Roles And Mechanism Of MHV-68Induces Combined Pulmonary Fibrosis And Emphysema Syndrome

PartⅠ MHV-68induces epithelial-mesenchymal transition of A549Objective: To study the epithelial-mesenchymal transition of A549induced bymurine gammaherpes virus68and to further provide evidence to suggest thatgammaherpes virus infected epithelial cell may play a role in CPFE.Methods: four groups are established: group A: control group; group B: interventiongroup by MHV-68; gourp C: TGF-β positive control group; group D: interventiongroup by RSV. Each group is detected after24hours,48hours,72hours and96hoursrespectively. We use phase contrast microscope to observe the cell morphologychange of A549, immunofluorescence and western blot to detect EMT relatedproteins—E-cadherin and α-SMA. Finally, we use ELISA to measure cytokineexpression of cell culture medium.Results: By using microscope, we find that in intervention group by MHV-68, cellmorphology of A549changes after48hours, and this change become significant after72hours with characters of long spindle cells and loose cell junction. In control group,A549cells have the typical epithelial morphology—cobble-shaped appearance, andtight junctions are observed. The immunofluorescence and western blot results showthat marker protein of epithelial cells-E-cadherin expression decrease obviously afterincubation with MHV-68for72hours, in the meanwhile, marker protein ofmesenchymal cells—α-SMA expression decrease obviously（.P<0.05）. ELISA resultshows that in the supernatant of culture medium, higher level of TGF-β1are detectedin intervention group by MHV-68（P＜0.05）.Conclusion: in vitro MHV-68can induce pulmonary epithelial cell line A549to gothrough EMT, and the possible mechanism may involve the increasing TGF-β1secreted by A549. In contrast, RSV in vitro cannot induce A549to go through EMT. Part II Establish a CPFE model by using MHV-68to infect agedsmoking miceObjective: To establish a CPFE model by using MHV-68to infect aged smokingmice to probe into the possible role of MHV-68playing on CPFEMethods:48SPF C57BL/6mice are randomly divided into8groups, with6mice foreach one: group A: control group，group B: COPD，group C：group C1（COPDcombined with MHV68for7days），group C2（COPD combined with MHV-68for14days），group C3（COPD combined with MHV-68for21days），group C4（COPDcombined with MHV-68for28days），group D（COPD combined with RSV for28days）,group E (COPD combined with bleomycin group for28days). Then we useparaffin section of lung tissues for HE staining and Masson staining to judge collagedeposition, and by using bronchoalveolar lavage fluid for counting all the cells andinflammatory cells respectively we judge pathological change and inflammationinfiltrates of lungs. Finally, we use ELISA to detect protein expression of fibrosisremodeling related cytokine--TGF-β1in BALF.Results: By observing HE-staining of lung tissue section, we find that comparedwith group A, lungs of other groups have broken alveolar structure, alveolus fusionand bullae of lung, that compared with other groups, group E has the most severeinflammation, and group C takes second place, group B and group D have nodifference with group A on inflammation. By Masson staining, we observe thatcompared with group A, group C and group E have more collage deposition, what’smore, group E has much more than group C. Group A, B and E have no obviousdifference. The result of ELISA shows that compared with group A, the TGF-β1protein expression of group C and E has increased obviously (P<0.05), though theTGF-β1protein expression of group B and D also increase, the data has nosignificantly different. Conclusion: CPFE model can be successfully copied by using MHV-68infectingold-age smoking mice. Pathological change in different stages indicate that increasingTGF-β1, which may promote airway remodeling and MHV-68that causesinflammation of airway may both contribute to fibrosis. On the other hand, RSV invivo cannot trigger CPFE pathological change of mice with COPD Part III The effect of treating with glucocorticoid DXMin CPFE modelObjective: To observe whether DXM treatment can reduce the severity of MHV-68induced fibrosis in aged smoking mice. and analyze the possible reasons.Methods: Twelve SPF C57BL/6mice were randomly divided into twogroups(n=6/group): A group(COPD+MHV-68), B group(COPD+MHV-68+DXM）..Inflammatory cell infiltrates in lung tissue were observed by H.E staining. The gradeof fibrosis were observed by Masson staining. While differential cell counts in BALFwere used to assess airway inflammatory cells. Finally, we use ELISA to detectprotein expression of fibrosis remodeling related cytokine--TGF-β1.Result: By observing HE staining lung tissue section and counting BALFinflammatory cells, we find that compared with group A the inflammation of group Bis obviously alleviated. Besides, Masson staining indicates that group B has much lesscollage deposition than group A does. The result of ELISA shows that the amount ofTGF-β1in group B has reduced obviously (P<0.05)Conclusion: DXM treatment can reduce the severity of MHV-68induced fibrosisand inflammation in aged smoking mice by suppressing the profibrogenic growthfactor:TGF-β1.