An Experimental Study On The Effect And Mechanism Of Astilbin On The Suppression Of Lung Allograft Rejection In Rat

SECTION ONE The Establishment of Orthotopic Left Pulmonary Allograft Model in RatsObjective: To establish rat orthotopic left lung allograft transplantation model by the"cuff-like"vessel and bronchial anastomosis technique. Methods: Sixty male Sprague-Dawley rats were randomly allocated to donors and recipients. Thirty left lung transplantation were performed by using the"cuff-like"vessel and bronchial anastomosis technique. Results: All rats were successfully performed orthotopic left lung allograft transplantation. The mean time for the donor lung (from perfusion to the harvest) was (10±1)mins; the mean time for cuff the vessel and bronchus was (10±4)mins; the mean time for anastomoses was (15±2)mins. The mean operative time was (80±10)mins. The 15d survival rates of rats who received transplants were 86%. Among the rats who received transplants, 2 died of pulmonary edema, 1 died of pulmonary thrombosis, the rest one dies of pneumothorax of the right side. Conclusion: Rat orthotopic left lung transplantation with successive anastomosis in cavity is a valid, reproducible, single and cheap model for studying donor lung preservation, ischemia-reperfusion, acute rejection and chronic rejection about lung transplantation.SECTION TWO Suppression effect of Astilbin on lung allograft rejection in ratsObjective: Using histopathological rejection grade and observeing the local fibrogenesis to study the suppression effect of Astilbin on lung allograft rejection in rats. Methods: The model of rat left lung transplantation was set up.Sixty lung transplanted rats were divided in to 4 groups randomly: group A were fed with normal saline 1ml per day, group B were fed with CsA 5mg/kg per day, group C were fed with Astilbin 1mg/kg per day, and group D were fed with Astilbin 1mg/kg per day aud CsA 2.5mg/kg per day. Histopathological rejection grade of the lung graft were analyzed in after 30 days (5 rats of each group were chosen). BALF(10 rats of each group were chosen) collected from recipients rats treated with drugs while undergoing rat orthotopic left lung transplantation model was used to stimulate human lung fibroblast cells in vitro. Fibroblast proliferation and typeⅠcollagen synthesis was dectected respectively by MTT and ELISA to evaluate the effect of astilbin. Results: (1) In group B , C and D, the histopathological rejection grade of the lung graft was significantly lower as compared with group A. The histopathological rejection grad of the grafts in group D was significantly lower as compared with group C (P<0.05) and group B (P<0.01). (2) In vitro, the BALF induced fibroblast proliferation and typeⅠcollagen synthesis; (3) Group B, C and D significantly decreased human lung fibroblast cells profliferation for stimulation of the BLAF, especially in Group D (P<0.05) Conclusions: Astilbin significantly decreased human lung fibroblast cells proliferation and typeⅠcollagen synthesis for stimulation of BLAF collected from recipients rats treated with drugs while undergoing lung transplantation, suggesting astilbin may inhibit obliterating bronchiolitis after lung transplantation. Astilbin can suppress lung allograft rejection, and has synergistic reaction with CsA.SECTION THREE The Mechanism of Astilbin on the Suppression of Lung Allograft Rejection PART ONEExpression and Implication of CD69, CD25 and CD71 on CD3+ lymphocytes in peripheral blood in the rats with lung transplantation using astilbinObjectives: To investigate the expression and implication of CD69, CD25 and CD71 on CD3+ lymphocytes in peripheral blood in the rats with lung transplantation using astilbin. Methods: The model of rat left lung transplantation was set up. Ten rats were taking peripheral blood as the control group. Forty lung transplanted rats were divided in to 2 groups randomly: group A were fed with normal saline 1ml per day, group B were fed with Astilbin 1mg/kg per day The lymphocytes from the peripheral blood in rats in different periods and were immunologically labeled by CD3·PE and CD69/CD25/CD71·FIFC. CD3/ CD69, CD3 /CD25 and CD3/CD71 were determined by flow cytometry (FCM). Results: The levels of CD3/ CD69, CD3 /CD25 and CD3/CD71 were markedly higher in the 2 days after the lung transplantation, which means the activated T cells were related to the lung allograft acute rejection. On the 5 days after the lung transplantation, the levels of CD3/ CD69 in group B were significant lower in the group A,(P<0.01), which means the astilbin act on the activated T cells and protect the allograft. Conclusion: Astilbin can act on the activated T cells and suppress lung allograft rejection maybe partially related to its effect in activated T cells.PART TWO Effect of astilbin on the inducement of apoptosis in activated T cells to suppress acute rejection on rat lung transplantation model.Objective:To study the suppression effect of Astilbin on lung allograft rejection in rats. Methods: The model of rat left lung transplantation was set up. sixty lung transplanted rats were divided in to 2 groups randomly: control group were fed with normal saline 1ml per day, astilbin group were fed with Astilbin 1mg/kg per day. Survival time of each group, activity of IL-2 in spleen lymph cells , transforming rate of T cells in spleen and apoptosis of T cells were observed. Results: Transforming rate of T cells in spleen was significant lower in the astilbin group than the control group. Activity of IL-2 in spleen lymph cells was 4.25±2.65UI/ml and 23.46±1.82UI/ml respectively in the astilbin group and the control group. There were significant difference between two groups (P<0.05). The astilbin group can effectively derive apoptosis of activated T cells in acute rejection. Conclusions: Astilbin can suppress lung allograft rejection maybe partially related to its lower the IL-2 concentration and IFN-γmRNA of activated T cells , which inducement of apoptosis in activated T cells.PART THREE Effect of astilbin on activation of p38MAPK and suppression of PI3K/Akt signal pathway in activated T cells of rat lung transplantation model with acute rejection.Objective: To investigate the effect of astilbin on activation of p38 mitogen activated protein kinase (p38MAPK) and suppression of PI3K/Akt in activated T cells of rat lung transplantation model with acute rejection. Methods: The model of rat left lung transplantation was established. At the day30, activated T cells were taken and cultured. The cultured activated T cells were divided into four groups: Group A: control group, were cultured with PBS; Group B: astilbin group, add astilbin (15mg/L); Group C: add Astilbin(15mg/L) and SB203580(5μmol/L); Group D, Astilbin+LY294002(10μmol/L). Apoptosis of T cells were observed by TUNEL. The expression of p38MAPK and PI3K/Akt were measured by RT-PCR and Western blot. Results: Apoptosis indexes and the expression of p38MAPK in activated T cells in Group B were found significant higher than those of the Group A, while PI3K/Akt was significant lower than Group A.(P<0.01). Apoptosis indexes and the expression of p38MAPK in activated T cells in Group B were found significant higher than those of the Group C, while PI3K/Akt was significant lower than Group C.(P<0.01). Apoptosis indexed and the expression of PI3K/Akt in Group D was significant higher than Group A, while the p38MAPK was significant lower than Group A.(P<0.01).Conclusion: Astilbin induces apoptosis of activated T cells of lung transplantation maybe partially related to its activation expression of p38MAPK and suppression of PI3K/Akt signal pathway. Key words: lung transplantation; astilbin, p38MAPK, PI3K/Akt...