| Objective:We were aimed to establish an animal model of autologous orthotopic liver transplantation ischemia-reperfusion injury with SD rats,and to explore the changes of fecal microflora in SD rats at different time after liver transplantation ischemia-reperfusion injury.The potential mechanism of fecal microflora changes caused by liver transplantation ischemia-reperfusion injury was analyzed,so as to provide guidance for the intervention and treatment of liver transplantation ischemia-reperfusion injury from the perspective of intestinal microflora in the future.Methods:The animal model of hepatic ischemia-reperfusion injury in rats was established by AOLT.According to the different time after operation,the model group(group M)was divided into seven subgroups:M1,M4,M7,M10,M14,M21,M28,and the sham operation group(group S)was established for control.The blood and fecal samples of the 8 groups(n=5)were collected before operation.The liver,intestinal,blood and fecal samples were collected on the first day after operation in the sham operation group.In the model group(group M),the liver,intestinal,blood and fecal samples were collected on the 1st,4th,7th,10th,14th,21st and 28th day after operation,respectively.The indexes of ALT,AST,TBIL,WBC,NEUT and LYM were detected in the blood of rats,and the degree of hepatic damage,cholestasis and inflammation were evaluated from the point of view of hematology.Making pathological slices of liver and ileocecum and observing their pathological changes,in order to understand the damaged degree of liver and intestinal mucosa.The fresh feces of rats were detected by high-throughput gene sequencing for understanding the changes of fecal microflora after ischemia-reperfusion injury after AOLT.Results:1.In this study,the ischemia-reperfusion injury animal model of liver transplantation was established by AOLT,and the success rate of the model is 100%.On the first day after operation in the model group,we can find that the indexes of ALT,AST and TBIL in the model group were significantly higher than those in the sham operation group(S)on the first day after liver transplantation ischemia-reperfusion injury,the differences were significant(P<0.05).The pictures of hepatic pathology showed us that the hepatic tissue of group M1 presented severe injury,and the Suzuki’s score was 3.0.The above results confirmed that the AOLT animal model could reflect ischemia-reperfusion of the liver and which is an ideal animal model for the study of ischemia-reperfusion after liver transplantation.2.The changes of AST,ALT,TBIL and hepatic histopathology are the same basically after AOLT ischemia-reperfusion injury.Among them,the AST,ALT and TBIL in model group M1 were 776.20±298.79,241.00±66.21 and 2.90±2.00 respectively on the first day after operation,which were significantly higher than those in sham operation group(S)and other model groups(P<0.05).There was no significant difference between other model groups and sham operation groupin(S).From the trend charts of AST,ALT,TBIL and the pictures of hepatic pathology in each group,we can see that the degree of hepatic damage and cholestasis of SD rats after AOLT ischemia-reperfusion were outstanding in the early stage after operation(the 1st day).However,the damaged degree of liver is significantly alleviated in the middle stage after operation.At the late stage of operation(28th day),it had recovered to the normal level completely.3.After AOLT in SD rats,through the detection of WBC,NEUT and LYM in the blood of rats and the observation of intestinal pathological sections in the ileocecum,we found that ischemia-reperfusion injury could also lead to intestinal injury and inflammatory after AOLT,and the changes of both occurred in parallel.The values of WBC,NEUT and LYMD in each model group were significantly higher than those in the sham operation group(S)(P<0.05).From the charts of trend of each indexs and the pathological picture of ileocecal intestinal tissue,it can be seen that inflammatory and intestinal mucosal injury occurred in the early stage after transplantation,but both of them are mild.It goes through a process of increasing at first and then decreasing gradually in the middle stage after transplantation(the 4th-14th days).The inflammatory and intestinal mucosal injury were especially significant on the 7th-14th day.At the end of the late postoperative period(the 28th day),the index of inflammatory was still higher than the normal level,and the intestinal mucosa also showed slight injury,but all of them were significantly better than those in the middle stage after operation.4.Under normal conditions,from the phylum level,the fecal flora of SD rats are mainly firmicutes and Bacteroides,and the ratio is about 1.From the genus level,the fecal microflora of SD rats in the top five(abundance>1%)are Lactobacillus,Prevotella,Oscillospira,Ruminococcaceae_Ruminococcus,Bacteroides.The microflora in the feces of rats showed obvious changes after ischemia-reperfusion injury after AOLT.From the phylum level,the firmicutes increased significantly in the early stage(the 1th day),decreased gradually in the middle stage(the 4th day),and then began to increase again in the late stage(the 28th day).On the other hand,the change trend of Bacteroides is opposite to that of firmicutes.From the genus level,the abundance of Lactobacillus,Ruminococcaceae_Ruminococcus,Bacteroides and Blautia in the total fecal flora increased significantly in the early stage(the lth day)after liver transplantation ischemia-reperfusion,while the abundance of Prevotella and Oscillospira decreased significantly.The Alpha diversity analysis of fecal flora showed that the diversity of fecal flora decreased at this time.In the middle stage after operation(the 4th day),Lactobacillus,Ruminococcaceae_Ruminococcus,Bacteroides and Blautia in the total fecal flora increased significantly are began to decrease gradually,while Prevotella and Oscillospira began to increase.During this period,Bacteroides and Blautia showed a trend of increase at first and then decrease,and the diversity of bacteria also began to increase.At the end of late postoperative observation(the 28th day),Oscillospira,Bacteroides Ruminococcaceae_Ruminococcusand Blautia basically returned to the preoperative level,and the proportion of Prevotella in the total fecal flora was still higher than Lactobacillus,but the gap between them was further narrowed,showing a trend as the preoperative level.And the diversity of flora was still higher than the preoperative level.Conclusion(s):1.The ischemia-reperfusion injury animal model of AOLT in SD rats is an ideal model to study the ischemia-reperfusion injury of liver transplantation.2.Ischemia-reperfusion injury of AOLT in SD rats can cause the change of fecal microflora,and the change has obvious regularity.3.The liver ischemia-reperfusion injury in rats is the most serious in the early stage after AOLT,but they also recover quickly.The main manifestations in the middle and later stage after operation were inflammatory and intestinal mucosal injury secondary to ischemia-reperfusion injury of AOLT,the changes of fecal microflora in rats after ischemia-reperfusion injury of AOLT were related to intestinal injury and inflammation. |
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