The Mechanism Of The Change And The Regulation Of The Pro-Resolving Factor Lipoxin On The Mice Model During Acute Lung Injury Stage

Part ⅠThe establishment of a lipopolysaccharide induced self-limitation acute lung injury model and the expression of endogenous lipoxin A4Objectives:To establish a lipopolysaccharide (LPS) induced acute lung injury model, and then to inspect the dynamic change of the model. Detect the change of endogenous lipoxin synthesis and the expression of the5-lipoxygenase.Method:Male BALB/c mice,6-8weeks old,20-25g in weight, were randomly divided into3groups:①zero point:sacrificed the mice as soon as we got them;②Control group(C):given normal saline1.5μl/g through the tracheal;③Acute lung injury(ALI) group:given LPS3μg/g through tracheal. After the administration, mice were sacrificed at6hour,12hour,1day,2day,4day,7day point. To evaluated the pathological change of the lung tissue; detect the protein concentration of the BALF; evaluated the concentration of TNF-α, IL-1β and LXA4in BALF; Use the RT-PCR to detect the5-LO mRNA expression in lung and western blotting to detect the protein expression of5-LO.Results:①there were no significant changes in O group and the C group under the detection of microscopy; the congestion of the alveolar capillaries, infiltration of the inflammatory cells, and the thickness of the alveolar septum of ALI group, but this phenomena was attenuated as the time goes by; the degree of the acute lung injury showed the similar phenomena as the pathological changes of the lung;②compared to the0group or the C group, the wet/dry ratio of the ALI groups increased significantly;③comparing to the O group or the C groups, the total cell number of the ALI groups increased from6hour to1day, and then the number decreased;④comparing to the O group and the C group, the concentration of the protein in the BALF increased from6hour to1day in the ALI groups, and decreased from1day to7day;⑤comparing to the O group and the C group, the concentration of the TNF-a reached the peak at1day while the IL-1β reached it at12hour;⑥comparing to the O group and the C group, the production of LXA4reached the peak at1day, and then decreased to4day, but the production of LXA4reached another peak at7day;⑦comparing to the O group and the C group, the mRNA of 5-LO in lung tissue was a increasing statement from6hour to1day, and then with a decrease from1day to4day, finally increased at7day.Conclusion:The self-limitation acute lung injury model was succeed established; the lipoxin A4played a important role in the resolution of inflammation, and this may related to the expression of5-LO. From the results, we supposed the peak of inflammation situation of the model was1day. Part ⅡThe relationship between the pretreatment of Peroxisome Proliferator-activated receptor y agonist rosiglitazone and the lipoxin in the model of acute lung injuryObjectives:To demonstrate effect of the Peroxisome proliferator-activated receptor gamma (PPARy) agonist rosiglitazone on the acute lung injury model induced by LPS; and the relationship between the PPARy with the endogenous LXA4synthesis and5-LO expression changes during the ALI. Finally, explore the intrinsically link between PPARy agonist rosiglitazone and lipoxin A4and5-LO.Method:To establish the ALI model, the LPS was administrated by instillation. According to the administration, the animals were randomized divided into seven groups:(1) control group (C):with saline instillation intratracheal (1.5ml/kg);(2) Acute lung injury group (ALI):intratracheal instillation of LPS (3mg/kg).(3) Rosiglitazone solvent group (LPS+DMSO(1:10)):DMSO solution (DMSO:NS=1:10,Vol:Vol) gavage three days before intratracheal instillation of LPS;(4) GW9662solvent group (LPS+DMSO(1:100)):DMSO solution (DMSO:NS=1:100, Vol:Vol) injection (i.p) of1.5hours before given LPS;(5) Rosiglitazone pretreatment group(ROS+LPS):rosiglitazone (10mg/kg) gavage for three days, then the administration of LPS;(6) the GW9662group(GW9662+LPS):mice given GW9662(i.p) lmg/kg1.5hours before LPS instillation.(7) A rosiglitazone+GW9662group (ROS+GW9662+LPS):Rosiglitazone pretreatment for3days, the GW9662injection (i.p)1.5hours before the instillation of LPS. After24hours of the administration of LPS, mice were sacrificed to evaluations. The lung tissue was used to make the HE staining and examined by the microscopy; acute lung injury score was performed; the protein concentration in BALF was detected by BCA assay; the ELISA assay was used to evaluate the IL-1β and the TNF-α; The production of LXA4was detected by ELISA; the expression of5-LO in lung tissue was examined by Western blotting.Result:Rosiglitazone pretreatment reduced the LPS-induced acute lung injury in mice pathological changes and reduce acute lung injury score; rosiglitazone pretreatment reduced the total number of cells in BALF, reduced protein concentration in BALF. Rosiglitazone pretreatment also reduced the concentration of TNF-a and IL-1β in BALF; Rosiglitazone increased the concentration of lipoxin A4in BLAF. We found that the increase of5-LO expression in the lungs after the pretreatment of rosiglitazone.Conlusion:PPARy agonist rosiglitazone pretreatment exhibited a protective effect on acute lung injury; PPARγ can promote the synthesis of LXA4, while promoting the expression of5-LO. Part ⅢThe posttreatment of aspirin-triggered lipoxin attenuated the acute lung injury through increasing the expression of PPARy Objectives:The lipoxin A4was shown to have the anti-inflammatory and pre-resolution effect, so does to the aspirin-triggered lipoxin A4(ATL). And the PPARy can attenuate the inflammation in the acute lung injury. We supposed that the ATL may reduce the injury of inflammation through increasing the expression of PPARy.Methods:Male BALB/c mice aged6-8weeks were randomized divided into six groups:a control group received NS, a group received LPS only; two group received LPS plus DMSO and LPS plus saline with injection through tail vein respectively; a group received LPS and lhour later received ATL through tail vein; the last group which received GW9662injected1.5hours before LPS administration, and then ATL injected1hour later. Mice were sacrificed at24hour after the administration of LPS. The lung tissue was sued to make the pathologic analysis, the degree of acute lung injury and the western blot. The brochoalveolar lavage fluid (BALF) was used for the analysis of cell counts, protein, TNF-a and IL-1β.Results:After24hours of the administration of LPS, group received ATL had attenuated the inflammation of the lung and the degree of ALI; and ATL decreased the total cell number in the BALF; the protein concentration was down regulated by ATL; the TNF-aand the IL-1β decreased by ATL in the BALF compared to the ALI group. The ATL increase the expression of PPARy, based on the Western blot, and the group received GW9662partially reverse the effect of ATL.Conclusion:These results improved that ATL had anti-inflammatory and pre-resolution functions on the model of acute lung injury; and this effect through the increase the expression of PPARy.