摘要
目的研究活性维生素D3(VD3)对高糖介导的系膜细胞增殖、纤维化及自噬水平的影响及其相关作用机制。方法体外培养HBZY-1大鼠肾小球系膜细胞,转染小干扰RNA(siRNA)沉默细胞中维生素D受体(VDR),反转录PCR和Western blot实验检测干扰效率;将培养系膜细胞分为正常葡萄糖培养组、高糖组、高糖联合VD3组、敲低VDR后高糖联合VD3组、高糖联合VD3和mTOR激活剂MHY1485组;噻唑蓝(MTT)法和乙炔基脱氧尿苷(EdU)法检测系膜细胞增殖,ELISA检测纤连蛋白(FN)、Ⅰ型胶原蛋白(Col1)和Ⅳ型胶原蛋白(Col4)的分泌水平,透射电镜检测各组系膜细胞自噬体数量变化,免疫荧光细胞化学染色检测系膜细胞中自噬标志分子微管相关蛋白1轻链3(LC3)表达,Western blot法检测系膜细胞纤维化相关蛋白转化生长因子β1(TGF-β1)、α平滑肌肌动蛋白(α-SMA)和P62蛋白的表达以及哺乳动物雷帕霉素靶蛋白(mTOR)磷酸化水平及LC3Ⅱ/LC3Ⅰ比值。结果转染si-VDR后HBZY-1细胞中VDR mRNA和蛋白表达水平明显下调;与正常糖组相比,高糖组、高糖联合VD3组、敲低VDR后高糖联合VD3组和高糖联合VD3和mTOR激活剂MHY1485组系膜细胞的增殖能力、细胞因子FN、Col1和Col4的表达水平均显著增加,且细胞中p-mTOR、TGF-β1、α-SMA、P62的蛋白表达水平均显著增加,而LC3Ⅱ/LC3Ⅰ比值明显降低,自噬体数量显著降低;其中高糖联合VD3组较高糖组、敲低VDR后高糖联合VD3组、高糖联合VD3和mTOR激活剂MHY1485组的上述检测指标的变化趋势明显降低,后三组无明显差异。结论抑制VDR的表达或提高mTOR活化水平能有效抵消活性VD3抑制高糖介导的系膜细胞增殖及纤维化水平增加和自噬水平降低的效应。
Objective To investigate the effects of 1,25(OH)_(2)D_(3)on the proliferation,fibrosis,and autophagy of mesangial cells mediated by high glucose and its mechanisms.Methods Rat glomerular mesangial cell line HBZY-1 was cultured in vitro and transfected with small interfering RNA(siRNA)to silence vitamin D receptor(VDR),and the transfection efficiency was detected by reverse transcription-PCR and Western blot.The cultured mesangial cells were divided into five groups:normal glucose group(NG group),high glucose group(HG group),high glucose combined with 1,25(OH)_(2)D_(3) group(HG-VD group),high glucose combined with 1,25(OH)_(2)D_(3) and si-VDR group(HG-VD-si-VDR group),high glucose combined with 1,25(OH)_(2)D_(3) and mTOR activator MHY1485 group(HG-VD-MHY1485 group);the proliferation of mesangial cells was detected by MTT assay and EdU staining,and the levels of fibronectin(FN),collagen typeⅠ(Col1),and collagen typeⅣ(Col4)were detected by ELISA.The number of autophagosomes in mesangial cells of each group was observed by transmission electron microscopy.The protein expression of autophagy marker LC3 in mesangial cells was detected by immunofluorescence cytochemistry.The expressions of transforming growth factor-β1(TGF-β1),α-smooth muscle actin(α-SMA),and p62,the phosphorylated mTOR(p-mTOR),and the LC3Ⅱ/LC3Ⅰratio were detected by Western blot.Results The expressions of VDR mRNA and protein in HBZY-1 cells were significantly down regulated after si-VDR transfection.Compared with those in the NG group,the proliferation ability,the expression levels of cytokines FN,Col1,and Col4,and the p-mTOR,TGF-β1,α-SMA,and P62 protein expression levels were significantly increased,while the number of autophagosomes,the positive expression rate of autophagy marker LC3Ⅱ,and the LC3Ⅱ/LC3Ⅰratio were significantly decreased in mesangial cells of HG group,HG-VD group,HG-VD-si-VDR group,and HG-VD-MHY1485 group.The above changes were significantly reduced in HG-VD group than in HG group,HG-VD-si-VDR group,and HG-VD-MHY1485 group,and there was no significant difference in the latter three groups.Conclusion Inhibition of VDR expression or increase of mTOR activation can effectively counteract the inhibitory effect of 1,25(OH)_(2)D_(3)on the high glucose induced proliferation,fibrosis increase,and autophagy decrease of mesangial cells.
作者
刘刚
任国臣
杨晓萍
LIU Gang;REN Guochen;YANG Xiaoping(Department of Nephrology,the First Affiliated Hospital of School of Medicine,Shihezi University,Shihezi 832000,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2022年第3期224-230,共7页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金地区科学基金(81160090)
石河子大学医学院第一附属医院院级课题(YL2015-R003)。
