The Effect And Mechanisms Of Resveratrol And HIF-1?-VEGF-A Pathway On Lung Transplantation In Rats

SECTION 1:The Establishment of Orthotopic Left Lung Transplant Model by Modified Anastomosis MethodObjective:To establish the model of orthotopic left lung transplantation in rat by the modified tri-cuff vascular anastomosis method,and to draw the operator learning curve and observe the survival rate.Methods:One hundred male Sprague-Dawley rats were divided into 60 donors and 40 recipients.First,we performed 20 cases of donor operation and the tri-cuff technique.Then,the left lung orthotopic transplantation in rat was done with the modified tri-cuff anastomosis technique.Results:In 20 cases of donor surgery,the donor lung lavage was started to be satisfactory in the fifteenth case.In 40 cases of lung transplantation,the recipient operation time was gradually stable until the twenty-fifth case was carried out.The mean time for the donor lung was 9.28�2.01 minutes,and the separation of hilar and vascular cuff were 6.04�1.43 and 5.70�2.36 minutes.The recipient operation time was 33.40�8.44 minutes.And the time of warm and cold ischemia were 16.29�3.58 and 26.37�6.01 minutes.The success rate of all lung transplantation was 51.61%,and the survival rate could reach 80%after mastering the surgical technique.Conclusion:The model of orthotopic left lung transplantation in rat with modified cuff anastomosis was highly reproducible.By improving the operation details of anastomosis,the difficulty of lung transplant operation was reduced,and the learning cycle was shortened obviously.SECTION 2:The Early Protection of Resveratrol in Rat Lung TransplantationObjective:Resveratrol is a multifunctional bioactive substance that has been found to have anti-inflammatory effects and to play an important role in the prevention of ischemia-reperfusion injury.However,the effects of resveratrol on transplanted lung have not yet been reported.This study compared the inflammation and expression of related proteins during the early stages after transplantation to explore the effects and mechanisms of resveratrol on transplanted lung.Methods:Sprague-Dawley rats were randomized to receive an intraperitoneal injection of resveratrol suspension(60 mg/kg;RES group),dexamethasone(1 mg/kg;DEM group),or normal saline solution(2 mL/kg;control group)1 hour before lung transplantation.The cytokine concentration in the serum and bronchoalveolar lavage fluid(BALF)of the recipients was determined 24 hours after transplantation.Histopathologic evaluation,including lung injury score,was performed with hematoxylin-eosin staining.The expression of inducible nitric oxide synthase(iNOS),nuclear factor-KB P65,receptor-interacting serine/threonine protein kinase 3(RIPK3),and mixed-lineage kinase domain-like(MLKL)was assessed by immunohistochemical analysis.Results:Histopathologic evaluation showed pulmonary epithelium damage and endothelialitis associated with hemorrhage in the alveoli in the control group,the severity of which was greater than that in the other two groups.The levels of interleukin-6 and tumor necrosis factor-a in the serum and BALF of the RES and DEM groups were lower than those in the control group.The expression of iNOS,RIPK3 and MLKL in the RES group was 50%,50%,and 37.5%lower than that in the control group,respectively,and was inversely proportional to lung injury.Conclusions:Based on these findings,we propose that pretreatment with resveratrol affords a certain degree protection to transplanted rat lung in the early stages after transplantation.We also determined a relationship between necroptosis-associated proteins and transplanted lung injury,which suggests that the mechanism of lung transplantation-associated ischemia-reperfusion injury may be related to necroptosis.SECTION 3:The Role of HIF-1?-VEGF-A Pathway in Bronchiolitis Obliterans after Lung TransplantationObjective:Hypoxia-inducible factor-1?(HIF-1?)plays a transcriptional activity in hypoxia.Recent findings have indicated that graft function would be affected if the organ is in hypoxia for a long time.We hypothesized that bronchiolitis obliterans(BO)after lung transplantation is associated with HIF-1?.This study compares the expression of HIF-1? and its downstream proteins in allograft and isograft to explore the relationship between this pathway and BO in rats.Methods:We performed an orthotopic left pulmonary transplant model using the tri-cuff vascular anastomosis method.The left lungs from Fischer 344 rats were transplanted into Lewis recipients.The recipients were treated with ciclosporin and lipopolysaccharide at a certain time.The grafts were collected 90 days after transplantation.The histopathology,including the severity of fibrosis(SF),were evaluated by hematoxylin-eosin and Masson's trichrome staining.The expression of HIF-la,vascular endothelial growth factor-A(VEGF-A),and VEGF receptor-2(VEGFR-2)was accessed by immunohistochemistry.Results:The imageology and pathology showed that the allotransplant model developed BO 90 days after the operation.The percentages of a high expression of HIF-1?,VEGF-A,and VEGFR-2 in the allograft group were 77.27,63.64,and 68.18%higher than in the isograft group,respectively.The SF score was highest in the allograft and was positively correlated with the expression of the proteins.Conclusions:This model could simulate human BO after lung transplantation.The expression of HIF-1? and its downstream proteins in post-transplantation was up-regulated,suggesting that activation of the HIF-1?-VEGF pathway might be involved in the occurrence and prognosis of BO.