中文摘要
乳腺炎是奶牛常见、多发的疾病之一，其导致的经济损失居于奶牛各类疾病之首。细菌在乳腺内感染是引起奶牛乳腺炎的最主要原因，其中大肠杆菌（Escherichia coli, E.coli）等革兰氏阴性菌是引起临床型乳腺炎的主要致病菌。
该类菌所产生的内毒素脂多糖（Lipopolysaccharide, LPS）会激活乳腺组织中相应细胞上的Toll-like reporter 4（TLR4）受体，从而产生大量促炎介质，导致严重的乳腺炎症反应。因此，临床治疗乳腺炎时，在进行抗菌治疗的同时，有效控制乳腺中的炎症反应对乳腺炎的病后转归起到重要作用。然而，受药物残留等因素的影响，目前临床上尚缺乏安全、有效的抗乳腺炎类药物。烟酸作为奶牛饲料添加剂已经在奶牛养殖中广泛应用，近年来的研究表明其受体GPR109A在体内起到广泛的抗炎作用。但是，烟酸是否能通过GPR109A起到抗奶牛乳腺炎的作用，目前尚无报道。因此，本课题以烟酸及其受体GPR109A作为研究对象，首先采取健康奶牛和患有乳腺炎的奶牛乳腺组织通过检测GPR109A表达量，来分析其与乳腺炎的相关性；然后，利用LPS诱导的奶牛乳腺上皮细胞（Bovine mammary epithelial cells, BMECs）炎症反应模型，系统的研究烟酸通过GPR109A抗炎症反应的作用及其机制；最后通过饲喂过瘤胃烟酸来观察烟酸对奶牛乳腺炎的治疗效果，以期为奶牛乳腺炎抗炎药物的筛选提供靶标。
首先，收集健康奶牛和患有乳腺炎奶牛的乳腺组织，利用H&E染色检测病理变化；利用实时荧光定量PCR（q RT-PCR）检测炎性因子表达情况；利用q RT-PCR和免疫组织化学检测GPR109A基因和蛋白水平。结果发现，与健康奶牛相比，患乳腺炎奶牛的乳腺组织病理损伤严重，乳腺组织中炎性因子白介素-6（Interleukin-6, IL-6）、肿瘤坏死因子-α（Tumor necrosis factor-α,TNF-α）、白介素-1β（IL-1β）基因水平显着增加，说明乳腺样本来源可靠。GPR109A基因和蛋白水平结果显示，与健康奶牛相比，患乳腺炎奶牛的乳腺组织中GPR109A基因和蛋白水平都显着增加。这些结果表明GPR109A在乳腺炎奶牛乳腺组织中高表达，提示GPR109A可能在乳腺炎的发生发展中发挥重要作用。
其次，为了进一步研究烟酸是否能通过GPR109A起到抗乳腺炎作用，我们在LPS诱导的BMECs炎症反应模型中进行了系统研究。结果发现烟酸在BMECs中能够抑制炎症反应，利用si RNA敲低GPR109A后抑制烟酸的抗炎作用。核因子E2相关因子2（Nuclear factor E2-related factor 2, Nrf2）和自噬过程在炎症中起到重要作用，利用RA和3-MA分别抑制Nrf2和自噬后也能抑制烟酸的抗炎功能。提示烟酸在LPS诱导的BMECs炎症反应模型中通过GPR109A、Nrf2和自噬抑制炎症反应。为了证实烟酸的抗炎机制，我们在BMECs中检测了GPR109A、Nrf2和自噬过程的上下游关系，结果发现在BMECs中烟酸通过GPR109A激活Nrf2信号，进一步活化自噬过程。AMPK依赖的蛋白激酶（Adenosine 5’-monophosphate (AMP)-activated protein kinase,AMPK）信号是自噬调控的关键枢纽，接着我们在BMECs研究了该信号是否参与烟酸调控自噬过程。结果发现烟酸在BMECs中激活GPR109A后能通过上调(AMP+ADP)/ATP而活化AMPK信号，AMPK活化后可以促进P62与胞浆蛋白伴侣分子（Keleh-like ECH-associated protein 1, Keap1）的相互作用而激活Nrf2。进一步我们在LPS诱导的BMECs炎症反应模型中检测了烟酸是否激活AMPK、Nrf2和自噬过程，结果发现烟酸能够激活AMPK、Nrf2和自噬过程。这些结果表明烟酸在BMECs中通过GPR109A/AMPK/Nrf2信号通路激活自噬过程而起到抗炎作用。
最后，选取临床型乳腺炎奶牛并饲喂过瘤胃烟酸后，收集血清和乳清，检测血清和乳清中促炎细胞因子IL-6、TNF-α和IL-1β蛋白水平和乳汁中体细胞数（Sometic cell count, SCC），来初步分析烟酸对临床型乳腺炎的治疗效果。结果发现，饲喂过瘤胃烟酸后，乳腺炎奶牛血清和乳清中IL-6、TNF-α和IL-1β蛋白水平明显下降，乳汁中SCC量显着降低。这些结果表明，烟酸对奶牛乳腺炎具有一定的疗效。
以上结果表明，烟酸在BMECs中通过GPR109A/AMPK/Nrf2信号激活自噬来抑制炎症反应；同时，在临床治疗奶牛乳腺炎症反应中，烟酸表现出良好的抗炎作用。因此，烟酸的推广使用对奶牛乳腺炎的临床治疗具有重要意义。
关键词： 奶牛乳腺炎，GPR109A，烟酸，AMPK，Nrf2，自噬。
Abstract
Mastitis is one of the most common and frequent diseases and its economic loss is the first of all kinds of diseases in dairy cows. Bacterial infection in the mammary gland is the main cause of mastitis, among which Gram-negative bacteria such as Escherichia coli (E.coli )are the main pathogens of clinical mastitis. The endotoxin lipopolysaccharide (LPS) produced by these bacteria can activate toll like reporter 4 (TLR4) in corresponding cells of mammary inflammation, thus producing a large number of proinflammatory mediators, leading to serious mammary inflammation.
