硒对金黄色葡萄球菌诱导的巨噬细胞和奶牛乳腺上皮细胞炎性损伤的保护作用研究

发布时间：2018-08-19 17:06

【摘要】：奶牛乳腺炎过程中伴随着免疫细胞在乳腺内的聚集、炎症因子释放等病理学特征,同时造成产奶量下降与淘汰率升高,给奶牛养殖业带来不可估量的损失。在动物体内,硒主要以硒蛋白的形式存在,除作为一种重要的饲源性微量元素外,硒在机体防御体系中也发挥着重要作用。临床研究表明饲料中添加适量的硒,能显著降低奶牛乳腺炎的发病率或减轻乳腺的病变程度。因此,本实验体外研究了硒的抗炎作用以及硒对炎症相关信号通路的影响,以丰富奶牛乳腺炎的发病机理并为乳腺炎的防治提供理论基础。金黄色葡萄球菌是乳腺炎的主要致病菌,揭示其致病机理以及机体对其感染的防御机制,具有重要的现实意义。实验采用经乳导管灌注金黄色葡萄球菌的方法构建干奶期奶牛乳腺炎急性感染模型,通过血液常规检查、生化相关指标和相关炎症细胞因子基因的检测,观察金黄色葡萄球菌侵袭奶牛乳腺组织后奶牛血液和乳腺组织局部的变化。结果显示,金黄色葡萄球菌侵入奶牛乳腺组织24 h后血液白细胞数显著增加；血清总蛋白、球蛋白的含量以及乳酸脱氢酶的活性升高；乳腺组织中TNF-α、IL-1β、IL-6和IL-8的mRNA表达均出现显著或极显著的升高,这些结果表明金黄色葡萄球菌诱导奶牛乳腺组织发生炎性损伤,并诱发全身性先天免疫应答反应。采集处于泌乳期的健康荷斯坦奶牛乳腺组织培养奶牛原代乳腺上皮细胞,构建金黄色葡萄球菌感染奶牛原代乳腺上皮细胞(bMEC)模型。检测在金黄色葡萄球菌感染后bMEC细胞因子表达的变化。结果表明,金黄色葡萄球菌感染乳腺上皮细胞后,IL-1β、TNF-α、IL-6和IL-8的mRNA表达于4～6 h内均出现显著或极显著升高(p0.01)且所有基因的表达均于8h达到峰值,说明金黄色葡萄球菌诱导奶牛乳腺上皮细胞炎症反应存在时间依赖性。为研究硒对奶牛乳腺上皮细胞活性的影响,采用不同浓度(0、1、2、4、8、16、32和64 μmol/L)的硒处理奶牛乳腺上皮细胞12 h, MTT法检测细胞活性。结果表明,0-16μM的硒不影响乳腺上皮细胞活性,当硒浓度在32 μmol/L和64 μmol/L时细胞活性极显著低于对照组(p0.001),表明高浓度的硒对乳腺上皮细胞有毒性作用。为研究硒在细胞炎症反应过程中对TLR2和Nod2信号通路的调控作用,在培养液中添加不同浓度的硒(2、4和8 μmol/L检测硒对TLR2和Nod2信号通路的影响。实验结果表明,硒对TLR2信号通路的传导具有抑制作用。金黄色葡萄球菌感染乳腺上皮细胞后,TLR2基因表达上升,在感染后8h,4μmol/L和8 μmol/L的硒能极显著抑制这种反应(p0.01)；在感染后8 h,金黄色葡萄球菌极显著提高Myd88基因的表达量(p0.001)；2、4和8 μmol/L的硒均能显著抑制金黄色葡萄球菌诱导的奶牛乳腺上皮细胞Myd88的表达；金黄色葡萄球菌极显著(p0.01或p0.001)提高乳腺上皮细胞Irak4和Irak1基因的表达量,但是硒对这一反应抑制作用微弱；金黄色葡萄球菌显著或极显著提高Traf6基因的表达(p0.05或p0.001)；硒能显著或极显著抑制这一反应(p0.01或p0.001)。硒可减弱Nod2信号通路的传导。在金黄色葡萄球菌感染后,乳腺上皮细胞Nod2基因表达升高或极显著升高,但是硒对这一效果的抑制作用微弱；金黄色葡萄球菌感染乳腺上皮细胞后RIP2基因的表达升高或极显著提高,硒能显著或极显著的抑制这一效果(p0.05～p0.001)。硒对乳腺上皮细胞炎症细胞因子基因的表达具有调控作用。金黄色葡萄球菌能显著或极显著的提高乳腺上皮细胞炎症细胞因子TNF-a、IL-1β和IL-6基因的表达,硒能显著或极显著抑制这种表达(p0.05～p0.001)。同时,金黄色葡萄球菌能极显著提高激活蛋白AP-1/c-jun和c-fos的表达,硒通过降低c-jun和c-fos异源二聚体的形成而减弱AP-1的表达。为从蛋白水平揭示硒对炎症信号通路的影响,采用Western Blot技术对NF-κB和MAPK信号通路中关键蛋白的表达进行检测。实验分5组,以不添加亚硒酸钠和金黄色葡萄球菌的细胞作为空白对照组,以金黄色葡萄球菌刺激0.5 h的细胞作为阳性对照组,以不同浓度的亚硒酸钠(2、4和8 μmol/L)预孵育12h并用金黄色葡萄球菌刺激0.5 h的细胞作为实验组,分别检测IκBα、p65、p38和Erk蛋白的磷酸化表达。结果显示,金黄色葡萄球菌刺激乳腺上皮细胞0.5 h后,IκBα、p65、p38和Erk蛋白的磷酸化水平与空白对照组相比极显著上升(p0.001)；与阳性对照组相比,4pM和8 μM的硒能极显著抑制金黄色葡萄球诱导的NF-κB IκBα和p65的磷酸化(p0.001),同时极显著降低金黄色葡萄球菌诱导的MAPK p38和Erk的磷酸化(p0.001),这表明4gM和8 μM的硒通过对TLR2以及Nod2信号通路的调控进而达到对NF-κB以及MAPK的抑制效果,最终下调了金黄色葡萄球菌诱导奶牛乳腺上皮细胞炎症细胞因子基因的表达,减弱了金黄色葡萄球菌诱导的奶牛乳腺上皮细胞的炎症损伤。最后,本实验构建了金黄色葡萄球菌感染巨噬细胞模型,通过向培养基内添加不同浓度的硒(1、1.5和2μmol/L),并采用荧光定量PCR和ELISA的方法检测硒对感染金黄色葡萄球菌感染巨噬细胞而引起的细胞因子表达与释放的影响。结果显示,与空白对照组相比,阳性对照组TNF-α、IL-1β和IL-6的表达与释放在10h内均极显著上升(p0.001)；与阳性对照组相比,不同浓度的硒下调或显著下调了巨噬细胞炎症细胞因子的表达与释放。为检测NF-μB以及MAPK信号通路是否参与巨噬细胞炎症反应过程中,采用Western Blot技术在蛋白水平上对IκBα、p65、p38、Erk和Jnk蛋白的磷酸化进行检测。结果显示,金黄色葡萄球菌刺激巨噬细胞0.5 h后,与空白对照组相比IκBα、p65、p38、Erk和Jnk蛋白的磷酸化水平极显著升高,说明NF-κB和MAPK信号通路被激活,与阳性对照组相比,硒能极显著抑制NF-κB和MAPK信号通路的传导,这种影响是通过抑制IκB、p65、p38和Jnk以及Erk的磷酸化完成的。
[Abstract]:With the accumulation of immune cells in the mammary gland and the release of inflammatory factors in the process of mastitis in dairy cows, the milk yield decreased and the elimination rate increased, which brought inestimable losses to the dairy cattle breeding industry. Selenium also plays an important role in the body defense system. Clinical studies have shown that dietary selenium supplementation can significantly reduce the incidence of mastitis or reduce the degree of breast lesion in dairy cows. Staphylococcus aureus is the main pathogen of mastitis, revealing its pathogenic