Effect Of Protectin DX On The Regulation Of Neutrophil Apoptosis On Acute Lung Injury In Sepsis Mice And Its Mechanism

Part one Effects of different doses of PDX on acute lung injury in septic miceObjective: To observe the effects of different doses of Protectin DX(PDX)on acute lung injury in sepsis mice.Method: 28 male C57 mice,6-8 weeks old,were randomly divided into 4 groups,with 7 mice in each group :(1)Sham operation group(Sham): Mouse sham operation,cut the mouse's abdominal cavity with scissors,suture each layer,1 hour later,take 100?l PBS,and intraperitoneally inject the mouse;(2)Sepsis group(CLP): To establish a CLP sepsis animal model with mice,1 hour later,100 ?l of PBS was taken and injected into mice intraperitoneally;(3)CLP+LD-PDX group: CLP was performed,1 hour later,500 ng PDX was dissolved in 100 ?l of PBS and injected intraperitoneally Into the mice;(4)CLP+HDPDX group: CLP was performed.After 1 hour,1000 ng PDX was dissolved in 100?l PBS and injected into the mice intraperitoneally.Arterial blood was taken 24 hours after operation for blood gas detection,and lung tissue and bronchoalveolar lavage fluid were taken.The HE staining method was used to observe the pathological changes of lung tissue and the score of lung injury.The wet/dry weight ratio of lung tissue and the protein concentration in BALF were measured.Enzyme-linked immunosorbent assay(ELISA)was used to determine the levels of inflammatory cytokine including interleukin-1?(IL-1?),interleukin-6,tumor necrosis factor(TNF-?)and macrophage inflammatory protein 2(MIP-2)in BALF.Results: Compared with Sham group,the oxygenation index in CLP group significantly decreased,and lung tissue pathological damage was serious,and lung injury score,wet/dry weight ratio and protein concentration in BALF were significantly increased,and the expression of IL-1?,IL-6 TNF-? and MIP-2 inflammatory factors were significantly upregulated.Using PDX increased oxygenation index significantly,alleviated the pathological changes of lung,decreased the wet/dry weight ratio,lung injury score and the protein concentration in BALF,and significantly down-regulated the expression of inflammatory factors IL-1?,IL-6,TNF-? and MIP-2.CLP+HD-PDX group has higher oxygenation index,more reduction of lung tissue pathological damage,and wet/dry weight ratio,lung injury score,BALF protein concentration and inflammatory factors IL-1?,IL-6,TNF-?,MIP-2 were lower than that of CLP+LD-PDX group.Conclusion: PDX can improve oxygenation index,reduce the pathological injury of lung tissue,wet/dry weight ratio,lung injury score and the protein concentration in BALF,downregulate the expression of IL-1?,IL-6,TNF-? and MIP-2 inflammatory factors,thus realize the protective effect on acute lung injury of sepsis mice,and the effect of high dose PDX is better than that of low dose PDX.Part two PDX protects acute lung injury caused by sepsis by regulating neutrophil apoptosisObjective: The effect of PDX on the apoptosis of neutrophils was observed by using acute lung injury model induced by CLP in vivo and lipopolysaccharide(LPS)stimulating neutrophils in vitro.Method: Animal model : 28 male C57 mice of 6-8 weeks were randomly divided into 4 groups,with 7 mice in each group :(1)Sham operation group(Sham): Mouse sham operation,cut the mouse's abdominal cavity with scissors,suture each layer,1 hour later,take 100?l PBS,and intraperitoneally inject the mouse;(2)Sepsis group(CLP): To establish a CLP sepsis animal model with mice,1 hour later,100 ?l of PBS was taken and injected into mice intraperitoneally;(3)CLP+LD-PDX group: CLP was performed,1 hour later,500 ng PDX was dissolved in 100 ?l of PBS and injected intraperitoneally Into the mice;(4)CLP+HDPDX group: CLP was performed.After 1 hour,1000 ng PDX was dissolved in 100?l PBS and injected into the mice intraperitoneally.After 24 hours,BALF was collected and leukocyte count was performed.Flow cytometry was used to detect neutrophil apoptosis.In vitro cell experiment: Firstly,the peripheral blood of healthy adult volunteers was collected,and neutrophils were inoculated into the culture plate at a density of 1×106 /ml.The cells were divided into 4 groups according to the different concentrations of PDX used:(1)Control group: neutrophils were treated with the medium containing 0.038%PBS(PDX solvent);(2)PDX low-dose group: neutrophils were treated with medium containing PDX 10 n M;(3)PDX medium dose group: neutrophils were treated with medium containing PDX 100 n M;(4)PDX high-dose group: Neutrophils were treated with medium containing PDX 1000 n M.At least 3 