| 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. |
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