柚皮素抑制DN系膜细胞炎症因子表达的分子机制研究

Molecular mechanism of naringenin inhibiting the expressions of inflammatory factors of mesangial cells in diabetic nephropathy

摘要:

目的：研究柚皮素（naringenin，NAR）对糖尿病肾病（diabetic nephropathy，DN）小鼠病情影响和肾小球系膜细胞炎症因子肿瘤坏死因子-α（tumor necrosis factor alpha，TNF-α）和白细胞介素-6（interleukin-6，IL-6）表达的作用。方法：将10只雄性db/db DN小鼠随机分成柚皮素组和DN组，每组各5只；5只雄性db/m正常对照小鼠为正常组进行实验。其中柚皮素组小鼠给予50 mg/（kg×d）柚皮素连续灌胃2周处理，DN组小鼠给予等量生理盐水连续灌胃2周处理，正常组小鼠给予正常饮食。检测体质量、血糖、尿白蛋白排泄率（urinary albumin excretion，UAE）等一般指标；HE染色和电镜检测小鼠肾脏组织肾小球形态学改变。将小鼠肾小球系膜细胞给予不同葡萄糖浓度（5 mmol/L和25 mmol/L）的DMEM培养基培养。其中5 mmol/L葡萄糖浓度培养的细胞为正常组，25 mmol/L葡萄糖浓度培养的细胞为高糖组，25 mmol/L葡萄糖浓度培养基中加入柚皮素400 μmol/L培养的细胞为柚皮素组。ELISA法检测炎症因子TNF-α和IL-6表达；Western blot检测细胞质和细胞核的NF-κB p65蛋白表达情况。结果：DN小鼠给予柚皮素后，UAE[（51.48±8.54） μg/24 h]较DN组[（99.3±5.15） μg/24 h]降低（F=254.302，P=0.000）；肾小球面积[（2 098±571.84） μm2]较DN组[（4 240±536.66） μm2]减少（F=24.468，P=0.000）；电镜下系膜增生抑制。同时，给予柚皮素的肾小球系膜细胞炎症因子TNF-α和IL-6表达水平（1.99±0.29，1.76±0.15）较高糖组（2.82±0.14，2.53±0.32）下降（F=111.303，P=0.000；F=65.127，P=0.000）；细胞核NF-κB p65（0.29±0.01）较高糖组（0.81±0.02）降低（F=1579.764，P=0.000）。结论：柚皮素可缓解DN小鼠尿白蛋白和减少系膜增生，其机制可能是在系膜细胞中通过抑制NF-κB介导的炎症因子表达。

Abstract:

Objective：To study the effect of naringenin（NAR） on diabetic nephropathy（DN） mice and the expressions of inflammatory factors tumor necrosis factor alpha（TNF-α） and interleukin-6（IL-6） in glomerular mesangial cells. Methods：Totally 10 male db/db DN mice were randomly divided into two groups：NAR group and DN group. Five db/m control mice were set up as normal group. Mice in NAR group were treated with NAR [50 mg/（kg×d）] by gavage for two weeks. Mice in DN group were treated with the same volume of saline by gavage for two weeks. Mice in normal group were treated with normal diet. Body weight，blood glucose and urinary albumin excretion（UAE） were tested in mice，and the morphology of the renal glomerular was tested by HE staining and electron microscope. Moreover，the glomerular mesangial cells were cultured in DMEM medium with different glucose concentrations（5 mmol/L and 25 mmol/L glucose）. Cells in normal group were cultured with 5 mmol/L glucose. Cells in high glucose group were cultured with 25 mmol/L glucose. Cells in NAR group were cultured with 25 mmol/L glucose containing 400 μmol/L NAR. Then，the expressions of inflammatory factors TNF-α and IL-6 were tested by ELISA，and the expression of NF-κB p65 was tested by Western blot in the cytoplasm and nucleus of the cells. Results：After the treat－ment of NAR in mice，UAE was decreased[（51.48±8.54） μg/24 h] when compared with that in DN group[（99.3±5.15） μg/24 h]（F=254.302，P=0.000）；glomerular mesangial area was smaller[（2 098±571.84） μm2] than that in DN group[（4 240±536.66） μm2]（F=24.468，P=0.000），and the mesangial proliferation was lessened. Additionally，after the treatment of NAR in mesan－gial cells，the expressions of TNF-α and IL-6 were lower（1.99±0.29，1.76±0.15） than those in high glucose group（2.82±0.14，2.53±0.32）（F=111.303，P=0.000；F=65.127，P=0.000），the expression of NF-κB p65 was decreased（0.29±0.01） when compared with that in high glucose group（0.81±0.02）（F=1579.764，P=0.000）. Conclusion：Naringenin may play roles in relieving albu－minuria and mesangial proliferation via inhibiting the expressions of inflammatory factors through NF-κB in DN.