The Effect Of DcR3on Fas/FasL Induced Pulmonary Fibrosis Animal Model

Objective: Pulmonary fibrosis can induced by alveolarepithelial cell apoptosis and this has happend in the early stage ofdisease. Many factors can cause this disease. Since Fas/FasL became oneof those members, and is universally accepted that it could induceapoptosis which make an important role in the occurrence and developmentof the disease.This research is mainly working on1) Established a newpulmonary fibrosis animal model: Rat tail injected with Fas antibodyto induce pulmonary fibrosis which is not only closer to the pathogenesisstatus of the disease but easier to operate and for further research.2) DcR3is an antagonist of Fas/FasL apoptotic signal way. We tend toinject DcR3on the pulmonary fibrosis animals, and try to figure out ifthe drug could alleviate the alveolar epithelial cells apoptosis andpulmonary fibrosis induced by Fas/FasL.Methods: Firstly, animal models are established and grouped. Rattail vein injected with saline, IgG antibody or anti-Fas antibody (withthree different concentrations1ug/ml,5ug/ml and10ug/ml) separatelyand grouped by each injection drugs or drug concentrations.Secondly, DcR3injected through intravenous to the anti-Fas antibody group for lateranalysis. Rats are sacrificed at the time of7d,21d and28drespectively.Lungs are separated and fixed for HE and Masson stainingto observe the pulmonary fibrosis formation and fibroblast proliferation.We evaluate the degree of the change including the alveolitis andpulmonary fibrosis according to Szapiel et. And the content ofhydroxyproline which is a signal of pulmonary fibrosis,TNF-αat kinds oftime point, the expression of TGF-β by ELISA. Also Western blot andimmune histochemistry are used to detect the expression of Fas and FasL and the apoptosis related factors such as Caspase3.Lung tissue apoptosisis assessed by TUNEL.Results:(1) pathology results showed: saline control group, lungsstrcture are normally,no effusion shows in alveolar cavity.Massonstaining do not see deposition of the green collagen.IgG antibody group:no significant differences comparing with the saline control group.1ug/ml anti-Fas antibody concentration groups: with the time going,pathological changes emerge，first comes the focal distribution, lungcongestion, mild edema, inflammatory cells decreased, alveolar structure,alveolar walls are markedly thickened, alveolar space is reduced,fibroblasts, pulmonary interstitial fibrosis, scarring, occasionalcapillary lumen occlusion;Masson staining of alveolar interval has alarge number of green collagen deposition. anti-Fas antibody5ug/ml and10ug/ml concentration groups: in the early stages of inflammation theyshows the similar pathological changing with the concentration of1ug/mlanti-Fas antibody group;Masson Staining did not see the green collagendeposition.at28days is visible in the rats lung tissue structure is clear,each observation point has not been a significant change, Masson stainingoccasional green collagen deposit.(2) the degree of alveolitis comparison:saline and IgG antibody controls the extent of alveolar inflammation atall time points have no obvious change;Resistance groups:1ug/ml anti-Fasantibody concentration with time extension of alveolitis and pulmonaryfibrosis, after inflammation gradually reduce, the degree of inflammationat each time point higher than that of control group, there was significantdifference compared with the control group (p <0.05); there was nosignificant difference of the alveolar inflammation between Anti-Fasantibody of10ug/ml,5ug/ml of and1ug/ml group, but no fibrosis formation,with statistical difference (p <0.05), and higher than the control groupwith significant difference (p <0.05),(3)the degree of pulmonaryfibrosis comparison: normal pulmonary fibrosis degree and IgG antibody control group in all time points have no obvious change;Resistance to1ug/ml anti-Fas antibody concentration group of pulmonary fibrosis degreeaggravating gradually, each time point compared with control group hassignificant difference (p <0.05);the concentration of anti-Fas antibody5ug/ml and of10ug/ml group, there was no significant difference comparedwith the control group (p>0.05), the degree of lung fiber at each timepoint below1ug/ml anti-Fas antibody concentration groups, there aresignificant differences (p <0.05).