线粒体外膜蛋白VDAC1在动脉粥样硬化斑块巨噬细胞极化中的作用及机制研究

Macrophage (Mφ) polarization into M1 phenotype is the preliminary cause that leads to inflammation exacerbation of vulnerable atherosclerotic plaque and its rupture, while the underlying mechanisms governing Mφ polarization are still not well defined. Our previous studies demonstrated that (i) ox-LDL incubation could up-regulate outer mitochondrial membrane protein VDAC1 expression. (ii) increased VDAC1 expression could drive Mφ to polarize towards M1 phenotype, while VDAC1 inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) could reverse this process. (iii) inhibiting mitochondrial energy metabolism could drive Mφ to polarize towards M1 phenotype. Further studies showed that increased VDAC1 expression could enhance the interaction with endoplasmic reticulum protein inositol 1,4,5-triphate receptors (IP3Rs), which leaded to mitochondrial calcium overload. Thus, we propose that accumulated ox-LDL could up-regulate Mφ VDAC1 expression and strengthen its interaction with IP3Rs, which leads to mitochondrial calcium overload and energy metabolic disorders, and eventually promotes Mφ to polarize towards M1 phenotype. By applying mouse-derived and human-derived Mφ, and apolipoprotein E knockout (ApoE-/-) mice model, we are going to use Vdac1 shRNA and over-expression skills to verify the hypothesis, on the cellular biology and molecular biology basis. Execution of the project is going to preliminarily illustrate the molecular mechanism of Mφ polarization during atherosclerosis (AS) progression from a novel aspect, which will provide new theoretical foundation and a practical intervention target to atherosclerotic plaque applying inflammation-regulation treatment aiming at Mφ polarization.

巨噬细胞（Mφ）向M1极化是动脉粥样硬化（AS）易损斑块炎症加剧并发生破裂的主要原因，但Mφ极化的调控机制仍尚未完全阐明。我们前期研究发现：ox-LDL可上调线粒体外膜蛋白VDAC1表达量，促进Mφ向M1极化，VDAC1抑制剂DIDS可逆转此过程，且抑制线粒体能量代谢可促使Mφ向M1极化。进一步研究发现上调VDAC1可增强与内质网IP3Rs相互作用，引起线粒体钙超载。据此我们提出假说：积聚的ox-LDL上调VDAC1表达量，促进与IP3Rs相互作用，引起线粒体钙超载及能量代谢障碍，最终促使Mφ向M1极化。项目拟应用小鼠和人来源分化成的Mφ及ApoE-/-小鼠模型，采用Vdac1 shRNA和过表达等手段，在细胞生物学和分子生物学层面证实上述假说。项目的实施将从新的角度初步阐明在AS发展过程中Mφ极化的分子机制，为将来针对Mφ极化进行AS斑块炎症调节治疗提供新的理论依据和切实可行的干预靶点。

巨噬细胞（Mφ）向M1极化是动脉粥样硬化（AS）易损斑块炎症加剧并发生破裂的主要原因，但Mφ极化的调控机制仍尚未完全阐明。我们在前期研究基础上提出假说：积聚的氧化低密度脂蛋白（ox-LDL）上调线粒体外膜蛋白VDAC1表达量，促进与IP3Rs相互作用，引起线粒体钙超载及能量代谢障碍，最终促使Mφ向M1极化。项目当前主要应用小鼠骨髓来源分化成的Mφ，ox-LDL孵育诱导成泡沫细胞，应用VDAC1抑制剂DIDS及Vdac1 shRNA慢病毒技术，前期基础及后期研究发现：ox-LDL可上调Mφ VDAC1表达量，用Western blot发现M1相关蛋白p-STAT-1表达量增加，用流式细胞术发现VDAC1含量增加促进Mφ向M1极化，用Vdac1 shRNA慢病毒干扰技术可抑制此过程；同时发现ox-LDL可诱导Mφ线粒体能量代谢减弱，而用抑制剂DIDS或Vdac1 shRNA可逆转此过程；同时，用ATP合成酶抑制剂oligomycin可促进Mφ向M1极化。最后，还发现VDAC1表达量的增加可增加线粒体内钙含量。项目的实施从新的角度发现了AS发展过程中Mφ极化的另一分子机制，可能为将来针对Mφ极化进行AS斑块炎症调节治疗提供新的理论依据和可行的干预靶点。