Autophage Is Involved In Rat Traumatic Brain Injury Combined Femoral Fracture-induced Indirect Acute Lung Injury

PartⅠ：The effects of traumatic brain injury combined with unilateral femoral shaft fractureson rat pulmonary tissue and inflammatory mediators.Objective：To build an animal model of closed traumatic brain injury combined with unilateralfemoral shaft fractures,then to observe the changes and differences in lung histopathology andlocal expression of inflammatory mediators in the four groups: Group Sham,Group FemoralShaft Fracture,Group Traumatic Brain Injury, Group Traumatic Brain Injury Combined WithUnilateral Femoral Shaft Fractures.Methods：Build an animal model of closed traumatic brain injury combined with unilateralfemoral shaft fractures induced acute lung injury,the HE（Hematoxylin and Eosin）staining isused to observer the histopathological changes of lung, meantime,the double antibody sandwichenzyme-linked immunosorbent assay(ELISA) is used to determinate the expression of TNF-a,IL-6 inflammatory mediators of pulmonary tissue.Results：Closed traumatic brain injury combined with unilateral femoral shaft fractures resultsin alveolar edema and pulmonary interstitial edema,and alveolar septum is widen.Theexpression level of local inflammatory factor and the histopathological scores have significantdifference between different groups(p＜0.05).Conclusion：1.Establish an animal model of closed traumatic brain injury combined withunilateral femoral shaft fractures induced acute lung injury.2.The histopathological changes of Group closed traumatic brain injury combinedwith unilateral femoral shaft fractures is more than the Group Sham,Group Femoral ShaftFracture,and Group Traumatic Brain Injury. PartⅡ：The Expression of autophagic genes in lung tissues after closed traumatic brain injurycombined with unilateral femoral shaft fractures.Objective：To investigate whether autophagy occur in the lung tissues after closed traumaticbrain injury combined with unilateral femoral shaft fractures.We also aim to compare theexpression value of autophagy in different time point.Methods：Seperated the rats into two groups randomly:trauma group and control group.Eachgroup had five time point. We built the trauma model using the way mentioned in thePartⅠ,while the rats in control group experience the same operation without injurying the brain.At every time point, we used the transmission Electron Microscope to observe theultrastructure of lung tissue in order to find the autophagosome. The mRNA expression ofBeclin1 and LC3B were also detected by the Real Time-PCR technique.TheImmune-Histochemical(IHC) is used to measure the protein expression of Beclin1 and LC3B inevery time point.Results：We found that the autophagosome could be seen in the alveolar type II epithelial cellsat every time point after trauma.The mRNA expression value of Beclin and LC3B were raisedsince 6H and peaked at 48H,and they could be detected until 5D after trauma.The expressioncurve of Beclin1 and LC3B were the same as the gene’s,and express in the cytoplasm.Conclusion：1.After closed traumatic brain injury combined with unilateral femoral shaftfracture, autophagosome could be seen in the alveolar epithelial cells.2.The autophagiccorrelative gene:Beclin1 and LC3B were found up-regulation from 6H to 5D after trauma,whilepeaked at 48H.3.The protein of Beclin1 and LC3B express in the cytoplasm. PartⅢ：The influence of 3-MA in rat pulmonary tissue and inflammatory mediators aftertraumatic brain injury combined with unilateral femoral shaft fractures.Objective：Aim to evaluate how the 3-MA ,which is a accredited autophagic inhibitor,effectedthe genic and proteinic expression of Beclin1 and LC3B.Meanwhile we wished to investigatethe effect of pulmonary tissue and inflammatory mediators after traumatic brain injurycombined with unilateral femoral shaft fractures by the 3-MA.Methods：We separated the rats into three groups randomly, there were Group control,Grouptrauma,Group 3-MA.Each group had five time period, they are 6H,12H,24H,48H,5D.In thistime point,we used the Real Time-PCR technique to measure the mRNA’s differences ofmTOR,Beclin1 and LC3B within the three groups. The proteinic differences of Beclin1 andLC3B within this three groups were evaluated by the Fluorescence immunohistochemistry.TheHE staining is used to observer the histopathological changes of lung,the ELISA is used todeterminate the expression of TNF-a, IL-6 inflammatory mediators of pulmonary tissue.Results：In the five time periods we observed, the genic expression of Beclin1 and LC3B werelower in the group 3-MA than in the group trauma,and they are also higher than in the groupcontrol Also the differences between each two groups had significant difference(p<0.05).Pulmonary pathological scores and expression of TNF-a, IL-6 in Group 3-MA is lower thanGroup trauma,the difference in groups had significant difference(p<0.05).Conclusion：3-MA as a accredited autophagic inhibitor,could both inhibited the autophagygenic and proteinic expression of Beclin1 and LC3B.Meanwhile,3-MA can reduce pulmonary histopathological change and expression of TNF-a, IL-6.In conclusion,autophage is involved inrat traumatic brain injury combined femoral fracture-induced indirect acute lung injury.