【作者】 王飞；

【导师】 李勇； 吴文；

【作者基本信息】 江西农业大学 ， 兽医（专业学位）， 2021， 硕士

【摘要】 肠屏障功能和肝脏自身免疫系统在防止抗原入侵中发挥重要作用,目前对内毒素诱导的瓦灰鸽肝肠免疫病理和显微结构改变的研究鲜有报道。因此,本试验采用沙门氏菌脂多糖诱导瓦灰鸽,探讨其对瓦灰鸽肝肠组织形态结构的变化以及损伤机理。本试验选取50只28日龄体重相近(0.47±0.05 kg)、健康状况良好、雌雄各半的瓦灰鸽,平均分为两组,分别为试验组和对照组,采取腹腔注射的给药方式分别对两组瓦灰鸽每日注射相同体积的脂多糖(100μg/kg)溶液和生理盐水,整个试验过程持续5天。在整个试验期的第1、3、5天从每组中选取6只瓦灰鸽,心脏采血,进行离心得到血清,以测量其炎性相关分子(炎症因子:TNF-α、HMGB1和抗炎因子:IL-10)、抗氧化指标(SOD、CAT、GSH、MDA)以及分别反应肠道(D-乳酸)和肝脏(AST、ALT)损伤指标的含量。颈部放血至死后采集回肠和肝脏组织,分成两份,一份固定于4%多聚甲醛中,分别通过HE染色和免疫组织化学染色来观察回肠和肝脏病理形态结构以及回肠中炎性因子TNF-α和IL-10阳性细胞的表达和分布情况;另一份冻存于超低温冰箱中,通过RT-q PCR以及蛋白质印记技术研究LPS诱导对瓦灰鸽回肠和肝脏组织中炎症信号通路、炎症因子、细胞凋亡、细胞自噬相关分子的mRNA以及蛋白表达的影响,同时研究肠道紧密连接蛋白mRNA的表达情况,以探讨LPS诱导瓦灰鸽肝肠损伤相关分子机制。本试验获得如下结果:1.LPS诱导引起了瓦灰鸽氧化应激。与对照组相比,持续注射LPS后第1天降低了瓦灰鸽血清中CAT以及GSH活性,升高了MDA含量(P<0.01,P<0.01);在第3次注射LPS后,与对照组相比,SOD、CAT以及GSH的活性更低(P<0.05或P<0.01),MDA的含量则高于对照组(P<0.05);在第5次注射LPS后,与生理盐水组相比,CAT以及GSH的活性更低(P<0.01,P<0.05)。2.LPS刺激引起瓦灰鸽炎症反应。与对照组相比,在LPS诱导瓦灰鸽的第1、3、5天血清中TNF-α、IL-10的表达量均升高;而HMGB1的表达量在后期(第3、5天)高于生理盐水组。3.LPS诱导引起了瓦灰鸽回肠损伤。ELISA结果显示:LPS组血清中D-LA含量在1、3、5天时都明显高于生理盐水组(P<0.01)。HE染色结果表示:LPS刺激引起瓦灰鸽回肠绒毛明显缩短,隐窝深度加深,二者的比值降低。免疫组织化学染色结果显示,LPS刺激引起瓦灰鸽TNF-α和IL-10阳性细胞表达均高于对照组。RT-q PCR结果显示,LPS刺激诱导瓦灰鸽回肠紧密连接蛋白Occludin、Claudin3和ZO-1的mRNA表达降低;炎症信号通路中相关分子在第1天时与对照组相比,LPS组NF-κB、TNF-α、IL-10的mRNA表达量升高,而TLR4 mRNA表达水平降低,在第3天时与对照组相比,LPS组TLR4、My D88、NF-κB、TNF-α、HMGB1 mRNA的表达水平增高;细胞凋亡通路相关分子在第1天时与对照组相比,LPS组Caspase-3 mRNA表达降低,第3天Apaf-1、Bcl-2和Caspase-3 mRNA表达升高。与生理盐水组进行比较,细胞自噬通路中LPS组在第1、3天时Atg5的mRNA表达量以及在第3、5天时Beclin1的mRNA表达水平更高。蛋白质印记试验结果表明,LPS组在第1天时LC3、Beclin1蛋白表达量显著高于生理盐水组,在第3天时TLR4蛋白高于生理盐水组,在第5天时TNF-α、Beclin1蛋白表达高于生理盐水组。以上结果表示:LPS刺激引起瓦灰鸽回肠紧密连接蛋白(Occludin、Claudin3和ZO-1 mRNA)的表达降低,炎症通路相关分子(TLR4、My D88、NF-κB、TNF-α、HMGB1、IL-10 mRNA及TLR4、TNF-α蛋白)、凋亡通路相关分子(Apaf-1、Bcl-2、Caspase-3mRNA)、自噬通路相关分子(Atg5、Beclin1 mRNA和LC3、Beclin1蛋白)的表达升高,从而导致瓦灰鸽回肠损伤。4.LPS刺激导致瓦灰鸽肝脏损伤。HE结果显示:瓦灰鸽腹腔第1次注射LPS后,肝细胞出现脂肪变性,中央静脉周围淋巴细胞浸润和充血,细胞核固缩;在连续注射LPS后的第3天,肝细胞脂肪变性加重,中央静脉周围大量炎性细胞浸润并出现淤血,肝窦扩张明显;在第5天时,中央静脉周围出现少量的炎性细胞浸润和轻度淤血。血清检测结果显示:LPS刺激瓦灰鸽后ALT和AST活性均明显高于对照组。RT-q PCR结果显示:LPS诱导瓦灰鸽肝脏炎症通路相关分子TLR4、My D88、NF-κB、TNF-α和IL-10 mRNA表达在第1天时均明显高于对照组,在第3次注射LPS后均达到峰值,在5天时有所下降,但始终高于对照组表达水平;细胞凋亡通路相关分子Apaf-1、Bcl-2和Caspase-3 mRNA表达均在第1、3天明显高于对照组;细胞自噬通路相关分子Beclin1和Atg5 mRNA表达在整个试验期均明显高于对照组。Western Blot结果显示:注射LPS后的第1天,瓦灰鸽肝脏中Caspase-3蛋白表达明显升高,第3次注射LPS后TNF-α、Caspase-3、LC3和Beclin1蛋白表达均达到峰值,第5天注射LPS后均表现下降的趋势。以上结果表示:LPS刺激引起瓦灰鸽肝脏炎症通路相关分子(TLR4、My D88、NF-κB、TNF-α、IL-10 mRNA及TNF-α蛋白)、凋亡通路相关分子(Apaf-1、Bcl-2、Caspase-3 mRNA和Caspase-3蛋白)、自噬通路相关分子(Atg5、Beclin1 mRNA和LC3、Beclin1蛋白)的表达升高,从而造成瓦灰鸽肝损伤。综上所述,沙门氏菌LPS刺激瓦灰鸽引起全身氧化应激与炎症反应,破坏肠道屏障,肠道通透性增加,以及回肠大量炎性因子释放并引起细胞凋亡和自噬,导致肠损伤。肠道受损导致LPS通过门静脉系统流入到肝脏中,使肝脏组织发生病变,产生大量炎性因子,从而促使细胞凋亡和自噬,最终造成肝损伤。更多还原

【Abstract】 Intestinal barrier function and liver’s own immune system play an important role in preventing antigen invasion.At present,there are few reports on the immunopathological and microstructure changes of liver and intestine induced by endotoxin in Wahui pigeon.Therefore,in this experiment,salmonella lipopolysaccharide was used to induce Wahui pigeon to explore the morphological changes and injury mechanism of liver and intestine.In this experiment,fifty 28-day-old Wahui pigeons with similar body weight,good health,half male and female were equally divided into the experimental group and the control group.Lipopolysaccharide(100 μg/kg)solution was administrated to the experimental group by intraperitoneal injection with daily when the same volume of normal saline was used to the control group by the same way,and the whole experiment lasted for 5 days.Six Wahui pigeons were selected from each group on the 1st,3rd and 5th day of the experimental period.The