The Study Of Toll-like Receptor 4-mediated Signal Activation On Ischemia Reperfusion Injury In Rats' Orthotopic Lung Transplantations And Inhibitory Effect Of PDTC On This Pathway

PART 1 Establishment and refining of rat' left orthotopic lung transplantation model.Objective To build up and refine rat left lung transplantation modelMethod 100 male Sprague-Dawley rats,weight ranging from 200 to 250 grams,were selected.After general anesthesia with intubation,donor left lungs were harvested individually after perfusing with LPD solution through left main pulmonary artery, then immersed into prepared fresh LPD solution.Muscle-sparing incision was adopted in Recipient lung operations.Blunt dissection was carried out during dissect hilum of left lung.Cuff techniques were introduced in anastomosis of bronchus, pulmonary artery,and pulmonary vein.Results All cuffs of the donor lungs were accomplished successfully.In all the 50 transplantations,we yielded a 90 percent success rate.Time for harvesting and cuff techniques was 25±2min,and anastomosis time was 30±4min.The reseasons of 5 failed cases were as follows:disrupture of pulmonary vein in 3,disrupture of pulmonary artety in 1,and massive bleeding in 1.Conclusion Rat lung transplantation model was successfully built up and refined on some techniques.The animal model could be duplicated easily and the success rate was high.PART 2 Dynamic change of the type of cell death during donor lung preservationObjective To investigate the dynamic change of cell death during donor lung preservationMethod Forty-two male Sprague-Dawley rats,weight ranging from 250 to 300 grams, were randomized into seven groups.After en bloc harvesting,the donor lung and heart was immersed into fresh prepared LPD solution.At different preservation time points(0,2,4,6,12,18,24hours),the donor lung was perfused with 20 ml 500mM trypan blue,20ml 0.9%saline and 10ml 4%paraformaldehyde through the main pulmonary artery.TUNEL and Hoechst staining were made on the section cuttings. Pathological studies were performed under light microscope.Varies of death cells during preservation were counted under fluorescence microscope by two individual researchersResults Death cells increased gradually,along with the time of donor lung preservation.At the early stage of preservation,apoptosis was the major type of cell death,the peak of apoptosis appeared in the 12 hours' group,which occupied 9.17±2.17%of the whole.After that point,apoptosis decreased gradually until it almost diminished in 18 hours' group.While necrosis started at 4 hours after preservation, and increased gradually with the peak at 24 hours' group,which occupied 33.0±2.87 %of the whole. Conclusion During donor lung preservation,apoptosis of the parenchyma cells can be observed in majority at early stage,while necrosis at the late stage.Cell necrosis increased gradually along with the preservation time.PART 3 Activation of TLR4 signal system during donor lung preservation and after transplantationObjective Based on the research of the expressions of TLR4 and its downstream inflammation factors during donor lung preservation and after transplantation,the possible mechanism of TLR4 signal system activation correlated with ischemia and reperfusion injury during rat lung transplantation was investigated.Method Forty-two male Sprague-Dawley rats were randomized into seven groups. The donor lung was harvested and preserved at the same manner.TLR4 expression on donor lungs were detected by western blot at different preserve time(0,2,6,8,12,18, 24hours),and downstream inflammation factors TNF—αand iNOS were detected by RT-PCR.Another thirty male Sprague-Dawley rats were randomized into another five groups for transplantation.Orthopedic left lung transplantation were performed by using the donor lungs at different preserve time(0,2,6,8,12hours).30 minutes after lung transplantations,TLR4 expression in donor lungs were detected by western blot, and downstream inflammation factors TNF—αand iNOS by RT-PCR.Results Expression of TLR4 increased along with the time of lung preservation,the peak was appeared in 24 hours' group.The dynamic change was similar to the cell necrosis.The correlation between them showed significantly difference(r=0.874, P＜0.01).The same phenomenon can be seen in the relationship of inflammation factors (TNF—αand iNOS) with TLR4(TNF-α,r=0.779,P＜0.01;iNOS,r=0.769,P＜0.01). After transplantation,the expressions of TLR4 and inflammation factors(TNF—αand iNOS) were significantly increased,compared with those at the correspondent time point of preservation.30 minutes after transplantation,the relationship of TLR4 expression with TNF—αand showed significant difference(TNF—α:r=0.617, P＜0.05:iNOS:r=0.460,P＜0.05).Conclusion Activation of TLR4 and its downstream inflammation factors and increasement along with the preserve time can be revealed during donor lung preservation.This phenomenon was parallel with cell necrosis at the same time.The expression of TLR4 and inflammatory factors were increased 30 minutes after transplantation and yielded positive relation with the time of donor lung preservation.PART 4 Influence of PDTC on TLR4 signal system and function of the transplanted lungObjective Based on the former research,Pyrrolidine dithiocarbamate(PDTC ) which is the specific blocker of NF-κB were added into preservation solution to observe the possible protective effect of transplanted lung.Method sixty male Sprague-Dawley rats,weigh ranging between 200-300g were randomized into two groups(PDTC group and control group).Each group contained 5 sub-groups according to its preservation time(0,2,6,8,12 hours).Six success lung transplantations were carried out in every subgroup.PDTC were added in the reseach group(Sigma,40mmol/L).30 minutes after the lung transplantations,the expression of TLR4 and P50 were detected by western blot,DNA binding activity of NF-κB by EMSA.Expression of iNOS and TNF—αwere detected by RT-PCR.In the aspect of lung function,airway pressure,oxygen indexes,wet/dry ratio,manifestation of lung injury on HE staining and transmission electron microscopy were compared between the two groups.Results No significant difference in expression of TLR4 and P50 were yielded between the PDCT group and control group(TLR4:p=0.661,p＞0.05;P50:p=0.276, p＞0.05).DNA binding activity of NF-κB of PDTC group reduced dramatically, compared with corresponding control groups(p=0.000,p＜0.01 ) as well as the expression of TNF—α(p=0.000,p＜0.01 ) and iNOS(p=0.000,p＜0.01 ) decreased dramatically,compared with corresponding control groups.In PDTC and control groups,Correlation analysis between decreasing DNA binding activity of NF-κB and down regulation of TLR4 signal downstream inflammation factors TNF—αand iNOS showed significant difference(TNF—α:r=0.636,P＜0.01;iNOS:r=0.633, P＜0.01 ) ).As to lung function after transplantation,in PDTC groups,the peak air pressure 30 minutes after lung transplantation deceased(p=0.038,p＜0.05),the oxygen indexes increased(P=0.042,P＜0.05),wet/dry ratio deceased(P=0.001, P＜0.05),as well as lung injury assessment(P=0.017,P＜0.05),compared with corresponding control groups.Conclusion PDTC pretreatment of donor lung could not block the expression of TLR4 and P50,but it could down regulate the TLR4 downstream inflammation factors dramatically.The process was approached by decreasing the DNA binding activity of NF-κB,not by down regulate NF-κB expression.The blockage was almost completely.The declined expression of inflammation factors was close correlation with the improvement of transplant lung function.In a word,PDTC pretreatment could improve the function of transplanted rat lung.