The Role Of TDO Regulating Trp-Kyn-Ahr Pathway In The Immune Response Of Rat Liver Transplantation

Objective:We established stable model of rat orthotopic liver transplantation,and then explored the role of the tryptophan 2,3-dioxygenase(TDO)mediating tryptophan(Trp)-kynurenine(Kyn)-aryl hydrocarbon receptor(Ahr)pathway in liver immune rejection after rat liver transplantation,the Kyn-Ah R involved the immune tolerance of liver cells in vitro was also verified.Metholds:Based on Kamada’s "two cuff method",combined with related literature and previous studies,we established a stable ROLT immune rejection model with Wistar rats(n=50)as donors and DA rats(n=50)as receptors,a total of 50 cases,DA→DA,25 cases as A group.The immune rejection group was randomly divided into 2 groups before operation,the experimental group(680C91 inhibition C group)and the control group B group,25 cases in each group.The TDO inhibitor 680C91 was intraperitoneally injected into the experimental group one day before the receptor operation,and the drug concentration in the receptor was maintained after the operation.Each group was divided into five subgroups according to the time after transplantation(hours 0,24,72,and 144),then peripheral blood and tissues of each subgroup(n=5 rats)were collected.In addition,the last subgroup(n=5 rats)of both groups were used for survival analysis.The changes of liver function(ALT,AST,TBIL),Kyn and Trp were detected at 0,24,72,144 hours of each group.Observation of the pathological changes of liver tissue at various time points and evaluation of the immune rejection activity coefficient(RAI)by Banff scheme,detected the expression of TDO and Ahr target genes CYP1A1,CYP1B1 in liver tissue.To verify the in vitro immune tolerance mediated by Kyn in liver cells,we divided the experiment groups into three,including normal rat hepatocyte group(BRL-3A),BRL-3A+680C91 group,BRL-3A+680C91+DMF group,BRL-3A+680C91 +Kyn group.Dimethoxyflavone(DMF)is a specific inhibitor of Ahr.Western blot was used to detect the expression of CYP1A1 and CYP1B1 in each group.The concentration of Kyn in the supernatant of each group was detected and co cultured with peripheral blood lymphocytes from DA rats,the stimulation index SI was calculated.Then construct sh Ahr lentiviral transfection of BRL-3A,downregulation of Ahr expression,the experiment is divided into 4 groups,including rat normal liver cells(BRL-3A)group,BRL-3A+680C91 group,BRL-3A+680C91+Kyn group,each group was divided into sh Ahr lentivirus transfection group(sh-Ahr)and empty vector transfected negative group(sh-NC),and then verified the knockdown efficiency by PCR.The expression of CYP1A1,CYP1B1 and the concentration of Kyn in the supernatant were detected by Western blot and HPLC,calculated the stimulation index SI.Results:The orthotopic liver transplantation model in rats was successfully established.There was no significant difference in donor operation,liver repair cannula,recipient operation and total time of liver transplantation bewteen DA→DA and Wistar→DA groups.The success rate of operation was 86.7% and 88.3% respectively(P>0.05).In group B and C,serum AST,ALT and TBIL gradually increased at 0,24,72,144 hours,while the A group rapidly increased in 24 hours and then slowed down to normal level.The pathology of liver graft detection results showed that the there was no obvious immune rejection after DA→DA and the receptors could survive for a long time.In the group Wistar→DA,the immune rejection was gradually increased in 72 h,and the RAI score in the C group was higher than that of the B group.In the C group,RAI increased from 6.24±1.65(72h)to 8.11± 0.72(144h),and the B group increased from 4.43±1.39(72h)to 7.11±0.49(144h).The average survival time of the C group was 13.5 days,and the B group was 20 days(P<0.05).In the B group,the expression of TDO increased with the increase of postoperative survival time and was more obviously at 3 days after the operation.In group A,the expression of TDO increased slightly with the increase of postoperative survival time,after TDO was inhibited,the expression of TDO was similar to group B.However,with the increase of postoperative survival time,TDO increased slightly comparing the results of western blot at 0h and 24h(P<0.05).In the group C,the serum Trp was inhibited by TDO.For the catabolism of Trp was weakened,the concentration increased obviously and the concentration of Kyn was significantly lower than that of the control group.The 72 h concentration decreased significantly after Trp in group B,the concentration of Kyn increased in DA→DA group;the trend of Trp and Kyn was significantly weaker than that of Wistar→DA in group B,which coincided with the expression trend of TDO,Ahr downstream gene CYP1A1 and CYP1B1 and the expression of TDO in each group was similar(P<0.05).BRL-3A cells were divided into different treatment groups.In each group of them,after being treated by 680C91 and DMF,the decrease of Kyn concentration was more obvious when the inhibition of TDO+Ahr than TDO alone suppressed,and the two were all lower than those of the untreated group.In group 680C91+Kyn,Kyn could maintain a certain concentration ratio.The expression of Ahr downstream gene CYP1A1 and CYP1B1 in 680C91+DMF group was lower than that in 680C91 group,while the expression in 680C91 group was lower than that in untreated group and 680C91+Kyn group(P<0.05).The value of SI in each group was consistent with the trend of Kyn concentration,and that of group 680C91+DMF and 680C91 was significantly higher than that of 680C91+Kyn and that of untreated group(P<0.05).After knocking down Ahr,the SI in the normal group,680C91 group and 680C91+Kyn increased significantly compared with the blank group,but after knocking down,the Kyn transformation trend was also consistent among the treatment groups(P<0.05).Conclusions:Using modified Kamada “two cuff methods”,the rat liver transplantation immune rejection model could be stablely established with Wistar rats as donors and DA rats as receptors.The liver function and pathological changes conform to the characteristics of acute immune rejection.TDO mediating Trp metabolic pathway TrpKyn-Ahr has a certain effect on immune rejection after liver transplantation in rats.The expression of TDO increased in liver tissue after liver transplantation in rats,and inhibition of TDO may aggravate the immune rejection of liver transplantation.This research suggests that TDO may be involved in mediate immune rejection in liver transplantation by regulating the Trp-Kyn-Ahr pathway.At present,due to there is a lack of TDO knockout rats,further exploration of the role of TDO in liver transplantation remains necessary.