| ObjectiveSepsis is defined as the systemic inflammatory response that occurs duringsevere infection, and it represented an uncontrolled inflammatory response.The pathophysiological process of sepsis is excessive activation of inflammatoryresponse induced by pathogens and subsequent immune disorder orimmunosuppression, which implicates multiple organs, many types of cells and variespathways. Macrophage play a significant role during each stage of sepsis.Resolvin D1（RvD1）, which is derived from docosahexaenoic acid (DHA), is apotent pro-inflammatory lipid absorption regulator. Resolvin D1can regulatingmacrophage phagocytosis by receptor-dependent mechanism. Our paper intends toobserve the influence of Resolvin D1on the immune function of murine sepsisinduced by cecal ligation and puncture(CLP), and to explore the mechanism of theaction of Resolvin D1in vitro cultured mouse macrophage.Methods1. Murine sepsis model induced by cecal ligation and puncture(CLP) wereestablished.32male C57/BL6mice which receiveing CLP were randomly dividedinto two groupsto receive intravenously Resolvin D1(100ng/mouse) or the sameamount of placebo-treated after operation, the8days survival rate were observed.2.16male C57/BL6mice were randomly divided into Sham group(SHAM, n=4),Cecal ligation and puncture group(CLP, n=6), and Resolvin D1treatmentgroup(CLP+RvD1, n=6). Lung of mice, peripheral blood plasma, peritoneal lavage,spleen and thymus of mice were harvested at24h after CLP. Pathological changes inthe lung tissue of mice were observed by HE staining; the bacterial colony formingunits(CFUs) of peripheral blood and peritoneal lavage of mice were detected bynutrient agar plate assay; cytokines TNFα, IFN-γ, IL-6and IL-10of peripheral bloodof mice were detected by ELISA; The number of neutrophils in peripheral blood andperitoneal lavage, the number of macrophages in peritoneal lavage and the number oflymphocytes and macrophages in the spleen were analysised by Flow cytometry andthe apoptosis rate of CD3+T lymphocytes in the thymus were analysised by Flowcytometry.3.15male C57/BL6mice were randomly divided into Sham group(SHAM, n=3),Cecal ligation and puncture group(CLP, n=3), after receiving cecal ligation and puncture surgery, other mice were treated with different dose of Resolvin D1(1ng、10ng、100ng) by tail vein injection. The phosphorylation of NF-κB and total NF-κBwere determined by western blot.4.9male C57/BL6mice were randomly divided into Sham group(SHAM, n=3)and cecal ligation and puncture group(CLP, n=6). The lung of mice were harvested at6h and24h after CLP. The expression of NLRP3inflammasome were detected byRT-PCR.12male C57/BL6mice were randomly divided into Sham group(SHAM,n=3), Cecal ligation and puncture group(CLP, n=3), Resolvin D1(10ng, n=3)treatment group and Resolvin D1(100ng, n=3) treatment group. The lung of micewere harvested at24h after CLP. The expression of NLRP3inflammasome weredetected western blot.5.12male C57/BL6mice were randomly divided into Sham group(SHAM, n=3),Cecal ligation and puncture group(CLP, n=3), Resolvin D1(10ng, n=3) treatmentgroup and Resolvin D1(100ng, n=3) treatment group. Peripheral blood of mice wereharvested at24h after CLP. Peripheral blood mononuclear cells were separated byFicoll density gradient centrifugation, and the expression of NLRP3inflammasomewere detected by western blot.6. Mouse peritoneal macrophages were isolated and cultured in vitro. To establistNLRP3inflammasome activation model in vitro, macrophages were pretreated withLPS and then with ATP(1mM). Resolvin D1(10ng/ml) was added before or afterLPS-stimulated murine peritoneal macrophages, the expression of NLRP3andcaspase-1(40kd,20kd) were detected by western blot.7. Mouse peritoneal macrophages were isolated and cultured in vitro. To establistNLRP3inflammasome activation model in vitro, macrophages were pretreated withLPS and then with ATP(1mM). Different dose of Resolvin D1(1ng/ml,10ng/ml,100ng/ml) was added after LPS-stimulated murine peritoneal macrophages, theexpression of NLRP3and caspase-1(40kd,20kd) were detected by western blot.8. Mouse peritoneal macrophages were isolated and cultured in vitro. ResolvinD1(10ng/ml) was added after LPS-stimulated murine peritoneal macrophages, with orwithout ATP for further stimulating mouse peritoneal macrophages, the expression ofNLRP3and caspase-1(40kd,20kd) were detected by western blot and RT-PCR.9. Mouse peritoneal macrophages were isolated and cultured in vitro.Macrophages were pretreated with LPS and then stimulated withKCl(75mM)/NaCl(75mM), Resolvin D1(10ng/ml) and ATP(1mM) successively. The expression of NLRP3and caspase-1(40kd,20kd) were detected by western blot, thelevels of IL-1βin cell supernatants were measured by ELISA.10. Mouse peritoneal macrophages were isolated and cultured in vitro.Macrophages were pretreated with LPS and then stimulated withN-acetylcysteine(10μM), Resolvin D1(10ng/ml) and ATP(1mM) successively. Theexpression of NLRP3and caspase-1(40kd,20kd) were detected by