Role Of Vasoactive Intestinal Peptide In Protecting The Grafts From Ischemia/Reperfusion Injury And Inducing Immune Tolerance After Rat Orthotopic Liver Transplantation

PartⅠ:Effects of Vasoactive Intestinal Peptide on the Expression of Toll-Like Receptor 4 in the Kupffer CellsObjectives:To explore the effects of vasoactive intestinal peptide on the activation of the kupffer cells and the expression of Toll-like receptor 4 on its surface, and to elucidate the probable mechanism of the effects.Methods:Kupffer Cells were isolated and purified by situ collagenase perfusion. The isolated and purified cells were than divided into 3 groups:Group A, control group in which kupffer cells were cultured normally; Group B, LPS group in which 10mg/l LPS was added into the nutrient solution; Group C, VIP+LPS group in which 1×10-8 mol VIP was added into the nutrient solution 24h before 10mg/l LPS was added. After 6h of stimulating to each group, the morphology of kupffer cells were measured by an inverted microscope, the activity of kupffer cell was measured by carbon phagocytic test, the purity was measured by anti ED-2 immunohistochemical staining, the level of TLR4 and NF-κB mRNA were measured by RT-PCR, the level of protein of TLR4 and NF-κB were measured by Western blot, the NF-κB DNA binging activity were measured by EMS A, the concentration of IL-1 and TNF-αin the supernatant were measured by ELISA. Treatments in each group were repeated for 8 times to obtain the mean value of each index as the final results.Results:The shape of newly isolated kupffer cells were sphere like, suspending in the nutrient solution; Most of them became flat and adhered to the wall, pseudopodium formation occurred in few cells; Pseudopodium formation occurred in most of the cells after 24h; After about 36h, all cells were extended totally with bigger size in star-shape or polygonal shape; Under a microscope after stained by Hematoxyhn-Eosin, typical star-shape were observed with renal type nuclear. The purity ratio of each group was about 90%, and the activity ratio was about 96%. The phagocytic ability of group C was higher than that of group B with smaller cell shape change; Group B was higher both in mRNA and protein level of TLR4 and NF-κB and in DNA binding activity of NF-κB than that of group A and C(P<0.05); Concentration of TNF-αand IL-1 of the nutrient solution in group B was much higher than that of group A and C (P<0.05).Conclusions:(1) Situ collagenase perfusion is a good method for kupffer cell isolation and purifying, by which the purity ratio and the activity ratio respectively reaches 90% and 96%;(2) VIP inhibits the phagocytic ability of kupffer cells and as a result reduce its ability of releasing cytokines such as TNF-a and IL-1;(3) VIP also inhibits the expression of TLR4 and the activation of NF-κB.PartⅡ:Modeling of the orthotopic liver transplantation in the ratObjectives:To establish a stable model of rat orthotopic liver transplantation.Methods:The operation was made following the modified Kamada's two-cuff technique, and the test animal was Sprague-Dawley (SD) male rats with closed colony, recipient rats were a little larger in size than the donor.10% glucose was given to the recipient rat after transplantation, and Penicillin was injected on the following 3 days without immunosuppressor.Results:30 cases were made in the early stage of modeling with 2 cases completed the operation, while other 28 cases died during the transplantation. The causes of death were:massive hemorrhage in 9 cases, anesthetic accident in 8 cases, pneumothorax in 5 cases, vascular injury by accident in 4 cases and with unknown reason in 2 cases. The 2 cases which completed transplantation died from anastomotic hemorrhage of the SIVC 0.5h and 2h after the operation respectively; In the stable stage of modeling 100 cased were made with donor harvest time 45-60min, warm ischemic duration 0-0.5min, donor preparation time 12-15min, cold storage of donor duration 2h, recipient operation time 85-100min, anhepatic phrase 20-23min,24h survival rate 92.0%(92/100), 1w survival rate 83.0%(83/100);8 cases died during the operation or within 24h after transplantation by:anesthetic accident in 3 cases, massive hemorrhage in 4 cases and pneumothorax in 1 case; 9 cases died successively in the following 7 days after transplantation from:infection in 4 cases, biliary leakage in 3 case and with unknown reason in 2 cases.Conclusions:(1) The modified Kamada's two-cuff technique can be used to made a stable model of the rat orthotopic liver transplantation for the advanced research in the future;(2) It is difficult to perform a liver transplantation in the rat and the sticking point to success is to shorten the time of operation and anhepatic duration, in addition the refined anastomosis techniques is also important.PartⅢ:Role of Vasoactive Intestinal Peptide in the Hepatic Ischemia/Reperfusion Injury in Rats After Transplantation Objectives:To explore the role of vasoactive intestinal