二甲双胍通过激活AMPK通路拮抗E.coli性乳腺炎的分子机制研究

E.coli是引起奶牛乳腺炎的常见致病菌，其主要致炎因子LPS可导致乳腺上皮细胞的急性炎性反应。前期研究发现AMPK通路参与炎性反应的调控；二甲双胍可有效激活AMPK通路并抑制炎性反应，但具体作用机制尚不清楚。因此，本项目以奶牛原代乳腺上皮细胞为体外研究模型，应用二甲双胍激活AMPKα，并以AMPKα为研究对象，利用CRISPR/Cas9技术敲除/敲入AMPKα，协同应用CHART-PCR、ChIP-PCR、EMSA、信号通路分析等技术，验证激活AMPK通路后调控E.coli诱导的细胞炎性作用，深入解析AMPK下游调控途径；在此基础上，以奶山羊为体内研究模型，明确二甲双胍通过激活AMPK通路调控E.coli性乳腺炎的作用机制。本项目旨在揭示应用二甲双胍有效激活并通过AMPKα/SIRT1/NF-κB通路拮抗E.coli性乳腺炎的分子机理，为发掘新的药物靶点防控奶牛乳腺炎提供理论依据。

E. coli is a common pathogen causing mastitis in dairy cows, and its main inflammatory factor LPS can cause acute inflammatory reaction of mammary epithelial cells. Previous studies have found that AMPK pathway is involved in the regulation of inflammatory response and metformin can effectively activate AMPK pathway and inhibit inflammatory response, but the specific mechanism is not clear. Therefore, this project takes primary bovine mammary epithelial cells as an in vitro model, uses metforminto activate AMPKα as the research object, using CRISPR / cas9 technology to knock out / in the AMPKα, together with application of CHART-PCR, ChIP-PCR, EMSA, signaling pathway analysis and other technologies to verify the regulation of E.coli induced cellular inflammation after activating AMPK pathway, and further analyzes the downstream regulators. On this basis, the mechanism of metformin regulating E.coli mastitis by activating AMPK pathway will be studied in dairy goats as verification of the results in study in vitro. This project aims to reveal the molecular mechanism of metformin effectively activating AMPKα / SIRT1 / NF-κB pathway and antagonizing E.coli mastitis, and provide theoretical basis for exploring new drug targets to prevent and treat bovine mastitis.