SARA状态下瘤胃产生的LPS对反刍动物乳腺炎症反应和酪蛋白合成的影响及其调控

发布时间：2018-01-07 21:11

  本文关键词：SARA状态下瘤胃产生的LPS对反刍动物乳腺炎症反应和酪蛋白合成的影响及其调控　出处：《南京农业大学》2016年博士论文　论文类型：学位论文

  更多相关文章： SARA LPS 酪蛋白 丁酸钠 肠系膜灌注 MAC-T

【摘要】：奶业是我国畜牧业的重要组成部分,牛奶质量安全问题严重制约我国奶业的健康发展。乳蛋白和乳脂肪是构成牛奶营养品质的主要物质基础,既涉及质量安全与消费者的健康,又决定着牛奶的经济价值与核心竞争力。在现代畜牧业生产中,为了提高奶产量,同时满足泌乳早期和中期不断增加的能量需求,奶牛或奶山羊需要饲喂高精料日粮,这种饲喂方式会引起瘤胃异常发酵,进而引起亚急性瘤胃酸中毒。瘤胃内LPS蓄积并透过胃肠道进入全身循环系统到达乳腺,到达乳腺的LPS可激活NF- κ B信号通路,抑制mTOR信号通路,导致乳腺炎症反应以及乳蛋白合成功能的抑制。面对我国现有粗饲料品质差的现实,如何在目前饲养模式下,既提高动物生产性能,又能维持动物健康,已成为当前高强度饲养中急需解决的问题。丁酸钠有促进胃肠道组织发育和增强其屏障功能的作用,能够抑制LPS介导的NF- κ B信号通路,阻止促炎性细胞因子的释放,一定程度的治疗乳腺的炎症反应,恢复乳腺酪蛋白的合成功能。本试验还从外源灌注和体外细胞实验进一步验证了 LPS对乳腺组织天然免疫反应和酪蛋白合成功能的影响。1.消化道产生的LPS对奶牛乳腺免疫功能和乳蛋白合成功能的影响为了满足泌乳的营养需求,奶牛通常饲喂高精料日粮。但是高精料饲喂会引起亚急性瘤胃酸中毒(SARA),导致乳蛋白下降。本研究旨在探明饲喂高精料日粮的奶牛瘤胃中产生的脂多糖(LPS)对乳腺炎性基因表达和酪蛋白合成的影响。12头体重为455±28kg的泌乳中期荷斯坦奶牛随机分为两组:一组饲喂高精料日粮(含60%的精料和40%粗料,HC); 一组饲喂低精料日粮(含40%的精料和60%粗料,LC),饲喂周期为18周。高精料日粮饲喂后瘤胃PH值小于5.6每天超过3小时,说明高精料日粮成功诱发了 SARA。我们发现在高精料日粮饲喂15周以后,乳蛋白显著下降,体细胞数显著升高,且瘤胃液和乳腺动静脉血浆中的LPS含量显著升高。乳腺血浆中的促炎性细胞因子IL-1B、IL-6和TNF-α含量显著升高。通过乳腺组织转录组芯片技术的分析,发现与炎症相关的信号通路的表达被激活了,而与酪蛋白合成相关的信号通路表达受到抑制。同时,高精料组炎症基因如LBP、TLR4、NF-κB、IL-1B和IL-6等的mRNA表达上调,而αs1酪蛋白和β酪蛋白及其合成相关基因mTOR、S6K等的表达下调。本试验还检测了相关信号通路上的关键蛋白表达,发现其表达与基因的mRNA表达一致,说明了饲喂高精料后,乳腺TLR4介导的炎症信号通路被激活,酪蛋白合成相关的mTOR信号通路受到抑制。因此,泌乳奶牛饲喂高精料日粮后,瘤胃产生的LPS增强了乳腺的炎症基因的表达,抑制了乳蛋白的合成。2. 丁酸钠对高精料日粮模式下奶山羊乳腺免疫功能和酪蛋白合成功能的影响本试验采用18头体重为38.86±2.06 kg泌乳中期的萨能奶山羊,随机分为三组,每组6只。三组的饲喂方案如下:第一组饲喂低精料日粮,即日粮中含有40%的精料和60%的粗料,精粗比为4:6, LC组;第二组饲喂高精料日粮,精粗比为6:4, HC组;第三组饲喂高精料日粮,精粗比为6:4,并且在日粮中添加丁酸钠,BHC组。饲喂周期为20周,试验开始前为所有奶山羊安装瘤胃瘘管和乳腺动静脉血管插管,试验后期采集瘤胃液、奶、乳腺动静脉血和乳腺组织样品。高精料日粮引起了瘤胃pH的下降,诱导了 SARA的产生。高精料日粮中添加丁酸钠后瘤胃pH值有一定回升;HC组外周血中LPS的含量显著高于LC组和BHC组(p0.01) ; HC组外周血中的促炎性因子IL-1 β、IL-6和TNF-α的含量都显著高于LC组和BHC组(p0.05 ) ; 丁酸钠组奶山羊乳腺炎症基因LBP、TLR4、NF-κB、IL-1B和IL-6等表达相比于高精料组显著下调(p0.05),乳蛋白合成相关基因CSN2、mTOR和S6K等表达显著上调(p0.05)。相关基因的蛋白表达的水平和mRNA水平是一致的,说明丁酸钠可以抑制LPS介导的TLR4炎症信号通路,缓解乳蛋白的合成抑制。因此,高精料日粮中添加丁酸钠可以一定程度上治疗高精料日粮诱导的SARA状态下的奶山羊乳腺炎症反应,恢复酪蛋白合成功能。3.肠系膜静脉灌注LPS对奶山羊乳腺天然免疫功能和泌乳功能的影响8只分娩3周以内、体重在42 ± 2.8 kg萨能奶山羊,分栏饲喂。试验中根据泌奶山羊饲养标准设计相同的日粮,所有试验动物均安装肠系膜血管插管。试验动物被随机分为两组,一组肠系膜灌注LPS作为处理组(Treatment); 一组肠系膜灌注生理盐水为对照组(Control)。灌注后4h采集血样,处死后采集乳腺组织样品。肠系膜灌注LPS的奶山羊血液中的LPS含量远远高于灌注生理盐水的。LPS灌注组的奶山羊乳腺炎症基因LBP、TLR4、NF-κB、IL-1B和IL-6等表达远高于对照组,且乳蛋白合成相关基因CSN2、mTOR,S6K、JAK2和STAT5等表达表达下调。乳腺中TLR4启动子区域染色质处于开放状态,DNA去甲基化,导致TLR4基因表达增强;CSN2启动子区域的染色质处于压缩状态,DNA甲基化率升高,导致CSN2基因表达下调。