GPR109A在奶牛乳腺上皮细胞炎症反应中的作用及其分子机制

Mastitis causes the greatest economic loss to dairy farming around the world. Currently, the treatment of mastitis mainly depends on antibiotic. However, it may lead to drug resistant bacteria and also antibiotic residue in the human food chain. Therefore, looking for a novel alternative therapy for bovine mastitis is an urgent topic. G protein coupled receptor109A (GPR109A) is a seven-transmembrane G-protein-coupled receptor of the Gi family. In previous study, we found that GPR109A is expressed in mammary epithelial cells from dairy cow and mice, and it may regulate LPS-induced inflammation in mammary epithelial cells. However, little is known about whether GPR109A could inhibit LPS-induced inflammation in mammary epithelial cells. Therefore, we attempt to clarify regulatory mechanisms of GPR109A on LPS-induced inflammation in mammary epithelial cells as well as its potential roles in development of mastitis, using LPS-induced inflammation in mammary epithelial cells from dairy cow and WT/ GPR109A knockout mice, LPS-induced mastitis in GPR109A knockout mice and technology such as Western blotting, Realtime-PCR, immunofluorescence, ELISA, EMSA and so on. Our finding will provide a new target for the prevention and treatment of mastitis.

乳房炎给奶牛养殖业造成巨大的经济损失。目前抗生素仍然是其预防和治疗的主要手段，但是随之带来的细菌耐药性及食品抗生素残留等问题日趋严重。因此，迫切需要寻找安全、有效的防治乳房炎的方法。GPR109A是Gi蛋白偶联的受体。本课题组前期发现在奶牛和小鼠乳腺上皮细胞中均有GPR109A表达，并且其可能具有调节LPS诱导的乳腺上皮细胞炎症反应的功能。但是GPR109A能否抑制LPS诱导的乳腺上皮细胞炎症反应，迄今国内外尚无报道。因此，本项目拟应用体外乳腺上皮细胞炎症模型及在体GPR109A敲除小鼠乳房炎模型，利用Western blotting、荧光定量PCR、免疫荧光、ELISA、EMSA等技术，从分子、细胞和整体水平探讨GPR109A对LPS诱导的乳腺上皮细胞炎症反应的影响及其分子机制，揭示GPR109A在乳房炎发生发展中的作用，以期为乳房炎的防治提供新的靶点。

乳房炎给奶牛养殖业造成巨大的经济损失。目前抗生素仍然是其预防和治疗的主要手段，但是随之带来的细菌耐药性及食品抗生素残留等问题日趋严重。本项目以寻找安全、有效的防治乳房炎的方法，系统的研究了GPR109A抗乳房炎作用及其机制。首先收集正常奶牛和乳腺炎乳腺组织，通过检测GPR109A表达量和乳腺炎症状态，分析GPR109A与乳腺炎的相关性。结果发现乳腺炎奶牛乳腺组织中的GPR109A显著升高，且乳腺中组织的炎症水平也显著升高。这一结果表明GPR109A可能与乳腺炎密切相关。为了验证GPR109A与乳腺炎的关系，我们在WT和GPR109A-/-小鼠中利用乳腺内注射LPS诱导乳腺炎小鼠模型，并饲喂GPR109A激动剂烟酸。结果发现烟酸在WT小鼠中能抑制乳腺内损伤、炎症反应以及修复血-乳屏障，而在GPR109A-/-小鼠中烟酸没有这些作用。在小鼠乳腺上皮细胞系（Eph4-Ev）的机制研究发现GPR109A活化后通过AMPK/Nrf2/自噬途径抑制炎症反应和修复血-乳屏障。接着，我们进一步在原代奶牛乳腺上皮细胞中对GPR109A的这一作用及机制进行了验证。以上结果表明GPR109A能抑制乳腺的炎症反应和修复血-乳屏障。这些结果为乳房炎的防治提供了新的靶点，对安全、有效的防治奶牛乳房炎具有推动作用。