肾复康对TGF-β1诱导的大鼠成纤维细胞转分化模型的抑制作用及机制  

Inhibition and Mechanism of Shenfukang on TGF-β1-induced Transdifferentiation of Rat Fibroblasts

作　　者：齐亮 刘劲松 周琳[2] 黄上 罗运花 肖波飞 张晓白 林哲 Qi Liang;Liu Jingsong;Zhou Lin;Huang Shang;Luo Yunhua;Xiao Bofei;Zhang Xiaobai;Lin Zhe(Department of Nephrology,Chinese Medicine and Western Medicine Hospital affiliated to Hunan University of Chinese medicine,Changsha 410000;Key Lab of Kidney Disease and Blood Purification in Hunan,the Second Xiangya Hospital of Central South University,Changsha 410000)

机构地区：[1]湖南中医药大学附属中西医结合医院肾脏科,长沙410000 [2]中南大学湘雅二院肾脏疾病与血液净化湖南省重点实验室,长沙410000

出　　处：《中药药理与临床》2021年第4期143-147,共5页Pharmacology and Clinics of Chinese Materia Medica

基　　金：湖南省自然科学基金(编号:2019JJ50342);湖南省中医药研究院重点项目(编号:202015);湖南省中医药科研计划项目重点项目(编号:2021202)。

摘　　要：目的:探讨肾复康对转化生长因子-β1(TGF-β1)诱导的大鼠成纤维细胞转分化模型的抑制作用及机制。方法:采用噻唑兰(MTT)法检测不同浓度肾复康对NRK-49F细胞增殖的作用;试验随机设置空白对照组、模型对照组,糖酵解抑制剂2-脱氧-D-葡萄糖(2-DG)3 mmol/L组、肾复康0.25、0.5、1 g/mL组。试剂盒检测细胞上清乳酸含量;采用实时荧光定量PCR检测α-肌动蛋白(α-Sma)、I型胶原(Col1a1)、己糖激酶(hexokinase,Hk)、6-磷酸果糖激酶-2(Pfk-2)和葡萄糖转运蛋白4(Glut4)mRNA表达水平;免疫荧光检测α-SMA的蛋白表达水平;Western Blot法检测HK、PFK-2和GLUT4蛋白表达水平。结果:肾复康在0、0.125、0.25、0.5和1 g/mL浓度对NRK-49F细胞增殖无抑制作用;与空白对照组相比,模型对照组乳酸水平显著升高,α-Sma、Col1a1、Hk、Pfk-2和Glut4 mRNA表达水平显著上调,α-SMA荧光强度增强,HK、PFK-2和GLUT4蛋白表达水平显著上调(P<0.01);与模型对照组相比,2-DG组及肾复康0.5、1 g/mL组乳酸水平显著降低,α-Sma、Col1a1、Hk、Pfk-2和Glut4 mRNA表达水平明显下调(P<0.05或P<0.01),α-SMA荧光水平明显减弱,HK、PFK-2和GLUT4蛋白表达水平显著下调(P<0.05或P<0.01)。结论:肾复康能够抑制成纤维细胞的转分化纤维生成,且其机制可能与抑制糖酵解有关。Objective:To explore the inhibitory effect and mechanism of Shenfukang(SFK) on TGF-β1-induced transdifferentiation of rat fibroblasts. Methods: Methyl thiazolyl tetrazolium(MTT) assay was carried out to detect the effect of SFK of different concentrations on the proliferation of NRK-49 F cells. The experimental rats were randomly divided into a normal control group, a model group, a glycolytic inhibitor 2-deoxy-D-glucose(2-DG) group(3 mmol/L),and high-(1 g/mL), medium-(0.5 g/mL), and low-dose(0.25 g/mL) SFK groups. The lactic acid in the supernatant of cells was detected by the assay kit. The mRNA expression levels of alpha-smooth muscle actin(α-SMA), collagen Type Ⅰ alpha 1(COL1 A1), hexokinase(HK), 6-phosphofructo-2-kinase(PFK-2), and glucose transporter type 4(GLUT4) were detected by RT-PCR. The α-SMA protein expression was detected by the immunofluorescence assay. The protein expression of HK, PFK-2, and GLUT4 was detected by Western blot.Results: SFK showed no inhibitory effect on the proliferation of NRK-49 F cells at the concentrations of 0, 0.125, 0.25, 0.5, and 1 g/mL. Compared with the normal control group, the model group showed increased lactic acid level, up-regulated mRNA expression levels ofα-SMA, COL1 A1, HK, PFK-2, and GLUT4, enhancedα-SMA fluorescence, and elevated protein expression of HK, PFK-2, and GLUT4(P<0.01). Compared with the model group, the 2-DG group, and high-and medium-dose SFK groups showed reduced lactic acid level, down-regulated mRNA levels of α-SMA, COL1 A1, HK, PFK-2, and GLUT4(P<0.05 or P<0.01), weakened α-SMA fluorescence, and declining protein expression levels of HK, PFK-2, and GLUT4(P<0.05 or P<0.01). Conclusion: SFK can inhibit the transdifferentiation of fibroblasts, and the mechanism may be related to the inhibition of glycolysis.

关 键 词：肾复康 成纤维细胞 肾纤维化 Α-平滑肌肌动蛋白 糖酵解 

分 类 号：R285.5[医药卫生—中药学]