(4) saline and IgG antibody controlgroup there are a little hydroxyproline expression, each time pointcontent has no obvious change;anti-Fas antibody1ug/ml concentration ofhydroxyproline increased, reached a peak of28days, each time pointcompared with control group (p <0.05); there are significant differences(p <0.01) of anti-Fas antibody5ug/ml and10ug/ml group ofhydroxyproline content in each time point lower than1ug/ml anti-Fasantibody group (p <0.05), higher than the control group, and there aresignificant differences (p <0.05).(5) saline and IgG antibody controls:in all the time points TNF-αonly trace expression, the expression of twokinds of cytokines of anti-Fas antibody group are higher than the controlgroup at all time points, there are significant differences (p <0.01),while the expression of TGF-β compared with anti-Fas antibody group hadsignificant difference (p <0.05).(6) the expression of Fas,FasL andcaspase3in anti-Fas antibody group were higher than control group, therewas significantly different (p<0.05), The Immunohistochemistry resultsshowed: the expression of Fas, FasL and caspase3in the lungs of anti-Fasantibody group are higher than control group, and there were significantlydifferent (p<0.05).(7)The TUNEL results showed:the cell apoptosis indexof anti-Fas antibody group are higher than than control group, and therewere significantly different (p<0.05).Because the early experiments, we have used anti-Fas antibody1ug/mlset up pulmonary fibrosis model, so we mentioned anti-Fas antibody is1 ug/ml in the second part (1) the pathological results showed that anti-Fasantibody groups:28days pathological changes is believed thatdistribution with focal, lung congestion, mild edema, inflammatory cellsdecreased, alveolar structure, alveolar septum are markedly thickened,and there were large amount of fibroblasts proliferation and fibrosisproduced.Masson staining of alveolar interval has a large number of greencollagen deposition.(2) the degree of alveolitis comparison: anti-Fasantibody groups: three days mild alveolar inflammation,7days a typicalalveolar inflammation,14days alveolitis and pulmonary fibrosis, afterinflammation gradually reduce, the degree of inflammation at each timepoint higher than the DcR3group, there were no significantly differentcompared with DcR3group (p>0.05).(3) the degree of pulmonary fibrosis,anti-Fas antibody group pulmonary fibrosis degree increase gradually,there are significant difference each time point compared with DcR3intervention group (p <0.05);DcR3intervention group pulmonary fibrosisdegree is lighter than the anti-Fas antibody group.(4) The result ofhydroxyproline content in serum by ELISA showed: the hydroxyprolinecontent of DcR3group was lower than anti-Fas antibody group but higherthan control group, there were significantly different (p<0.05).(5) DcR3group compared with controls, the expression of TGF-βand TNF-α therewas significant difference (p <0.05), compared with anti-Fas antibodygroups have significant difference (p <0.05).(6). Western blot andImmunohistochemistry results showed：At28d,the expression of Fas,FasLand caspase3in anti-Fas antibody groups were higher than DcR3group,there was significantly different (p<0.05).(7)The TUNEL resultsshowed:the cell apoptosis index of DcR3group are higher than anti-Fasantibody group but higher than control group, and there were significantlydifferent (p<0.05).Conclusion:(1) We successfully establish a new pulmonary fibrosisanimal model by rat caudal vein injection anti-Fas antibody to induce Fas/FasL apoptosis signal way.(2) We observe that TNF-α is aninflammatory factor working at the early stage of inflammation while TGF-βcould play a role through the whole pathological process. Otherwise,DcR3can inhibit the production of inflammatory markers in both.(3) DcR3may effectively by inhibiting Fas/FasL mediated signaling pathwaysleading to apoptosis of alveolar epithelial cells and reduce thedevelopment of pulmonary fibrosis, and there are a great significance tothe clinical treatment of the formation of Fas/FasL of pulmonaryfibrosis pathways.