基于PI3K/Akt通路调控DNA甲基化研究参元丹胶囊治疗动脉粥样硬化胰岛素抵抗的分子机制

A large number of studies have indicated that DNA methylation is one of important mechanisms for the occurrence of atherosclerosis (AS). Aberrant methylation can affect the expression of insulin resistance (IR)-related genes and their regulatory pathways, and further result in the occurrence of AS. Our previous studies showed that the promoter regions of IR related genes (IRS-1 and Pde3B) in AS mouse aorta were highly methylated, and were regulated by PI3K/Akt pathway. Promoting Qi and removing blood stasis is an important treatment on coronary heart diseases in traditional Chinese medicine, Shen-yuan Dan capsule has the inhibitive effects on AS and increased the level of DNA methyl transfer enzyme (DNMT) in atherosclerosis mice serum and decreased the insulin level, but it remains unclear whether Shenyuan-Dan can inverse aberrant methylation of IR-related genes to exert anti-atherosclerotic effects through regulating PI3K/Akt pathway. Therefore, we put forward the hypothesis: "Shen Yuan Dan capsule, one of the typical prescription with the effect of promoting Qi and removing blood stasis, can regulate PI3K/Akt pathway, reverse the DNA methylation status of IR-related genes, and further exert anti-atherosclerotic effects ". Therefore, this study is designed to adopt ApoE-/- mice fed with high-fat diet as AS experimental model to study the effect of Shen Yuan Dan capsule on PI3K/Akt pathway and IR gene methylation status in the process of exerting anti-atherosclerotic effect and adopt adipose cells as target cells, the DNMT inhibitors and inhibitors of the PI3K/Akt pathway were combined used to study the roles of DNA methylation and PI3K/Akt pathway in the process of Shen Yuan Dan improving IR. It provides a new way to study the mechanism oif Shen Yuan Dan capsule preventing AS, one of the typical prescription with the effect of promoting Qi and removing blood stasis.

大量研究表明，DNA甲基化是动脉粥样硬化(AS)发生的重要机制。甲基化异常可影响胰岛素抵抗（IR）相关基因及通路表达，导致AS发生。前期发现，AS小鼠主动脉IR相关基因启动子区发生高度甲基化，并受PI3K/Akt通路调控。益气逐瘀是中医治疗冠心病的重要治法，既往发现益气逐瘀代表方参元丹胶囊可抑制AS，与提高血清甲基化转移酶和降低胰岛素水平有关，但其是否通过调控PI3K/Akt通路逆转IR基因甲基化发挥抗AS作用尚不清楚。因此提出假说：“益气逐瘀代表方参元丹可调控PI3K/Akt通路，逆转IR相关基因甲基化异常，发挥抗AS作用”。本课题以高脂喂养的ApoE-/-小鼠为观察对象，研究参元丹抗AS过程对IR基因甲基化的影响，并以脂肪细胞为靶细胞，用DNMT抑制剂和通路抑制剂联合干预，研究PI3K/Akt通路在参元丹改善IR基因甲基化异常过程中的作用，为明确参元丹防治AS的作用机制提供实验学依据

大量研究表明，DNA甲基化是动脉粥样硬化(AS)发生的重要机制。甲基化异常可影响胰岛素抵抗（IR）相关基因及通路表达，导致AS发生。前期发现，AS小鼠主动脉IR相关基因启动子区发生高度甲基化，并受PI3K/Akt通路调控。益气逐瘀是中医治疗冠心病的重要治法，既往发现益气逐瘀代表方参元丹胶囊可抑制AS，与提高血清甲基化转移酶和降低胰岛素水平有关，但其是否通过调控PI3K/Akt通路逆转IR基因甲基化发挥抗AS作用尚不清楚。因此提出假说：“益气逐瘀代表方参元丹可调控PI3K/Akt通路，逆转IR相关基因甲基化异常，发挥抗AS作用”。研究结果显示：参元丹干预6周后，与模型组相比，参元丹胶囊可显著改善AS小鼠血脂水平、AS斑块面积、AI指数、血清NEFA、FBG、FIns和HOMA-IR水平。参元丹胶囊显著降低AS小鼠血清基因组DNA甲基化水平和DNMT1水平，可显著增加AS小鼠主动脉斑块内GLUT-4和PPAR-γ表达面积和蛋白水平、可明显改善AS小鼠胰岛素抵抗基因IRS-1mRNA水平和启动子DNA甲基化水平，可抑制AS小鼠主动脉胰岛素抵抗信号通路PI3K/Akt通路的活化。参元丹胶囊可减轻AS小鼠肝脏内的脂滴形成，可显著增加AS小鼠肝脏内GLUT-4和PPAR-γ表达面积和蛋白水平，可显著抑制AS小鼠肝脏内胰岛素抵抗信号通路PI3K/Akt通路的活化。参元丹胶囊可通过促进胰岛素抵抗脂肪细胞PI3K/Akt信号通路的活化，明显改善脂肪细胞胰岛素抵抗基因IRS-1mRNA水平和启动子DNA甲基化水平，从而促进脂肪细胞对葡萄糖的吸收能力。在课题执行过程中，发表SCI论文4篇、核心期刊论文2篇。获得中华中医药学会一等奖1项，北京市科学技术进步三等奖1项，中国中西医结合学会心血管专委会科技奖一等奖1项。获得国家发明专利授权1项。培养研究生5名。做国内外学术汇报交流5次。