Therefore, in the clinical treatment of mastitis, effective control of the inflammatoryresponse in the mammary gland plays an important role in the prognosis of mastitiswhile conducting antibacterial treatment. However, due to the influence of drugresidues and other factors, there is still a lack of safe and effective anti-mastitis drugsin clinical practice. GPR109A belongs to Gi protein-coupled receptor, which plays awide range of anti-inflammatory effects in vivo. Its ligand niacin has been widely usedin dairy cattle breeding as feed additive. However, whether niacin can play an anti-mastitis role in dairy cows through GPR109A has not been reported. Therefore, niacinand its receptor GPR109A were taken as the research object in this paper. Firstly, thecorrelation between GPR109A and mastitis was analyzed by detecting the expressionof GPR109A in mammary tissue of healthy and mastitis dairy cows. Secondly, in theinflammation model of bovine mammary epithelial cells (BMECs) induced by LPS,the role and mechanism of niacin through GPR109A in the inflammatory weresystematically studied. Finally, the therapeutic effect of niacin on mastitis wasobserved by feeding rumen niacin, so as to provide a target for the screening of anti-inflammatory drugs for cow mastitis.
Firstly, the mammary tissues of healthy and mastitis cows were collected, andthe pathological changes were detected by H&E staining; the expression of inflammatory factors was detected by q RT-PCR; GPR109A gene and protein levelswere detected by q RT-PCR and immunohistochemistry. The results showed thatcompared with healthy cows, the mammary tissue of mastitis cows was seriouslydamaged by pathology, and the levels of inflammatory factors interleukin-6 (IL-6),tumor necrosis factor-α(TNF-α) and IL-1βgenes in mammary tissue weresignificantly increased, which indicated that the source of mammary samples wasreliable. The results of GPR109A gene and protein level showed that GPR109A geneand protein levels were significantly increased in mammary tissue of mastitis cowscompared with healthy cows. These results indicate that GPR109A is highlyexpressed in mammary tissue of mastitis cows, suggesting that GPR109A may playan important role in the occurrence and development of mastitis.
