甘草酸对TGF-β1诱导的NRK-49F细胞活化及分泌细胞外基质的影响

Effect of glycyrrhizic acid on the activation and extracellular matrix secretion in renal interstitial fibroblast cells induced by transforming growth factor β1

目的探讨甘草酸对转化生长因子β1(TGF-β1)诱导的大鼠肾间质成纤维细胞(NRK-49F)中纤维化相关因子表达的影响。方法体外培养NRK-49F细胞,不同浓度甘草酸干预细胞48 h后,CCK-8法检测其对细胞增殖的影响,确定对细胞活性无显著影响的作用浓度。以10μg/L TGF-β1刺激NRK-49F细胞构建肾间质纤维化细胞模型,加入不同浓度甘草酸进行干预,免疫荧光法检测α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原(COL1)表达情况。实时荧光定量PCR、Western Blot法检测纤维化相关因子α-SMA、COL1、Ⅲ型胶原(COL3)mRNA及蛋白表达情况,以评价甘草酸对成纤维细胞活化及细胞外基质分泌的影响。结果CCK-8结果显示甘草酸在100~600μmol/L浓度范围内对NRK-49F细胞活性无影响。免疫荧光结果表明,加入甘草酸后可抑制α-SMA表达及COL1分泌,随甘草酸浓度增加,抑制作用增强。Western Blot结果显示,与模型组相比,甘草酸干预可下调α-SMA、COL1、COL3的蛋白表达,其中甘草酸150μmol/L组COL1的蛋白表达水平降低(P<0.01),甘草酸200μmol/L组COL1、COL3表达均降低(P<0.01)。RT-PCR结果表明,与模型组相比,甘草酸干预可下调α-SMA、COL1、COL3表达,甘草酸150μmol/L组COL1 mRNA的表达水平降低(P<0.01),甘草酸200μmol/L组COL1、COL3 mRNA表达均降低(P<0.01)。结论甘草酸能够有效抑制TGF-β1诱导的NRK-49F细胞中α-SMA、COL1、COL3的mRNA及蛋白表达,其作用机制有待进一步探讨。

Objective To investigate the effect of glycyrrhizic acid(GA)on the expression of fibrosis-related factors in the renal interstitial fibroblastcells(NRK49F)of rats induced by transforming growth factorβ1(TGF-β1).Methods NRK-49F cells were cultured in vitro.After 48 h of intervention with different concentrations of GA,Cell Counting Kit-8 was used to detect their effect on cell proliferation,so as to determine the concentration with no obvious effect on cell viability.The model was established by stimulating NRK-49F cells with 10μg/L TGF-β1.Then different concentrations of GA were used for intervention.The expression ofα-SMA and type 1 collagen(COL1)was detected with immunofluorescence method.The mRNA and protein expression of fibrosis-related factors ofα-SMA,COL1,COL3 was detected with RT-PCR and WB methods,so as to evaluate the effect of GA on the activation of fibroblasts and the secretion of extracellular matrix.Results CCK-8 results showed that within the concentration range of 100~600μmol/L,GA had no effect on the activity of NRK-49F cells.According to immunofluorescence results,the addition of GA intervention inhibited the expression ofα-SMA and the secretion of COL1,with the increase of GA concentration,the inhibitory effect being enhanced.According to Western Blot results,GA intervention could down-regulate the expression ofα-SMA,COL1 and COL3 compared with the model group;the expression level of COL1 protein in the GA 150μmol/L group was reduced(P<0.01),and the level of COL1 and COL3 in the GA 200μmol/L group was reduced(P<0.01).RT-PCR results showed that GA intervention could down-regulate the expression ofα-SMA,COL1 and COL3 compared with the model group;among them,the mRNA expression level of COL1 in the GA 150μmol/L group was reduced(P<0.01),and the expression of COL1 and COL3 in the GA 200μmol/L group was reduced(P<0.01).Conclusion GA seems to inhibit the mRNA and protein expression ofα-SMA,COL1 and COL3 in NRK-49F cells induced by TGF-β1,but its mechanism still needs to be further explored.