基于Notch信号通路探讨益肾散结复方拆方组合对肾小球系膜细胞凋亡的抑制作用及配伍机制

Glomerulosclerosis is the primary pathological basis for CKD to ESRD. It is found that GMCs apoptosis is the main pathological feature of glomerulosclerosis, and Notch signaling pathway participates in the process of cell apoptosis. Kidney-reinforcing and Mass-disintegrating Compound may treat and prevent glomerulosclerosis but its compatibility mechanism is still to be discussed, which will be the focus of this study. (1) In vivo test: establish Adriamycin kidney disease mat models and divide them into normal group, benazepril hydrochloride group, warming the kidney to replenish essence group, replenishing Yin to tonify Kidney and remove blood stasis group and Kidney-reinforcing and Mass-disintegrating Compound group; pharmacodynamic indicator will be tested after drug intervention; the expression of Notch signaling pathway related proteins will be tested with RT-PCR and Western-Blot; the expression of related proteins will be verified by immunohistochemistry; and the morphological changes will be obsereved with electron microscope ;(2) in vitro test: culture mat mesangial cells and divide them as above; after drug intervention,CCK-8 method will be applied to test their proliferation vitality, flow cytometry will be used to test apotosis and periodic changes and Western-Blot, ELISA and RT-PCR will be employed to test Notch signaling pathway related protein expression; morphologic changes will be observed with electron microscope; the target and mechanism of Kidney-reinforcing and Mass-disintegrating Compound on treating and preventing glomerulosclerosis will be studied on cellular and molecular level, so that its compatibility mechanism can be revealed.

肾小球硬化是CKD进展至ESRD的主要病理基础。研究发现，GMCs凋亡是肾小球硬化的主要病理特征，且Notch信号通路参与细胞凋亡的发生过程。益肾散结复方可防治肾小球硬化，但配伍机制尚待研究。因此，本课题展开研究：①体内实验：建立阿霉素肾病大鼠模型，将其分为正常组、盐酸贝那普利组、温肾益精化瘀组、滋阴益肾化瘀组、益气化瘀组及益肾散结复方组，药物干预后检测药效学指标；RT-PCR、Western-Blot测定肾组织Notch信号通路相关蛋白的表达；免疫组化验证相关蛋白表达；电镜观察形态学改变;②体外实验：培养大鼠肾系膜细胞，分组同上，药物干预后，CCK-8法检测增殖活力；流式细胞术检测凋亡和周期变化；Western-Blot、ELISA、RT-PCR测定其Notch信号通路相关蛋白表达情况；电镜观察形态学改变。从细胞分子水平探讨益肾散结复方防治肾小球硬化的作用靶点及机制，揭示配伍的科学内涵。

慢性肾脏病是指各种原因引起的慢性肾脏结构和功能障碍。肾小球硬化是慢性肾脏病进展至终末期肾衰竭的主要病理基础。研究发现，系膜细胞凋亡是肾小球硬化的主要病理特征，且Notch信号通路参与细胞凋亡的发生过程。益肾散结复方可防治肾小球硬化，但配伍机制尚待研究。因此，本课题展开研究：(1)体内实验：建立阿霉素肾病大鼠模型，将其分为正常组、盐酸贝那普利组、温肾益精化瘀组、滋阴益肾化瘀组、益气化瘀组及益肾散结复方组，药物干预后检测药效学指标；RT-PCR、Western-Blot测定肾组织Notch信号通路相关蛋白的表达；免疫组化验证相关蛋白表达；电镜观察形态学改变；(2)体外实验：培养大鼠肾系膜细胞，分组同上，药物干预后，RTCA法检测增殖活力；流式细胞术检测凋亡和周期变化；Western-Blot、ELISA、RT-PCR测定其Notch信号通路相关蛋白表达情况；电镜观察形态学改变。实验初步证实：①阿霉素肾病模型可诱导系膜细胞凋亡发生；②阿霉素肾病模型可激活Notch信号通路，进而加重系膜细胞凋亡；③益肾散结复方及拆方均通过调节Notch信号通路中Notch1、Jagged1、TGF-β1、Hes1、MCP-1、Hey1相关蛋白的表达，进而抑制肾小球系膜细胞凋亡，证明益肾散结复方可有效延缓肾小球硬化。研究结论：通过体内和体外展开实验，从细胞分子水平，证明益肾散结复方防治肾小球硬化的作用靶点及作用机制，进一步揭示出配伍的科学内涵。