Secondly, in order to further investigate whether niacin play an anti-mastitis rolethrough GPR109A, we conducted a systematic study in the LPS-inducedinflammatory response model of BMECs. The results showed that niacin could inhibitthe inflammatory response in BMECs and the anti-inflammatory effect of niacin wasinhibited by knocking down GPR109A with si RNA. Nuclear factor E2-related factor2(Nrf2) and autophagy play an important role in inflammation, and inhibition of Nrf2and autophagy by RA and 3-MA can also inhibit the anti-inflammatory function ofniacin. It was suggested that niacin inhibited the inflammatory response in the LPS-induced BMECs model by GPR109A, Nrf2 and autophagy. In order to confirm theanti-inflammatory mechanism of niacin, we detected the upstream and downstreamrelationship between GPR109A, Nrf2 and autophagy process in BMECs. The resultsshowed that niacin activated Nrf2 signal through GPR109A and further activatedautophagy process in BMECs. Adenosine 5’-monophosphate (AMP)-activated proteinkinase (AMPK) signal is a key point of autophagy regulation and then we studiedwhether AMPK signal is involved in the process of niacin regulation autophagy inBMECs. The results showed that niacin activated GPR109A in BMECs and activatedAMPK signal by up-regulating (AMP + ADP) / ATP, and the activation of AMPKpromoted the interaction between p62 and Keleh-like ECH-associated protein1(Keap1) to activate Nrf2. Furthermore, we tested whether niacin activates AMPK,Nrf2 and autophagy in LPS-induced BMECs inflammatory response model, and found that niacin can activate AMPK, Nrf2 and autophagy. These results indicatedthat niacin plays an anti-inflammatory role in BMECs by activating autophagy viaGPR109A / AMPK / Nrf2 signaling pathway.
Finally, serum and whey were collected from clinical mastitis cows fed withrumen niacin. The protein levels of pro-inflammatory cytokines IL-6, TNF-α andIL-1β in serum and whey were detected, as well assometic cell count (SCC) in milk, topreliminarily analyze the therapeutic effect of niacin on clinical mastitis. The resultsshowed that after feeding with rumen niacin, the levels of IL-6, TNF-α and IL-1βprotein in serum and whey of mastitis cows significantly decreased, and the amount ofSCC in milk significantly decreased. These results indicate that niacin has certaineffect on cow mastitis.