duplicate holes shall be set for different treatment groups.After treating with PDX for 24 h,the PDX toxicity was detected by MTT assay.Secondly,neutrophils were inoculated into the culture plate at a density of 1×106 /ml.,and the cells were divided into 5 groups according to different drugs:(1)Control group: neutrophils were treated with the medium containing 0.038%PBS(PDX solvent);(2)LPS group:neutrophils were treated with 1?g/ml LPS medium;(3)LPS+ PDX10 n M group: Cell therapy 1 ?g/ml LPS for 1 h and PDX added to the final concentration of the medium PDX10 n M;(4)LPS+ PDX100 n M group: 1 ?g/ml LPS medium is used for 1 h and PDX is added to the final concentration of the medium PDX100 n M;nLPS+ PDX1000 n M group: 1 g/ml LPS medium is treated for 1h,and PDX is added to the final concentration of PDX1000 n M.At least 3 duplicate holes shall be set for different treatment groups.The apoptosis of neutrophils was detected by flow cytometry after treatment for 24 h.DCFH-DA was used to assess the expression of ROS in neutrophils.Using ELISA to detecte the levels of inflammatory cytokines IL-1?,IL-6,TNF-?,MIP-2.Results: In the animal model,compared with the Sham group,the number of total cells,neutrophils and monocytes/macrophages in BALF were significantly increased in CLP group,and the neutrophils apoptosis was significantly decreased.Compared with CLP group,the using of PDX significantly reduced the number of total cells and neutrophils,and significantly promoted the neutrophils apoptosis.Compared with CLP+LD-PDX group,total cells number and neutrophil number decreased more in CLP+HD-PDX group,and neutrophil apoptosis was more.In cell test,different doses of PDX had no toxic effect on neutrophils.LPS significantly reduced neutrophil apoptosis.Compared with LPS group,LPS+PDX100n M group and LPS+PDX1000n M group significantly promoted neutrophils apoptosis,but the effect of LPS+PDX10n M was not obvious.Compared with LPS+PDX10n M group,LPS+ PDX100 n M group and LPS+ PDX1000 n M group promoted neutrophils apoptosis more significantly.Compared with the LPS+PDX100n M group,the PDX1000 n M group had a stronger effect on promoting neutrophils apoptosis.Therefore,in our subsequent cell experiments,the LPS+PDX10n M group was no longer added.In the LPS group,the production of ROS was significantly increased comparing with the control group,and LPS stimulated neutrophils to produce a large number of inflammatory cytokines;LPS+PDX100n M group and LPS+PDX1000n M group significantly reduced LPS induced ROS production and significantly reduced the concentration of cytokines(IL-1?,IL-6,TNF-?,MIP-2).Compared with the LPS+PDX100n M group,the production of ROS and the level of L-1?,IL-6 and TNF-? were significantly decreased in the LPS+PDX1000n M group.Conclusion: PDX plays a protective role in acute lung injury caused by sepsis in mice by promoting neutrophil apoptosis.Part three PDX regulates neutrophils apoptosis through PI3K-Akt and MAPK-ERK signaling pathwaysObjective: To explore the possible molecular mechanism of PDX regulating neutrophil apoptosis.Method: The peripheral blood of healthy adult volunteers was collected,and neutrophils were inoculated into the culture plate at 106 / ml.The cells were divided into four groups according to different drugs:(1)Control group: neutrophils were treated with the medium containing 0.038%PBS(PDX solvent);(2)LPS group:neutrophils were treated with 1?g/ml LPS medium.(3)LPS+ PDX100 n M group: 1 ?g/ml LPS medium is used for 1 h and PDX is added to the final concentration of the medium PDX100 n M;(4)LPS+ PDX1000 n M group: 1 ?g/ml LPS medium is treated for 1h,and PDX is added to the final concentration of PDX1000 n M.At least 3 duplicate holes shall be set for different treatment groups.After 24 hours,the cells were isolated and the expressions of p-AKT,p-ERK,p-p38,Bcl-2 and Bax were detected by Western blot.Results: LPS significantly up-regulated the expression of p-AKT,p-ERK,p-p38 and Bcl-2,and down-regulated the expression of Bax in neutrophils.Compared with LPS group,PDX100 n M group and PDX100 n M group significantly decreased the expression of p-AKT,p-ERK,p-p38 and Bcl-2,and significantly up-regulated the expression of Bax.Compared with LPS+ PDX100 n M group,1000 n M PDX significantly decreased the expression of pERK and p-p38,and up-regulated the expression of Bax in neutrophils.Conclusion: PDX promoted the neutrophils apoptosis in sepsis model by down-regulating the expression of Bcl-2 and up-regulating the expression of Bax.The mechanism may be mediated by PI3K/ AKT and p38/ERK signaling pathways.