏通及肾康丸高低组分别按5mg/kg,2.4g/kg和1.2g/kg体质量灌服相应药物混悬液。正常血清组灌服等容积生理盐水。连续灌胃7d,麻醉,分离药物血清。②体外培养大鼠系膜细胞,用不同浓度葡萄糖(10,20,30和40mmol/L)分别刺激24,48,72和96h后,四甲基偶氮唑盐法测增殖。③培养的系膜细胞随机分为6组,低糖组(10mmol/L),高糖组(30mmol/L),正常血清组(30mmol/L葡萄糖),开搏通(50g/L开搏通药物血清)组(30mmol/L葡萄糖)及肾康丸高低高糖(30mmol/L葡萄糖)。培养72h后,采用四甲基偶氮唑盐法测定各组细胞增殖,酶联免疫法测系膜细胞分泌纤连蛋白水平,反转录-聚合酶链反应法测系膜细胞重纤连蛋白mRNA的表达。主要观察指标:①不同浓度葡萄糖对大鼠系膜细胞增殖的影响。②各组系膜细胞增殖及分泌纤连蛋白和纤连蛋白mRNA表达情况。结果:①不同浓度的葡萄糖对系膜细胞增殖的影响:与低糖组(10mmol/L)比较,20mmol/L葡萄糖在实验的96h里能促进系膜细胞增殖,但仅在72h和96h具有统计学意义(P<0.05)。30mmol/L葡萄糖在实验的24h至96h里均能显著促进系膜细胞增殖(P<0.05或P<0.01),72h内随着作用时间的延长,促进增殖作用越明显,72h后作用减弱,但仍有统计学意义;40mmol/L浓度的葡萄糖在48h内对系膜细胞增殖有促进作用(P<0.05),48h后作用减弱,甚至转为抑制。②药物血清对系膜细胞增殖的影响:高糖组吸光度值明显高于低糖组(P<0.01)。与高糖组比较,开搏通、肾康丸高低剂量组吸光度值均明显降低(P<0.01或P<0.05)。正常血清组与高糖组比较差异无显著性(P>0.05)。③药物血清对系膜细胞分泌纤连蛋白的影响:高糖组细胞上清中纤连蛋白含量显著高于低糖组(P<0.01)。与高糖组比较,开搏通及肾康丸高低剂量组纤连蛋白含量均显著降低(P<0.01或P<0.05),而正常血清组纤连蛋白含量变化无显著性差异(P>0.05)。④药物血清对系膜细胞中纤连蛋白mRNA表达的影响:高糖组纤连蛋白条带明显比低糖组明亮,且其与β-actin条带吸光度比值明显升高(P<0.01)。与高糖组比较,开搏通及肾康丸高低剂量组纤连蛋白条带亮度显著降低,与β-actin条带吸光度比值也显著减少(P<0.01),而正常血清组上述变化无显著性差异(P>0.05)。结论:高糖可以促进系膜细胞增殖,增加系膜细胞分泌纤连蛋白,提高系膜细胞中纤连蛋白mRNA的表达。肾康丸能明显抑制上述作用。肾康丸可以抑制高糖诱导的系膜细胞增殖及细胞外基质分泌。

BACKGROUND： Diabetic nephropathy is one of the most serious vascu- lar complications of diabetes mellitus. Compound preparation of huangqi and dahuang, a traditional Chinese medicine, has been used to preventing or treating diabetic nephropathy for several years, and has a certain protective effect on the kidney of diabetes mellitus patients. But its exact mechanism remains unknown and needs to be studied more. OBJECTIVE： To investigate the effect of compound preparation shenkang wan on the proliferation and secretion of extracellular matrix in cultured rat mesangial cells induced by high glucose. DESIGN： Randomized and controlled study. SETTING： Center of Integrated Traditional and Western Nephrology of Zhujiang Hospital and Medicine Department of Nanfang Hospital, Southern Medical University. MATERIALS： The serum pharmacological experiment was performed in Animal Experimental Center of Southern Medical University in April 2005. The cell culture dxperiment was conducted in Cell culture room of Southern Medical University from April 2005 to July 2005. Totally 16 normal Wistar male rats, weighted varied from 190 g to 220 g, were used in the study. METHODS： Sixteen normal Wistar male rats were randomly divided into 4 groups： normal serum group, capoten group, shenkang wan group （high dose and low dose）; shenkang wan was mainly constituted of huangqi, dahuang, leech, gordon guryale seed and corn stigma and made in Pharmacy Department of Zhujiang Hospital of Nanfang Medical University, agent number. 20031214）.① The rats in capoten group and high and low dose shenkang wan group were given the corresponding drugs respectively according to 5 mg/kg, 2.4 g/kg, 1.2 g/kg weight. The rats in normal serum group were given the same volume water. After treated 7 days, all rats were hocused and separated medication serum.