Establishment And Research Application Of Concanavalin A-Induced Acute Liver Failure Model In Pigs

Background&Aims:Acute liver failure is a clinically critical and severe disease.The conventional medical treatment is not ideal and the mortality rate is high.Although liver transplantation has been proven effective,it is difficult to carry out widespread problems due to lack of donors,high costs,technical difficulties,and severe postoperative immune rejection..Bio-type artificial liver can help remove toxins from patients,reduce liver burden,supplement essential substances,and stabilize physiological and biochemical indicators.It is an important treatment method for patients to restore liver function,and it is also a bridge to help transition to liver transplantation.In view of the current state of understanding of ALF and the difficulty of researching patients,animal models have an important substitute role in future basic research on the disease.It is important to establish an animal model that accurately reflects the clinical symptoms of human ALF.Objectives:1.Establish a large animal model that is close to the common acute liver failure in my country in terms of pathophysiological mechanism,so as to be used in preclinical research of bioartificial liver treatment.2.Through sequential monitoring of pig models of acute liver failure,a suitable time window for therapeutic intervention of bioartificial liver was found.3.To detect the expression of autophagy in the liver tissues of pig models of acute liver failure induced by Concanavalin A(ConA),analyze the mechanism of autophagy in immune liver injury,and use this animal model to find the treatment of acute liver failure.New means of liver failure.Methods:1.A large animal model of acute liver failure was established by injecting ConA through the ear vein with small-ear pigs in southern Yunnan province as the experimental research object.The experimental animals were randomly divided into 2 groups,namely ConA drug model(3,intravenous injection of ConA)and normal control group(3,intravenous injection of the same amount of 5%GS),before administration(0h)and after administration 2,4,6,8,10,12,and 24h to take blood samples for examination of liver function,blood sugar,blood coagulation and blood ammonia.The general conditions of the two groups of pigs were observed and recorded.After the pigs died,the liver tissues were taken for hematoxylin-eosin(HE)staining to observe the pathological changes.2.Real-time fluorescence quantitative PCR was used to detect the mRNA expression of autophagy-related genes Atg5,Atg7,Beclin-1,Lamp-1 and LC3a in the liver tissues of the two groups.3.Western blot was used to detect the protein expression levels of the abovementioned autophagy-related genes in the liver tissues of the two groups.Results:1.Successfully established a Diannan small-ear pig model of acute liver failure similar to human in clinical manifestations,biochemical indicators,and histopathological changes:1.1 General situation:After intravenous injection of ConA in model pigs,they showed significant appetite loss,reduced urination and defecation,darker urine,faster breathing rate,some vomiting,and the vomit was white secretions.1.2 Nervous system changes:Compared with the control group,the model pigs were sluggish,sluggish,slow to painful stimuli,unstable gait,and sometimes irritable,and showed drowsiness and coma over time.1.3 Survival time:ConA-induced model pigs died 24 hours after administration without any intervention,with an average survival time of 28.5±1.8h.1.4 Biochemical indicators related to liver function:serum total bilirubin,total bile acid,alanine aminotransferase,aspartate aminotransferase,blood ammonia,prothrombin time and INR of model pigs were all after administration There are different degrees of increase,6h and 8h,there is a significant difference compared with the control group(P<0.05).The blood glucose level of the pigs of the model building was lower than that of the control group,but the difference was not statistically significant.There was no significant difference in serum albumin between the two groups of pigs.Total bile acid and aspartate aminotransferase increased at 2h after administration and reached a peak at 24h;total bilirubin,alanine aminotransferase,blood ammonia and INR increased at 4-6 hours after administration,Reaching the peak at 24h.Compared with alanine aminotransferase,aspartate aminotransferase increased significantly,and the difference between the two was about 3 to 5 times.1.5 Histopathological manifestations of the liver:the model pig liver cells are spotted and bridging necrosis,the necrotic area is accompanied by inflammatory cell infiltration,the structure of the liver lobules is unclear,and interface inflammation can be seen in some areas,even with hemorrhage and acute cholangitis.Histopathological examination of pig liver in the control group showed no abnormalities.2.The expression of mRNA and protein of autophagy-related genes Atg5,Atg7,Beclin-1,Lamp-1 and LC3a in the liver tissues of the two groups of pigs was detected by Realtime PCR and western blot,and it was found that the autophagy expression of model pigs was significantly enhanced.Conclusions:1.The Diannan small-ear pig acute liver failure model induced by ConA well simulates the pathogenesis of common clinical causes of acute liver failure.2.The production method of the model is simple and reproducible.The circulation path established by the anterior vena cava catheter can meet the needs of sequential blood sampling and extracorporeal circulation of bioartificial liver.3.The indicators related to liver failure between the model and the control group are significantly different 6-8h after ConA administration.If no intervention is performed at this time,the experimental animals will die 24h after administration,so 24h after administration Inner is the best time window for artificial liver treatment.4.The expression of autophagy-related genes in the liver tissue of the Diannan smallear pig model of acute liver failure induced by ConA is enhanced,which is consistent with the autophagy expression level of viral hepatitis and autoimmune liver disease.Therefore,this model can also be used for immune liver injury-related autophagy.Further research on phagocytosis.