从TLR4/MyD88/NF-κB通路探讨丹参酮ⅡA干预动脉粥样硬化易损斑块的抗炎及免疫调节机制

With the in-depth research, the inflammation and immunity doctrine of atherosclerosis (AS) has been gradually recognized. Toll-like receptor (TLR) is a bridge between the immune reaction and chronic inflammation, which plays an important role in the occurrence and development of AS, and is considered as a new target for anti-inflammation and immunotherapy. Nevertheless, due to the particularity of immunologic function, the effectiveness and safety of TLR-blocker in clinical applications has not been confirmed. Tanshinone Ⅱ A is a natural cardio-protective agent with pleiotropic effect. However, yet it has not been reported whether Tanshinone Ⅱ A can play anti-inflammation and immune-regulation effects through intervening TLR signal transduction pathways, which lead to stabilization of vulnerable AS plaque. In this study, with the starting point of immunity and inflammatory pathway associated with TLR4/MyD88/NF-κB, and on the basis of a combined studies of in vivo and in vitro, we observe the effect of Tanshinone Ⅱ A on aorta vulnerable AS plaque of ApoE-deficient mice, and its relationship with endothelial cells, macrophage cells TLR4/MyD88/NF-κB pathway and the expression of downstream inflammatory cytokine including TNF-α, MCP, ICAM-1, MIP-1α. We aim to explore Tanshinone Ⅱ A's anti-inflammation and immune-regulation mechanism in stabilizing AS vulnerable plaque, and provide new ideas and interventions in anti-inflammation and immunotherapy for AS.

随着研究深入，动脉粥样硬化(AS)的炎症、免疫学说逐渐得到公认。Toll样受体(TLR)是免疫反应和慢性炎症之间的桥梁，在AS的发生、发展中至关重要，成为AS抗炎免疫治疗的新靶点。然而，由于免疫功能的特殊性，针对TLR的特异阻断剂在临床应用的效果及安全性尚难确定。丹参酮ⅡA是具有多效性的天然心血管保护剂，在国际上引起广泛关注。但能否通过干预TLR信号转导通路发挥抗炎及免疫调节作用，从而抗AS、稳定易损斑块？尚未见报道。本研究以免疫炎症相关TLR4/MyD88/NF-κB通路为切入点，在体与体外研究相结合，观察丹参酮ⅡA对ApoE基因缺陷小鼠主动脉AS易损斑块的影响及其与内皮细胞、巨噬细胞TLR4/MyD88/NF-κB通路和下游炎性细胞因子TNF-α、MCP、ICAM-1、MIP-1α表达的关系，探讨其稳定AS易损斑块的抗炎、免疫调节机制，以期为AS的抗炎及免疫治疗提供新的思路和干预措施。

动脉粥样硬化（atherosclerosis，AS）的炎症、免疫学说逐渐得到公认。Toll 样受体是免疫反应和慢性炎症之间的桥梁，在AS 的发生、发展中至关重要。丹参酮ⅡA（Tan ⅡA）是具有多效性的天然心血管保护剂。本研究以免疫炎症相关TLR4/MyD88/NF-κB 通路为切入点，在体与体外研究相结合，观察Tan ⅡA对ApoE基因缺陷小鼠主动脉AS 易损斑块的影响及其与巨噬细胞、内皮细胞TLR4/MyD88/NF-κB 通路和下游炎性细胞因子表达的关系，探讨了其稳定AS 易损斑块的抗炎、免疫调节机制。.1 在体实验研究：雄性ApoE-/-小鼠高脂饲料喂养和雄性C57BL/6 小鼠普通饲料喂养，共计26 周。其中ApoE-/-小鼠在13 周高脂饲料喂养AS 初步形成后，被随机分为模型组，高中低剂量Tan ⅡA组，阿托伐他汀组，C57BL/6小鼠作为正常对照组。采用苏丹IV大体染色，直接肉眼观察AS病变程度及分布范围；对ApoE-/-小鼠心脏主动脉根部制成的石蜡切片行HE法、Movat五色套染法观察AS斑块的病理形态学；免疫组化染色检测TLR4、MyD88和NF-κB；采用酶法和免疫比浊法检测总胆固醇、高密度脂蛋白、低密度脂蛋白和甘油三酯水平；采用流式高通量多因子检测技术检测炎症因子TNF-α，MCP-1；采用RT-RCR法检测TLR4、MyD88 和ICAM-1的RNA 表达水平。经过实验证实Tan ⅡA可以延缓高脂饲料喂养的ApoE-/-小鼠的AS进程，Tan ⅡA不仅降低血脂，同时也抑制炎症反应、稳定斑块，作用呈现剂量依赖性。此外，Tan ⅡA延缓AS 的作用可能涉及TLR4/MyD88/NF-κB信号通路。.2 离体实验研究：采用脂多糖诱导的RAW264.7巨噬细胞/EA.hy926内皮细胞建立炎性细胞模型，模拟巨噬细胞/内皮细胞在AS中的作用，实验分为6组，正常对照组，模型组，Tan ⅡA高中低剂量组，特异阻断剂组。用CCK-8 法检测各组细胞存活率，Western-blot法定量分析TLR4、MyD88、NF-κB 蛋白表达水平，ELISA 法检测血清炎性因子TNF-α水平。Tan ⅡA可抑制巨噬细胞炎症反应，其机制与抑制TLR4/MyD88/NF-κB 通路有关，但TLR4/MyD88/NF-κB 通路并非Tan ⅡA 免疫调节和抗炎干预的唯一通路