Inhibition Of B-box Binding Protein1Prolong Skin Graft Wurvival In Mice

Objective X-box binding protein1(XBP1), a master regulator of theadaptive UPR during endoplasmic reticulum stress, participated in theregulation of Homeostasis of endoplasmic reticulum and the integrity ofcellular structure. Recently，several studies has been shown the role ofXBP-1to increase responsiveness towards inflammatory and immunesystem modulation. However，the exact mechanism of XBP-1in theformation of immune tolerance after tissue or organ transplantation remainunclear. The aim of this study was to investigate whether inhibition ofx-box binding protein1can reduce acute rejection of skin allograft.Methods Healthy BALB/c mice and C57BL/6mice were used asdonors and recipients in an acute rejection skin graft model according tothe MacFarlane’s methods, respectively. Animals were randomly dividedinto3groups. Knocking down XBP1using si-XBP1(small interferenceRNA targeted XBP1) was injected into transplant recipients post-surgicallywhich named transfected group; the non-target vector group received thesame dose of vector；the control group received the same dosage ofphosphate buffered saline (PBS). Skin sample and peripheral blood werecollected for further testes. Graft survival and histopathologic changes was evaluated; the weight in every individuals were documented and the liverand renal function were tested by Auto-analyzer; the mRNA expression andserum levels of related cytokines were assessed by Real-Time PCR andELISA respectively.Results (a) The acute rejection model of BALB/c to C57BL/6mice"back to back" skin transplantation were stably established with a less than5%failure rate.(b) Allograft in transfected group showed new hair growtharound5-8days post-transplantation with no observation of detachment,shrinking and hyper pigmentation.(c) No significant changes in bodyweight in all groups during the experimental period. The white blood cellcount decreased after skin transplantation and then rose gradually in allgroups. There were no significant differences in white blood cell countsamong groups at each time point.(d) plasma ALT levels, and creatininelevels, in all groups mice increased slowly up but no statistics differenceswere observed.(e) the effects of si-XBP1treatment on skin graft survivalwas evaluated in comparison with controls and PBS groups, found thatsi-XBP1treatment significantly increased graft survival compared withcontrol treatment and PBS groups (*P<0.05); the mean survival days oftransfected groups was more than30days, but it were10days and12daysin control group and PBS group respectively; The histopathology showedmild changes in the transfected group compared with the other2groups.(f) the mRNA expression of XBP-1was0.41±0.07,1.08±0.08and 1.11±0.51, the expression was inhibited in transfected groups comparedwith the other2groups; IFN-γ and IL-6were0.28±0.012，0.84±0.02,0.79±0.015and0.47±0.019,0.95±0.02,0.98±0.017, whichshowed the relative expression of IFN-γ and IL-6to be signifcantly lowerin the transfected group compared with the other2groups, while IL-10wasmuch higher otherwise.(g) the ELISA results showed a consistent tendencywith PCR results.Conclusion Inhibition of XBP1treatment significantly couldprolonged skin allograft survival in mice by regulate Th cell relatedcytokines.