松弛素对高糖环境下肾小球系膜细胞分泌细胞外基质的影响及其作用机制研究

Study of the effect and mechanism of relaxin on the ECM excretion of Human renal mesangial cells cultured in high ambient glucose

目的 观察人松弛素（RLX）对高糖培养人肾小球系膜细胞分泌细胞外基质的影响,初步探讨其可能作用机制.方法 将人肾小球系膜细胞株分组：正常糖组：含5.5 mmol/L葡萄糖;高糖组：含30 mmol/L葡萄糖;松弛素干预组：高糖（30 mmol/L葡萄糖）＋松弛素（10 ng/ml）;α-平滑肌动蛋白（α-SMA）的检测增加TGF-β1阳性对照组,即5.5 mmol/L葡萄糖＋TGF-β1（5μg/L）.CCK-8法检测松弛素对系膜细胞增殖的影响,ELISA法检测细胞上清中Ⅳ型胶原（ColⅣ）、纤连蛋白（FN）的表达.qPCR检测系膜细胞TGF-β1 mRNA表达.免疫印迹技术（Western blot）检测α-SMA表达,确定肾小球系膜细胞表型转化情况.结果 松弛素对正常糖组及高糖组系膜细胞活力均未发现有影响;与正常糖组相比,高糖组各时间点肾系膜细胞分泌ColⅣ、FN显著增加,48 h尤为明显（57.28±0.59 vs 41.85 ±0.03、56.52±0.88 vs 33.80±0.24,P＜0.01）,α-SMA表达增加（P＜0.05）.与高糖组比较,松弛素干预组ColⅣ、FN表达在48 h下降最为明显（47.08±0.03 vs 57.28±0.59、36.16±0.52 vs 56.52±0.88,P＜0.01）,α-SMA表达减少（P＜0.01）,TGF-β1 mRNA表达下降（P＜0.01）.结论 松弛素能抑制高糖培养的系膜细胞分泌细胞外基质,部分作用机制是通过抑制细胞表型转分化及抑制TGF-β1基因表达,具有抗纤维化作用.

Objective To explore the effect and mechanism of relaxin on the production of extracellular matrix （ECM） excreted by high glucose stimulated human renal mesangial cells.Methods Cultured human mesangial cells （HMCs） were divided into three groups：（1） normal glucose group （NG,5.5 mmol/L D-glucose）,（2） high glucose group （HG,30 mmol/L D-glucose）,and （3） high glucose ＋ relaxin group.Cell count kit （CCK8） was used to examine the cell proliferation.The levels of fibronectin and collagen type Ⅳ in the culture supernatants were examined with a solid-phase enzyme-linked immunoadsorbent assay （ELISA）;Western blot method was used to detect the expression of α-smooth muscle actin （α-SMA） protein.The transforming growth factor-β1 （TGF-β1） mRNA expression was detected with quantitative polymerase chain reaction （qPCR） method.Results No proliferation and inhibition effects were observed in both normal and high glucose group.Compared to the normal glucose group,the levels of fibronectin,and collagen type Ⅳ increased significantly （57.28 ± 0.59 vs 41.85 ± 0.03,56.52 ± 0.88 vs 33.80 ± 0.24,P 〈 0.01）after cultured 48 h in high concentration of glucose.Compared to the high glucose group,a significantly decreases of fibronectin and collagen type Ⅳ （47.08 ± 0.03 vs 57.28 ± 0.59,36.16 ± 0.52 vs 56.52 ±0.88,P 〈0.01） were observed in the relaxin treated group.The expressions of α-smooth muscle actin and TGF-β1 were decreased （P 〈0.01）.Conclusions Relaxin can suppress the overproduction of ECM excreted by HMC cultured in high ambient glucose,and its mechanism is partly due to the inhibition of TGF-β1.