SARA产生的内源性LPS对乳腺上皮细胞增殖和凋亡的影响

Subacute ruminal acidosis（SARA）is a common metabolic disease. Abnormal elevation of lipopolysaccharide (LPS) in dairy cows can cause systemic inflammatory response, result in declined milk production when SARA occurs. At present, there is less study relating to the structure and function of mammary gland under the condition of SARA. In recent years, researchers found that toll like receptor 4(TLR4)/nucleur factor κB(NF-κB) signaling mediated by LPS involved in the regulation of cell apoptosis and proliferation. It remained uncovered whether milk performance decline caused by SARA is related to proliferation and apoptosis of mammary epithelial cell through TLR4/NF-κB signaling. The project intends to select early lactating Saanen dairy goat as animal model, induce SARA by feeding high grain feed, in combination with external pudic artery perfusion LPS and culture primary mammary epithelial cells treated by LPS.To explore the effect of LPS on mammary blood-milk barrier, proliferation and apoptosis of mammary epithelial cell, and TLR4/NF-κB pathway and apoptosis signal interaction will be studied. The aim of this project is to disclose the effect of endogenous LPS on proliferation and apoptosis of mammary epithelial cell and its mechanism based on the TLR4/NF-κB pathway in the state of SARA, to provide a new therapeutic target and theoretical basis for improving milk production in the state of ruminant SARA.

亚急性瘤胃酸中毒（SARA）是一种常见的代谢性疾病。当泌乳奶牛患SARA时，体内异常升高的脂多糖（LPS）会引起全身炎性反应，导致产乳性能下降。目前关于SARA发生时乳腺结构和功能的研究较少。近年来发现LPS介导的TLR4/NF-κB信号通路参与细胞增殖和凋亡的调节，那么SARA引起产乳性能下降是否与TLR4/NF-κB调节的细胞增殖和凋亡有关。本项目拟用LPS处理培养的乳腺上皮细胞，检测TLR4/NF-κB与凋亡信号的变化与互作，揭示LPS对乳腺上皮细胞增殖和凋亡的影响及分子机制，为改善反刍动物SARA发生时的产乳性能提供新的治疗靶点和理论依据。

亚急性瘤胃酸中毒（SARA）是一种常见的代谢性疾病。当泌乳奶牛患SARA时，体内异常升高的脂多糖（LPS）会引起全身炎性反应，导致产乳性能下降。目前关于SARA发生时乳腺结构和功能的研究较少。近年来发现LPS介导的TLR4/NFκB信号通路参与细胞增殖和凋亡的调节，那么SARA引起产乳性能下降是否与TLR4/NFκB调节的细胞增殖和凋亡有关。本项目基于TLR4/NFκB与凋亡信号的变化与互作关系，从细胞水平探讨LPS调节乳腺上皮细胞增殖和凋亡的机理，为改善反刍动物SARA发生时的产乳性能提供新的治疗靶点和理论依据。利用不同浓度LPS处理细胞，MTT和LDH法检测细胞的增殖，发现LPS能明显抑制细胞生长，提高细胞生长的抑制率，且依赖于LPS浓度，与作用时间有关；DNA Ladder结果显示，LPS作用细胞后，DNA出现多条大小不等的片段，呈梯形图谱。凋亡相关基因检测结果显示LPS能上调凋亡关键酶Caspase3/4的转录水平。提示LPS促进细胞发生凋亡，抑制细胞的增殖。利用荧光定量PCR和Western blot方法检测信号通路分子、炎性和凋亡基因的变化，初步阐明LPS对TLR4信号通路关键分子、NFκB下游信号和凋亡信号变化的影响，分析NFκB与Caspase-3/4的关系。发现LPS能提高TLR4、MyD88、TRAF6、IRAK4、NFκB、IRF3转录水平，同时提高NFκB下游炎性信号分子转录水平，尤其是TNFα和IL-8的转录水平，提示LPS激活TLR4的MyD88依赖性信号通路，即激活NFκB介导的炎性信号通路，启动炎性反应；凋亡基因检测结果表明：凋亡基因Bax、Caspase3/4的转录水平提高，Bcl-2水平下调，即LPS启动细胞凋亡发生；Western blot结果也证实了Q-PCR的结果，提示NFκB在炎性反应和凋亡发生中扮演了“桥梁”作用。利用NFκB阻断剂PDTC和转染NFκB质粒探讨NFκB在LPS调节上皮细胞增殖和凋亡中的作用，PDTC逆转LPS诱导的细胞凋亡结果提示，LPS能通过NFκB信号引起细胞发生炎性反应和促进细胞凋亡，过表达NFκB质粒的细胞在LPS处理后，TLR4的MyD88依赖性信号通路、炎性信号、凋亡信号的变化与PDTC的处理基本相反，再次证明和佐证了NFκB在LPS诱导的炎性和凋亡信号中起“桥梁”的作用。