基于ERK1/2通路MicroRNAs调控探讨泽泻汤对AS VSMC迁移增殖的机制研究

Atherosclerosis(AS) has the great dangerous effects on human health. Lipid deposition, inflammation, and the abnormal secretion of matrix metalloproteinases (MMPs) are closely related with AS. The macrophages and vascular smooth muscle cells (VSMC) play an indispensable role in the process. Our previous studies showed that Alisma Decoction could attenuate lipid deposition and inhibit inflamation in macrophages-derived foam cells. However, the impact of Alisma Decoction on VSMC has not been reported until now.Several studies show that MicroRNAs(MiRNA-143 145 29b 31)related with ERK1/2 singal are associated with VSMC migrant and proliferation. This project utilizes Alisma Decoction to treat Ox-LDL loaded VSMC model. Then we detect the phenotypic transformation marker proteins SM22α and Osteopontin expression levels, cell migrant and proliferation, MMPs/TIMPs, cell cycles and other indicators, aiming to clarify the effect of Alisma Decoction on the changes of VSMC biological behavior, such as phenotype transformation, migration and proliferation . At the same time we explore its effects on anti-AS on MiRNAs levels and its correlation with ERK1/2 pathway, in order to elucidate the mechanisms of Alisma Decoction blocking atherosclerosis.Furthermore we construct the promoter-luciferase by amplification of Alisa Decoction target gene promoter sequences to clarify the Alisa Decoction anti-AS function.

动脉粥样硬化（AS）严重危害人类健康，与脂质沉积和炎性反应相关，巨噬细胞和血管平滑肌细胞（VSMC）在其中起着重要作用。泽泻汤出自《金匮要略》，在临床上广泛用于治疗高脂血症、AS等，本项目前期研究提示泽泻汤能改善巨噬细胞源性泡沫细胞的脂质沉积，抑制炎性反应，但是泽泻汤对AS VSMC的作用及其机制尚未见报道。近来研究表明ERK1/2通路的MicroRNAs（MiRNA-143、145、 29b、 31）与VSMC迁移增殖密切相关。本项目通过泽泻汤含药血清干预Ox-LDL负荷VSMC的AS细胞模型，检测VSMC表型标志蛋白、迁移增殖、MMPs/TIMPs、EMMPRIN、细胞周期等指标，探讨泽泻汤对VSMC迁移增殖的作用，并基于ERK1/2通路MicroRNAs调控深入探索泽泻汤抗AS的作用机制；同时通过扩增泽泻汤靶基因启动子序列，构建启动子-荧光素酶报告基因，验证泽泻汤抗AS的作用靶点。

近来研究提示ERK1/2通路调控MicroRNA在VSMCs的生物学行为（表型转化、迁移增殖）有重要的作用，本研究立足前期研究基础上探讨泽泻汤通过ERK1/2通路-microRNA调控VSMCs的生物学行为。.我们成功了从大鼠主动脉分离并纯化到VSMCs，经SMA-α抗体免疫荧光鉴定证实为VSMCs；制备了泽泻汤含药血清，经MTS检测提示20%泽泻汤含药血清是干预细胞的最佳浓度；本实验采用Ox-LDLC诱导VSMCs建立AS细胞模型，我们发现泽泻汤含药血清具有抑制Ox-LDLC诱导VSMCs的迁移和表型转化，同时发现泽泻汤能显著抑制Ox-LDLC诱导VSMCs的MMP-2和MMP-9表达，抑制ERK1/2的磷酸化。在泽泻汤对VSMCs增殖的实验中，采用Brdu标记活细胞的方法，免疫组化检测Brdu标记阳性细胞实验中，发现泽泻汤在12h，24h和48h三个时间点具有明显抑制Ox-LDLC诱导的VSMCs增殖，其机制与调节MMP-2/9、Cyclin D1、Cyclin E、P27的表达有关，并与抑制ERK1/2的磷酸化密切相关，与JNK和p38信号通路未见明显相关性。采用miR 分析技术发现，与Ox-LDLC组相比，泽泻汤能够上调22个miR,下调33个miR；U0126具有类似的作用；序列分析发现ox-LDL 诱导VSMCs过表达miR-17~92 cluster (miR-17-5p, miR-18a-3p_R+2, miR-19b-1 -5p_R+1, miR-20a- 3p_R+1, miR-20a-5p, miR-92a-3p_R+1)、miR−128−1−5p_R+2、PC−3p−42332_32，泽泻汤和U0126能够抑制Ox-LDLC诱导VSMCs的miR过表达，提示泽泻汤可能作用于ERK1/2通路抑制这些miR过表达，从而发挥其抗增殖的作用。在泽泻汤主要成分-24乙酰泽泻A的研究中，发现其亦能抑制VSMCs的表型转化和迁移增殖，抑制ERK1/2的磷酸化，提示泽泻汤及其主要成分具有多途径多靶点的抗AS作用。