益气化瘀清热方及其拆方对嘌呤霉素氨基核苷损伤小鼠足细胞表达TRPC6的影响

Effect of Yiqi Huayu Qingre Prescription and Its Separations on Expression of Podocyte TRPC6 of Mice Injuried by Puromycin Aminonucleoside

目的：观察益气化瘀清热方及其拆方对嘌呤霉素氨基核苷（Puromycin aminonucleoside,PAN）损伤体外培养的小鼠足细胞表达TRPC6的影响,探讨不同类中药的作用强度。方法：应用PAN（50μg/mL）刺激体外培养的小鼠足细胞,形成PAN足细胞损伤模型,造模成功后的足细胞随机分为益气组、化瘀组、清热组、复方组、对照组、模型组、空白组,采用RT-PCR和Western Blot法检测各组血清对小鼠足细胞表达TRPC6 mRNA及蛋白的影响。结果：（1）RT-PCR结果显示：空白组与PAN刺激24、48 h的模型组TRPC6mRNA表达差异显著（P〈0.05）。含药血清作用24 h后,化瘀组、清热组、复方组表达TRPC6mRNA明显低于对照组（P〈0.01）和益气组（P〈0.05）,而3组之间无显著差异（P〉0.05）,益气组与对照组也无显著差异（P〉0.05）。含药血清作用48 h后,益气组、化瘀组、清热组及复方组表达TRPC6mRNA均显著低于对照组（P〈0.05）,且4组之间无明显差别（P〉0.05）。（2）Wersten印迹结果显示：空白组与PAN刺激24、48 h的模型组TRPC6蛋白表达差异显著（P〈0.05）。含药血清作用24 h后,益气组、化瘀组TRPC6蛋白表达低于对照组（P〈0.05）,清热组、复方组与对照组相比无显著差异（P〉0.05）,且4组间差异不明显（P〉0.05）。各含药血清作用48 h后,益气组、清热组、复方组TRPC6蛋白表达低于对照组（P〈0.01）,化瘀组与对照组相比无差别（P〉0.05）;复方组、益气组、清热组3者在TRPC6蛋白表达上无差别（P〉0.05）;化瘀组TRPC6表达水平高于复方组（P=0.008〈0.01）,而与益气组、清热组相比无显著差异（P〉0.05）。结论：益气化瘀清热方及其拆方均能抑制造模足细胞TRPC6mRNA和蛋白的表达,但不同药物作用于造模足细胞的时间不同,抑制TRPC6表达的作用强度不同。

Objective： To observe the effect of Yiqi Huayu Qingre Prescription and its separations on the expression of mice podocyte TRPC6 and study the drug potency of those traditional Chinese medicine in the treatment of podocyte in vitro stimulated by Puromycin Aminonucleoside（ PAN）. Methods： We stimulated the mice podocyte with PAN（ 50 μg/mL） in vitro to make the model of the damaged podocytes which were divided randomly into seven equal groups： Yiqi group,Huayu group,Qingre group,Yiqi Huayu Qingre group,control group,model group,blank group,and we observed the effect of those drug-containing serum on the expression of the mRNA and protein of TRPC6 by using RT-PCR and Western Blot. Results：（1）RT-PCR results： The blank group and the model group had significant differences on the expression of TRPC6 mRNA by PAN stimulated for 24 hours and 48 hours（ P 〈 0. 05）. After intervening for 24 hours,the expression of TRPC6 mRNA in Huayu group,Qingre group and Yiqi Huayu Qingre group were lower than the expression in the control group（ P 〈 0. 01） and Yiqi group（ P 〈 0. 05）,meanwhile,there was no significant difference among the three groups（ P 〉 0. 05） and Yiqi serum group and the control group had no significant difference. After intervening for 48 hours,the expression of TRPC6 mRNA in Yiqi group,Huayu group,Qingre group and Yiqi Huayu Qingre group were lower than the expression in the control group（ P 〈 0. 05）,and there was no significant difference among the four groups（ P 〉 0. 05）.（2）Western Blot results： The blank group and the model group had significant differences on the expression of protein of TRPC6 by PAN stimulated for 24 hours and 48 hours（ P 〈 0. 05）. After intervening for 24 hours,the level of the expression of the protein of TRPC6 in the Yiqi group and Huayu group were lower than that of the control group（ P 〈 0. 05） and Qingre group,Yiqi Huayu Qingre group and the control group had no significant difference（ P 〉0. 05）. Meanwhile,there was no difference between the drug-containing serum groups（ P 〉 0. 05）. After intervening for 48 hours,the level of the expression of the protein of TRPC6 in Yiqi group,the Qingre group and Yiqi Huayu Qingre group were lower than those of the control group（ P 〈 0. 01）. Huayu group and Yiqi Huayu Qingre group had no significant difference on the level of expression of the protein of TRPC6（ P 〉 0. 05）. Yiqi Huayu Qingre group,Yiqi group and Qingre group had no significant difference（ P 〉 0. 05）. The level of the expression of the protein of TRPC6 in Huayu group was obviously significant（ P 〈 0. 01） and had no significant difference with Yiqi group and Qingre serum group（ P〉 0. 05）. Conclusion：Yiqi Huayu Qingre Prescription and its separations could improve the expression of the mRNA and protein of TRPC6,but the work of the different drugs on improving the expression of the mRNA and protein of modeling podocyte TRPC6 had different effect in the different time.