色氨酸调节慢性不可预知应激动物肠道屏障及免疫功能的机制研究

发布时间：2018-04-29 21:02

本文选题：慢性不可预知温和应激 + 肉鸡　；参考：《中国农业大学》2017年博士论文

【摘要】：本论文主要研究了慢性不可预知温和应激对肉鸡肠道屏障及免疫功能的影响,并通过小鼠进行进一步验证;通过日粮添加L-色氨酸研究色氨酸对慢性应激诱导的肉鸡肠道屏障及免疫功能的影响。再结合体外试验,进一步确定L-色氨酸调节慢性应激诱导的动物肠道屏障结构及免疫功能紊乱的机制。试验一:为探讨色氨酸对慢性不可预知温和应激(CUMS)肉鸡肠道屏障功能的调节作用,试验选用320只1日龄爱拔益加AA肉仔鸡,随机分为4个处理,每个处理8个重复,每个重复10只鸡。采用玉米-豆粕型基础日粮,基础日粮中色氨酸水平为0.21%(0～21日龄)及0.18%(22～42日龄)。然后在基础日粮中分别添加L-色氨酸,使其分别达到0.42%(0～21日龄)及0.36%(22～42日龄),并分为正常饲养和应激条件下饲养,试验期为6周。应激程序包括:2h黑暗,抓鸡,红或黄旗挥舞101min,陌生人走动101min,驱赶鸡群5min,针刺等。结果显示,CUMS处理显著降低肉鸡生产性能,增加血清皮质酮、β-肾上腺素、去甲肾上腺素水平;日粮添加色氨酸可增加肉鸡生产性能,降低应激激素释放;CUMS处理促进肉鸡空肠吲哚胺2,3双加氧酶(IDO)的基因表达,降低色氨酸羟化酶1(TPH1)的表达,使血清5-羟色胺(5-HT)水平下降;日粮添加色氨酸可明显提高空肠TPH1的基因表达,抑制IDO基因表达,提高5-HT水平,缓解机体应激反应。另外,日粮添加色氨酸抑制了 CUMS处理诱导的空肠IL-1β、IL-6、TNF-α、IL-8、NF-κB基因表达,降低IL-10表达,提高空肠紧密连接蛋白表达,降低肠道通透性。另,以小鼠为研究对象对上述试验进一步验证,试验结果表明,色氨酸可能是通过增加其代谢酶TPH1的表达,进而提高外周血中5-HT水平,降低应激反应;色氨酸通过抑制肠道促炎症细胞因子表达,进而提高紧密连接蛋白表达,保障肠道上皮细胞的完整性,维持了正常的肠道屏障功能。试验二:为探讨CUMS处理影响动物肠道屏障功能的机制,揭示L-色氨酸保护肠道屏障免于应激影响的机理。本试验通过分离培养鸡肠道上皮细胞,并利用外源添加地塞米松(DEX)、肾上腺素(AD)、去甲肾上腺素(NE)模拟应激反应,研究上述应激激素对鸡肠上皮细胞炎症相关细胞因子基因表达及紧密连接蛋白的影响。结果发现,应激激素处理不同程度的提高了鸡肠道上皮细胞内IL-6、IL-1β、TNF-α等细胞因子的基因表达;且DEX处理显著降低了鸡肠道上皮细胞中紧密连接蛋白ZO-1及Occludin表达;然而,L-色氨酸及应激激素相应受体抑制剂预处理可明显降低应激激素诱导的细胞因子基因的高表达,提高紧密连接蛋白的表达。另外,本试验研究了DEX、AD、NE处理对Caco-2细胞跨膜电阻值的影响,结果显示上述激素处理16、24h后可不同程度的降低Caco-2细胞的跨膜电阻值,而L-色氨酸预处理可缓解应激激素诱导的电阻值下降。上述结果表明,L-色氨酸可能是通过降低应激激素与其相应受体结合,从而削弱激素的效应,降低激素引起的细胞因子的释放,提高紧密连接蛋白的表达,维持肠道屏障结构的完整性,进而保护肠道屏障功能。试验三:为探究色氨酸对CUMS处理动物免疫功能的调节作用,本试验以肉鸡为研究对象并采用小鼠进一步验证肉鸡试验结果。肉鸡试验设计同试验一,小鼠试验设计如下:试验选用6周龄雄性健康Balb/c小鼠48只,随机均分为4个处理,每个处理12只。分别是对照组(Ctrl),色'氨酸组(L-Trp),饲喂基础日粮且应激组(Stress),饲喂高色氨酸日粮且应激组(L-Trp+Stress)。所有小鼠饲养在环境控制的鼠房内,自由采食和饮水。试验基础日粮中色氨酸水平为0.2%。然后在基础日粮中分别添加L-色氨酸,使其达到0.4%,并分为正常饲养和应激条件下饲养,每周记录小鼠体重,试验期为8周。小鼠应激程序包括:换笼饲养,垫料潮湿,无垫料,冷水浸泡,空水瓶刺激,24h光照/黑暗,黑白颠倒,笼子倾斜45°。结果显示,CUMS处理降低肉鸡新城疫抗体滴度,抑制外周血淋巴细胞的增殖;提高脾脏中应激激素受体的基因表达;日粮添加色氨酸可提高外周血淋巴细胞增殖、NDV抗体滴度,降低脾脏细胞中应激激素受体的基因表达,进而抑制了激素与受体的结合,从而增强机体的免疫机能。以上结果表明,色氨酸可能是通过阻断应激激素与其受体的结合,从而缓解应激,进而提高机体免疫细胞功能,增强机体免疫机能的。试验四:为探究CUMS处理影响动物机体免疫机能的机制,揭示L-色氨酸缓解应激调节免疫机能的机理。本试验通过小鼠外周血分离获得T淋巴细胞,并利用外源添加DEX、AD及NE模拟应激反应,研究上述应激激素对T淋巴细胞免疫活性的影响。结果发现,应激激素处理显著抑制了 T淋巴细胞增殖,而通过对上述应激激素受体特异性抑制则明显缓解应激激素对T淋巴细胞增殖的影响,表明CUMS处理引起的免疫机能紊乱可能是通过促进了应激激素如皮质酮CORT、NE、AD等激素的释放及这些应激激素相对应受体在免疫细胞上的表达,进而促进了激素与其受体的结合,增加了激素的敏感性,从而破坏了机体的免疫机能稳态。为进一步揭示L-色氨酸缓解应激反应,调节机体免疫机能机理。本试验利用外源添加L-色氨酸及其代谢产物5-HT对T淋巴细胞进行预处理,研究其对应激激素引起的T淋巴细胞活性抑制的影响。结果显示,L-色氨酸及5-HT预处理均可不同程度的缓解应激激素对T淋巴细胞增殖的影响。L-色氨酸及5-HT可能是通过降低应激激素的释放及其受体的表达而阻断二者的结合,从而钝化应激激素的敏感性,进而保护动物免于应激引起的免疫机能紊乱,而结合体内试验结果,提示L-色氨酸发挥作用可能是通过激活了色氨酸代谢途径中的5-HT代谢途径,使更多的L-色氨酸分解成为5-HT。综上所述,L-色氨酸通过降低应激激素的分泌并阻断应激激素与其受体结合而缓解应激引起的动物肠道屏障结构及免疫紊乱。
[Abstract]:The effect of chronic unpredictable mild stress on intestinal barrier and immune function of broilers was studied in this paper, and the effects of tryptophan on intestinal barrier and immune function induced by chronic stress were studied by adding L- tryptophan in diet. In addition, the L- tryptophan modulation was further determined in vitro. The mechanism of intestinal barrier structure and immune dysfunction induced by chronic stress. Test 1: To investigate the regulatory effect of tryptophan on the intestinal barrier function of chronic unpredictable mild stress (CUMS) broilers, 320 1 day old AA broilers were randomly divided into 4 treatments, each treatment was 8 repetitions, and 10 repeated each. The level of tryptophan in basal diet was 0.21% (0~21 days old) and 0.18% (22~42 days old) with corn soybean meal base diet. Then L- tryptophan was added to basal diet to make it 0.42% (0~21 days old) and 0.36% (22~42 days old) respectively, and were kept under normal feeding and stress conditions for 6 weeks. The stress program was 6 weeks. Including: 2h dark, catch chicken, red or yellow flag waving 