背景:肾素-血管紧张素-醛固酮系统(RAAS)激活和氧化应激的肾纤维化是高血压的主要并发症之一。2-苯乙酰胺(PA),一种主要的活性成分。(L.A),具有多种药理作用。其类似物具有抗肾脏纤维化和减轻肾脏损伤的作用。本研究旨在基于MAPK通路介导的RAAS和氧化应激,探讨PA调控SHR肾纤维化的潜在机制。
方法:以SHR大鼠为高血压模型,以WKY大鼠为对照组。血压(BP),每周检测尿量。PA治疗4周后,RAAS的水平,通过酶联免疫吸附测定(ELISA)测量炎症和细胞因子。苏木精-伊红染色(HE),Masson和免疫组化(IHC)观察肾脏病理,胶原沉积和纤维化。Westernblot用于检测肾脏中的MAPK通路。最后,在高NaCl诱导的NRK52e细胞中使用SB203580(p38MAPK抑制剂)拮抗试验,与In-CellWestern(ICW)一起,流式细胞仪(FCM),高含量筛选(HCS)和ELISA来确认潜在的药理机制。
结果:PA降低了血压,RAAS,炎症和细胞因子,促进尿液,减轻肾脏病理损伤和胶原沉积,修复的肾纤维化,降低NADPH氧化酶4(NOX4)的表达,转化生长因子-β(TGF-β),SHR大鼠的SMAD3和MAPK信号通路.同时,,p38拮抗剂SB203580可以有效阻断PA在高NaCl诱导的NRK52e细胞中的作用。此外,分子对接表明PA占据了p38MAPK的配体结合位点。
结论:PA通过MAPK信号通路介导的RAAS和氧化应激抑制SHR大鼠肾纤维化。
BACKGROUND: Renal fibrosis with Renin-angiotensin-aldosterone system (RAAS) activation and oxidative stress are one of the major complications in hypertension. 2-phenylacetamide (PA), a major active component of
Lepidium apetalum Willd. (L.A), has numerous pharmacological effects. Its analogues have the effect of anti-renal fibrosis and alleviating renal injury. This study aims to explore the underlying mechanism of PA for regulating the renal fibrosis in SHR based on the MAPK pathway mediated RAAS and oxidative stress.
METHODS: The SHR rats were used as the hypertension model, and the WKY rats were used as the control group. The blood pressure (BP), urine volume were detected every week. After PA treatment for 4 weeks, the levels of RAAS, inflammation and cytokines were measured by Enzyme-Linked Immunosorbnent Assay (ELISA). Hematoxylin-Eosin staining (HE), Masson and Immunohistochemistry (IHC) were used to observe the renal pathology, collagen deposition and fibrosis. Western blot was used to examine the MAPK pathway in renal. Finally, the SB203580 (p38 MAPK inhibitor) antagonism assay in the high NaCl-induced NRK52e cells was used, together with In-Cell Western (ICW), Flow Cytometry (FCM), High Content Screening (HCS) and ELISA to confirm the potential pharmacological mechanism.
RESULTS: PA reduced the BP, RAAS, inflammation and cytokines, promoted the urine, and relieved renal pathological injury and collagen deposition, repaired renal fibrosis, decreased the expression of NADPH Oxidase 4 (NOX4), transforming growth factor-β (TGF-β), SMAD3 and MAPK signaling pathway in SHR rats. Meanwhile,,the role of PA could be blocked by p38 antagonist SB203580 effectively in the high NaCl-induced NRK52e cells. Moreover, molecular docking indicated that PA occupied the ligand binding sites of p38 MAPK.
CONCLUSIONS: PA inhibited renal fibrosis via MAPK signalling pathway mediated RAAS and oxidative stress in SHR Rats.