Experimental Study Of Expression Of XAF1 Following Lung Contusion In Rabbit

Objectives: The purpose of the study was to investigate the expression of XAF1 following lung contusion(LC) in rabbit and to explore the role of XAF1 in cell apoptosis following LC in order to confirm a new mechanism existing in cell apoptosis of lung tissue following LC.Methods: 20 healthy adult New Zealand white rabbits were used to establish LC animal model by a free-falling weight impacted device hitting the right chest wall,Four groups of LC each containing 5 rabbits were established according to the time point such as 6h,24h,48h,72h after contusion, In addition , the control group comprised of 5 normal healthy rabbits which were not impacted. Every rabbit in each group was killed by over anesthesia,and did gross observation on lung specimens generally by Postmortem. Lung tissue samples were prepared in all groups. light microscopy and Transmission electron microscopy(TEM) were examined. Apoptotic lung tissue cell was examined using TUNEL staining and TEM. XAF1mRNA was examined using semiquantitative RT-PCR. XAF1 protein was examined using immunohistochemistry.Results:(1)Each rabbit by hitting had lung contusion, the right lung was injuried mainly and severely,Some together with the left lung injuried,but the left injuries were very lingt and limited.(2) lung injuries following LC were very obvious by light microscopy , the pathological changes of pulmonary tissue showed diffuse areas of intra alveolar hemorrhage with disruption of alveoli,interstitial edema,leukocyte infiltration in the alveolar spaces. (3) The apoptotic pulmonary tissue cell were mainly vascular endothelial and alveolar epithelial cells by TEM examination.(4) In situ apoptotic cells detection: Very few apoptosis cells of pulmonary tissue(TUNEL staining positive)were found in control group. apoptosis cells increased obviously following LC,mainly bronchial,vascular endothelial and alveolar epithelial cells,and partly a few macrophagocytes,etc. inflammatory cells. Compared with the control group,the apoptotic index of 6h group following LC increased,and reached a peak at 48h,decreased at 72h(P<0.05). Compared with the 6h group,the apoptotitic cell increase of 24h,48h,72h group was significant(P<0.05). 48h group had no significant difference compared with 24h group(P>0.05),but each of the two groups was greater than 72h group(P<0.05).(5) Expressions of XAF1mRNA by RT-PCR:normal lung tissue of control group had a little expression of XAF1 mRNA ,but in comparion with control group,the expressions of each group following LC increaced significantly(P<0.05),XAF1mRNA levels inreaced from 6h at first, continued at 24h , reached maximum at 48h,then decreaced at 72h following lung contusion. XAF1mRNA of 72h were still significantly higher than 6h group(P<0.05),but lower than 24h and 48h groups (P<0.05) .there were no significant diffrence between 24h and 48h groups(P>0.05).(6) Expressions ,distribution and location of XAF1 protein by immunohistochemistry: normal lung tissue of control group had certain levels XAF1 protein expression. Staining positive cells mainly distributed at bronchial epithelial cells(almost all cells appear staining positive,only few cells expressed negatively or weakly),some staining positive cells showed in vascular endothelial and alveolar epithelial cells,a few of macrophagocytes,lymphocytes,bronchial and vascular smooth muscle cells. If primary antibody was replaced by PBS , all positive cells were negative absolutely. XAF1protein positive cells increased from 6h following LC,continued to increace at 24h,after reach maximum at 48h then lessen,in comparion with control group,the diffrence of each group of LC is significant(P<0.05).The positive cells percentage of 24h,48h,72h group each is significantly higher than that of 6h group(P<0.05). No significant diffrence was found between 24h and 48h group(P>0.05),The positive cells of 72h group decreaced significantly compared with 24h or 48h group(P<0.05). XAF1 protein almost located in cell nucleus in all groups,there were no definite signal of cytoplasm staining positive,demonstrated that LC inducing apoptosis did not alter subcellular localization of XAF1 protein.Conclusion:(1) overapoptosis of lung tissue cells following LC demonstrated that,apoptosis abnormality participated in pathologic process following LC.(2) XAF1mRNA and protein expressed certain levels in normal lung tissue,LC induced them to overexpress,that indicated XAF1 participated in regulating apoptosis induced by LC,therefore participated in pathologic change following LC.(3)XAF1 protein almost located in cell nucleus in the control or each group of LC,demonstrated that LC inducing cell apoptosis did not alter subcellular localization of XAF1 protein.