mechanism and the body's defense mechanism against its infection has important practical significance. The changes of blood and mammary gland tissue in dairy cows were observed by routine blood test, biochemical indexes and related inflammatory cytokine gene detection. The expression of TNF-a, IL-1beta, IL-6 and IL-8 mRNA in mammary gland tissues were significantly or extremely significantly increased. These results indicated that Staphylococcus aureus could induce inflammation injury in mammary gland tissues of dairy cows and induce systemic innate immune response. The expression of bMEC cytokines in primary mammary epithelial cells of cows infected with Staphylococcus aureus (S. aureus) was detected after S. aureus infection. The expression of - 8 mRNA increased significantly or extremely significantly within 4 to 6 hours (p0.01) and all the genes reached the peak at 8 hours, indicating that the inflammation of dairy cow mammary epithelial cells induced by Staphylococcus aureus was time-dependent. To study the effect of selenium on the activity of dairy cow mammary epithelial cells, different concentrations (0, 1, 2, 4, 8, 16, 32 and 64) were used. The results showed that selenium of 0-16 Mu did not affect the activity of mammary epithelial cells. When selenium concentration was 32 and 64 mu mol/L, the cell activity was significantly lower than that of the control group (p0.001), indicating that high concentration of selenium was toxic to mammary epithelial cells. TLR2 and Nod2 signaling pathways were regulated by selenium (2,4 and 8 micromol/L). The results showed that selenium inhibited the transmission of TLR2 signaling pathways. TLR2 gene expression was observed in breast epithelial cells infected with Staphylococcus aureus. Selenium at 4, 4 and 8 micromol/L significantly inhibited this reaction (p0.01), Staphylococcus aureus significantly increased Myd88 gene expression (p0.001), 2, 4 and 8 micromol/L selenium significantly inhibited Myd88 expression in dairy cow mammary epithelial cells induced by Staphylococcus aureus. Staphylococcus aureus significantly or extremely significantly increased the expression of Irak4 and Irak1 genes in mammary epithelial cells (p0.01 or p0.001), but selenium had a weak inhibitory effect on this reaction; Staphylococcus aureus significantly or extremely significantly increased the expression of Traf6 gene (p0.05 or p0.001); selenium could significantly or extremely significantly inhibit this reaction (p0.01 or p0.001). Selenium could weaken the Nod2 message. The expression of Nod2 gene in mammary epithelial cells was elevated or significantly elevated after S. aureus infection, but the inhibition effect of selenium on this effect was weak. The expression of RIP2 gene in mammary epithelial cells infected by S. aureus was elevated or significantly elevated after S. aureus infection, and selenium could significantly or significantly inhibit this effect (p Selenium can regulate the expression of inflammatory cytokines in mammary epithelial cells. Staphylococcus aureus can significantly or extremely significantly increase the expression of inflammatory cytokines TNF-a, IL-1beta and IL-6 in mammary epithelial cells. Selenium can significantly or extremely