基于IRE1/NF-κB通路的丹皮萜苷组分调控肾系膜微炎症作用及机制研究

The imbalance of endoplasmic reticulum stress (ERS) is one of the important mechanisms of microinflammatory response in mesangial cell early damage of diabetic nephropathy (DN). Studies demonstrated that IRE1/NF-κB pathway is one of the most sensitive targets of the intervention mechanism. Our preliminary research suggested that the terpene glycoside component of Mudanpi might reduce mesangial microinflammation response through regulating ERS imbalance, however, the underlying mechanism is still unknown. Based on our preliminary studies, we proposed a scientific hypothesis on “Regulatory of terpene glycoside of Mudanpi on glomeruli mesangial micro-inflammation caused by ERS imbalance may be associated with IRE1/NF-κB pathway”. This project intends to adopt AGEs-induced mesangial HBZY-1 cell microinflammatory model and STZ-induced DN mice model, uses siRNA and other techniques to intervene specifically IRE1/NF-κB pathway, reveals the regulation features and molecular mechanism of terpene glycoside component of Mudanpi on ERS imbalance-mediated glomeruli mesangial microinflammation, and to verify its action characteristics for JNK/AP1 and IKK/IκB signaling events in IRE1/NF-κB pathway, and finally reveal its underlying mechanisms and targets. This research project investigates innovatively the mechanism of terpene glycoside component of Mudanpi on regulating glomeruli mesangial pathology from microinflammation and endoplasmic reticulum stress imbalance, and it has an important theoretical and practical significance in depth understanding for the prevention and treatment mechanism of Mudanpi on DN pathological lesions and promoting the development of innovative drugs for targeting interventions for DN.

内质网应激(ERS)失衡是糖尿病肾病(DN)系膜细胞损伤早期微炎症反应的重要机制之一。 IRE1/NF-κB通路是该机制最为敏感的干预靶标之一。我们前期研究提示牡丹皮萜苷组分可能通过调控ERS失衡减轻系膜微炎症反应，但作用机制不明确。本项目在前期研究基础上，以“丹皮萜苷组分调控肾系膜ERS失衡所致的微炎症作用可能与IRE1/NF-κB通路密切相关”为科学假说，采用AGEs诱导HBZY-1肾系膜细胞的微炎症模型和STZ诱导的DN小鼠模型，借助siRNA等技术特异性干预IRE1/NF-κB，研究丹皮萜苷组分调控系膜ERS失衡所致的微炎症作用特点与分子机制，验证其对IRE1/NF-κB通路的作用特点，揭示其潜在作用机制和靶标。本项目创新地从微炎症和ERS失衡角度研究丹皮萜苷组分调控肾系膜病理的作用机制，对于深入理解丹皮防治 DN机制和推动靶向干预DN的创新药物研发具有重要理论和实际意义。

本课题基于IRE1/NF-κB信号通路，采用AGEs诱导系膜ERs模型和STZ诱导的DKD大鼠模型，借助siRNA，活化工具药物、荧光标记、质粒转染等技术，阐明丹皮萜苷组分（MCTG）调控系膜内质网应激失衡所致的微炎症作用机制。研究发现如下：.1.MCTG 能够有效降低DKD大鼠的血清和肾组织中GRP78/Bip、XBP-1(s)的异常表达。电镜观察了MCTG可以显著改善AGEs作用下的内质网的结构损伤情况，改善系膜细胞中内质网的超微结构以及粗面内质网扩张、囊泡化，部分出现脱颗粒现象，抑制滑面内质网增生。MCTG可以显著降低肾组织、外周血及细胞培养液中炎症因子IL-6、MCP-1、ICAM-1的表达水平。研究结果揭示出内质网与炎症反应的密切关系以及MCTG通过抑制ERs作用减缓炎症损伤。.2. MCTG干预后，对p-IRE1a的抑制作用较ATF-6、PERK效果更为显著，抑制p-NF-kB p65的表达。MCTG与4-PBA联合干预时，抑制NF-κB p65的磷酸化更为显著。结果表明MCTG可通过调节IRE1/NF-κB通路抑制ERs介导的炎症反应发挥保护DKD大鼠肾损伤的作用。ATF6抑制剂AEBSF及PERK抑制剂GSK2656157的干预并未显著降低AGEs引起的炎症因子异常表达。在阻断PERK、ATF6通路的情况下，IRE1α siRNA能够显著抑制AGEs诱导的炎症反应。尤其在IRE1α siRNA、NF-κBp65 siRNA的联合干预下，异常表达的炎症因子被有效降低。.3. 毒胡萝卜素(TG)能够明显提高HBZY-1细胞IRE1α及NF-κBp6的磷酸化水平，能够显著促进IL-6、MCP-1及ICAM-1的表达；MC-TG干预能显著抑制TG诱导的炎症因子的异常水平。NF-κB抑制剂PDTC能够显著下调IL-6及MCP-1的过度表达。以上结果表明，MCTG能够通过抑制IRE1α/NF-κBp65通路的激活，改善内质网应激介导的炎症反应。MCTG不能显著降低JNK磷酸化水平；但是MCTG能够显著降低 NF-κBp65、IκBα、IκKα 的磷酸化表达。.本课题研究表明MCTG能介导IRE1/IκK/IκB/NF-κB通路抑制AGEs诱导的系膜细胞的内质网应激及微炎症反应，减缓糖尿病肾病的病理进程，为有效地防治糖尿病肾病提供了依据和支撑。