基于SIRT3调控能量敏感通路AMPK/PCG-1/CPT1A促进肾纤维化PTCs生成机制研究“益肾活血法”抗肾纤维化效应

Renal fibrosis is the final common pathway of all chronic kidney disease (CKD) developing to end-stage renal disease (ESRD),and the peritubular capillary(PTC) injury is the key factor of RIF. A latest study has found that the energy metabolism disorder is the vital factor which cause Vascular endothelial cells (EC) damage. Preliminary studies confirm that the tonifying the kidney and activating blood resisting herbal formulas can promot angiogenesis and antifibrosis.We propose that the tonifying the kidney and activating bloodresisting herbal formulas can promote PTC damage repair by improving the EC energy metabolism. With the purpose of researching the regulatory mechanism of sirt3-AMPK/PCG-1/CPT1A signal pathway and testing the intervention effect of tonifying the kidney and activating blood resisting prescription，we’ll use SIRT3 knockout mice as the experimental animal model, and establish the ischemia/hypoxia HUVECs cultured system in vitro. This study aims to exploring PTC damage mechanisms in RIF from the perspective of EC energy metabolism, demonstrating the renal fibrosis "toxic stasis" biological essence from the molecular level, and testifying that the method of tonifying the kidney and activating blood plays a role of intervention in RIF and microvascular damage by regulating sirt3-AMPK/PCG-1/CPT1A signal pathway, to provides objective basis to further the reserch on the relations among energy metabolic disorders, endothelial cells and RIF.

肾间质纤维化（RIF）是所有CKD发展至终末期肾病的最后共同通路，而肾周毛细血管（PTC）损伤是RIF发生、发展的关键环节。最新研究发现能量代谢障碍是导致血管内皮（EC）损伤的重要因素。前期研究证实益肾活血方具有抗RIF和促血管新生作用。我们假说益肾活血方可以通过改善EC能量代谢促进PTC损伤修复。本研究以SIRT3基因敲除小鼠作为实验动物模型，并建立体外类缺血/缺氧HUVECs培养体系，研究SIRT3对能量敏感通路AMPK/PCG-1/CPT1A的调控机制，以及益肾活血方对RIF内皮细胞能量代谢和血管新生的干预作用。本研究旨在从分子水平阐释肾纤维化“久病入络”的生物学本质，从能量代谢障碍与EC损伤关系探索肾纤维化PTC受损新机制，证实益肾活血方通过调控SIRT3-AMPK/PCG-1/CPT1A通路发挥抗RIF作用，为进一步研究能量代谢-内皮细胞-肾纤维化三者的内在联系提供重要实验依据。

肾间质纤维化（RIF）是各种慢性肾脏疾病（Chronic kidney disease, CKD）和进行性肾脏疾病的最后因素，而肾周毛细血管（PTC）损伤是RIF发生、发展的关键环节。最新研究发现能量代谢障碍是导致血管内皮（EC）损伤的重要因素。本研究以UUO小鼠作为实验动物模型，予以益肾活血方（低、中、高）剂量及白藜芦醇灌胃给药，并建立体外类缺血/缺氧HUVECs培养体系，予以含药血清、白藜芦醇进行干预，分别检测肾脏及HUVECs的Ang-1/Tie-2、VEGF、NOTCH3、VE-cadherin、occludin、SIRT3、AMPK、PCG-1、CPT1A mRNA及蛋白表达 ；免疫荧光技术检测Ang-1/Tie-2、VEGF、NOTCH3、VE-cadherin、occludin、SIRT3、AMPK、PCG-1、CPT1A；MTT比色实验检测细胞活性和流式细胞仪检测细胞凋亡情况；采用HE、Masson染色及检测BUN、Scr指标观察肾组织纤维化情况与肾功能状况；免疫组化检测CD31及第Ⅷ因子；免疫共沉淀检测SIRT3、AMPK、PCG-1、CPT1A；观察益肾活血方对RIF血管内皮功能结构及细胞能量的影响。益肾活血方治疗减少了UUO模型小鼠的BUN、Scr浓度、ROS含量、α-SAM、Col-Ⅳ表达，增加了CD31、Ang-1/Tie-2、VEGF、VE-cadherin、Occludin、AMPK、SIRT3、CPT1A、PGC-1α表达。在体外，益肾活血方增加了细胞活性，并增加了在缺氧条件下的耐受性。益肾活血方通过激活AMPK/PGC-1α/Sirt3信号抑制ROS的生成。本研究旨在从分子水平阐释RIF“久病入络”的生物学本质，从能量代谢障碍与EC损伤关系探索RIF的PTC受损新机制，证实益肾活血方通过调控SIRT3-AMPK/PCG-1/CPT1A通路发挥抗RIF作用，为进一步研究能量代谢-内皮细胞-肾纤维化三者的内在联系提供重要实验依据，为益肾活血方在临床上治疗RIF提供科学依据。