中药复方制剂消可宁对高糖诱导大鼠肾小球系膜细胞增殖的影响

Compound Chinese medicine Xiaokening in the proliferation of rat mesangial cells induced by high glucose

目的:观察中药复方制剂消可宁对高糖培养的大鼠肾小球系膜细胞增殖的抑制作用。方法:实验于2004-04/11在南京医科大学第一附属医院内分泌代谢研究中心细胞培养研究室完成。①中药复方制剂消可宁(由制大黄、制附子、生黄芪组成,南京军区南京总医院制剂科生产,药品批号:院临(2003)第01017号,规格为90mL/袋)。大鼠系膜细胞株HBZY-1购自武汉大学中国典型培养物保藏中心。②将购入的系膜细胞株消化培养,取培养4～8代的细胞,吸去培养瓶中的上清液,消化离心后混匀,锥虫蓝染色观察细胞活性。③不同刺激时间系膜细胞增殖实验设立3组,正常糖浓度组每孔加入5.6mmol/L葡萄糖溶液200μL,高糖对照组每孔加入30mmol/L葡萄糖溶液200μL,高糖+消可宁组每孔加入30mmol/L葡萄糖与0.06g/mL消可宁混合液200μL,6孔/组。各组分别于培养24,48,72h后向板孔中加5g/L的4-甲基偶氮四唑蓝20μL,于酶标仪492nm波长处检测吸光度值。④不同浓度消可宁刺激高糖环境下系膜细胞增殖实验设立7组,高糖对照组每孔加入30mmol/L葡萄糖溶液200μL,消可宁20,40,60,80,120,200g/L组每孔加入30mmol/L葡萄糖+对应浓度的消可宁混合液200μL,6孔/组。培养72h后各组向板孔中加5g/L的4-甲基偶氮四唑蓝20μL,同法检测吸光度值。结果:①系膜细胞活性观察:原代培养的4～8代系膜细胞,经锥虫蓝染色,着色的死亡细胞数<5%,未着色活细胞数>95%。②不同刺激时间各组系膜细胞增殖情况的比较:与正常糖浓度组比较,高糖对照组于高糖刺激24h即可促进系膜细胞的增殖,且这种作用在刺激48h时最为明显,至72h细胞数量开始减少,但仍明显高于正常糖浓度组(0.527±0.047,0.659±0.018,P<0.05)。与高糖对照组比较,高糖+消可宁组在24h内并未表现出抑制系膜细胞增殖的作用,而刺激48h后显示出抑制趋势,但差异无显著性意义,于72h后表现出较强的抑制系膜细胞增殖的效应(0.659±0.018,0.538±0.023,P<0.05)。③不同浓度消可宁刺激对高糖环境下系膜细胞增殖的影响:刺激72h后与高糖对照组比较,20,40g/L的消可宁能够部分阻止系膜细胞的增殖;60,80g/L的消可宁则表现出明显阻止高糖对系膜细胞的过度刺激作用,且随着浓度的增加,抑制作用逐渐增强;浓度升至120g/L时出现细胞脱落死亡,高达200g/L时则不见存活的细胞。结论:消可宁能够有效抑制高糖诱导的系膜细胞增殖,可能是其早期防治糖尿病肾病的细胞学基础。

AIM： To observe the effect of compound Chinese medicine Xiaokening in suppressing the proliferation of mesangial cells in rats cultured with high glucose. METHODS： The experiment was conducted in Cultural Department of Cells, Center for Endocdnology and Metabolism, First Hospital Affiliated to Nanjing Medical University between Apdl and November 2004. （1） Xiaokening （made from Dahuang, Zhifuzi and Shenghuangqi, and was prepared by the Department of Pharmaceutic,s, General Hospital of Chinese PLA of Nanjing Military Area＇with the batch number of （2003） 01017, 90 mL/pack. （2） Intercapillary cell strains were cultured to obtain the cells from the 4^th to the 8^th passage, and the supematant in cultural bottles was gotten dd off, and samples were mixed after digestion and centdfugation. Trypan blue stain was performed to study the activity of cells. （3） Intercapillary cells stimulated at different times were divided into 3 groups with 6 pores in each group. Normal glucose group was added with 200 μL of glucose solution （5.6 mmol/L for each pore）, high-concentration glucose groupwas added with 200μL glucose solution （30 mmol/L for each pore）, and high-glucose and Xiaokening group was added with 200 p,L mixed solution （30 mmol/L glucose and 0.06 g/mL of Xiaokening for each pore）, Samples in 3 groups were added with 20 p,L of MTT （5 g/L） respectively at 24, 48 and 72 hours after culture. And the absorbance at 492 nm of meter tech. （4） There were 7 groups for proliferation test of intercapillary cells under high-glucose stimulated by different concentrations of Xiaokening. 30 mmol/L glucose solution was added into each pore of high-glucose control group, while 20, 40, 60, 80, 120 and 200 g/L Xiaokening groups were added with 200 μL mixed solution of 30 mmol/L glucose and Xiaokening of corresponding dose with 6 pores in each group. 72 hours after the culture, 20 μL of MTT （5g/L） was added into each group, and the absorbance was determined with the same method. RESULTS： （1） Observation on the activity of intercapillary cells： The apeptotic rate of stained cells from the 4^th to 8^th passage after trypan blue staining was below 5%, and that of unstained cells was greater thatn 95%. （2） Compadson in the proliferation of intercapillary cells stimulated at different time among all groups： Compared with normal glucose group, the proliferation can be promoted at 24 hours after high-glucose stimulation in high-glucose control group, moreover, the effect was the best at the 48^th hour, and cells began to decrease since the 72^th hour, which was still higher than that in normal glucose group （0.527±0.047,0.659±0.018,P 〈 0.05）. Compared with high-glucose control group, there was no inhibitory effect in the proliferation of intercapillary cells within 24 hours in high-glucose and Xiaokening group, while it worked at 48 hours later. However, there were no remarkable differences. 72 hours tater, the effect was the strongest （0.659±0.018,0.538±0.023,P 〈 0.05）. （3） Effects of Xiaokening of different concentrations op-the proliferation of intercapillary cells under high-glucose： Compared with high-glucose control group, 20 and 40 g/L Xiaokening could block part proliferation at 72 hours after the stimulation, and the effects were reinforced while at the dose of 60 and 80 g/L. With the concentration increasing, the inhibitory effect was gradually reinforced, and cell apeptosis was found at the concentration of 120g/L. No survival cell was＂ found at the concentration of 200 g/L. CONCLUSION： Xiaokening can effectively suppress the proliferation of intercapillary cells induced by high glucose, which might be the cytological basis of its early treatment of diabetic nephropathy.