基于Notch信号通路探讨益肾活血法治疗慢性移植肾功能不全的机制

Chronic renal allograft dysfunction(CARD) is due to the synergistic effects of both immune and non-immune factors, which has a high incidence after renal transplantation with an unsatisfactory treatment outcome. The symptoms of CARD mainly are renal deficiency and blood stasis, and its major pathology is renal interstitial fibrosis (IF). The Chinese herbal mixture, which can improve renal microcirculation, ShenKangLin can effectively treat CRAD, however, the mechanism is unclear. Previous studies showed that TGF-β1 can modulate Notch signaling pathway and regulate immune responses to induce renal IF. Our study revealed that Notch signaling pathway takes part in the immune responses of CARD; and ShenKangLin can treat renal IF and modulate its immune responses. The results suggest that ShenKangLin has effects on Notch signaling pathway, cellular immunization and renal IF parameters in CARD rat. We predict that ShenKangLin can regulate Notch signaling pathway through TGF-β1, so as to increase the resistance towards renal IF and/or modulate immune responses to induce tolerance to eventually cure CARD. This project plans to establish a CARD rat model first, and then use ShenKangLin as an intervention for the measurements on the cell, protein and gene levels in vivo and in vitro. The investigation will start with TGF-β1 and Notch signaling pathway to analyze IF and T cell proliferation, differentiation as well as functional parameters. In order to verify this hypothesis and clarify the mechanisms of the effects of ShenKangLin in CARD treatments, our work can provide experimental and theoretical basis for the application of ShenKangLin in CARD treatments.

慢性移植肾功能不全(CRAD)是免疫与非免疫因素协同作用的结果，在肾移植术后高发但疗效差，主要病理表现为肾间质纤维化(IF)，辩证为肾虚血瘀。益肾活血法中药肾康灵治疗CRAD有效，但机制不明。据报道TGF-β调控Notch信号通路，并调节免疫、致IF。我们研究发现：Notch信号参与CRAD的免疫调节；肾康灵下调TGF-β、调节免疫并改善IF；肾康灵影响CRAD大鼠的Notch信号、细胞免疫和肾纤维化指标。我们推测肾康灵可能通过调节TGF-β，以调控Notch信号，继而抗肾纤维化和/或调节细胞免疫、诱导免疫耐受，从而治疗CRAD。本研究拟建立CRAD大鼠模型，予肾康灵干预，在体内和体外，从细胞、蛋白、基因水平检测；从TGF-β及Notch受、配体入手，分别分析IF指标及T细胞增殖、分化、功能等免疫指标。以验证该假说，阐明益肾活血法（肾康灵）治疗CRAD的机制，为该应用提供理论、实验依据。

慢性移植肾功能不全(CRAD)在肾移植术后高发，是晚期移植肾丧失功能的主要原因，目前疗效欠佳。研究发现益肾活血法中药肾康灵治疗CRAD有效，但机制不明。我们设想肾康灵可能通过抑制TGF-β/Notch信号通路，诱导免疫耐受和/或抑制肾纤维化而改善CRAD。为证实这一设想，我们通过体内、外实验，研究肾康灵在TGF-β/Notch信号通路中的作用，及调节细胞免疫和肾纤维化在CRAD发生中的作用。为揭示肾康灵治疗CRAD的分子机制提供实验依据。.结果发现，构建的CRAD大鼠模型中，血清尿素氮、尿蛋白浓度和血清肌酐浓度显著高于正常大鼠，结合病理结果显示成功构建CRAD大鼠模型。正常大鼠、CRAD大鼠和经过肾康灵治疗的CRAD大鼠各组血清尿素氮：模型组高于治疗组、对照组，尿蛋白浓度模型组较高，治疗组和正常组水平接近。血清白蛋白各组没有表现出明显差异。血清肌酐：模型组和治疗组都明显高于正常组。模型组、正常组和治疗组大鼠肾脏组织分别行HE染色、Masson染色、C4d染色和PAS染色，结果提示肾康灵治疗能够减轻CRAD大鼠肾脏纤维化病变。模型组、正常组和治疗组大鼠肾脏组织中Notch1、Jagged1、MMP-9和Vimentin基因的mRNA和蛋白表达均为模型组大于治疗组大于正常组，免疫组化结果也具有相同的趋势。此外检测三组大鼠血清中TGF-β1、IL-17、INF-γ和组织中NotchL、NotchR以及外周血T、B淋巴细胞表面Foxp3的含量，发现其中NotchL、NotchR和FOXP3在治疗组中的表达高于模型组，而TGF-β1、IL-17和INF-γ在模型组中的表达高于治疗组。.本研究提示肾康灵通过抑制TGF-β/Notch信号通路，进而诱导细胞免疫耐受和抑制肾纤维化，从而改善CRAD。本研究为深入认识肾康灵治疗CRAD的分子机制提供了新的思路。