肾衰泄浊汤及其拆方干预周细胞阻抑UUO大鼠肾小管间质纤维化的机制研究

Renal interstitial fibrosis (RIF) is a common pathway of chronic kidney disease progression to end-stage renal disease and is closely related to the degree of renal function impairment. These were very little method for treating chronic kidney disease in clinic, in particular, almost no effective anti RIF drug. The latest research shows that pericyte-muscle fibroblast differentiation is an important way of RIF. Vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) these signaling pathways are involved in the differentiation of pericyte. Inhibition of pericytes myofibroblast transdifferentiation and prevention of RIF have now become a hot research topic. Our preliminary study had confirmed that the traditional Chinese medicine compound prescription Shenshuaixiezhuo Decoction can regulate the BMP7/TGF beta 1/Smads signal pathway and inhibite expression of TGF beta 1 protein and excessive deposition of collagen type I and type III, so as to achieve a reduction of RIF. We We take "regulating peripheral cell and inhibiting RIF for research" as the research point.This project through the establishment of UUO rat model of renal interstitial fibrosis and through whole, organ, cell, molecule these many levels to observe the intervention effect of Shenshuaixiezhuo Decoction and its disassembled prescription in RIF degree, pericyte muscle fibroblast and related signaling pathway in rats, to lay a new objective basis for the application of the new theory of "micro - form syndrome" in RIF and provide a new method and therapeutic target for the clinical anti-RIF of TCM and treatment of chronic kidney disease.

肾间质纤维化（RIF）是多种慢性肾脏病进展至终末期的共同通路，与肾功能损害程度相关。临床上治疗慢性肾脏病的方法很少，特别是几乎不存在有效的抗ＲIF药物。最新研究显示周细胞-肌成纤维细胞转分化是RIF的重要途径，血管内皮生长因子（VEGF）、血小板衍生生长因子（PDGF）等信号通路参与了周细胞转分化。阻抑周细胞-肌成纤维细胞转分化防治RIF目前已成为研究的热点。我们前期研究证实肾衰泄浊汤可通过调节BMP7/TGFβ1/Smads信号通路，抑制TGFβ1蛋白表达及Ⅰ、Ⅲ型胶原过度沉积,达到减轻RIF。我们以它为研究切入点，通过构建UUO大鼠RIF动物模型，从整体、器官、细胞、分子等多层次观察肾衰泄浊汤及其拆方对大鼠RIF程度、周细胞-肌成纤维细胞转分化标志物及相关信号通路的干预效应。为“微形癥积”中医病机学创新理论在RIF的应用奠定新的客观依据，为临床中医药抗RIF提供新的可能途径及干预靶点。

目的：研究肾衰泄浊汤及其补虚，祛邪组分对单侧输尿管梗阻（UUO）大鼠肾间质纤维化的影响。方法：通过结扎单侧输尿管建立UUO大鼠模型。将SD大鼠随机分为假手术组、模型组、贝那普利组、肾衰泄浊汤组、补虚方组、袪邪方组6组，每组24只。手术后第3天各组大鼠开始灌胃，术后第7、14、21d各组大鼠分别处死，检测大鼠肌酐（SCr）、尿素氮（BUN）及24h尿蛋白定量（UP）水平。取肾脏组织行HE、Masson染色，并在光镜、电镜下观察；Western blot和Real-time PCR法检测肾组织中周细胞及相关信号通路标志物表达情况。结果：同时期各组对比，模型组大鼠24hUP、BUN、SCr较其他组升高明显（P<0.05）；治疗组24hUP、BUN、SCr较其他组明显下降（P<0.05）；其中肾衰泄浊汤组疗效最明显（P<0.05）。HE染色、Masson染色及电镜结果：除假手术组外，术后7d各组大鼠均可出现炎症和坏死病变，术后14、21d出现炎症和纤维化，模型组纤维化程度最重。与模型组比较，中药各组术后第14，21天间质损伤评分和间质胶原积累均显著降低（P<0.05），α-平滑肌肌动蛋白（α-SMA），血小板衍生生长因子A（PDGFA）、神经/胶质细胞2型硫酸软骨素糖蛋白（NG-2）、血管内皮生长因子A（VEGFA）、血管内皮生长因子受体（VEGFR1）、血小板源性生长因子β（PDGF-β）、血小板源性生长因子受体β（PDGFR-β）的蛋白表达均较第7天显著增加（P<0.05）。与模型组比较，术后第21天，肾衰泄浊汤组的肾组织中NG-2，VEGFA，VEGFR1，PDGFR-β 蛋白表达均显著低于模型组（P<0.05）；α -SMA，PDGFA，NG-2，VEGFA，VEGFR1，PDGF-β，PDGFR-β mRNA表达量均显著降低（P<0.05）。且肾衰泄浊汤组术后第21天α-SMA，NG-2，VEGFA，VEGFR1，PDGFR-β mRNA相对表达量也显著低于贝那普利组（P<0.05）。结论：肾衰泄浊汤及其补虚，祛邪组分对UUO大鼠肾间质纤维化均具有拮抗作用，其作用机制与抑制周细胞及其相关细胞信号通路激活有关。