The Role Of PDE4B In LPS Induced Acute Lung Injury

Objective:In our previous research,high level of PDE4B was found in the lung of LPS-induced acute lung injury Knockout of PDE4B can significantly suppress the inflammatory response induced by LPS.But the mechanism is still not clear.In the present study,we investigate the role of PDE4B in acute lung injury induced by LPS in vitro and in vivo to clarify the possible mechanism.Methods:The model of acute lung injury(ALI)was reproduced by intratracheal instillation of lipopolysaccharide(LPS)with a dosage of 2mg/kg,in PDE4B(-/-),PDE4B(?/-)and PDE4B wild-type C57/BL6 mice(PDE4B(+/+)).After LPS or saline solution instillation 6h,the right side of the lungs was ligated and harvested,the alveolar lavage fluid(BALF)of the left side was collected.The levels of inflammatory cytokine as TNF-a.IL-6 and IL-1? in BALF were measured with ELISA kits.The mRNA expression level of PDE4B?NF-?B and inflammatory cytokines in lungs were detected by fluorescent quantitative PCR.The protein level of cell signaling pathways in lungs,such as NF-?B and IKK were detected by western blot analysis.To silence the expression of PDE4B in MH-S cells,we used electric transfection of PDE4B-siRNA,fluorescence quantitative PCR to detect the efficiency of silence.MH-S cells stimulated with LPS 500ng/ml 6h,then collect the cell culture supernatants and cell pellets respectively.Detected parameters were same with above.To over expression of PDE4B in MH-S cell,we used Fugene6 to transfect PDE4B expression plasmid,fluorescence quantitative PCR to detect the efficiency.18 hours after transfection,collect the cell culture supernatants and cell pellets respectively.Detected parameters were same with above.Results:In vivo experiments,wild type mice is sensitive to LPS stimulation.After LPS stimulation,the inflammatory cell number and neutrophil number in BALF of wild type mice are sharply increased.The mRNA and protein expression of inflammatory cytokines,such as TNF-a,IL-6 and IL-1?,are obviously rised.Meanwhile,the phosphorylation of IKK protein is high,the mRNA and protein expression of NF-?B are also increased.PDE4B knockout mice is not sensitive to LPS stimulation compared with wild type mice.After LPS stimulation,the inflammatory cell number and neutrophil number in BALF of PDE4B knockout mice was lower than wild type mice.The mRNA and protein expression of inflammatory cytokines,such as TNF-?,IL-6 and IL-1?,are not obviously increased compared with control.At the same time,the phosphorylation of IKK protein is low,the mRNA and protein expression of NF-?B are also decreased.In vitro experiment,It is so sensitive for MH-S cell to LPS stimulation that the cell showed many signs of inflammation.After LPS stimulation,the mRNA and protein expression of inflammatory cytokines in MH-S cell are sharply increased.The phosphorylation of IKK protein is high at the same time,the mRNA and protein expression of NF-?B are also obviously increased.After LPS stimulation,the mRNA and protein expression of inflammatory cytokines in PDE4B silenced MH-S cell are not obviously increased compared with control.The phosphorylation of IKK protein is low at the same time,the mRNA and protein expression of NF-?B are also decreased.While the mRNA and protein expression of inflammatory cytokines in MH-S cell with PDE4B over-expression increased significantly.The phosphorylation of IKK protein increase at the same time,the mRNA and protein expression of NF-?B are also increased.The profile of PDE4B over-expression MH-S cell was similar with the MH-S stimulated by LPS.Conclusion:In our present study,our data suggested that PDE4B regulate inflammation by adjusting the IKK phosphorylation via JNK in the NF-?B pathway.It provide insights into the pathway of inflammation is tightly regulated via PDE4B and may also lead to the development of anti-inflammatory therapeutics that down-regulated PDE4B expression.