Role And Mechanism Of Ly6Chigh Monocyte In Ventilator-Induced Lung Injury In Mice

OBJECTIVE:To investigate the role and mechanism of Ly6Chigh monocyte in mice with ventilator-induced lung injury(VILI).METHODS:Forty-eight healthy male SPF C57BL/6 mice were divided into spontaneous breathing group(n=8),normal tidal volume(VT)group(VT was 8 mL/kg,n=8),and high VT group(VT was 20 mL/kg,n=32).The mice in the high VT group were subdivided into 1,2,3 and 4 hours subgroups,with 8 mice in each subgroup.All mice underwent direct tracheal intubation,those in the spontaneous breathing group maintained spontaneous breathing,and those in the normal VT group and high VT group were mechanically ventilated with different VT.After ventilation for 4 hours,bronchoalveolar lavage fluid(BALF)was collected to determine total protein,and the levels of inflammatory factors including tumor necrosis factor-α(TNF-α)and interleukin-1 β(IL-1β)were determined by enzyme-linked immune sorbent assay(ELISA).The lung tissues were harvested to determine the wet/dry(W/D)ratio,and lung tissue injury was assessed in terms of lung histopathologic examination after hematoxylin-eosin(HE)staining under the light microscope.The protein expressions of monocyte chemotactic protein-1(MCP-1)and CC-chemokine receptor 2(CCR2)in lung tissues were determined by Western Blot.Flow cytometry was used to detect the proportion of Ly6Chigh monocyte in lung tissue.RESULTS:Successful establishment of a model of ventilator-induced lung injury.Light microscopy showed that the lung tissue structure of the spontaneous breathing group and the normal VT group was normal.Compared with the spontaneous breathing group,inflammatory reaction began to appear at 2 hours of high VT ventilation,and gradually increased with the extension of mechanical ventilation time.The lung injury was the most serious in the 4 hours of high VT group,there was alveolar structure disordered,alveolar septum widened,pulmonary interstitial thicken and numerous inflammatory cells and red blood cell infiltration in the sight.The lung injury score difference was statistically significant(P<0.05).Compared with the spontaneous breathing group,the total protein levels of BALF was increased from 2 hours of high VT ventilation,the difference was statistically significant(P<0.05).Compared with the spontaneous breathing group,the lung W/D ratio was increased from 2 hours of high VT ventilation,the difference was statistically significant(P<0.05).Compared with the spontaneous breathing group,TNF-α and IL-1β levels in BALF were increased from 2 hours of high VT ventilation,the difference was statistically significant(P<0.05).Western blot showed that compared with the control group,MCP-1 expression were increased from 2 hours of high VT ventilation,CCR2 expression were increased from 3 hours of high VT ventilation,the difference was statistically significant(P<0.05).Flow cytometry analysis showed that compared with the spontaneous breathing group,the proportion of Ly6Chigh monocytes in the 3h tidal ventilation group and the 4h tidal ventilation group gradually increased,and the difference was statistically significant(P<0.05).There was no significant difference in the parameters mentioned above among the spontaneous breathing group,normal VT group and high VT ventilation 1-hour group.The lung injury score difference was statistically significant(P<0.05).CONCLUSION:Ly6Chigh monocytes are involved in VILI.