Liu Gang, Ren Guochen, Yang Xiaoping
Department of Nephrology, the First Affiliated Hospital of School of Medicine, Shihezi University, Shihezi 832000, China.
Department of Nephrology, the First Affiliated Hospital of School of Medicine, Shihezi University, Shihezi 832000, China. *Corresponding author, E-mail:
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2022 Mar;38(3):224-230.
Objective To investigate the effects of 1, 25(OH)D 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)D group (HG-VD group), high glucose combined with 1, 25(OH)D and si-VDR group (HG-VD-si-VDR group), high glucose combined with 1, 25(OH)D 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 type I (Col1), and collagen type IV (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 LC3 II/LC3 I ratio 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 LC3 II, and the LC3 II/LC3 I ratio 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)D on the high glucose induced proliferation, fibrosis increase, and autophagy decrease of mesangial cells.
目的 探讨1,25(OH)D对高糖介导的系膜细胞增殖、纤维化及自噬的影响及其机制。方法 体外培养大鼠肾小球系膜细胞系HBZY-1，转染小干扰RNA（siRNA）沉默维生素D受体（VDR），通过逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测转染效率。将培养的系膜细胞分为五组：正常葡萄糖组（NG组）、高糖组（HG组）、高糖联合1,25(OH)D组（HG-VD组）、高糖联合1,25(OH)D及si-VDR组（HG-VD-si-VDR组）、高糖联合1,25(OH)D及mTOR激活剂MHY1485组（HG-VD-MHY1485组）；采用MTT法和EdU染色检测系膜细胞增殖情况，酶联免疫吸附测定（ELISA）法检测纤连蛋白（FN）、Ⅰ型胶原（Col1）和Ⅳ型胶原（Col4）水平。通过透射电子显微镜观察各组系膜细胞自噬体数量。采用免疫荧光细胞化学法检测系膜细胞中自噬标志物微管相关蛋白1轻链3（LC3）的蛋白表达。采用蛋白质免疫印迹法检测转化生长因子-β1（TGF-β1）、α-平滑肌肌动蛋白（α-SMA）、p62、磷酸化mTOR（p-mTOR）以及LC3Ⅱ/LC3Ⅰ比值的表达。结果 si-VDR转染后，HBZY-1细胞中VDR mRNA和蛋白表达显著下调。与NG组相比，HG组、HG-VD组、HG-VD-si-VDR组和HG-VD-MHY1485组系膜细胞的增殖能力、细胞因子FN、Col1和Col4的表达水平以及p-mTOR、TGF-β1、α-SMA和P62蛋白表达水平均显著升高，而自噬体数量、自噬标志物LC3Ⅱ阳性表达率以及LC3Ⅱ/LC3Ⅰ比值均显著降低。HG-VD组上述变化较HG组、HG-VD-si-VDR组和HG-VD-MHY1485组明显减轻，后三组间差异无统计学意义。结论 抑制VDR表达或增加mTOR激活可有效对抗1,25(OH)D对高糖诱导的系膜细胞增殖、纤维化增加及自噬减少的抑制作用。
