TLR4信号通路介导游离脂肪酸诱导血管平滑肌细胞氧化应激的分子机制

Inflammation and oxidative stress play a critical role in the development of the macrovascular complications in type 2 diabetes, and free fatty acids (FFA) is the major cause of this progress. TLR4 is a key molecular link between vascular inflammation, insulin resistance and innate immune system. For the first time we want to clarify whether TLR4 signaling pathway mediate the oxidative stress induced by FFA in HVSMC, and further, we will investigate the underlying molecular mechanisms of how FFA induce oxidative stress and inflammation via TLR4 pathway in HVSMC. The contents of this study including: First, through blocking TLR4 signaling with siRNA method, to clarify whether TLR4 pathway is involved in oxidative stress induced by FFA in HVSMC. Second, to identify which pathway that downstream of the TLR4 signaling plays crucial role in generation of reactive oxygen species (ROS) induced by FFA in HVSMC. Third, to study how TLR4 signaling pathway regulate the oxidant and antioxidant system, and further to find out the critical oxidant and antioxidant enzymes that responsible for generation of ROS during oxidative stress stimulated by FFA in HVSMC. Fourth, to investigate how ROS regulate nuclear factor-КB （NF-КB ） and its downstream inflammatory cytokines in HVSMC. Finally,to observe the effects of blocking TLR4 signaling using siRNA method on proliferation, migration and apoptosis of HVSMC induced by FFA. This study will eluciate the important role played by TLR4 pathway in inflammation of HVSMC and the development of macrovascular complications in type 2 diabetes, and will be helpful to explore a new anti-inflammation therapy that targeting TLR4 for treatment of type 2 diabetes in the future.

氧化应激和炎症是2型糖尿病大血管并发症的主要病理机制，游离脂肪酸（FFA）是主要致病因素。TLR4很可能是联系2型糖尿病天然免疫、血管炎症反应和胰岛素抵抗的关键因子。本项目以TLR4信号通路对血管平滑肌细胞(HVSMC)氧化应激的调控为切入点，深入研究FFA诱导HVSMC产生氧化应激和炎症的分子机制。内容：①利用siRNA技术阻断TLR4信号，研究TLR4是否介导FFA诱导HVSMC氧化应激；②TLR4信号通路介导FFA诱导HVSMC氧化应激的下游信号途径；③TLR4信号通路对HVSMC氧化/抗氧化酶系统的调节作用；④氧化应激对NF-κB及其下游炎症因子的调节作用；⑤阻断TLR4信号对HVSMC增殖、迁移和凋亡等功能的影响。意义：有助于阐明TLR4信号通路在HVSMC炎症反应和2型糖尿病大血管并发症发病机制中的重要作用，为今后将TLR4做为2型糖尿病抗炎靶标和探索新的干预途径提供理论依据。

研究背景：氧化应激和炎症是2型糖尿病大血管并发症的主要病理机制，游离脂肪酸（FFA）是主要致病因素。本项目以TLR4信号通路对血管平滑肌细胞(VSMCs)氧化应激的调控为切入点，深入研究FFA诱导VSMC产生氧化应激和炎症的分子机制。.研究内容：①TLR4信号通路是否介导FFA诱导VSMC氧化应激；② TLR4信号通路介导FFA诱导VSMC氧化应激的下游信号途径；③ TLR4信号通路对VSMCs氧化/抗氧化酶系统的调节作用；④ 氧化应激对NF-κB及其下游炎症因子的调节作用；⑤ 阻断TLR4信号对VSMCs增殖、迁移和凋亡等功能的影响。.研究结果：① TLR4敲除和ROS抑制剂 apocynin和二苯基碘（DPI）显著阻断Palmitate刺激的ROS生成，而MAPK抑制剂PD98059和SB203580，PKC抑制剂chelerythrine和calphostin C, PI3K抑制剂wortmannin对Palmitate诱导VSMCs的ROS生成无明显作用。② TLR4 敲除显著抑制Palmitate诱导VASMCs 的NOX1和NOX4 mRNA和蛋白的表达。③ TLR4敲除及NF-κB阻断剂SM-7638及apocynin和DPI显著抑制Palmiate刺激的VASMCs NF-κB p65活性及MCP-1、IL-6和TNF-a的表达。④ TLR4敲除和SM-7638 则显著抑制Palmitate（50 μmol/L）刺激的VSMCs 增殖和迁移。TLR4敲除及apocynin和DPI则显著抑制Palmitate（100 μmol/L）诱导的ROS产生和VSMCs 凋亡。结论：Palmitate通过激活TLR4/ROS/NF-κB通路诱导VSMCs产生炎症反应；较低浓度（50 μmol/L）促进VSMCs 增殖和迁移，而较高浓度的Palmitate（100 μmol/L）则促进VSMCs 凋亡，阻断TLR4信号显著抑制Palmitate诱导的VSMCs细胞增殖、迁移和细胞凋亡。.研究意义：有助于阐明TLR4信号通路在VSMCs炎症反应和2型糖尿病大血管并发症发病机制中的重要作用，为今后将TLR4作为2型糖尿病抗炎治疗的新靶标和探索新的干预途径提供了有力的理论依据，具有较高的理论研究价值和一定的临床应用前景。