慢性心理应激促进动脉粥样硬化病变的HMGB1/TLR4途径依赖机制

Our previous studies indicated that chronic psychological stress accelerated the development of atherosclerosis(As),and increased the expressions of TLR4 and inflammatory cytokines. Preliminary studies suggested HMGB1/TLR4 signaling cascade might be associated with inflammation induced by psychological stress. Combined with recent literatures, we hypothesize that the HMGB1/TLR4 pathway promotes the development and plaque instability of As induced by chronic psychological stress through inflammation responses.The effect of chronic psychological stress on HMGB1 translocation, release and localization will be visualized with immunofluorescent staining, and the effects on the expressions of HNGB1/TLR4 in mice will be detected by RT-PCR, western blot and immunohistochemical analyses, respectively. Furthermore, we use an anti-HMGB1 neutralizing monoclonal antibody to determine whether endogenous HMGB1 is a critical signaling molecules which contributes to atherosclerosis and inflammation in TLR4 wild-type mice induced by the psychological stress, and the influences on leision area, plaque instability, and proinflammatory mediators(which are downstream cytokines of HMGB1/TLR4) will be assessed by oil red O, immunohistochemistry, and western bolt, respectively. Finally, to confirm whether HMGB1-mediated inflammation responses and the development of As are via HMGB1-TLR4 signaling, TLR4 over-expression ApoE-/-mice,TLR4-/-/ApoE-/- mice and ApoE-/-mice will be pretreated with rHMGB1 and underwent the chronic psychological stress, and then the effects on the degree of As lesion and inflammatory cytokines levels will be assessed with the methods mentioned above. This study will provide novel insights into the pathogenesis of atherosclerosis induced by chronic psychological stress. Targeting HMGB1/TLR4 pathway may be a novel therapeutic approach to As-related diseases.

我们前期研究发现：慢性心理应激加速动脉粥样硬化(As)发生、并增加TLR4与炎症因子表达；HGMB1/TLR4可能参与应激诱导的炎症反应。结合文献，我们提出慢性心理应激(以下简称应激)通过HMGB1激活TLR4而促进As发生发展的工作设想。本课题将观察应激As模型小鼠HMGB1、TLR4等的表达，以明确应激对HMGB1移位、释放与定位变化及HMGB1和TLR4相互作用的影响；探讨抗HMGB1中和抗体阻断对应激致As的抑制作用，以明确HMGB1是应激致As的关键信号分子；分析TLR4过表达与敲除对应激致炎症与As的影响，以证实应激通过TLR4途径促进As；探讨TLR4敲除与过表达对rHMGB1介导应激促As发生的影响，以阐明TLR4途径是HMGB1介导应激促As的重要信号级联。本项目的开展将揭示应激促进As病理进程的HMGB1/TLR4依赖机制，为As的防治拓展新思路和提供新靶点。

TLR4介导的信号途径在CUMS致As中作用机制尚不清楚。本研究探讨CUMS是否通过HMGB1/TLR4途径促进As病变。首先，我们在CUMS小鼠As模型观察HMGB1和TLR4表达变化，以明确应激对HMGB1的释放和TLR4激活的影响。随后，分别以HMGB1的选择性抑制剂丙酮酸乙酯（EP）和TLR4特异性抑制剂TAK-242干预，探讨抑制HMGB1释放和TLR4激活对慢性心理应激ApoE-/-小鼠As病变的影响。在体外慢性心理应激细胞模型，分别以EP、TAK-242和PPARγ抑制剂罗格列酮干预糖皮质激素共孵育的Raw 264.7巨噬细胞，观察阻断HMGB1/TLR4-PPARγ途径后对慢性心理应激巨噬细胞内脂质蓄积、HMGB1的释放、LXRα和ABCA1表达等的影响，以进一步阐明HGMB1/TLR4途径在CUMS促As中的作用及其机制。研究结果发现：（1）与对照组相比较，CUMS组ApoE-/-小鼠血清的皮质酮（273.36±41.65 ng/mL vs 178.01±37.73 ng/mL）、HMGB1(20.30±5.74 ng/mL vs 9.47±3.69 ng/mL)及其炎症因子IL-1β和TNF-α的水平显著升高，As病变面积（18.78% ± 5.08% vs 9.20% ± 3.61%）及HMGB1、TLR4和IL-1β蛋白的含量显著增加，而PPARγ、LXRα和ABCA1的表达则明显减低；（2）与应激组相比较，EP组和TAK-242组小鼠主动脉的As病变面积、巨噬细胞数量和炎症因子IL-1β蛋白的表达显著减少，而病变处的胶原和平滑肌细胞含量则显著增多；（3）与应激组相比较，EP组和TAK-242组小鼠血管壁PPARγ、LXRα和ABCA1的表达显著升高；（4）EP、TAK-242和罗格列酮干预均能显著抑制皮质酮诱导的巨噬细胞源性泡沫细胞形成，降低上清液中HMGB1的含量，但显著升高其PPARγ、LXRα和ABCA1的表达。综上所述，我们的研究结果表明，HMGB1激活TLR4介导的PPARγ/LXRα/ABCA1表达下调是CUMS促As病变的重要机制。本研究揭示了慢性心理应激促进As病理进程的HMGB1/TLR4依赖途径新机制，并为As的防治拓展新思路和提供新靶点。

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