heart blood was collected and the serum was separated.The contents of antioxidant indexes(SOD,CAT,GSH,MDA),inflammatory factors(TNF-α,HMGB1,IL-10)and intestinal(D-lactic acid)and liver(AST,ALT)injury indexes were determined.The ileum and liver tissues were collected from bloodletting in the neck until postmortem.One part was fixed in 4% paraformaldehyde.The pathological morphology of ileum and liver and the expression and distribution of TNF-α and IL-10 positive cells in ileum were observed by HE staining and immunohistochemical staining,respectively.The other sample was stored in refrigerator at-80℃.The effects of LPS on the expression of inflammatory signal pathway,inflammatory cytokines,apoptosis,autophagy related molecules mRNA and protein in ileum and liver of Wahui pigeon were studied by fluorescence quantitative PCR and Western Blot techniques,and the expression of intestinal tight junction protein mRNA was studied to explore the molecular mechanism of liver and intestine injury induced by LPS.The results of this experiment are as follows:1.The oxidative stress of Wahui pigeon was induced by LPS.On the first day after continuous injection of LPS,compared with the control group,the activities of CAT and GSH in serum were significantly lower,and the expression level of MDA was higher(P<0.01,P<0.01).After the third injection of LPS,the activities of SOD,CAT and GSH in serum were lower than those in the control group(P<0.05 or P<0.01).After the fifth injection of LPS,compared with the normal saline group,the activities of CAT and GSH decreased continuously(P<0.01,P<0.05),while there is no difference on the activity of SOD and the expression level of MDA.2.LPS stimulation caused inflammation in Wahui pigeon.On the 1st,3rd and 5th day of LPS-induced Wahui pigeon experiment,compared with the control group,the contents of serum inflammatory factors,such as TNF-α and IL-10,were constantly higher,but the expression level of HMGB1 was ceaselessly higher than that of normal saline group.3.The ileal injury of Wahui pigeon was induced by LPS.The results of ELISA showed that the content of D-LA in serum of LPS group was significantly higher than that of normal saline group on the 1st,3rd and 5th day.The results of HE staining showed that the ileal villi of the pigeons induced by LPS were significantly shortened,the ratio of villus length to crypt depth decreased,and the crypt depth deepened on the third day.The results of immunohistochemical staining showed that the expression of TNF-α and IL-10 positive cells in Wahui pigeon stimulated by LPS was higher than that in the control group.The results of RT-q PCR manifested that LPS stimulation could lead to lower the mRNA expression of tight junction proteins including Occludin,Claudin3 and ZO-1 in the ileum of Wahui pigeons.On the first day,compared with the saline group,the mRNA expression of NF-κB,TNF-α and IL-10 in LPS group was higher while the expression level of TLR4 mRNA was lower.On the third day,compared with the saline group,the expression of TLR4,My D88,NF-κB,TNF-α and HMGB1 mRNA in LPS group was higher.On the fifth day,could lead to higher level of HMGB1 and IL-10 mRNA.In the apoptotic pathway,LPS stimulation reduced the expression of Caspase-3 mRNA on day one,and the contents of Apaf-1,Bcl-2 and Caspase-3 mRNA was significantly higher compared with control group on the third day.In the autophagy pathway,in LPS group,the mRNA expression level of Atg5 on the first and third day and the mRNA content of Beclin1 on the third and5 th day were higher when compared with the saline group.The results