western blot, thelevels of IL-1βin cell supernatants were measured by ELISA.11. Mouse peritoneal macrophages were isolated and cultured in vitro.Macrophages were pretreated with LPS and then stimulated with cathepsin B inhibitorCA-074-Me(10μM), Resolvin D1(10ng/ml) and ATP(1mM) successively. Theexpression of NLRP3and caspase-1(40kd,20kd) were detected by western blot, thelevels of IL-1βin cell supernatants were measured by ELISA.12. Mouse peritoneal macrophages were isolated and cultured in vitro.Macrophages were pretreated with LPS and then stimulated with Endocytosisinhibitor cytochalasin D(5μM), Resolvin D1(10ng/ml) and ATP(1mM) successively.The expression of NLRP3and caspase-1(40kd,20kd) were detected by western blot,the levels of IL-1βin cell supernatants were measured by ELISA.13. Mouse peritoneal macrophages were isolated and cultured in vitro. Theexpression of NLRP3were knocked down by si-RNA, then macrophages were treatedwith Resolvin D1(10ng/ml). The phagocytosis of macrophages were measured byflow cytometry.14. Mouse peritoneal macrophages were isolated and cultured in vitro.Macrophages were pretreated with LPS and then with ATP(1mM). The survivalnumber of macrophages were measured by CCK8cell counting assay kit, and theapopotosis rate of macrophages were detected by flow cytometry.Result1. The8days survival rate of sepsis mice induced by CLP were signicantlyimproved by tail vein injection of Resolvin D1compared with the control group.2. Resolvin D1treatment will mitigate the severity of pathological lung injury ofmice24h after CLP; decreased bacterial burden was found in the blood and peritoneallavage fluid samples in the mice treated with Resolvin D124h after CLP comparedwith the control group; the levels of TNF-α, IFN-γ, IL-6and IL-10in peripheral bloodof mice were significant increased at24hours after operation, but Resolvin D1treatment can dilute the levels of TNF-α, IFN-γ, IL-6and IL-10; treatment with Resolvin D1suppressed the increase in the numbers of neutrophils in peritoneallavage fluid but have no significant effect on the numbers of neutrophils andmonocytes in peripheral blood of mice or the macrophages and lymphocytes numberon mouse spleen24h after CLP; resolvin D1significantly decrease the apoptosis rateof CD3+T lymphocytes of thymus in mice24h after CLP model.3. The phosphorylation of NF-κB were increased in the lung tissues24h afterCLP, but injection of Resolvin D1by teil vein to mice could inhibit thephosphorylation of NF-ΚB(P65) by dose-dependent.4. The expression of NLRP3inflammasome was significantly higher than that ofSham groups at6hours after CLP challenge. Resolvin D1could inhibit the expressionof NLRP3inflammasome dose-dependently in lung tissues of septic mice.5. Resolvin D1enhanced the expression of NLRP3mRNA in peripheral bloodmononuclear cells(PBMCs)of septic mice24h after CLP.6. Resolvin D1pretreatment of murine peritoneal macrophage before LPSpriming and the second activation step of ATP represent a inhibitory action to NLRP3inflammasome. However, when macrophages were treated with Resolvin D1afterLPS priming, it can not inhibit the activation of NLRP3inflammasome, but canenhance the expression of NLRP3inflammasome.7. When macrophages were treated with Resolvin D1after LPS priming, it candose-dependently enhance the expression of the components of NLRP3inflammasome.8. Resolvin D1can exhibit the same effect of activating the NLRP3inflammasome after LPS priming with the presence of ATP or not.9. Pretreatment of cultured mouse peritoneal macrophage with extracellularpotassium can partly dilute the activating role of Resolvin D1on NLRP3inflammasome.10. Pretreatment of cultured mouse peritoneal macrophage with the antioxidantN-acetylcysteine can partly dilute the activating role of Resolvin D1on NLRP3inflammasome.11. Pretreatment of cultured mouse peritoneal macrophage with the cathepsin Binhibitor CA-074-Me can partly dilute the activating role of Resolvin D1on NLRP3inflammasome.12. Pretreatment of cultured mouse peritoneal macrophage with the phagocytosisinhibitor cytochalasin D can not reduce the activating role of Resolvin D1on NLRP3 inflammasome.13. The phagocytosis of macrophages were impaired after NLRP3beingknocked down. Resolvin D1could enhance the phagocytosis of macrophages.14. Resolvin D1did not reduce the survival number nor increase the apoptosisrate of cultured mouse peritoneal macrophage.ConclusionResolvin D1can reduce the systemic inflammatory response, enhance theclearance of pathogens in septic mice induced by cecal ligation and puncture, therebyincrease the survival rate of mice with sepsis. The role of Resolvin D1enhanced thescavenging of pathogenic microorganisms may be related to the activation of NLRP3inflammasome of mouse peritoneal macrophages. The activation role of Resolvin D1on NLRP3inflammasome of mouse peritoneal macrophages may depend on themechanisms of potassium efflux and the generation of mitochondria-derived reactiveoxygen species (ROS). |
PDF Full Text Request