Role Of Pyroptosis In Intestinal Congestion-reperfusion Injury In Mice

Background : In the treatment of related clinical diseases such as liver transplantation,intestinal transplantation,and intestinal obstruction,partial blood flow obstruction has clearly caused ischemia reperfusion injury(ICI,Ischemia Reperfusion Injury);and intestinal tissue damage has not been relieved when blood flow reperfusion.At the same time,the toxins and translocated bacteria produced by tissue damage circulate to the remote organs and the whole body with the blood flow,causing damage to remote tissues and organs,that is,intestinal congestion-reperfusion injury(ICRI,Intestinal Congestion-reperfusion Injury).Related research shows that apoptosis plays an important role in the pathogenesis of ICRI.Apoptosis is a classic way of cell death,and recent studies have shown that pyroptosis that characterized by inflammatory reactions and lysis of cells is also a way of cell death.There are two pathways for the occurrence of pyroptosis,the classic pathway mediated by caspase1/4/5/11 in the caspase family and the non-classical pathway mediated by lipopolysaccharide(LPS).In the classic pathway of pyroptosis,pattern recognition receptors(pattern recognition receptors,PRRs)are used to identify pathogen associated molecular patterns(pathogen associated molecular patterns,PAMPs)or danger associated molecular patterns(danger associated molecular patterns,DAMPs)when the body's cells are subjected to various internal and external harmful stimuli,and these complex combined with/or linker protein ASC to form a multi-protein complex inflammatory body and recruit and activate caspase-1 precursor,activated caspase-1 cuts react substrate GSDMD and the formed N-terminal GSDMD can perforate the cell membrane and cause lytic death of the cell,releasing a large number of inflammatory cells and chemokines and then aggravating the inflammatory response and further tissue damage;in non-classical pathways,LPS cleave GSDMD by activating caspase-4/5(human)and caspase-11(mouse)and then entering the next reaction.This method of pyroptosis has been extensively studied in varieties of tissue injuries and inflammatory bowel disease has also been confirmed to be involved in pyroptosis.But there is no clear report that pyroptosis is involved in occurrence and development of ICRI as so far.Based on the above,we speculate that pyroptosis also participates in the mechanism of ICRI and plays an important role.Therefore,we established the animal model of ICRI to verify the above conjecture and explore the mechanism of ICRI.Objective and aim: In this experiment,the animal model of intestinal congestion reperfusion injury in mice was established to detect the expression of cell pyroptosis related genes and to explore whether cell pyroptosis was the mechanism of ICRI injury in mice.Methods: 142 male C57BL/6 mice(8-10 weeks old,weight 18-22 g,SPF level)were used in this experiment.First,we established the model of intestinal congestion injury(intestinal congestion injury,ICI)and got the best congestion length and time.Then we established the model of intestinal congestion reperfusion injury(ICRI).Thedetails were as follows.1.Established ICI model.(1)Survival analysis and observation: According to the total length of the small intestine of the mice,the intestines with the length of 10 cm,15 cm and 20 cm were selected for the experimental group,including sham operation control group(control group,n=10),congestion 10 cm group(C10 group,n=10),congestion 15 cm group(C15 group,n=10)and congestion 20 cm group(C20 group,n=10),,11-0 needle thread was used to ligate the veins on the mesentery margin of C10 group,C15 group and C20 group respectively among 40 mice and observed 48 hours to record the survival of the mice and draw the survival curve(kaplan-meier method),which was used to compare among groups(log rank test);2)Obtaining of specimens: another 60 mice were ligated using the above method,C10 group(n=15),C15group(n=15),C20(n= 15)The mesenteric limbal venous blood vessels of the experimental.Treatment of the control group was the same as above 1)(n=15),and three samples of the intestinal tract samples of congestion at different time points(Control,0.5h,1h,1.5h,2h)were taken into considerations and recorded respectively.The pathological changes of the small intestine tissue were observed after conventional HE staining.Chiu's intestinal mucosal injury score method was used to evaluate the damage of the small intestinal mucosa.Spearman correlation analysis method was used to analyze the correlation between different groups and different time points.Finally,the optimal parameters of the congestion length and time of the mouse intestinal congestion model were obtained,which