二氢杨梅素对高糖诱导的肾小球系膜细胞增殖及纤维连接蛋白堆积的影响

摘要

OBJECTIVE:To study the effect of dihydromyricetin(DMY)on high glucose(HG)-induced glomerular mesangial cell(MCs)proliferation and fibronectin(FN)accumulation,and explore its mechanism for diabetic nephropathy glomerulosclero-sis. METHODS:Cells were divided into normal group(5.5 mmol/L glucose),HG group(30 mmol/L glucose),DMY low-concen-tration,medium-concentration,high-concentration groups (30 mmol/L glucose+22.5,45,90 μmol/L DMY). After incubating 48 h,MTT was used to detect the proliferative activity [reflected by the optical density(OD)value] of cells;molecular docking meth-od was adopted to conduct simulation analysis for DMY binding state with Smad2. Cells were divided into normal group(5.5 mmol/L glucose),HG group (30 mmol/L glucose),DMY group (30 mmol/L glucose+45 μmol/L DMY) and DMY control group (5.5 mmol/L glucose+45 μmol/L DMY). After incubating 5 h,Western blot was used to detect the expression levels of phosphorylated Smad2 (p-Smad2) and extracellular matrix protein FN. RESULTS:Results of MTT detection showed,compared with normal group,OD values in HG group were significantly increased(P<0.05);compared with HG group,OD values in DMY each con-centration group were significantly reduced(P<0.05). The Gibbs free energy(ΔG)of DMY and Smad2 protein was -5.64 kJ/mol, Ki was 73.53 μmol/L,and there were hydrogen bond donor and receptor binding in No. 465,464,461,458 amino acid residues. Results of Western blot showed,compared with normal group,expression levels of p-Smad2 and extracellular matrix protein FN in HG group were significantly increased (P<0.05);compared with HG group,expression levels of p-Smad2 and extracellular ma-trix protein FN in DMY group were significantly decreased(P<0.05). CONCLUSIONS:DMY inhibits HG-induced MCs prolifera-tion and improves diabetic nephropathy glomerulosclerosis by combining with Smad2 and inhibiting Smad2 phosphorylation to re-duce the extracellular matrix protein FN expression.%目的:研究二氢杨梅素(DMY)对高糖(HG)诱导的肾小球系膜细胞(MCs)的增殖及纤维连接蛋白(FN)堆积的影响,探讨其对糖尿病肾病肾小球硬化的作用机制.方法:将细胞分为正常组(5.5 mmol/L葡萄糖)、HG组(30 mmol/L葡萄糖)和DMY低、中、高浓度组(30 mmol/L葡萄糖+22.5、45、90μmol/L DMY),培养48 h后采用MTT法检测细胞的增殖活性[以光密度(OD)值反映];采用分子对接法对DMY与Smad2的结合状态进行模拟分析;将细胞分为正常组(5.5 mmol/L葡萄糖)、HG组(30 mmol/L葡萄糖)、DMY组(30 mmol/L葡萄糖+45μmol/L DMY)和DMY对照组(5.5 mmol/L葡萄糖+45μmol/L DMY),培养5 h后采用West-ern blot法检测细胞中磷酸化Smad2(p-Smad2)及细胞外基质蛋白FN的表达水平.结果:MTT检测结果显示,与正常组比较,HG组细胞的OD值显著升高(P<0.05);与HG组比较,DMY各浓度组细胞的OD值均显著降低(P<0.05).DMY与Smad2蛋白分子结合的吉布斯自由能(ΔG)为-5.64 kJ/mol,抑制常数Ki为73.53μmol/L,在第465、464、461、458这4个氨基酸残基位点有氢键供体与受体的结合.Western blot结果显示,与正常组比较,HG组细胞中p-Smad2及细胞外基质蛋白FN表达水平均显著升高(P<0.05);与HG组比较,DMY组细胞中p-Smad2及细胞外基质蛋白FN表达水平显著降低(P<0.05).结论:DMY可抑制HG诱导的MCs增殖,并通过与Smad2结合,抑制Smad2的磷酸化,继而降低细胞外基质蛋白FN的表达,从而改善糖尿病肾病肾小球硬化.