【作者】 李慧；

【导师】 宋立群；

【作者基本信息】 黑龙江中医药大学 ， 中医内科学， 2014， 博士

【摘要】 目的：观察虫草益肾颗粒对C-BSA所致膜性肾病大鼠肾脏的保护作用,并探讨该方增加激素敏感性的机制。方法：实验一：将72只雄性SD大鼠,除12只作为空白对照外,其余60只采用多次尾静脉注射阳离子化牛血清白蛋白(C-BSA)的方法进行造模,最后一次尾静脉注射后,收集造模大鼠24h尿液,根据24hUPQ分为模型组、西药对照组、虫草益肾颗粒低、中、高剂量组,每组12只。造模结束后开始灌胃给药,虫草益肾颗粒低、中、高剂量组分别给予中药溶液3.24g/kg/d,6.48g/kg/d,12.96g/kg/d(含颗粒量),西药组给予环孢素A(31.5mg/kg/d)联合醋酸泼尼松混悬液(0.945mg/kg/d)。空白组及模型组给予等体积生理盐水。连续灌胃4周。观察大鼠一般状态、测定24h尿蛋白定量(24hUPQ)；治疗结束麻醉状态下采血检测各组大鼠血清总蛋白(TP)、白蛋白(ALB)、甘油三酯(TG)、胆固醇(CHOL)等生化指标；光镜及电镜观察肾脏组织病理改变。Real Time PCR方法检测各组肾组织中糖皮质激素受体a(GRa)mRNA、多药耐药基因(MDR1)mRNA的表达情况；免疫组化法检测各组肾组织中GRα、MDR1基因表达产物P糖蛋白170(P-gp170)的表达情况。实验二：48只雄性SD大鼠,分为空白组、单纯模型组、激素干预后模型组(简称激素组)、激素干预后中药治疗组(简称激素加中药组),每组12只。其中单纯模型组同实验一方法造模,激素干预后模型组是在单纯模型基础上给予醋酸泼尼松混悬液(6.3mg/kg/d)灌胃,制备经激素干预后的C-BSA肾病大鼠模型。激素加中药组给予虫草益肾颗粒中剂量(6.48g/kg/d)溶液灌胃,其余三组给予等体积生理盐水,连续给药4周。观察大鼠一般状态,24hU PQ,给药结束后麻醉状态下采血检测各组大鼠血清TP.ALB.TG.C HO L等生化指标,光镜及电镜观察肾脏组织病理改变；Real Time PCR方法检测各组肾组织中GRamRNA、MDR1mRNA的表达情况；免疫组化法及Western Blot法检测各组肾组织中GRα、P-gp170的表达情况结果：(1)模型组大鼠皮毛松散,无光泽,活动少、精神萎靡,足趾皮肤松弛处可观察到水肿,摄食量少,皮毛脱落。虫草益肾颗粒各剂量治疗组大鼠状态明显改善,活动增多、摄食量增加,水肿消退。西药组大鼠水肿消退,但状态不及中药治疗组,且出现死亡。(2)虫草益肾颗粒各剂量治疗组大鼠的24hUPQ低于模型组(P<0.01)；血清ALB、TP水平高于模型组(P<0.01)；TG.CHOL水平低于模型组(P<0.01)。(3)激素加中药组大鼠的24hUPQ低于单纯模型组及激素组(P<0.01)；血清ALB、TP水平高于单纯模型组及激素组(P<0.01)；TG、CHOL水平低于单纯模型组及激素组(P<0.01)。(4)Real Time PCR及免疫组化结果显示,单纯模型组大鼠肾组织中GRα及P-gp170的表达在基因与转录水平上均与空白组无明显差异(P>0.05)。(5)Real Time PCR.免疫组化及Western Blot结果显示,与空白组相比,激素组大鼠肾组织中GRamRNA以及GRa的表达减少(P<0.01),MDR1mRNA及P-gp170的表达升高(P<0.01)。(6)Real Time PCR.免疫组化及Western Blot结果显示,与激素组相比,激素加中药组肾组织中GRαmRNA以及GRα表达上调(P<0.01)而MDR1mRNA及P-gp170表达降低(P<0.01)。结论：(1)虫草益肾颗粒可改善C-BSA肾病大鼠的一般状态,该方面的作用优于西药组。(2)虫草益肾颗粒可减少C-BSA肾病大鼠蛋白尿,改善低蛋白血症、调节脂质代谢紊乱,并可减轻C-BSA肾病大鼠肾脏病理改变,保护肾小球。(3)虫草益肾颗粒协同激素治疗C-BSA肾病大鼠起效快,效果显著,较单纯应用激素治疗效果明显,缩短病程。(4)单纯的C-BSA肾病大鼠模型肾组织中可能不存在GRα及P-gp170表达的改变。(5)糖皮质激素可能会减少C-BSA肾病大鼠肾组织中GRa的表达,上调P-gp170的表达。(6)虫草益肾颗粒可增加激素敏感性,其机制可能与上调肾组织中GRα、下调P-gp170的表达有关更多还原