The above results indicate that niacin activates autophagy through GPR109A /AMPK / Nrf2 signal to inhibit the inflammatory response in BMECs. At the sametime, niacin showed a good anti-inflammatory effect in the clinical treatment of cowmastitis. Therefore, the popularization of niacin is of great significance to the clinicaltreatment of mastitis in dairy cows.
Key words: Dairy cow mastitis, GPR109A, Niacin, AMPK, Nrf2, Autophagy 。
前 言
奶牛乳腺炎(Mastitis)是奶牛业最常见、危害最严重的疾病之一。乳腺炎不仅影响奶牛的产奶量，还会影响奶牛的乳品质，最终甚至会给养牛场造成巨大的经济损失。目前大量研究表明病原菌在乳腺内感染是引起乳腺炎的主要原因。大肠杆菌等革兰氏阴性菌引起的乳腺炎约占临床型乳腺炎病例中的40%。而脂多糖（LPS）是革兰氏阴性菌的主要组成成份之一，也是导致乳腺炎加剧的重要因素。在大肠杆菌型乳腺炎发生过程中，细菌大量增殖所产生的LPS能被乳腺组织内的模式识别受体识别，并能引起动物机体急性炎症反应或败血性休克。因此，在临床中治疗乳腺炎时抗菌和消炎需要双管齐下，而且目前的研究表明控制乳腺中的炎症反应对乳腺炎的病后转归起到重要作用。因此抑制奶牛乳腺中的炎症反应对治疗奶牛乳腺炎具有重要意义。但是目前牛场中抗炎类的药物种类有限，迫切需要寻找新的、安全、有效的抗炎药物。
在乳腺组织中防御病原微生物入侵的第一道防线为乳腺上皮细胞，该细胞膜表面存在Toll样受体-4(TLR-4)和TLR-2等多种模式识别受体，能够识别病原微生物的病原相关分子模式，参与乳腺的局部防御反应，又因其和乳汁中的巨噬细胞一样处于乳腺组织外围，发挥哨兵的作用，因此乳腺上皮细胞的局部免疫功能已成为当前的研究热点。目前，在LPS诱导的乳腺上皮细胞炎症反应模型中进行的抗炎药物的筛选已经非常普遍，但是针对乳腺上皮细胞本身具有的抗炎分子筛选研究相对较少。
G蛋白偶联受体（GPCRs）是一类哺乳动物体内广泛存在的受体家族，有研究表明其在体内参与抑制炎症反应、脂代谢和传递细胞外信号等多个生物调控环节。因此，在乳腺上皮细胞中寻找能起到抑制炎症作用的GPCRs可能为乳腺炎的防治提供新的靶点。
烟酸作为饲料添加剂在奶牛饲养中已经被广泛应用。目前的研究表明其对奶牛的生产性能、脂代谢、预防早期能量缺乏和冷热应激方面都有一定的作用。近年来发现烟酸的受体G蛋白偶联受体109A（GPR109A）激活后在体内起到广泛的抗炎作用。在单核细胞中烟酸能通过GPR109A/NF-κB信号通路抑制TLR-4和TLR-2激动剂诱导的肿瘤坏死因子（TNF）-α、白介素（IL）-6和单核细胞趋化因子（MCP）-1的合成和分泌。而我们课题组首次发现小胶质细胞的细胞膜上存在GPR109A的受体，并且其受体激动剂能通过GPR109A/NF-κB信号通路抑制LPS诱导的小胶质细胞炎症反应。在巨噬细胞中GPR109A能通过抑制促炎细胞因子、低密度脂蛋白摄入和化学趋化作用来抑制LPS诱导的炎症反应。但是，目前关于烟酸通过GPR109A缓解奶牛乳腺炎的研究尚未见相关报道。
鉴于上述，本研究以烟酸及其受体GPR109A作为研究对象，首先采取健康奶牛和乳腺炎奶牛乳腺组织通过检测GPR109A表达量，来分析GPR109A与乳腺炎的相关性；然后，再利用LPS诱导的奶牛乳腺上皮细胞（BMECs）炎症反应模型，系统的研究烟酸抗BMECs炎症反应的作用；最后通过饲喂过瘤胃烟酸来观察烟酸对奶牛乳腺炎的治疗效果，以期为推广烟酸在牛场中的应用提供数据支持。
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第一篇 文献综述.
第一章 奶牛乳腺炎的研究进展.
1.1、 奶牛乳腺炎的分类
1.2、奶牛乳腺炎的发病原因.
1.3、奶牛乳腺炎的防治
1.4 、小结.
第二章 烟酸的研究进展
2.1、烟酸 的理化性质
2.2、烟酸 的生物学功能
2.3、烟酸的来源
2.4、烟酸对畜禽的影响
2.5、烟酸与炎症
第三章 GPR109A 受体的研究进展
3.1 、GPR109A 的结构与分布
3.2、GPR109A的生物学功能
3.3、小结.
第二篇 研究内容.
第一章 GPR109A与奶牛乳腺炎相关性分析
1.1、 材料与方法.
1.2、结果
1.3、讨论.
1.4、小结.
第二章 烟酸通过GPR 109A缓解BMECs炎症反应.
2.1、材料与方法
2.2、结 果.
2.3、讨论
2.4、小结
第三章 烟酸缓解奶牛乳腺炎的临床研究
3.1、材料与方法
3.2、结 果.
3.3、讨论.
3.4、小结.
结 论
1、GPR109A在临床型乳腺炎奶牛的乳腺组织中呈现高表达，提示GPR109A可能在乳腺炎中发挥重要作用。
2、烟酸在BMECs中通过GPR109A/AMPK/Nrf2/自噬途径抑制LPS诱导的炎症反应。
3、临床型乳腺炎奶牛饲喂过瘤胃烟酸后可有效缓解乳腺炎症反应。
参考文献.
