The Role Of P38MAPK On H9N2-SIV Induced Acute Lung Injury In Mice

Acute lung injury(ALI) refers to acute and progressively increased dyspnea, hypoxemiaand pulmonary edema caused by pathogenetic factors inside and outside the lung excludingcardiogenic ones. As the disease worsens, ALI develops into Acute respiratory distresssyndrome(ARDS), and even can be fatal to severe patients. ALI is one of the main symptomsof mammals infected by influenza virus. Its precise pathogenesis still remain unknown now.But recently, the research shows that AlI caused by influenza virus results in damages of manyorgans. These recent reseaches of pathogenesis focus on the function of cytokines andinflammatory mediator. p38MAPK is closely related to the regulation of the inflammatoryresponse. And in vitro studies shows that it is involved in regulating the expression of a varietyof inflammatory cytokines. According to data, p38MAPK plays an important regulatory role inthe process of lung injury. It is found that all previous pandemic influenza is associated withpigs, and the swine influenza virus(SIV) has a closer relationship in the process of humaninfluenza pandemic and virus variation than the avian influenza virus(AIV). But these relatedstudy that p38MAPK can cause ALI of mice infected by H9N2-SIV has not been reported.To study the role of p38MAPK on ALI of mice infected H9N2-SIV, We take6to8weeksof SPF BALB/c female mice as the research objects, use A/swine/HeBei/012/2008(H9N2)(H9N2-SIV) separated by this research group, and adopt the method of chicken embryoallantoic cavity inoculation to multiply the virus. And mice are infected through intranasalinoculation. The standard depends on change of clinical symptoms, histopathological changesin lung disease, pulmonary edema, the inflammatory cells in bronchoalveolar lavagefluid(BALF), and hemodynamics of each group. A mouse model of ALI induced byH9N2-SIV is built, and p38MAPK specific inhibitor——SB203580treatment group is alsobuilt. The different periods in the process of ALI of mice are measured. The principle methodsare as follows:①Observe clinical symptoms and count the mortality of mice, weigh body weight and feed intake of living mice regularly, determination of lung coefficient, W/D,the inflammatorycells in BALF, and hemodynamics of each group;②Bulid mice model ofALI induced by H9N2-SIV;③Dynamically observe the pathological changes of lung by the HE staining technique,immunohistochemical technique and transmission electron microscopy technology;④Measure the expression and regulation of inflammatory factor(TNF-α、IL-1β、IL-6、IL-10)by ELISA;⑤Measure the distribution and expression level of phospho-p38MAPK in lung tissue byusing immunohistochemistry and Western-blot.The results showed:①The mice of infected group got ill on2or3days post-infection (p.i.). The earlysymptoms were that back fur gets rough and unpolished, activities were less, and they loved toflock together; that they were depressed in spirit and slow in response; that they decreased inappetite, and the eyes were constricted or even closed, and covered with secretion. Then atlater stage, they appeared shortness of breath, reverse stand of back fur, retraction of heads andbend of backs, loss of appetite, and thinness etc.. Additionally, they crouched in a corner in thecage, and ran slowly or stumbled. And even some mice appeared obviously neurologicsymptoms. The death rate reached to60%. Body weight descended firstly then ascended, andmice were attacked by severe pulmonary edema. From8day p.i., the symptoms graduallyreduce, and basically recovered on14day p.i..On2day p.i.,the partial pressure of arterial oxygen(PaO2) for infected mice began toreduce compared with that of control group,which showed significant difference(P<0.01) on4day p.i.,and only6.79kPa±1.27kPa on6day p.i.,infected group appeared serioushypoxemia,moreover, the partial pressure of arterial carbon dioxide(PaCO2) rosesignificantly(P＜0.05).Inflammatory cells in BALF increased significantly,especially thealveolar macrophage and polymorphonuclear leukocytosis,and from4-8days after infectionshowed extremely significant difference(P<0.01) compared with that of control group.Physical symptoms of SB group were lighter than infection group, but severer thancontrol group, between infected and control group.②By the observation of the pathological histology, there were inflammatory cells mainlycontaining neutrophils, erythrocyte, a great deal of pink fibrinous exudate and edema fluid in the pulmonary alveoli of infected mice. And alveolar space became less, alveolar wall wasthickened, alveolar interstitial was widened to varying degree, Around bronchioles and smallvessels showed interstitial edema and loosen, moderate levels of lymphocytes and monocytewere infiltrated can be seen, and part of epithelial cells of bronchial mucosa underwentnecrosis and fall off; blood vessels and bronchus wall of SB group were slightly thickeningand edema, alveolar size were slightly irregular, and injury degree was also less thaninfected group.③Compared with the control group at each time point, the concentration of TNF-α、IL-1β、IL-6and IL-10in Lung tissue homogenate increased significantly in infection groupand SB group (P<0.05). TNF-α and IL-1β of infection group were remarkably increased on2day p.i., reached maximal levels on4day p.i. and then decreased, and finally almost returnedto the control levels on14day p.i.. IL-6and IL-10increased on2day p.i., reached maximallevels on6day p.i., then decreased on8day p.i., but drop slightly, and remained higher thancontrol group on14day p.i.. Cytokine levels of SB group decreased compared with infectiongroup at each time point, but were still higher than control group.④By the determination of immunohistochemistry, mice lungs in control group werenormal, but Positive expression of phospho-p38MAPK exsited in epithelial cell of alveolarsepta and airway in small quantities. Positive expression of phospho-p38MAPK in infectiongroup increased obviously, especially almost all of alveolar septa epithelial cells were positive,and myoid cells of the alveolar ducts nodular enlargement were positive too. Meanwhile,Positive expression of phospho-p38MAPK also exsited in cytoplasm and karyon of airwayepithelial cells, vascular endothelial cells, infiltrating inflammatory cells, and express quantityis higher in infiltrating inflammatory cells. The positive expression in SB group droppedsignificantly compared with infection group. It can only be founded in the cytoplasm ofAlveolar septa epithelial cells in small quantities. So positive expression of capillaryendothelial cells in alveolar septa disappeared. Positive expression of phospho-p38MAPKrecovered to level of the control group on14day p.i..⑤By the determination of Western-blot, mice lungs of control group have lightphospho-p38MAPK protein bands at different time point when mice were infected byH9N2-SIV. However, no significant changes were found in control group at any time point.Compared with control group, the expression of phospho-p38MAPK protein in infection group was stronger with significant difference(P<0.05) on2day p.i.,；it reached to the top withextremely significant difference (P<0.01) on6day p.i.,，decreased on8day p.i., but remainedhigher than control group on14day p.i.. The expression of phospho-p38MAPK protein in SBgroup was lower than infection group, but higher than control group.The following conclusions were drawn by this study. A/swine/HeBei/012/2008(H9N2)could infect BALB/c mouse. The lung is the main target organs, and it can cause ALI ofmouse. BALB/c mouse can be used as good animal model to study mammal infected byinfluenza virus. TNF-α and IL-1β play the role of inflammatory factors in the process of ALI,IL-6may play a role of anti-inflammatory action in this study, and IL-10also has the role ofanti-inflammatory factors and play an important part in the sequelae process of ALI.p38MAPK can be activated by H9N2-SIV and cause ALI, and p38MAPK takes part in theoccurrence and development of ALI induced by H9N2-SIV. p38MAPK specificinhibitor——SB203580can lower the expression of phospho-p38MAPK, and decrease theexpression of inflammation factors, lighten pathological injury of lung in ALI.