101min, strangers walk 101min, drive chicken group 5min, acupuncture and so on. The results showed that CUMS treatment significantly reduced the production performance of broiler, increased serum corticosterone, beta adrenalin, norepinephrine level; dietary tryptophan added to broiler production performance, reduce stress hormone release; CUMS treatment The gene expression of indolamine 2,3 dioxygenase (IDO) was promoted in the jejunum of broiler, and the expression of tryptophan hydroxylase 1 (TPH1) was reduced, and the level of serum 5- hydroxytryptamine (5-HT) decreased. The diet supplemented with tryptophan could obviously improve the gene expression of jejunum TPH1, inhibit the expression of IDO gene, improve the level of 5-HT and alleviate the stress reaction of the body. In addition, the dietary tryptophan added tryptophan to the diet. Inhibiting the expression of IL-1 beta, IL-6, TNF- a, IL-8, NF- kappa B induced by CUMS treatment, reducing the expression of IL-10, improving the expression of the jejunum tight connexin and reducing the permeability of the intestines. 5-HT level in high peripheral blood decreased stress response; tryptophan increased the expression of inflammatory cytokines by inhibiting intestinal proinflammatory cytokines, and then enhanced the expression of tight connexin, guaranteed the integrity of intestinal epithelial cells and maintained normal intestinal barrier function. Test two: To explore the mechanism of CUMS treatment for the intestinal barrier function of the animal and reveal L- tryptophan To protect the intestinal barrier from the effect of stress, this experiment was conducted by separating and cultivating intestinal epithelial cells, and using exogenous dexamethasone (DEX), adrenaline (AD) and norepinephrine (NE) to simulate stress response, and to study the expression of cytokine gene and close connexin in chicken intestinal epithelial cells. The results showed that stress hormone treatment increased the gene expression of IL-6, IL-1 beta, TNF- alpha and other cytokines in the intestinal epithelial cells of chicken, and DEX treatment significantly reduced the expression of closely connexin ZO-1 and Occludin in the intestinal epithelial cells of chicken; however, the pretreatment of L- tryptophan and stress hormone corresponding receptor inhibitors could be obvious. In addition, the effect of DEX, AD and NE on the transmembrane resistance of Caco-2 cells was studied. The results showed that the transmembrane resistance of Caco-2 cells could be reduced to varying degrees after the treatment of 16,24h, and the pretreatment of L- tryptophan could be alleviated. The resistance values induced by stress hormones decrease. The results show that L- tryptophan may reduce the effect of hormone and reduce the release of cytokines caused by hormone, increase the expression of tight connexin, maintain the integrity of the intestinal barrier structure and protect the intestinal barrier function by reducing the hormone effect, reducing the release of cytokines caused by hormone. Test three: in order to explore the effect of tryptophan on the immune function of CUMS in treating animal immune function, this experiment took broiler as the research object and used mice to further verify the results of broiler test. The design of broiler test was the same as the experiment. The experiment was designed as follows: 48 6 weeks male healthy Balb/c mice were selected and divided into 4 treatment randomly, each place was divided into each place. 