A Research About The Mechanism Of Histone Deacetylase Sirt1 Inhibitor Preventing Allograft Rejection Reaction

Backgroud:Organ transplantation is the final therapeutic schedule for most patients with end-stage organ disease.Numerous studies have shown that calcineurin inhibitors(CNIs)significantly improve short-term solid organ graft survival in transplantation.The rate of acute rejection has dramatically reduced and 1-year survival clearly increased since CNIs have been administered to patients after transplantation.But long-term used of CNIs might result in various side-effect,such as renal toxicity,neurotoxicity and increased risk of infection and cancer.CNI agents are now thought to be necessary for prevention of allograft rejection but reduced doses are preferred.Traditional transplantation immunology shows that the APC from donor or recipient activates T cell through direct recognition and indirect recognition,respectively.Next T helper cell releases interferon(IFN)-?,interleukin(IL)-2,tumor necrosis factor(TNF)and oTher types of cytokines to mediate rejection.Based on This,CNIs combine wiTh FKBP12 to inhibit the activation of nuclear factor of activated T-cells(NF-AT),and then stop transcription of IL-2 gene to prevent the reject reaction.Some recent studies have shown the balance of Th17/Treg is strongly associated with allograft rejection.Th17 are important for mediate acute and chronic rejection,while Treg contribute to the induction and maintenance of tolerance of recipients to allografts.However,CNIs cannot effectively restrain the reject reaction mediated by Th17,and some researches have found That CNIs caused an imbalance of Th17/Treg.Therefore,therapeutic strategies aiming at manipulating Th17/Treg balance are now considered the most promising approach for preventing allograft rejection.In recent years,the immunologists pay more attention to the epigenetic regulation which is thought to be the precise regulation mechanism of immune cells.Transplant scientists focus on the mechanism of epigenetic regulation of rejection,particularly the effect of preventing chronic rejection.Sirtuins are NAD-dependent class III histone deacetylase(HDAC)that play critical roles in diverse physiological processes such as prolonging life-span,metabolism,cell senescence,cell autophagy/apoptosis,autoimmunity,oxidative stress,and inflammation.Sirtuins include seven members known as Sirtuin1-Sirtuin7.Among them,Sirtuin1(Sirt1)in particular acts as an epigenetic regulator that modulates the activity of several transcription factors important for immune function.Initial studies suggested That Sirt1 has a primarily anti-inflammatory function.But recent researches focusing on T cells showed that Sirt1 play a proinflammatory role in immune response.One hand,Sirt1 was identified as a negative regulator of Treg cell function,via deacetylation Foxp3,The signature transcription factor of Treg cells.The other hand,Sirt1 might positively regulate the function of Th17 cells by modulating the activity of ROR?t.Based on these researches above,Sirt1 may be a new potential therapeutic target to immunosuppression for transplantation.As a Sirt1 inhibitor,sirtinol has been shown to have anti-tumor and anti-inflammatory properties,but its impact on allograft rejection and its molecular mechanisms of action have not yet been reported.In this present study,we established a cervical heterotopic heart transplantation mouse model and evaluated the impact of sirtinol on cardiac allograft rejection.We noted that sirtinol synergizes with tacrolimus to prevent mouse cardiac allograft rejection,in which sirtinol inhibit the rejection mediated by Th17 and enhanced the proportion of Treg cells.Part One:A research about what the role Sirt1 inhibitor sirtinol play in immune reject reactionIn order to assess the potential influence of sirtinol for immune responses,we developed a fully MHC-mismatched murine cervical heterotopic cardiac transplant model,in which C57BL/6 mouse were transplanted with BALB/C heart and randomized to treatment with sirtinol(1mg/kg/d i.p.after transplantation),or dimethylsulfoxide as a vehicle control.We found the maximum cardiac allograft survival time is 13d in sirtinol group,but only 8d in DMOS group.Histologic analysis showed obvious reject reaction in The DMSO group,whereas sirtinol group was characterized by less infiltration.Together,the date above suggest that sirtinol prolong the survival time of MHC fully mismatched cardiac allografts,but the exact mechanism is still unknown.Next,we examined the impact of sirtinol on the expression of inflammatory cytokines and regulatory molecules.Allografts from DMSO group and sirtinol group were harvested on day 7 after transplantation for quantitative PCR analysis.T cells play the main role in acute reject reaction,so we first detected the expression of IL-2,IFN-?,IL-4,IL-10,IL-17A and Foxp3.Compared with allografts derived from DMSO group,we found allografts from sirtinol-treated recipients showed significantly lower levels of IL-17A and higher levels of Foxp3 expression.In contrast,the expression of IL-2,IFN-?and IL-4,IL-10 between DMSO group and sirtinol group was failed to detect the significant difference.The results of immunofluorescence also show the same conclusion.Given the role of Foxp3 played in Treg cells,the above results remind us to test the number of Treg cells in host of DMSO and sirtinol group.We isolated cells from spleen at 7 days after transplantation and then analyzed cells through flow cytometer.Indeed,sirtinol treatment significantly rising the proportion of Treg cells among CD4~+T cells in spleen.Influence of sirtinol on cytokine production including IL-4,IFN-?and IL-17A was also assessed by intracellular staining.A significant decrease of IL-17A was observed in sirtinol group.There were no obvious changes in the expression of IL-4 nor IFN-?between DMSO group and sirtinol group.We also detected the level of IL-17A in serum with ELISA kits and got the same result.Sirtinol suppressed IL-17A expression in receptor,so we examined the impact of sirtinol on the differentiation of Th17 cells.Naive CD4~+T cells were isolated and then polarized to Th17lineage or Treg lineage in the presence of sirtinol or DMSO.Flow cytometry analysis of polarized T cells revealed that sirtinol significantly suppressed the production of Th17 cells and promoted the production of Treg cells.Next western blot analysis indicated that sirtinol reduced the expression of ROR?t,and by which it promoted Foxp3 expression.In line with these results,qPCR analysis further confirmed that sirtinol down regulated the expression of IL-17A and ROR?t,but upregulated Foxp3.According to the results above,we speculated that sirtinol might regulatethe balance between Th17 and Treg cells to prolong cardiac allograft survival.Part 2:Perliminary exploration of the clinical application of sirtinolBecause sirtinol and CNIs have the different mechanism to restrain rejection,we then made receptor receive treatment of sirtinol along with tacrolimus after transplantation and then we got an interestingly results.Administration of sirtinol(1mg/kg/d)along with low dose tacrolimus(0.5mg/kg/d)prolonged maximum allograft survival time to 24 days,while administration of low dose tacrolimus prolonged maximum allograft survival time to 18days and administration of normal dose tacrolimus(1mg/kg/d)prolonged maximum allograft survival time to 22 days.Then we established CAV model to research therapeutic effect of sirtinol in chronic rejection.We found sirtinol can prolong allograft survival time to 78 day while 48 day in control group and 52 day in FK506 group.This result imply that sirtinol can prevent chronic rejection and have tremendous potential to be a immune inhibitors and used in clinic.