② Mesangial cell was cultured in vitro with different concentrations of glucose （10, 20, 30 and 40 mmol/L）. The proliferation of mesangial cell was observed with the methyl-thiazoltelrazolium colorimetric assay at 23,, 48, 72 hours and 96 hours. ③ Then the cultured mesangial cells were divided into six subgroups ：Low glucose control group （10 mmol/L glucose）, high glucose group （30 mmol/L glucose）; normal serum group （30 mmol/L glucose）; capoten group （30 mmol/L glucose）; shenkang wan group （high dose and low dose, 30 mmol/L glucose）. After cultured 72 hours, the proliferation of mesangial cell was detected with the methyl-thiazol-/elrazolium colorime/ric assay, the secretion and mRNA gene expression of fibronetin levels in mesangial cell were respectively detected by enzyme linked immunosorbent assay （ELISA） and reverse iranscription-polymerase chain reaction （RT-PCR） method. MAIN OUTCOME MEASURES： ①Proliferation of mesangial cell induced by different concentrations glucose. ② Proliferation and secretion and mRNA gone expression of fibronectin in every group. RUSULTS： ① Effect of different concentrations glucose on the proliferation of mesangial cell： Compared with low concentrations glucose （10 mmol/L）, 20 mmol/L glucose could accelerate the proliferation of mesangial cell during 96 hours experiment period, but only had a statistically significant difference at 72 and 96 hours （P 〈 0.05）. 30 mmol/L glucose could significantly accelerate the proliferation of mesangial cell than that of 10 mmol/L glucose from 24 hours to 96 hours （P〈 0.05 or P〈 0.01）, and this effect was increasing with time in 72 hours and reduced after 72 hours. 40 mmol/L glucose could significantly increase the proliferation of mesangial cell than of low concentrations glucose in 48 hours （P 〈 0.05）, and this effect was reduced after 48 hours and even conversed to restrain effect. ② Effect of different medication serum on the proliferation of mesangial cell： The optical density value in high glucose group is obviously higher than that of low glucose control group （P 〈 0.01）. Compared with high glucose group, the optical density value in capoten, shenkang wan group （high dose and low dose） was decreased markedly （P 〈 0.01 or P 〈 0.05）. While the optical density value in normal serum group was showed no difference with the high glucose group （P 〉 0.05）. ③ Effect of different medication serum on secretion of fibronectin in mesang/al cell： Content of fibroneetin in high glucose group was increased more markedly than that of low glucose group （P 〈 0.01）. Compared with high glucose group, content of fibronectin in capoten and shenkang wan group （high dose and low dose） was showed a significantly decrease （P 〈 0.01 or P 〈 0.05）, while content of fibronectin in normal serum gronp was showed no difference with the high glucose group （P〉 0.05）. ④ Effect of different medication serum on expression of fibronectin mRNA in mesangial cell： The optical density value of fibronectin strip in high glucose group was brighter than that in low glucose group and the ratio of it and β-actin were increased markedly too （P 〈 0.01）. Compared with high glucose group, the optical density value of fibronectin strip in capoten and shenkang wan group （high dose and low dose） was showed a significantly decrease and the ratio of it and β-actin was reduced distinctly too （P 〈 0.01）, while the ratio of it and β-actin in normal serum group was showed no difference （P 〉 0.05）. CONCLUSION： High glucose could accelerate proliferation, increase the secretion and mRNA gene expression of fibronectin in mesangial cell, while shenkang wan could inhibit proliferation and secretion of the extracellular matrix in mesangial cell induced by high glucose.