12 subjects were the control group (Ctrl), the tryptophan acid group (L-Trp), feeding the basal diet and the stress group (Stress), feeding the high tryptophan diet and the stress group (L-Trp+Stress). All the mice were fed in the environment controlled rat room and were free to eat and drink. The level of tryptophan in the basal diet was 0.2%. and then added L- color to the basal diet. Ammonia, which was made up to 0.4%, and kept under normal feeding and stress conditions, recorded the weight of mice weekly for 8 weeks. The stress procedures of mice included cage rearing, wet padding, no cushion, cold water immersion, air water bottle stimulation, 24h light / dark, black and white reverse, and cages tilting 45 degrees. The results showed that CUMS treatment reduced the new antibody antibody to broilers. The titer, inhibiting the proliferation of peripheral blood lymphocyte and improving the gene expression of stress hormone receptor in the spleen, adding tryptophan in the diet can increase the proliferation of peripheral blood lymphocytes, the titer of NDV antibody, reduce the gene expression of stress hormone receptor in the spleen cells, and then inhibit the combination of hormone and receptor, thus enhancing the immune function of the body. The results show that tryptophan may be by blocking the combination of stress hormones and their receptors, thus alleviating stress and improving the immune function of the body and enhancing the immune function of the body. Experiment four: To explore the mechanism of CUMS treatment on the immune function of animal body and reveal the mechanism of L- tryptophan alleviating stress regulating immune function. T lymphocytes were isolated from the peripheral blood of mice, and the effects of stress hormones on the immune activity of T lymphocytes were studied by adding DEX, AD and NE, and the results showed that stress hormone treatment significantly inhibited the proliferation of T lymphocytes, and the stress hormone receptor specific inhibition was significantly relieved by stress. The effect of hormone on the proliferation of T lymphocytes indicates that the immune dysfunction caused by CUMS treatment may be by promoting the release of hormones such as corticosterone, such as corticosterone, CORT, NE, AD, and the expression of these stress hormone relative receptors on the immune cells, thus promoting the combination of hormones with their receptors and increasing the sensitivity of hormones. In order to further reveal the immune function homeostasis of the body, to further reveal the response of L- tryptophan to relieve stress response and regulate the mechanism of immune function, this experiment uses exogenous L- tryptophan and its metabolite 5-HT to pretreat the T lymphocyte, and studies its effect on the inhibition of T lymphatic cell activity induced by stress hormones. The results show that L- tryptophan The effects of acid and 5-HT preconditioning on the proliferation of T lymphocytes can be alleviated to a different degree,.L- tryptophan and 5-HT may be by reducing the release of stress hormones and the expression of their receptors and blocking the combination of the two, thus passivating the sensitivity of stress hormones and thus protecting the animals from immune disorders caused by stress, and the conclusion is that The results of the internal test suggest that L- tryptophan may play a role in activating the 5-HT metabolic pathway in the tryptophan metabolic pathway, making more L- tryptophan decomposed into 5-HT., and L- tryptophan alleviates stress induced intestinal barrier by reducing the secretion of stress hormones and blocking the binding of stress hormones to their receptors. Structural and immune disorders.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S816

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