本研究表明,肠系膜灌注LPS上调了乳腺炎性基因的表达,抑制的酪蛋白合成相关基因的表达,并从一定程度上通过表观遗传学机制调控TLR4和CSN2基因的表达,对乳腺的炎症反应和酪蛋白合成功能造成影响。4. LPS在MAC-T细胞上通过NF- κ B信号通路调节炎症基因的表达本试验以乳腺上皮细胞传代细胞系MAC-T细胞为研究对象,分别作5种处理:PBS 组;添加 100ng/mL LPS 组;NF- κ B 过表达组;LPS+BAY11-7085 组和 LPS+MG132组。本试验发现LPS浓度在0.1-1000 ng/mL之间时,TLR4表达随着浓度的升高的增加,LPS浓度大于1000ng/mL后,TLR4表达变化不大。最后我们选择了 100ng/mL的LPS浓度作为处理浓度。只有当LPS浓度很高时才会导致MAC-T细胞凋亡,因此其凋亡的生物学意义较小。 LPS处理的MAC-T细胞内NF-κB表达上调,且促炎性因子的表达也增加了。NF-κB的过表达上调了细胞的炎症基因蛋白的表达。NF-κB的两种抑制剂BAY11-7085和MG132都抑制了 NF- κ B蛋白的表达。BAY11-7085抑制了炎性基因的表达,特别是IL-1B; MG132同样也抑制了这些基因的表达,尤其是IL-8。因此,我们认为,NF-κB在LPS激活的TLR4炎症信号通路上具有重要的作用。
[Abstract]:The dairy industry is an important part of animal husbandry in China, the healthy development of milk quality and safety problems seriously restricting China's dairy industry. Milk protein and milk fat is an important material basis for the nutritional quality of milk, which relates to the quality and safety of the health of consumers, but also determines the milk economic value and core competitiveness in modern animal husbandry. In the production, in order to improve milk production, and meet the energy demand of the early and mid lactation dairy cows or goats to fed high concentrate diets, this feeding way will cause abnormal rumen fermentation, thereby causing subacute ruminal acidosis. The rumen LPS accumulation through the gastrointestinal tract into the systemic circulation system to reach the breast. Arrive at the breast LPS can activate NF- B signaling pathway, inhibition of the mTOR signaling pathway, leading to inhibition of inflammation and breast milk protein synthesis function. In the face of China's existing forage The poor quality of the reality, how in the current feeding mode, which can improve the production performance of animal, and to maintain animal health, has become the urgent problems in the breeding of high strength. Sodium butyrate can promote the development and enhance the barrier function of gastrointestinal tissue, NF- can inhibit B signaling pathway mediated by LPS. To prevent the release of pro-inflammatory cytokines, inflammatory reactions in the treatment of breast to a certain extent, the synthesis of functional recovery of casein. This experiment also breast from exogenous perfusion and in vitro experiments to further validate the effect of LPS on anti breast tissue should be natural immunity and casein synthesis function of digestive