疏利少阳法对HBV诱导HK-2细胞转分化pSmad3蛋白表达的影响  

Effects of shaoyang-dredging method on HBV-induced protein expression of pSmad3 in HK-2 Cells

作　　者：徐冰[1] 王耀光[2] 周慧杰 Xu Bing;Wang Yaoguang;Zhou Huijie(Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China;First Teaching Hospital of Tianjin University of Tradi- tional Chinese Medicine, Tianjin 300381, China;Affiliated Hospital of Hebei University of Engineering, Hebei 056002, China)

机构地区：[1]天津中医药大学,天津300193 [2]天津中医药大学第一附属医院 [3]河北工程大学附属医院

出　　处：《北京中医药大学学报》2018年第6期497-501,共5页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(No.81573888)~~

摘　　要：目的观察疏利少阳法代表方肾疏宁对乙型肝炎病毒(HBV)诱导人肾皮质近曲小管上皮细胞(HK-2)细胞转分化pSmad3蛋白表达的影响,以探讨该方治疗乙型肝炎病毒相关性肾炎(HBVGN)的相关机制。方法应用MTT法确定肾疏宁的体外给药浓度,设9组浓度梯度,在酶标仪OD570 nm处测量各孔的吸光值,并计算细胞存活率,根据绘制的生长曲线,计算半数抑制率(IC50),IC50对应的浓度即为下一步实验给药的浓度。分别用C基因型重组乙型肝炎病毒质粒pHY106-HBV及重组人生长转化因子-β1(TGF-β1)刺激剂干预HK-2细胞,实验分为6组:空白对照组(HK-2细胞常规培养),空质粒组(转染空质粒pHY106的HK-2细胞),HBV组(转染pHY106-HBV的HK-2细胞),TGF-β1组(HK-2细胞中加入12 ng重组人TGF-β1),HBV+肾疏宁组(HK-2细胞在转染pHY106-HBV后6 h加入肾疏宁),TGF-β1+肾疏宁组(HK-2细胞在重组人TGF-β1刺激6 h后加入肾疏宁)。药物干预48 h后,用Westernblot检测方法检测pSmad3的表达量。结果根据细胞存活率绘制的细胞生长曲线,计算IC50值为生药浓度1.313 g/m L按1/200稀释的加药浓度。Westernblot法检测各组pSmad3蛋白的表达,HBV组与空质粒组比较pSmad3蛋白表达上调,差异有统计学意义(P<0.05);TGF-β1组与空白对照组比较pSmad3蛋白表达上调,差异有统计学意义(P<0.05);HBV+肾疏宁组与HBV组比较pSmad3蛋白表达下调,差异有统计学意义(P<0.05);TGF-β1+肾疏宁组与TGF-β1组比较pSmad3蛋白表达下调,差异有统计学意义(P<0.05)。结论在生药浓度1.313 g/m L按1/200稀释的加药浓度下,肾疏宁能降低pSmad3蛋白表达,进而推测其可通过抑制Smad介导的信号转导通路而干预HK-2细胞转分化。Objective To observe the effects of shaoyang（ lesser yang）-dredging method on HBV-induced transdifferentiation and pSmad3＇s protein expression in HK-2 cells,with the attempt to explore the mechanism of shaoyang-dredging method in treating HBV associated glomerulonephritis（ HBV-GN）.Methods MTT assay was used to measure the concentration of chinese medicine（ SSN）,a representative formula of dredging lesser yang. 9 concentration gradients were set. Absorbance was measured at the OD570 nm of a microplate reader and the cell survival rate was calculated. Half inhibitory concentration（ IC50） was estimated according to the drawn growth curve,and this IC50 concentration would be used in the next steps. HK-2 cells were treated with pHY106-HBV agent and TGF-β1 stimulation. HK-2 cell lines were divided into 6 groups： blank control group（ routine culture of HK-2 cells）; empty plasnid group（ HK-2 cells transfected with empty plasmid pHY106）; HBV group（ HK-2 cells transfected with pHY106-HBV）; TGF-β1 group（ HK-2 cells with 12 ng of recombinant human TGF-β1）; HBV ＋ SSN group（ HK-2 cells treated with Chinese medicine 6 h after being transfected by pHY106-HBV）; and TGF-β1 ＋ SSN group（ HK-2 cells treated with chinese medicine 6 h after being stimulated by recombinant human TGF-β1）. 48 hours later,pSmad3 expression was evaluated with Western blot assay. Results Based on the cell growth curve plotted according to the cell survival rate,the IC50 value calculated was equivalent to 1/200-diluted 1. 313 g/ml crude drug concentration. The expression of pSmad3 protein increased in HBV group compared with empty plasmid group,and the difference was statistically significant（ P〈0. 05）. The expression of pSmad3 protein was also significantly increased in TGF-β1 group compared with blank control group（ P〈0. 05）. Compared with HBV group,the expression of pSmad3 was markedly reduced in SSN group（ P〈0. 05）. This reduction was also observed in TGF-β1 group reduced compared wi

关 键 词：疏利少阳 肾疏宁 乙型肝炎病毒相关性肾炎 HK-2细胞 pSmad3 

分 类 号：R285.5[医药卫生—中药学]