of Western Blot demonstrated that compared with the control group,the expression of LC3 and Beclin1 protein in LPS group significant higher on the 1st day,the expression level of TLR4 protein higher on the third day,and the expression of TNF-α and Beclin1 protein increased on the fifth day.The above results suggested that LPS stimulation decreased the expression of tight junction proteins(Occludin,Claudin3 and ZO-1 mRNA)in the ileum of Wahui pigeons,and increased the expression of inflammatory pathway related molecules(TLR4,My D88,NF-κB,TNF-α,HMGB1,IL-10 mRNA and TLR4,TNF-α proteins),apoptosis pathway related molecules(Apaf-1,Bcl-2,Caspase-3 mRNA),autophagy pathway related molecules(Atg5,Beclin1 mRNA and LC3,Beclin1 proteins),which resulted in ileal injury of Wahui pigeons.4.LPS stimulation caused liver damage in Wahui pigeons.The results of HE showed that after the first intraperitoneal injection of LPS,the hepatocytes showed steatosis,lymphocyte infiltration and hyperemia around the central vein,and nuclear pyknosis.On the third day after continuous injection of LPS,hepatocyte steatosis aggravated,a large number of inflammatory cells around the central vein infiltrated and congestion appeared,and the hepatic sinusoid dilated obviously.On the 5th day,there were a small amount of inflammatory cell infiltration and mild congestion around the central vein.The results of serum detection showed that the activities of ALT and AST in Wahui pigeons stimulated by LPS significantly increased compared with the control group.The results of RT-q PCR demonstrated that the expressions of inflammatory pathway related molecules TLR4,My D88,NF-κB,TNF-α and IL-10 mRNA in liver induced by LPS were significantly higher than those in the control group on the first day,reached the peak after the third injection of LPS,and decreased on the 5th day,but always higher than that in the control group.The expressions of apoptosis pathway related molecules Apaf-1,Bcl-2 and Caspase-3 mRNA were significantly increased compared with control group on the first and third day.The expression of autophagy pathway related molecules Beclin1 and Atg5 mRNA was significantly higher than that of the control group during the whole experimental period.The results of Western Blot presented that the expression of Caspase-3 protein in the liver of Wahui pigeon increased significantly on the first day after continuous intraperitoneal injection of LPS.The expression of TNF-α,Caspase-3,LC3 and Beclin1 protein reached the peak after the third injection of LPS,and decreased on the fifth day after injection of LPS.The above results showed that LPS stimulation increased the expression of inflammatory pathway related molecules(TLR4,My D88,NF-κB,TNF-α,IL-10 mRNA and TNF-α proteins),apoptosis pathway related molecules(Apaf-1,Bcl-2,Caspase-3mRNA and Caspase-3 proteins)and autophagy pathway related molecules(Atg5,Beclin1 mRNA,LC3,Beclin1 proteins)in the liver of Wahui pigeons,which resulted in liver injury of Wahui pigeons.To sum up,salmonella LPS stimulation of Wahui pigeons caused systemic oxidative stress and inflammation,which destruced intestinal barrier and increased intestinal permeability,and the release of a large number of inflammatory factors in ileum caused apoptosis and autophagy,which leaded to intestinal injury.The damaged intestinal tract causesd LPS to flow into the liver through the portal vein system,which caused pathological changes in the liver tissue and produces a large number of inflammatory factors,thus promoting apoptosis and autophagy,and which resulted in liver injury.更多还原