peptide in the hepatic ischemia/reperfusion injury in rats after transplantation and elucidate the probable mechanism of the effects.Methods:Donors and recipients were all male SD rats, while the recipients were a little larger in size than the donors. The rats were randomly divided into 3 groups, n=8:Group A, sham group in which only peri-hepatic ligaments were dissociated and then close the abdomen; Group B, VIP group in which 2nmol VIP was given by intraperitoneal injection every other day for 3 times before harvest the donor liver; Group C, control group in which same dose of normal saline was given by the same way. The transplantation was made by the modified Kamada's two-cuff technique.24h after the operation samples were collected for assay. The serum concentration of ALT,AST were measured by an automatic biochemical analyzer, the level of TLR4 and NF-κB mRNA were measured by RT-PCR, the level of protein of TLR4 and NF-κB were measured by Western blot, the NF-κB DNA binging activity were measured by EMS A, the concentration of IL-1 and TNF-αwere measured by ELISA, the cell apoptosis were measured by TUNEL and calculate the apoptosis index, hepatic tissue was stained by Hematoxyhn-Eosin for pathological observation and grading according to the Suzuki's schema.Results:There was no significant differences in dornor harverst time, warm ischemic duration, anhepatic phrase and recipient operation time between group B and C (P>0.05); Compared with group C, the serum concentration of ALT and AST, level of TLR4 and NF-κB mRNA and protein, the NF-κB DNA binging activity, the concentration of IL-1 and TNF-a, the apoptosis index and the Suzuki's score in group B were significantly decreased (P<0.05).Conclusions:(1) VIP down-regulates the expression of TLR4 on kupffer cells in vivo;(2) VIP decreases the NF-κB DNA binging activity in vivo;(3) VIP decreases the releasing of cytokines thus protect the liver against ischemia/reperfusion injury;(4) Inhibition the expression of TLR4 and the activity of NF-κB by VIP leads to the extenuation of hepatic IRI in rats.Part:IV Role of Vasoactive Intestinal Peptide in Inducing Immune Tolerance in Rats After TransplantationObjectives:To explore the role of vasoactive intestinal peptide in inducing immune tolerance in rats after transplantation and elucidate the probable mechanism of the effects.Methods:The SD rats and Wistar rats were used as donors and recipients. Rats were randomly divided into 4 groups, n=16,8 cases were used for observation the survival rate the rest 8 cases were used for sample collection. Group A:Sygeneic group, SD→SD,2nmol VIP was given by intraperitoneal injection to the donor rats every other day 3 days before harvest the donor liver and to the recipient rats on day l,day 3, and day 5 after the transplantation; Group B:Rejection group, SD→Wistar, without any treatment; Group C:VIP group, SD→Wistar, with the same treatments as group A; Group D:Control group, SD→Wistar, with the same treatments as group A but normal saline was used instead of VIP. Model was made by the method mentioned in partⅡ.8 rats in each group were sacrificed for sample collection; the other 8 were kept alive for survival rate observation. The serum concentration of ALT, AST and TBIL were measured by an automatic biochemical analyzer, the level of IL-2,IL-4,IL-10, IFN-γand TLR4 mRNA were measured by RT-PCR, the level of protein of IL-2,IL-4,IL-10, IFN-y and TLR4 were measured by Western blot, the concentration of IL-2,IL-4,IL-10 and IFN-y were measured by ELISA, the cell apoptosis were measured by TUNEL and calculate the apoptosis index, hepatic tissue was stained by Hematoxyhn-Eosin for pathological observation, and grading the degree of rejection according to Banff's schema as a rejection activity index.Results:Compared with group D, the survival rate of group C was increased (P<0.05); Compared with group D, the concentration of ALT, AST and TBIL in group C was decreased significantly (P<0.05); Compared with group A, the level of TLR4 mRNA and protein in group B and D was increased significantly, while no significant difference was found between group A and C (P>0.05); IL-2 and IFN-y mRNA showed no expression in group A; Compared with group D, level of IL-2,IFN-γmRNA and their protein were significantly decreased (P<0.05); Compared with group A, IL-4 and IL-10 mRNA and their protein expression level in group B and D were decreased significantly (P<0.05), while no significant difference was found between group A and C (P>0.05); Compared with group D, the rejection activity index and apoptosis index in group C were decreased significantly (P<0.05).Conclusions:(1) VIP induces graft immune tolerance by up-regulating the level of Th2 cytokines and protects the graft against acute rejection injury after rat liver transplantation.(2) The ability of up-regulating the level of Th2 cytokines by VIP might attribute to its inhibition the expression of TLR4.(3) TLR4 is involved in the regulation of differentiation of Th1/Th2.