Protectin DX Ameliorates Sespsis-induced Acute Lung Injury By Suppressing Neutrophils Activation And Its Mechaism

Part one Protectin DX ameliorated sespsis-induced acute lung injury in miceObjective: To investigate the effects of Protectin DX（PDX）on sespsis-induced acute lung injury in mice.Method: 28 male C57 mice were randomly divided into 4 groups,each group consisted of 7 mice:（1）sham operation group（sham）: the abdominal cavity of mice was opened along the midline of abdomen and then sutured layer by layer,100 μ l PBS was injected intraperitoneally at one hour after operation,;（2）sepsis group（CLP）: the sepsis model of mice was established by cecal ligation and puncture（CLP）,100 μ l PBS was injected intraperitoneally at one hour after operation;（3）PDX500ng group: 500 ng PDX（diluted in 100μl PBS）was adiministrated intraperitoneally at one hour after CLP;（4）PDX1000ng group: 1000 ng PDX（diluted in 100μl PBS）was adiministrated intraperitoneally at 1 h of operation;24 h after operation,mice were sacrificed and lung tissue and bronchoalveolar lavage fluid（BALF）were collected 24 h after operation.The pathological changes of lung tissue and lung injury scores were observed by HE staining;The lung wet/dry weight ratio（W/D）and BALF protein concentration were measured;the levels of cytokines（IL-1β,TNF-α,IL-6 and MIP-2）in BALF were detected by ELISA.Results: Compared with sham group,the lung tissue pathological damage of CLP group was obvious,lung injury scores and the dry wet ratio,protein concentration,total cell number,neutrophil number and inflammatory factor（IL-1 β,IL-6,TNF-α,MIP-2）concentration in BALF were significantly increased;compared with CLP group,PDX significantly improved lung tissue pathological damage,decreased lung injury scores,dry wet ratio,protein concentration in BALF,total cell number,neutrophil The quantity and the concentration of inflammatory factors（IL-1 β,IL-6,TNF-α,MIP-2）.Compared with the low-dose group,PDX high-dose group had lower pathological score,dry wet ratio and BALF protein concentration,and lower levels of IL-1 β,IL-6,TNF-α and MIP-2 in BALF.Conclusion: PDX reduced the acute lung injury and edema induced by sepsis,reduced the aggregation of inflammatory cells and the released of inflammatory factors.Part two Protectin DX inhibited neutrophil activation and its mechanismObjective: To investigate whether PDX has effect on acute lung injury induced by sepsis by regulating neutrophil activation.Methods: In the first set of experiments:28 male C57 mice were randomly divided into 4 groups,each group consisted of 7 mice:（1）sham operation group（sham）: the abdominal cavity of mice was opened along the midline of abdomen and then sutured layer by layer,100 μl PBS was injected intraperitoneally at one hour after operation,;（2）sepsis group（CLP）: the sepsis model of mice was established by cecal ligation and puncture（CLP）,100 μl PBS was injected intraperitoneally at one hour after operation;（3）CLP+ PDX500 ng group: 500 ng PDX（diluted in 100μl PBS）was adiministrated intraperitoneally at one hour after CLP;（4）CLP+ PDX1000 ng group: 1000 ng PDX（diluted in 100μl PBS）was adiministrated intraperitoneally at 1 h of operation;24 h after operation,mice were sacrificed and lung tissue samples were collected.The levels of ROS were detected by DCFH-DA.The activities of MDA and SOD were measured using TBA and WST-1 respectively.The levels of neutrophils accumulatinon were observed by immunohistochemistry and immunofluorescence staining.In the second set of experiments: the neutrophils extracted from the peripheral blood of healthy adults were cultured in vitro and randomly divided into 5 groups:（1）control group: PBS was added to the culture medium as the control group;（2）LPS group: isolated neutrophils were stimulated with 1μg/ml LPS;（3）LPS+PDX10n M group: isolated neutrophils were stimulated with 1μg/ml LPS for 1 h,and then coincubated with 10 n MPDX;（4）LPS+PDX100n M group: isolated neutrophils were stimulated with 1μg/ml LPS for 1 h,and then coincubated