鳖甲煎丸调控NLRP3炎症小体对糖尿病大血管病变内皮损伤保护作用的机制研究

Macroangiopathy complications are the leading cause of disability and mortality among patients with diabetes, while vascular endothelial cell injury is the initial factor and the key point of pathogenesis. The reaction mediated by NLPR3 inflammatory cell activation plays an important role during the whole porcess. Preliminary studies have shown that Biejiajian Pill has the effect of improving vascular endothelial function, anti-inflammatory and plaque stability, but the mechanism has not been elucidated. The following studies will be conducted as:①using ApoE-/- mice injected with STZ to establish AS model of diabetes mellitus as the study objects while applying techniques of electron microscope, QPCR, Western blotting in order to observe the vascular endothelial cell factor, tight junctions and the indicators of signaling pathway (NLRP3、caspase-1、IL-1β);②Using the NLRP3 gene silencing method on high glucose injury HUVECs, applying confocal laser and molecular biology technology, to oberve the permeability of endothelial cell, tight junctions(ZO-1、F-actin) and the NLRP3 inflammasome signaling pathway. Through this study, we want to confirm that Biejiajian pill could protect against vascular endothelial cell injury in diabetes mellitus by regulating NLRP3 inflammasome，which would provide a new scientific basis for the later clinical application.

大血管并发症是糖尿病患者致残、致死的首要原因，血管内皮细胞损伤是发病的始动因素和关键环节，NLRP3炎症小体激活介导的炎症反应在此过程中发挥了重要的作用。前期研究显示鳖甲煎丸具有改善血管内皮功能、抗炎及稳定斑块等作用，但机制尚未阐明。本课题拟开展①以ApoE-/-小鼠注射STZ建立糖尿病AS模型为研究对象，采用电镜、qPCR、Western blot等技术，观察血清内皮细胞因子（NO、ET、NOS等）、紧密连接、信号通路（NLRP3、caspase-1、IL-1β等）指标；②采用NLRP3基因沉默的方法，以高糖损伤HUVECs为对象，应用激光共聚焦、分子生物学等技术，观察内皮细胞通透性、紧密连接相关蛋白（ZO-1、F-actin）、NLRP3炎症小体信号通路等。围绕NLRP3炎症小体的活化与调控，阐释鳖甲煎丸治疗糖尿病大血管病变内皮损伤的机制，为临床应用提供新的思路和靶点。

大血管并发症是糖尿病患者致残、致死的首要原因，血管内皮细胞损伤是发病始动因素和关键环节，NLRP3炎症小体激活介导的炎症反应在此过程中发挥重要的作用。本研究提出“鳖甲煎丸可通过调控NLRP3炎症小体活化减轻血管内皮细胞损伤，从而治疗糖尿病大血管病变”的假说，从体内外两个层面开展研究。一是在动物实验方面，构建了糖尿病动脉粥样硬化小鼠模型，采用油红O染色和HE染色评价主动脉斑块和内皮损伤情况，生化分析仪检测血糖、血脂，ELISA法检测炎症因子和内皮因子水平，透射电镜观察线粒体和自噬，qPCR、Western blot检测NLRP3炎症小体信号通路相关基因和蛋白表达。结果显示鳖甲煎丸具有：①降低血清IL-1β、IL-18炎症因子水平；②降低TC、TG、LDL-C和升高HDL-C的水平，减低ET-1和升高eNOS水平；③缩小主动脉斑块面积；④降低TXNIP、NF-κB、NLRP3、ASC、caspase-1、IL-1β的mRNA和蛋白表达。二是在细胞实验方面，构建了高糖损伤HUVECs模型，制备了鳖甲煎丸含药血清，利用siRNA转染技术沉默NLRP3基因，采用ELISA方法检测炎症因子和内皮因子水平，Transwell技术测定内皮细胞通透性，免疫荧光分析紧密连接蛋白ZO-1、F-actin的表达，qPCR、Western blot技术检测NLRP3炎症小体信号通路相关基因和蛋白表达。结果显示鳖甲煎丸含药血清具有：①降低促炎因子IL-1β、IL-18释放水平；②降低ET-1、TXA2水平，升高NO、NOS、PGI2水平；③降低荧光素钠透过血管内皮细胞屏障的渗透率；④上调ZO-1、F-actin的表达水平；⑤降低TXNIP、NF-κB、NLRP3、ASC、caspase-1、IL-1β的mRNA和蛋白的表达。上述结果提示鳖甲煎丸通过调控NLRP3炎症小体活化减轻血管内皮细胞损伤，进而发挥治疗糖尿病大血管病变的作用。本研究结果将为丰富“脉积”理论的现代科学内涵和临床应用鳖甲煎丸治疗糖尿病大血管病变提供实验依据。