| Background：Small bowel transplantation (SBT) is considered one of the mosteffective treatment options for the end-stage small bowel dysfunction or shortbowel syndrome. However, the progress in small bowel transplantation haslagged well behind that of other types of solid organ transplantation. There aremany risks leading to the SBT failure such as the high incidence rate of acuterejection; serious infection; slow recovery of intestinal function; and chronicrejection. Among them, the high incidence of acute rejection is the majorfactor to hinder the development of SBT. As for its irreversibility, how toavoid the acute rejection becomes hot research area. Studies revealed thatacute graft rejection and graft damage mainly result from thecytokine-mediated complicated inflammatory response and antigen-specificimmune response. Therefore, study on cytokines arouses great concern in recent years.HMGB1is a non-histone chromosomal protein in eukaryotic cell nuclei.It is not only a high migratory nuclear binding protein, but also a powerfulextracellular factor. HMGB1plays a critical role in the occurrence andprogression of the inflammatory response and the immune response aftertissue damage or infection. It is also the downsteam treatment target of TNF.In organ transplantation, the immune rejection usually occurs after IRI. Thefunction of HMGB1in autoimmune diseases and ischemia-reperfusion injuryof kidney or heart has been proved, but its function in organ transplantationremains unclear.With rat heterotopic segmental SBT model, the change of HMGB1level inserum and tissue was detected and the relationship between HMGB1andimmunological responce was studied. Furthermore, the mechanism andsignificance of HMGB1in SBT were explored by detecting serum IL-2andTNF-α and observing the pathological changes ofthe heterogeneic SBT modelwhich were intervened by HMGB1A box.Objectives：1. To establish the model of rat segmental heterotopic SBT, and tooptimize the techniques of construction and perioperative treatment of SBT.2. To observe the changes of HMGB1and to explore the role andsignificance of HMGB1in injury after SBT.3. To investigate the possible protective function of HMGB1in acutegraft rejection.Methods：1.144male inbred SD rats and inbred Wistar/A rats were adopted forestablishing segmental heterotopic SBT models. The rats were randomly divided into four groups: group A (control group, n=36), in which the ratsunderwent abdominal operation and surgical removal of their left kidneys;group B (the syngenic SBT group, n=36), in which both the donors andreceptors were SD rats; group C (heterogenic SBT group, n=36), in which thedonors were SD rats while the receptors were Wistar/A rats; and group D(HMGB1A box-treated group, n=36), in which the heterogenic SBT modelswere given HMGB1A box protein600ug/g(POD0,3,5) by abdominalinjection.2.36rats in every group were respectively killed at2hr,12hr,1d,3d,5dand7d after the operation. The blood and graft samples were colle cted for thefurther study. The level of HMGB1, IL-2and TNF-a in the surum with ELISA,the protein levels of HMGB1, IL-2and TNF-α in intestinal graft withWestblot, immunohistochemical staining and flow cytometry, and the mRNAlevels with RT-PCR assay were detected. At the same time, the rejectionresponse and its strength were investigated by observing the pathologicalchanges of the transplanted intestine tissues.Results：1. The models of segmental heterotopic SBT were successfully established,and the modelling method and perioperative care were optimized.2. Histopathology assessments: No obvious difference of mucosa wasdetected in group A. The pathological changes in intestinal graft in group Bwere also unconspicuous. No evidence of acute rejection and inflammatoryresponse was detected in group C and D at2hr,12hr and1d postoperatively,just with infiltration of a small amount of lymphocyte and plasmocyte wasobserved. Mild， moderate and serious outcome of acute rejection wasobserved in group C in POD3,5,7, respectively, and acute rejection aggravated with time extension. Compared with group C, unconspicuousevidence of acute rejection was observed on POD3and POD5in group D,while mild outcome of acute rejection was observed in POD7.3. HMGB1: No obvious rise of HMGB1was detected in group A afterthe operation both in serum and graft. In group B, HMGB1level in serum roseafter small bowel transplantation, reached the top after12h, and then declinedto the relatively normal level on POD3. In group C, two peaks of HMGB1level in serum were showed:12h after the operation and on POD5, which kepthigher in POD7. The difference was significant compared with group A(P<0.05).But in the graft tissue, the change of HMGB1showed the different trendcompared with that in serum. In group B, HMGB1level in the graft slightlyelevated, but it was not significantly increased compared with that in groupA(P>0.05).On POD5, the western blot assay showed significant elevation ofHMGB1protein level in group C. The immunohistochemical staining andflow cytometry confirmed the elevation of HMGB1expression.4. IL-2: The trend of IL-2change in serum and intestinal graft isbasically the same. No obvious difference showed in group A and B. In groupC, the level of IL-2rose sharply in POD3, reached top in POD5, and kepthigher in POD7(P<0.05). In group D, the level of IL-2was low with noobvious peak showed compared with group C. It also showed one peak ingroup D, but it was significantly lower compared with that in group C at sametime(P<0.05).5. TNF-α: The trend of TNF-α change rule is basically the same in serumand intestinal graft. In group B, the level of TNF-α rose immediately at2hrafter the operation, reached the top at12hr and then declined to normal level in POD3. In group C, two peaks were showed: at12hr after SBT and onPOD5, which kept higher in POD7. It also showed two peaks in group D, butit was significantly lower compared with that in group C(P<0.05).Conclusions:1. The close relationship between the serum HMGB1level and the graftinjury and immunological reaction indicates that serum HMGB1may be apotential and available marker for the evaluation of the occurrence and degreeof the acute rejection after SBT.2. In this study, HMGB1A box protein could significantly inhibite theallograft rejection reaction after SBT, which indicates that HMGB1might bean important damage factor and a potential therapeutic target forSBT-associated tissue damage. |