were used in subsequent experiments2.The establishment of an ICRI model: performing a congestion reperfusion loss(ICRI)study with a congested intestinal tube length of 15 cm and a congestion time of 1 h was done.Forty-two male C57BL/6 mice(8 to 10 weeks old,18 to 22 g in weight,SPF grade)were divided into 7 groups according to the different reperfusion time,6 in each group: sham operation Control group(Control group),ICR 30 minutes group(reperfusion 30 minutes),ICR 1hr group(reperfusion 1hr),ICR 1hr and 30 minutes group(reperfusion 1hr30minutes),ICR 2hrs group(reperfusion 2h),ICR 2hrs 30 mins Group(reperfusion 2hrs 30minutes)and ICR 3hrs group(reperfusion 3hrs).Using a 11-0 silk thread with a needle was done,ligating the intestinal tube of 15 cm in length,congestion time 1hr.The removal of the ligation line,observation and the collection and drawing of the mouse blood and intestinal tissue samples at different time points of the above reperfusion was also carried out.Extraction of the serum in the blood sample,using a biochemical detector for Liver function and myocardial enzyme levels were detected.HE staining was used to observe the histopathological changes of the small intestine in each group of mice.RT-PCR and Western Blot were used to detect the genes related to cell pyrolysis and inflammation in the intestinal tissue of each group.Results:(1)The ICI model of mice was established successfully by ligating mesenteric vein with silk thread,and the best model conditions were obtained as follows: the length of the congestion bowel was 15 cm,the time of congestion was 1h,and then the ICRI model was established successfully;(2)The detection results of liver function and myocardial enzymes revealed that the expression levels of each experimental group increased with the reperfusion time compared with the sham operation control group,among which ALT(ICR 2h group,p<0.001),AST and LDH(ICR 1.5h group,p <0.0001),AST/ALT(ICR 2.5h,p <0.05),CK(ICR 2.5h group,p<0.0001)and CK-MB(ICR 1.5h group,p <0.0001)The expression level was significantly increased,and the difference was statistically significant.(3)HE staining among groups: the structure of intestinal mucosa in the sham operation control group was intact under the light microscope,the villi were arranged in order,and the cells were as usual;the intestinal mucosa and villi structure in the ICI group and the ICRI group were damaged in varying degrees under the light microscope.There were expansion of the hypodermic space,the subepithelial space,the loss of the top of villi,and the inherent membrane exfoliation,hemorrhage or local ulcer formation,which accompanied by a large number of inflammatory cell infiltration with the extension of time.This phenomenon began to relieve in ICR 2.5h group or ICR 3h group,and there was lymphoid follicle formation in ICR 3h group.(4)After ICR,the expression of genes and proteins related to cell scorch increased with the prolongation of reperfusion time,and gradually decreased after reaching the peak.1)The expression of related mRNA:(1)The expression of Caspase-1(ICR 1.5h,p < 0.05),caspase-11(ICR 2.5h,p < 0.05)and NLRP3(ICR 1/1.5/2/2.5/3h,p < 0.05)related to cell scorch was significantly higher compared with the control group;GSDMD was significantly lower in ICR 0.5h(p < 0.05)compared with the control group.(2)TNF-?(ICR1.5/2.5h group,p < 0.05),IL-1?(ICR 2/2.5h group,p < 0.001),MCP-1(ICR 2H group,p < 0.0001)and cxcl-1 / 2(ICR 2h group,p < 0.0001)were significantly higher compared with the control group.2)The expression of related proteins:(1)The expression of AIM2(ICR 1h,p < 0.05),ASC(ICR 2.5h,p < 0.05),caspase-1(ICR 2h,p < 0.05),GSDMD(ICR 1h,p < 0.01)and caspase-11(ICR 1h,p < 0.01)were increased significantly compared with the control group,which are involved in the nonclassical pathway of charring,.(2)The expression of IL-1?(ICR 2h group,p <0.001),IL-18(ICR 1.5/3 h group,p < 0.01),TNF-?(6 groups,p < 0.01),MCP-1(ICR2h group,p < 0.0001),cxcl-1/2(ICR 2h group,p < 0.0001)was significantly higher in the experimental group compared with the control group.Conclusion:(1)Success in the established the mice ICI models and ICRI models with the parameters of congestion intestinal tube length of 15 cm and congestion time 1h was well noticed.(2)Success in the established the mice ICI models and ICRI models with the parameters of congestion intestinal tube length of 15 cm and congestion time 1h was well noticed.(3)The classic pro-inflammatory factors TNF-?,interleukin family IL-1? and IL-18,MCP-1 and chemokine family members CXCL1/2 were also involved in ICRI inflammatory process.The occurrence and development of the reaction was well noted.(4)The ICRI process can indeed cause functional damage to remote organs such as liver and heart.