tract.1. produced LPS of bovine mammary immune function and milk protein synthesis function in order to meet the nutritional needs of lactating cows, usually fed high concentrate diets. But high concentrate feeding can cause subacute ruminal acidosis (SARA), lead Milk protein caused by lipopolysaccharide decline. This study aims at exploring the rumen of dairy cows fed high concentrate diets in (LPS) on the expression of inflammatory genes and casein synthesis.12 head weight was 455 + 28KG in lactating Holstein cows were randomly divided into two groups: one group fed high concentrate diets (containing 60% concentrate and roughage 40%, HC); a group fed low concentrate diets (containing 40% concentrate and 60% coarse material, LC), with a period of 18 weeks. High concentrate diets after rumen pH is less than 5.6 of more than 3 hours a day, the high concentrate diets was successfully induced by SARA. we found that in the high concentrate diet after 15 weeks, milk protein decreased significantly, somatic cell count increased significantly, and LPS content of rumen fluid and breast venous plasma increased significantly. Inflammatory cytokines in plasma of breast IL-1B, IL-6 and TNF- were significantly increased by alpha. Breast tissue transcription group chip technology The analysis showed that the expression of signaling pathways associated with inflammation is activated, and casein synthesis related signaling pathway expression was inhibited. At the same time, the high concentrate group of inflammatory genes such as LBP, TLR4, NF- kappa B, upregulation of IL-1B and IL-6 mRNA, and S1 alpha and beta casein and casein synthesis related gene mTOR, expression of S6K and so on. This experiment also detected key protein related signaling pathway on the expression, we found that the expression and gene expression of mRNA, the fed high concentrate, the inflammatory signaling pathway mediated by activation of breast TLR4, casein synthesis related signaling pathway by mTOR suppression. Therefore, fed high concentrate diets of lactating dairy cows, rumen produced LPS enhanced the expression of inflammatory genes in breast milk protein, inhibit the synthesis of.2. sodium butyrate on high concentrate diets of dairy goat mammary gland model of immune function and casein synthesis 鑳界殑褰卞搷鏈�璇曢獙閲囩敚�

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