基于IKK/IκB/NF-κB信号通路的苓桂术甘汤干预心梗后心室重构的机制研究

Ventricular remodeling is the important pathophysiology foundation in the occurrence and development of chronic heart failure（CHF）. IKK/IκB/NF-κB signal pathway is closely related to the activation of neuroendocrine inflammatory factor of ventricular remodeling after myocardial ischemia, and it is an important target for intervention of ventricular remodeling. Traditional Chinese Medicine believes that CHF belongs to the syndrome of deficiency of Ben and repletion of Biao. Deficiency of Heart Yang and Qi runs through the occurrence and development of the whole process of CHF. Warming-yang and benefiting-qi is the basic therapeutic methods of CHF. The previous findings showed, as the representive prescription of warming-yang and benefiting-qi, Lingguizhugan Decoction could inhibit the over-expressions of the neuroendocrine inflammatory factor of TNF-α, IL-1β, IL-6, AngⅡ, Ald, ET-1, BNP and the nuclear transcription factor of NF-κB, and also inhibit ventricular remodeling of rats with CHF and improve the diastolic and systolic function of heart. Based on the preceding research, this program will use research methods of the combination of in vitro and in vivo. Serum pharmacological method, Western-blot, RT-qPCR, ELISA, immunofluorescence and flow cytometry and other modern molecular biology techniques will be used to intensively study the relationship between the effect of Lingguizhugan Decoction on ventricular remodeling and IKK/IκB/NF-κB signal pathway, find its target further reveal the biological foundation of Lingguizhugan Decoction in the prevention and treatment of CHF to provide theoretical evidence of its clinical application.

心室重构是CHF发生发展的重要病理生理学基础，心室重构过程中神经内分泌细胞因子的激活与IKK/IκB/NF-κB信号通路关系密切，阻断IKK/IκB/NF-κB信号通路，是阻抑心室重构、防治心衰的重要策略之一。中医认为，CHF属本虚标实之证，心阳（气）亏虚贯穿CHF发生发展的始终，温阳益气乃其基本治法。既往研究表明，温阳益气代表方苓桂术甘汤能够抑制CHF模型大鼠神经内分泌炎性细胞因子BNP、AngⅡ、AT1R、Ald、ET-1、TNF-α、IL-6、IL-1β和核转录因子NF-κB过度激活、阻抑心室重构、改善心功能。本项目拟在前期基础上，采用体内外相结合的研究方法，运用Western blot、RT-qPCR、ELISA、免疫荧光、激光共聚焦技术和流式细胞术等现代分子生物学技术，从IKK/IκB/NF-κB信号通路入手，深入探讨苓桂术甘汤干预心室重构的分子机制，为该方临床应用提供实验依据。

心室重构是慢性心衰发生发展的重要病理生理学基础，心室重构过程中神经内分泌细胞因子的激活与IKK/IκB/NF-κB信号通路关系密切，阻断IKK/IκB/NF-κB信号通路，是阻抑心室重构、防治心衰的重要策略之一。本项目在既往研究基础上，体内体外研究相结合深入研究苓桂术甘汤抑制炎性细胞因子过度激活、干预心室重构与IKK/IκB/NF-κB信号通路的相关性，寻找其调控靶点。.主要研究成果：.1. 苓桂术甘汤能明显减少AMI后心室重构模型大鼠心肌组织炎性细胞浸润，减轻心肌细胞损伤，减少胶原纤维沉积，降低模型大鼠血清TNF-α、IL-1β 及IL-6含量，增强模型大鼠心肌组织IKKβ和IκBα的蛋白表达、抑制p-IκBα和NF-κBp65蛋白表达。表明其能调控心肌组织IKK/ IκB/NF-κB信号通路相关蛋白表达及磷酸化水平、抑制NF-κB信号通路过度激活，发挥保护心肌细胞损伤、阻抑心室重构作用（in vivo）。.2．苓桂术甘汤能够显著降低TGF-β1 损伤的心肌细胞死亡率和凋亡率，下调caspase-3和caspase-8表达，保护心肌细胞损伤、抑制心肌细胞凋亡；能明显抑制LPS诱导的乳鼠心细细胞炎性细胞因子TNF-α、IL-1β和IL-6过表达，上调IKKβ、IκBα蛋白表达、下调p-IκBα、NF-κBp65蛋白表达和核移位；基因沉默实验显示：PDTC与苓桂术甘汤均能显著降低LPS诱导的乳鼠心肌细胞NF-κBp65蛋白表达及p65mRNA转录水平，抑制TNF-α、IL-1β和IL-6过表达；TPAC-1与苓桂术甘汤均能明显提升IKKβ表达水平，抑制p-IκBα及NF-κBp65表达（in vitro）。.以上研究结果表明，苓桂术甘汤抑制炎性细胞因子过度激活、保护心肌细胞损伤、抑制心室重构与其调控IKK/IκB/NF-κB信号通路蛋白表达及磷酸化水平关系密切；能拮抗心肌细胞IKKβ活化、降解，减少IκBα磷酸化，抑制NF-κB解离与核移位，调控对下游靶基因的转录复制，进而抑制炎性细胞因子过表达，IKKβ / IκBα / NF-κB信号通路可能是关键调控靶点之一。.本项目初步阐明了苓桂术甘汤阻抑AMI后心室重构与调控IKK/IκB/NF-κB信号通路的相关性，初步明确了IKKβ/IκBα/NF-κB可能是其抑制炎性细胞因子过度激活、阻抑心室重构的关键调控靶点之一。