IGF-1缓解高酮血症奶牛中性粒细胞免疫抑制的机制

发布时间：2018-05-06 03:01

本文选题：BHBA + NF-κB通路　；参考：《吉林大学》2017年硕士论文

【摘要】：血液中高浓度的β-羟丁酸(BHBA)是围产期酮病奶牛的主要临床特征。中性粒细胞是机体抵抗外源细菌感染的第一道防线,酮病奶牛表现为中性粒细胞免疫活性下降,吞噬作用、呼吸爆发作用减弱,从而导致酮病奶牛易于发生感染性疾病。因此,高浓度的BHBA可能与酮病奶牛免疫细胞免疫抵抗力下降密切相关。围产期酮病奶牛干物质摄入量减少而泌乳量增加所造成能量负平衡(NEB),可影响生长激素-胰岛素样生长因子(GH-IGF)轴内分泌调控系统,使胰岛素样生长因子1(IGF-1)分泌减少,干扰机体能量代谢,由此可加重酮病奶牛的先天免疫抑制。IGF-1在调控细胞代谢、增殖和分化方面起着非常重要的作用,因此,我们推测给予外源性IGF-1有可能改善酮病奶牛的免疫抑制。本实验通过体外培养奶牛中性粒细胞,分别添加不同浓度的BHBA、IGF-1和NF-κB通路抑制剂、PI3K抑制剂、自噬抑制剂,来探讨IGF-1调控围产期酮病奶牛中性粒细胞免疫抑制的作用与机制;通过分析酮病奶牛免疫抑制的原因,探究IGF-1调控中性粒细胞免疫抑制的信号通路,并通过测定中性粒细胞活性氧产生、吞菌实验和呼吸爆发,来揭示IGF-1在调控中性粒细胞免疫功能的作用,为提高酮病奶牛中性粒细胞免疫抑制提供理论依据。通过体内实验检测奶牛血液指标,结果显示,与健康奶牛相比,酮病奶牛血清中GH明显升高,而IGF-1的浓度显著下降,说明NEB导致GH调控IGF-1通路被阻断。通过Western blotting结果分析,表明酮病奶牛中性粒细胞发生严重的自噬现象,而自噬的过度积累会引发Ⅱ型细胞程序性死亡,这可能与细胞免疫抑制有关。体外培养中性粒细胞添加4.8 m M BHBA结果显示,在作用6 h时自噬关键蛋白LC3和磷酸化NF-κB p65蛋白表达量最高。添加不同浓度的BHBA及NF-κB通路抑制剂PDTC,结果表明,随着BHBA浓度的升高,磷酸化NF-κB p65的表达上调,加重了自噬的积累;而添加NF-κB通路抑制剂后,减轻了自噬的积累,说明高浓度的BHBA通过调控NF-κB通路上调了自噬的积累,所以,抑制NF-κB对调控中性粒细胞自噬尤为重要。随后在高浓度的BHBA的基础上,添加不同浓度的IGF-1,在IGF-1在100 ng/m L时,显著下调了NF-κB通路并抑制自噬的发生。IGF-1信号通路上的蛋白PI3K、AKT的测定结果表明,IGF-1可以上调PI3K和磷酸化AKT的表达,而添加PI3K抑制剂LY294002后,PI3K/AKT通路被抑制,磷酸化NF-κB p65和自噬关键蛋白LC3显著升高,说明IGF-1是通过调控PI3K/AKT通路和缓解NF-κB通路来抑制自噬的。通过体外添加高浓度的BHBA,检测其对中性粒细胞的免疫活性的影响,结果表明高浓度的BHBA使中性粒细胞活性氧的产生、吞菌实验和呼吸爆发作用显著下调,而添加自噬抑制剂3-MA后,抑制了高BHBA诱导的自噬积累,中性粒细胞活性氧的产生、吞菌实验和呼吸爆发作用显著上升,说明高BHBA诱发中性粒细胞自噬积累,导致细胞免疫活性下调。在高浓度的BHBA的基础上添加IGF-1(100 ng/m L)后,细胞免疫活性显著上调,说明IGF-1可以通过下调自噬来缓解由BHBA引起的中性粒细胞免疫活性低下。通过以上实验可以认为:高浓度的BHBA可以使中性粒细胞NF-κB通路激活,导致自噬过度积累,细胞免疫活性显著下降;IGF-1可以通过调控PI3K/AKT通路和缓解NF-κB通路抑制自噬的发生,从而上调中性粒细胞活性氧产生、吞菌能力和呼吸爆发作用,恢复中性粒细胞的免疫活性,进而提高围产期酮病奶牛先天免疫机能。
[Abstract]:The high concentration of beta hydroxybutyrate (BHBA) in the blood is the main clinical feature of perinatal ketosis cows. Neutrophils are the first line of defense against the external bacterial infection. The cows with ketosis show the decrease of neutrophil immune activity, phagocytosis and respiratory burst, which leads to the prone to infectious diseases of ketosis cows. Therefore, high concentration of BHBA may be closely related to the decrease of immune cell immunity in cows with ketodisease. The decrease of dry matter intake and the increase of lactating amount caused by the negative balance of energy (NEB) in perinatal ketosis cows may affect the regulation system of the growth hormone insulin-like growth factor (GH-IGF), and make insulin-like growth factor 1 (IGF-1). Decrease in secretion and interfere with the body's energy metabolism, thus can aggravate the congenital immunosuppressive.IGF-1 of cows with ketosis, which plays a very important role in regulating cell metabolism, proliferation and differentiation. Therefore, we speculate that giving exogenous IGF-1 may improve the immunosuppression of cows with ketosis. Do not add different concentrations of BHBA, IGF-1 and NF- kappa B pathway inhibitors, PI3K inhibitors, autophagy inhibitors, to explore the role and mechanism of IGF-1 regulation of neutrophilic granulocyte immunosuppression in perinatal cows. By analyzing the causes of immunosuppression in cows with ketoosis, the signal pathway of sexual granulocyte immunosuppression in IGF-1 regulation is explored, and the determination of the signal pathway in the regulation of sexual granulocyte immunity in IGF-1 regulation and regulation is determined. Neutrophil reactive oxygen species, bacteria swallowing experiment and respiratory burst to