【Abstract】 Objective:To investigate the effect of ChongCaoYiSheng granule on the membranous nephropathy(MN) induced by cationic bovine serum albumin(C-BSA) in rats,and to explore the mechanism of increased sensitivity to glucocorticoid.Methods:Experiment One:Except12rats were the comparison group,72wistar rats were divided into5groups according to24hUPQ:model group, western medicine group, ChongCaoYiSheng granule low dose group, medium dose group, and high dose group. Rats model were induced by repeatedly tail vein injection C-BSA. The western medicine group were fed with suspension liquid consisted of Prednisone acetate and cyclosporine A. While ChongCaoYiSheng group was fed with traditional Chinese medicine solution, and the normal group was fed with normal saline with the same volume as the western medicine group. Every group was fed one time one day for4weeks. The general conditions of rats were dynamically observed during treatment, and the24hUPQ were determined before and after treatment. ALB, TP, TG, and CHOL level of rats blood serum were measured after treatment which was taken from the anaesthetic rats. All rats were killed after4weeks, and pathological changes were observed by light microscopy and electron microscope. RealTime PCR was used to detect the expression of GRamRNA and MDRlmRNA. Immunohistochemistry and Western Blot method were used to examine the expression of GRa and P-gp170.Experiment Two:48wistar rats were randomly divided into4groups:normal group, pure model group, model group after hormonal intervention (glucocorticoid group), Chinese medicine treatment model after hormonal intervention (glucocorticoid combined with Chinese medicine group), with12rats in every group. The pure model group was founded by the same method as in the experiment one. The glucocorticoid group was founded by feeding the pure model group with Prednisone acetate suspension liquid (6.3mg/kg/d), by which the glucocorticoid C-BSA rats model are founded. The glucocorticoid combined with Chinese medicine group was fed with ChongCaoYiSheng medium dose solution (3.6g/kg/d). The normal group, pure model group and glucocorticoid group was fed with the same volume normal saline. Every group were fed one time each day for4weeks. The general conditions of rats were dynamically observed during treatment, and the24hUPQ were determined before and after treatment. After4weeks, pathological changes were observed by light microscopy and electron microscope, the same indexes (ALB, TP, TG and CHOL level) as experiment one were measured, and the same detecting measures (such as RealTime PCR, Immunohistochemistry and Western Blot method) were also used to examine the expression of GRamRNA, MDR1mRNA, GRa and P-gp170.Results:(1) The fur of the model group rats were loose and lackluster and they showed declining activity, listlessness, and lack of food intakes. There were edema and fur shedding which were observed in the toe slack skin of the model rats. The status of rats in ChongChao YiSheng granule low, medium, and high dose group were ameliorative distinctly, which demonstrated more activity, eating more and their edema were faded. The edema of rats in western medicine group were faded; however their status could not compete with those in Chinese medicine group, and someone were dead.(2) The24hUPQ of rats in ChongChao YiSheng granule group are lower than that model group (P<0.01); the ALB and TP level of blood serum were higher than that of model group (P<0.01); and the TG and CHOL level of rats were less than that model group (P<0.01);(3) The24hUPQ of rats with glucocorticoid combined with Chinese medicine group were lower than that of model group and glucocorticoid group (P<0.01); But the TG, CHOL level of rats were below that of the model group and glucocorticoid group (P<0.01);(4) The results of Real Time PCR and Immunohistochemical results indicated that the GRa and P-gp170expressions level of rats in model group were not obviously discrepancy from normal group (P>0.05);(5) Comparing to normal group, the results of Real Time PCR, Immunohistochemical and Western Blot indicated that the GRa expressions in nail tissue of rats in glucocorticoid group were decreased (P<0.01), whereas the expressions of P-gpl70were raised (P<0.01):(6) Comparing to glucocorticoid group, the results of Real Time PCR, Immunohistochemical and Western Blot indicated that the GRa expressions in nail tissue of rats in glucocorticoid combined with Chinese medicine group were raised (P<0.01), nevertheless the expressions of P-gp170were reduced (P<0.01);Conclusion:(1) ChongCaoYiSheng granule can ameliorate the status of C-BSA rats and improve their quality of life. Its effect in this respect is better than that of western medicine group;(2) ChongCaoYiSheng granule can reduce the albuminuria of C-BSA rats, meliorate Hypoproteinemia, and adjust disturbance of lipid metabolism. At the same time, it can alleviate the nephritic pathological changes of C-BSA rats and protect Glomerulus;(3) The effect of ChongCaoYiSheng granule combined with glucocorticoid treatment on C-BSA rats were fast and significant. It shortens the treating time and has better treatment effect than treatment only with glucocorticoid treatment;(4) Possibly, there were not changes of GRa and P-gp170expressions in renal tissue of the simple C-BSA model rats;(5) Glucocorticoid can reduce the GRa expressions in renal tissue of C-BSA rats and raise the expressions of P-gp170;(6) ChongCaoYiSheng paticle can enhance the glucocorticoid sensitive and improve the GC resistance of RNS. The mechanism may have to do with the up-regulation GRa expressions and down regulation P-gp170expressions of renal tissue.更多还原