with 100 n MPDX;（5）LPS+PDX1000nm group: isolated neutrophils were stimulated with 1μg/ml LPS for 1 h,and then coincubated with 1000 n MPDX.After 24 hours,the inflammatory factors（IL-1 β,IL-6,TNF-α,MIP-2）and myeloperoxidase（MPO）were detected by ELISA,the level of LDH was detected by microplate method,the activity of SOD was measured by WST-1,DCFH-DA was used to assess the expression of ROS in cells.Results: The first part of the results showed that compared with sham group,the levels of ROS and MDA were significantly increased and the activity of SOD was significantly reduced in CLP group.A major increased number of ly-6G+ neutrophils were observed in the CLP group.PDX significantly reduced the production of ROS and MDA in septic lung tissue.The activity of SOD and levels of neutrophil infiltration were observed significantly reduced when the mice received PDX.Compared with the low dose group,PDX high-dose group was lower in the levels of ROS and MDA,higher in the activity of SOD.The second part of the results showed that LPS stimulated neutrophils to secrete a number of inflammatory factors（IL-1 β,IL-6,TNF-α,MIP-2）,activities of MPO,LDH and ROS were increased significantly.In LPS group,the expression of SOD was decreased significantly compared with Control group.PDX decreased inflammatory factors（IL-1 β,IL-6,TNF – α,MIP-2）and activities of MPO,LDH,ROS in a dose-dependent manner.Besides,the expression of SOD was enhanced in a dose-dependent manner when treated with PDX.Conclusion: PDX reduced the acute lung injury by inhibiting the activation of neutrophils.Part three Protectin DX inhibitd LPS-induced neutrophil activation and its mechanismObjective: To explore the specific molecular mechanism of PDX regulating neutrophil activation.Methods:In the first part of experiment: 28 male SPF grade C57 mice of 6-8 weeks were randomly divided into 4 groups according to the operation mode and administration or not,Seven mice in each group:（1）sham operation group（sham）: the abdominal cavity of mice was opened along the midline of abdomen,then sutured layer by layer,and 100 μl PBS was injected into the abdominal cavity one hour after operation;（2）sepsis group（CLP）: the sepsis model of mice was established by CLP method,and 100 μl PBS was injected into the abdominal cavity one hour after operation;（3）CLP + PDX500 ng group: 500 ng was injected into the abdominal cavity one hour after the establishment of CLP model PDX（soluble in 100 μl PBS）;（4）CLP + PDX1000 ng group: after establishing CLP model,1000 ng PDX（soluble in 100 μl PBS）was injected intraperitoneally one hour after operation;24 hours later,mice were killed by bleeding through abdominal aorta,lung tissue samples were collected,and the levels of p-p38 MAPK,p-ERK1/2,p-NF-κBp65,p-p47^phox-ser345 were detected by immunoblotting.In the second part of experiment: the neutrophils extracted from the peripheral blood of healthy adults were cultured in vitro and randomly divided into five groups:（1）control group: PBS（PDX solvent）was added to the culture medium as the control group;（2）LPS group: 1μg/ml LPS was added to the culture medium;（3）PDX10nm group: after 1 μg/ml LPS stimulated cells for 1 hour,add 10 nm PDX;（4）PDX100nm group: after 1 μg/ml LPS stimulated cells for 1 hour,add 100 nm PDX;（5）PDX1000nm group: after 1 μg/ml LPS stimulated cells for 1 hour,add 1000 nm PDX.The expression of p-p38,p-ERK1/2,p-NF-κB p65 and p-p47^phox-ser345 were detected by Western blot.Results: The first part of the results showed that the phosphorylation level of p-p38 MAPK,p-ERK1/2,p-NF-κB p65,p-p47^phox-ser345 was increased by CLP,and PDX could significantly reduce the phosphorylation level.The second part of the results showed that LPS stimulated the expression of p-p38,p-ERK 1/2,p-NF-κB p65,p-p47^phox-ser345 in neutrophils,and PDX decreased the expression of p-p38,p-ERK 1/2,p-NF-κB p65,p-p47^phox-ser345 in a dose-dependent manner.Conclusion: PDX inhibited neutrophils activation through suppressing MAPK/NF-κB/p47^phox-ser345 signaling pathway.