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1、饲喂烟酸对奶牛乳腺炎的治疗效果探究,兽医硕士论文乳腺炎是奶牛常见、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，华而不实大肠杆菌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 4TLR4受体，进而产生大量促炎介质，导致严重的乳腺炎症反响。因而，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响对乳腺炎的病后转归起到重要作用。然而，受药物残留等因
2、素的影响，当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，近年来的研究表示清楚其受体GPR109A在体内起到广泛的抗炎作用。但是，烟酸能否能通过GPR109A起到抗奶牛乳腺炎的作用，当前尚无报道。因而，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表示出量，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炎症反响模型，系统的研究烟酸通过GPR109A抗炎症反响的作用及其机制；最后通过饲喂过瘤胃烟
3、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挑选提供靶标。 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PCRq RT-PCR检测炎性因子表示出情况；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-6Interleukin-6, IL-6、肿瘤坏死因子- Tumor necrosis factor- ,TNF- 、白介素-1 IL-1 基因水平显着增加，讲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
4、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。这些结果表示清楚GPR109A在乳腺炎奶牛乳腺组织中高表示出，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能否能通过GPR109A起到抗乳腺炎作用，我们在LPS诱导的BMECs炎症反响模型中进行了系统研究。结果发现烟酸在BMECs中能够抑制炎症反响，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2Nuclear factor E2-related factor 2, Nrf2和自噬经过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
5、抗炎功能。提示烟酸在LPS诱导的BMECs炎症反响模型中通过GPR109A、Nrf2和自噬抑制炎症反响。为了证实烟酸的抗炎机制，我们在BMECs中检测了GPR109A、Nrf2和自噬经过的上下游关系，结果发如今BMECs中烟酸通过GPR109A激活Nrf2信号，进一步活化自噬经过。AMPK依靠的蛋白激酶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研究了该信号能否介入烟酸调控自噬经过。结果发现烟酸在BMECs中激活GPR109A后能通过上调(AMP+ADP)/ATP而活化
6、AMPK信号，AMPK活化后能够促进P62与胞浆蛋白伴侣分子Keleh-like ECH-associated protein 1, Keap1的互相作用而激活Nrf2。进一步我们在LPS诱导的BMECs炎症反响模型中检测了烟酸能否激活AMPK、Nrf2和自噬经过，结果发现烟酸能够激活AMPK、Nrf2和自噬经过。这些结果表示清楚烟酸在BMECs中通过GPR109A/AMPK/Nrf2信号通路激活自噬经过而起到抗炎作用。 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- 和IL-1 蛋白水平和乳汁中体细胞数Sometic cell c
7、ount, 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- 和IL-1 蛋白水平明显下降，乳汁中SCC量显着降低。这些结果表示清楚，烟酸对奶牛乳腺炎具有一定的疗效。 以上结果表示清楚，烟酸在BMECs中通过GPR109A/AMPK/Nrf2信号激活自噬来抑制炎症反响；同时，在临床治疗奶牛乳腺炎症反响中，烟酸表现出良好的抗炎作用。因而，烟酸的推广使用对奶牛乳腺炎的临床治疗具有重要意义。 本文关键词语： 奶牛乳腺炎，GPR109A，烟酸，AMPK，Nrf2，自噬。 Abstract Mastitis is one of the
8、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 e
9、ndotoxin 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,
10、 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 clinica
11、l 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
12、 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 bo
13、vine 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-inflamm
14、atory 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 immunohisto
15、chemistry. 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- (TNF- ) and IL-1 genes in mammary tissue weresignificantly increased, which indicate
16、d 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 tiss
17、ue 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 B
18、MECs. 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 a
19、utophagy 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
20、 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 regula
21、tion 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 Kel
22、eh-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-inflammator
23、y 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- andIL-1 in serum and whey were detected, as well assometic cell cou
24、nt (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- and IL-1 protein in serum and whey of mastitis cows significantly decreased, and the amount ofSCC in milk significantly de
25、creased. 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
26、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宏大的经济损失。当前大
27、量研究表示清楚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经过中，细菌大量增殖所产生的LPS能被乳腺组织内的形式辨别受体辨别，并能引起动物机体急性炎症反响或败血性休克。因而，在临床中治疗乳腺炎时抗菌和消炎需要双管齐下，而且当前的研究表示清楚控制乳腺中的炎症反响对乳腺炎的病后转归起到重要作用。因而抑制奶牛乳腺中的炎症反响对治疗奶牛乳腺炎具有重要意义。但是当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
28、织中防御病原微生物入侵的文章为硕士论文，如需全文请点击底部下载全文链接】 文献综述. 研究内容. 第一章 GPR109A与奶牛乳腺炎相关性分析 1.1、 材料与方式方法. 1.2、结果 1.3、讨论. 1.4、小结. 第二章 烟酸通过GPR 109A缓解BMECs炎症反响. 2.1、材料与方式方法 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、材料与方式方法 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示出，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诱导的炎症反响。 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响。 以下为参考文献.