significantly inhibit the expression of inflammatory cytokines TNF-a, IL-1beta and IL-6 in mammary epithelial cells (p0.05-p0.001). In order to reveal the effect of selenium on inflammatory signaling pathway at protein level, Western Blot technique was used to detect the expression of key proteins in NF-kappa B and MAPK signaling pathway. The cells without sodium selenite and Staphylococcus aureus were used as blank control group, the cells stimulated by Staphylococcus aureus for 0.5 h were used as positive control group, and the cells pre-incubated with different concentrations of sodium selenite (2,4 and 8 micromol/L) for 12 h and stimulated by Staphylococcus aureus for 0.5 h were used as experimental group. I kappa B alpha, p65, p38 and Erk were detected respectively. The results showed that the phosphorylation levels of I-kappa B-alpha, p65, p38 and Erk proteins in breast epithelial cells stimulated by Staphylococcus aureus were significantly higher than those in the blank control group (p0.001); compared with the positive control group, selenium at 4pM and 8mu M significantly inhibited the phosphorus levels of NF-kappa B I-kappa B-alpha and p65 induced by Staphylococcus aureus. Acidification (p0.001) and significantly decreased the phosphorylation of MAPK p38 and Erk induced by Staphylococcus aureus (p0.001) suggested that 4gM and 8mu M of selenium could inhibit NF-kappa B and MAPK by regulating TLR2 and Nod2 signaling pathways, and ultimately down-regulated the inflammatory cytokines induced by Staphylococcus aureus in dairy cow mammary epithelial cells. The expression of subgene weakened the inflammation damage of dairy cow mammary epithelial cells induced by Staphylococcus aureus. Finally, the macrophage model of Staphylococcus aureus infection was established. Different concentrations of selenium (1,1.5 and 2 micromol/L) were added to the culture medium, and the effect of selenium on the infection of dairy cow mammary epithelial cells was detected by fluorescence quantitative PCR and ELISA. The results showed that the expression and release of TNF-a, IL-1beta and IL-6 in the positive control group were significantly increased within 10 hours (p0.001) compared with the blank control group. Compared with the positive control group, different concentrations of selenium decreased or significantly decreased the inflammation of macrophages. In order to detect the expression and release of cytokines, the phosphorylation of I-kappa B-alpha, p65, p38, Erk and Jnk proteins at protein level was detected by Western Blot technique. The results showed that Staphylococcus aureus stimulated macrophages for 0.5 hours and compared with the blank control group, I-kappa was detected. The phosphorylation levels of Ba, p65, p38, Erk and Jnk proteins were significantly elevated, suggesting that NF-kappa B and MAPK signaling pathways were activated. Compared with the positive control group, selenium could significantly inhibit the transmission of NF-kappa B and MAPK signaling pathways. This effect was achieved by inhibiting the phosphorylation of I-kappa B, p65, p38 and Jnk and Erk.
【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S858.23

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