Essential Role Of Protectin DX In Septic Mice And Its Molecular Mechanism

Part oneEffects of PDX on survival rate and multiple organ injury of septic miceObjective:To investigate the effects of Protectin DX (PDX) on survival rate and multiple organ injury of septic mice.Method:In the first set of experiments:Establish a mouse model of sepsis, and observe the effects of different doses of PDX on survival rate of septic mice.We performed cecal ligation and puncture(CLP) in mice to establish model of microbial sepsis:Sixty male C57BL/6J mice were divided into 5 groups randomly (n=12):(1) Sham group:sham operation was performed and 100?l vehicle was injected by i.p. 1h after the operation. (2) CLP group:cecal ligation and puncture (CLP) was performed to induce sepsis and 100?l vehicle was injected by i.p. 1h after the operation. (3)CLP+100ng PDX group:CLP was performed to induce sepsis and 100ng PDX was injected by i.p. 1h after the operation. (4) CLP+300ng PDX:CLP was performed to induce sepsis and 300ng PDX was injected by i.p. 1h after the operation; (5) CLP+1000ng PDX:CLP was performed to induce sepsis and 1000ng PDX was injected by i.p. 1h after the operation. Mice were observed every 24 hours to record the survival rate in each group until the 8th day after operation. The second set of experiments:Investigate the effects of PDX on the major organs damage and systemic inflammatory response in septic mice. Thirty male C57BL/6J mice were divided into 3 groups randomly (n=10):(1) Sham group:sham operation was performed and 100?l vehicle was injected by i.p. 1h after operation. (2)CLP group: cecal ligation and puncture (CLP) was performed to induce sepsis and 100?l vehicle was injected by i.p. 1h after operation. (3)CLP+PDX group:CLP was performed to induce sepsis and 300ng PDX was injected by i.p. 1h after operation.24 hours after operation, mice were euthanized. Blood and peritoneal lavage fluid (PLF) together with lung, kidney, liver and spleen were collected. Lung, kidney, liver, spleen were sectioned for hematoxylin-eosin staining to estimate pathological damage. ALT% AST?Cr and BUN were analyzed by automatic analyzer. Cytokines(TNF-?, IL-6, MCP-1 and IL-10) in serum and PLF were measured by enzyme- linked immunosorbent assay (ELISA) kits.Results:(1) The 8days survival rate of CLP induced mice was 25%; PDX300ng and 1000ng can respectively improve 8days survival rate of septic mice to 66.7% and 75%; In PDX100ng group, the 8days survival rate of mice was 41.67%, and there is no statistic significance between PDX100ng and CLP group. (2) In CLP group, the major organs such as lung, kidney, and liver were damaged seriously leading to the pathological scores significantly lowered; Levels of serum ALT, AST, Cr and BUN were prominently increased as well as the levels of pro-inflammatory mediators(TNF-a, IL-6, MCP-1,IL-10) in serum and PLF; PDX300ng can make the major organs injury induced by CLP significantly relieved, decrease the levels of serum ALT, AST, Cr, BUN as well as the pro-inflammatory mediators (TNF-a, IL-6, MCP-1, IL-10) in serum and PLF, and reversely increase the level of IL-10 relating to anti-inflammatory response.Conclusion:PDX improved survival rate of cecal ligation and puncture induced septic mice and mitigated systemic inflammatory response together with organ injury.Part twoPDX modulates the macrophage subsets in peritoneal cavity to promote the resolution of inflammationObjective:To investigate effects of PDX on the macrophage subsets and macrophage phagocytosis.Methods:Male C57BL/6J mice were divided into 3 groups randomly:(1) sham group: sham operation was performed and 100?l vehicle was injected by i.p. 1h after operation. (2) CLP group:cecal ligation and puncture (CLP) was performed to induce sepsis and 100?l vehicle was injected by i.p. 1h after operation. (3) CLP+300ng PDX group:CLP was performed to induce sepsis and 300ngPDX was injected by i.p. 1h after operation.24 hours after operation, mice were euthanized. Blood and peritoneal lavage fluid(PLF) were collected for related evaluations. Bacterial CFU were cultured to evaluate the blood and peritoneal bacterial load; The changes of the number of neutrophils and monocytes/macrophages in PLF were observed by Giemsa staining method; Flow cytometry was performed to detect the M1 and M2 phenotype of macrophages and macrophage phagocytosis in PLF. Western blot was performed to detect protein levels of alternative activation markers Arginase 1 (Argl), Chi-tinase 3-like 3 (Yml) and peroxisome proliferator-activated receptor gamma (PPARy).Results:, In CLP group, bacterial load in both blood and peritoneal lavage existed obviously compared to sham control. Concomitantly, the number of leukocytes, neutrophils and macrophages were prominently increased and macrophage phagocytosis was enhanced; The number of M1 macrophages were increased in PLF. Conversely, the number of M2 subtype were decreased. Meanwhile, the expression of Argl?Yml and PPAR-yin macrophages were inhibited. PDX dramatically decreased bacterial load in both blood and peritoneal lavage as well as the number of neutrophils. Furthermore, PDX increased the proportion of monocytes and neutrophils and enhanced the activation of macrophage phagocytosis. Compared to CLP group, the number of M1 macrophages were decreased. On the contrary, the number of M2sutype were dramatically increased and the expression of Argl?Yml and PPAR-? were increasingly promoted.Conclusion:PDX promoted macrophages polarized to M2, enhanced macrophage phagocytosis and improved bacterial clearance.Part threeThe effects and mechanisms of PDX on M2 polarization in AW264.7 cellsObjective:To investigate the effects and mechanisms of PDX on the M2 polarization in RAW264.7 cells.Methods:In the first set of experiments:To observe the effects of different doses of PDX on M2 polarization in RAW264.7 cells. RAW264.7 cells were divided into 4 groups randomly:(1) Control group:treated with vehicle. (2) PDX(10nM) group: treated with PDX to 10nM.3) PDX(100nM) group:treated with PDX to 100 (4) PDX (1000nM group:treated with PDX to 1000nM. Western blot and immunofluorescence were performed to detect protein levels of M2 markers Arginase 1 (Arg1), Chi-tinase 3-like 3 (Yml) and PPARy. The second set of experiments: Investigate the mechanisms of PDX on the M2 polarization in RAW264.7 cells. RAW264.7 cells were divided into 4 groups randomly:(1) Control group:treated with vehicle. (2) PDX(100nM) group:treated with PDX to 100nM. (3) PDX(1000nM) group:treated with PDX to 1000nM.(4) PDX+GW9662 group:Administrated PPAR? antagonist GW9662 1?M 30min before treated with PDX 1000nM. Western blot and immunofluorescence were performed to detect protein levels of Argl, Yml and PPARy.Results:PDX increased expression of Argl and Yml in RAW264.7 as well as upregulation of PPARy.And these effects were blocked by inhabiting the PPARy pathway with GW9662.Conclusion:PDX promotes M2 polarization in RAW264.7 cells via PPARy signaling pathway.