reveal the role of IGF-1 in regulating the immune function of neutrophils, and provide a theoretical basis for improving neutrophils immune suppression in cows with ketosis. The blood indexes of dairy cows were detected by in vivo experiments. The results showed that the serum levels of GH in cows of ketosis cows were significantly higher than those of healthy cows. The concentration of IGF-1 decreased significantly, indicating that NEB led to the blocking of the GH regulation IGF-1 pathway. Through the analysis of Western blotting results, the serious autophagy occurred in neutrophils of cows with ketodisease, and the excessive accumulation of autophagy could lead to programmed cell death of type II cells, which may be related to cellular immunity inhibition. Adding 4.8 m M BHBA results showed that the expression of autophagic key protein LC3 and phosphorylated NF- kappa B p65 protein was the highest at 6 h. The addition of BHBA and NF- kappa B pathway inhibitor PDTC, the results showed that the expression of phosphorylated kappa kappa was up up and increased the accumulation of autophagy with the increase of concentration. The accumulation of autophagy indicates that high concentration of BHBA increases the accumulation of autophagy by regulating the NF- kappa B pathway. Therefore, inhibition of NF- kappa B is particularly important for regulating autophagy in neutrophils. Then, on the basis of high concentration of BHBA, the addition of different concentrations of IGF-1 is added to IGF-1 at 100 ng/m L, which significantly reduces the NF- kappa B pathway and inhibits the occurrence of autophagy. The results of the protein PI3K on the F-1 signaling pathway, AKT showed that IGF-1 could up regulate the expression of PI3K and phosphorylated AKT, while the PI3K/AKT pathway was inhibited after the addition of the PI3K inhibitor LY294002, and the phosphorylated NF- kappa B p65 and autophagy key proteins were significantly increased, indicating that the inhibition of autophagy by regulating the pathway and alleviating the kappa pathway. The effect of high concentration of BHBA on the immune activity of neutrophils was detected in vitro. The results showed that high concentration of BHBA resulted in the production of reactive oxygen species in neutrophils, the inhibition of autophagy induced autophagy and the production of neutrophils after adding autophagic inhibitor 3-MA. BHBA induced autophagic accumulation of neutrophils and decreased cellular immune activity. After adding IGF-1 (100 ng/m L) on the basis of high concentration of BHBA, the cellular immune activity was significantly up-regulated, indicating that IGF-1 could alleviate neutrophils induced by BHBA by down regulation of autophagy. It is suggested that high concentration of BHBA can activate the NF- kappa B pathway of neutrophils, lead to excessive accumulation of autophagy, and significantly decrease the cellular immune activity; IGF-1 can inhibit the occurrence of autophagy by regulating the PI3K/AKT pathway and alleviating the NF- kappa B pathway, thus increasing the production of reactive oxygen species in neutrophils, the ability to swallow bacteria and the ability to swallow bacteria. The effect of respiratory burst can restore the immune activity of neutrophils and further improve the innate immunity of perinatal ketosis cows.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23

【参考文献】