己酮可可碱联合霉酚酸酯对高糖诱导人肾小球系膜细胞单核细胞趋化蛋白-1和纤维连接蛋白表达的影响

摘要

Objective To investigate the effect of high glucose on expression of MCP-1 and fibronectin in human mesangial cells and the intervention of pentoxifylline combined mycophenolate.Methods Cultured human mesangial cells were divided into eight groups:normal control group,high glucose group (HG,30 mmol/L glucose),mannitol osmotic pressure control group (Man,5 mmol/L glucose + 25 mmol/L mannitol),high glucose + pentoxifylline-0.03 group (P1 30 mmol/L glucose + 0.03 mg/mL PTX),high glucose + pentoxifylline-0.3 group (P2 30 mmol/L glucose + 0.3 mg/mL PTX),high glucose +MMF-10 group (M1,30 mmol/L glucose + 10 μg/mL MMF),high glucose+MMF-100 group (M2,30 mmol/L glucose + 100 μg/mL MMF); high glucose +PTX+MMF group (P+M,30 mmol/L glucose + 0.3 mg/mL PTX 100 μg/mL MMF).The mRNA and protein of MCP-1 were measured by RT-PCR and ELISA,and fibronectin in supernatant was detected by ELISA at 24,48,72 h.Results The mRNA and protein expression of MCP-1 and fibronectin increased significantly in high glucose (P ＜0.01).However,both pentoxifylline and mycophenolate inhibited the expression (P ＜ 0.01).The different suppression degree of MCP-1 and fibronectin depended on different concentration of mycophenolate and action time,submitting dosage and time dependence (P ＜ 0.05).While pentoxifylline groups were not.The combination of pentoxifylline and mycophenolate group was associated with a superior suppression in high glucosed-induced overexpression of MCP-1 mRNA,protein and fibronectin compared to pentoxifylline group at any time,also the combination had a superior suppression than mycophenolate (24 h,P ＜ 0.05),while as long as the time being,the difference between them was not significant:Conclusion Pentoxifylline and mycophenolate may through depressing the expression of MCP-1 and fibronectin delay the progression of glomerular sclerosis and interstitial fibrosis in DN.The combination group confered superiority over monotherapy groups,a mechanism which may be at least partly correlated with synergistic of improving hemodynamic and anti-inflammatory.%目的 探讨高糖对人肾小球系膜细胞(human mesangial cells,HMCs)单核细胞趋化蛋白-1 (monocyte chemoattractant protein-1,MCP-1)及细胞外基质的主要成分(extracellular matrix,ECM)纤维连接蛋白(fibronectin,FN)表达的影响及己酮可可碱(pentoxifylline,PTX)联合霉酚酸酯(mycophenolate,MMF)对上述指标的干预作用.方法 将培养的HMCs分为8组:正常对照组(NG,5 mmol/L葡萄糖);高糖组(HG,30 mmol/L葡萄糖);甘露醇渗透压对照组(Man,5 mmol/L葡萄糖+25 mmol/L甘露醇);高糖+PTX-0.03组(P1,30 mmol/L葡萄糖+0.03 mg/mL PTX);高糖+PTX-0.3组(P2,30 mmol/L葡萄糖+0.3 mg/mL PTX);高糖+MMF-10组(M1,30 mmol/L葡萄糖+10 μg/mL MMF);高糖+MMF-100组(M2,30 mmol/L葡萄糖+100 μg/mL MMF);高糖+PTX+MMF组(P+M,30 mmol/L葡萄糖+0.3 mg/mL PTX+100 μg/mL MMF),分别在24、48、72 h采用RT-PCR法和ELISA法检测PTX、MMF及PTX+MMF对高糖条件下MCs内MCP-1 mRNA及蛋白、FN表达的影响.结果 高糖组HMCs MCP-1 mRNA、蛋白的表达及FN的分泌较正常对照组显著增加(P＜0.01),且48 h表达最高;不同浓度的PTX、MMF均能下调MCP-1 mRNA、蛋白及FN的表达(P＜0.01);不同浓度的MMF对MCP-1 mRNA、蛋白的表达及FN分泌的抑制程度不同,呈时间剂量依赖性(P＜0.05);不同浓度的PTX则随着时间的延长对其抑制作用无明显差别;PTX联合MMF组比单独PTX各组在各时间点的抑制程度更明显(P＜0.01),比单独MMF各组在各时间点抑制作用增强,(24 h,P＜0.05),但随着时间延长,差异无统计学意义.结论 PTX及MMF可能通过阻抑MCP-1的表达及FN的分泌延缓糖尿病肾病(diabetic nephropathy,DN)中肾小球硬化及间质纤维化的进展,联合给药组作用优于单药,可能从改善血流动力及抗炎角度解释了PTX联合MMF对DN肾脏的协同保护作用.