The Study Of Mechanisms Of Docosahexaenoic Acid(DHA)-induced GSDME-mediated Pyroptosis

Backgrounds:Docosahexaenoic acid?DHA?is an indispensable?-3 polyunsaturated fatty acids??-3 PUFAs?that cannot synthesize by human body.DHA participates in a variety of physiological processes,including cell membrane fluidity maintenance,synaptic transmission,brain glucose uptake,neural development,memory and cognition.DHA was primarily known for its anti-inflammatory effects,which could achieve the purpose of inhibiting inflammation through various mechanisms,such as reducing the adhesion and extravasation of immune cells,inhibiting the production of reactive oxygen species?ROS?,and attenuating the expression of major inflammatory cytokine interleukin-1?IL-1?.Pyroptosis is a form of regulatory cell death that critically depends on the formation of plasma membrane pores by GSDM?Gasdermin?family,and the activation of GSDM is often?but not always?induced by the activation of inflammatory Caspase.Pyroptosis includes the canonical pathway which depends on Caspase-1 and the non-canonical pathway depends on Caspase-4/-5/-11,both rely on GSDMD to trigger pyroptosis,which plays an important role in immune defense.However,in recent years,it has also been indicated that Caspase-3 leaded to the cleavage of GSDME and mediated pyroptosis,which is of great significance in anti-tumor therapy and drug development.Recently,some studies have found that DHA could attenuate inflammatory by regulating inflammasomes in upstream of the pyroptosis signaling pathway in several disease models,such as atherosclerosis?AS?,diabetes,and non-alcoholic liver disease?NALD?.Additionally,there were also several studies proved that DHA could induce pyroptosis in microglial and breast cancer cells.Therefore,the regulatory effect of DHA on pyroptosis may also appear in many other cells,which is of general significance,but its specific mechanism is still unclear.Therefore,we intend to investigate whether DHA induce pyroptosis and its molecular mechanism on cell-based level,and expect to provide reference for DHA in disease prevention and treatment.Inwe performed THP-1 monocytes,THP-1 macrophages?differentiated from THP-1 monocytes induced by PMA?and human umbilical vein endothelial cells?Human umbilical vein endothelial cells,HUVECs?as our research objects.Part? DHA treatment blocks NLRP3/Caspase-1/GSDMD canonical pyroptosis pathway in THP-1 monocytes and THP-1 macrophagesObjective:We studied whether 10 anti-inflammatory substances,which have been reported,including DHA,Curcumin,Eicosapentaenoic acid?EPA?,Sulforaphane?SFN?,Atorvastatin,Puerarin,Honokiol,Artemisinin,sphingosine 1-phosphate 2 antagonist JTE013,Mucosa-associated-lymphoid-tissue lymphoma-translocation gene 1inhibitor 2?MI-2?could regulate the canonical pyroptosis pathway.And that,how DHA regulate the canonical pyroptosis pathway.Finally,we preliminarily explored whether there was any difference between the toxic effect of DHA on normal cells and tumor cells.Methods:1)Western blot was used to detect the pyroptosis related molecules NLRP3,Caspase-1,GSDMD,and the key executor of apoptosis Caspase-3 to screen 10anti-inflammatory substances including Curcumin,EPA,DHA,SFN,Atorvastatin,Puerarin,Honokiol,Artemisinin,JTE013 and MI-2,which anti-inflammatory substances could regulate pyroptosis in THP-1 monocytes and THP-1 macrophages.THP-1 monocytes and THP-1 macrophages were treated with different concentrations of DHA?0,10,25,50,100 and 200?M?for 16 hours and 50?M DHA for different time?0,2,4,8,16and 24 hours?,respectively.2)THP-1 monocytes and THP-1 macrophages were pretreated with 0,10,25,50,100 and 200?M DHA for 1 hour,and then treated with LPS?Lipopolysaccharide?for 3 hours,and Nigericin for another one hour?establish cell model of canonical pyroptosis?.Western blot was used to analyze the expression of NLRP3,Caspase-1,GSDMD and IL-1?;3)The morphological changes of THP-1 monocytes with DHA treatment at different concentrations?0,10,25,50,100 and 200?M?were observed by the microscope,and counted the positive cells by trypan blue staining,and then calculated the positive cell ratio;4)The toxicity of DHA in THP-1 monocytes at different concentrations?0,10,25,50,100,200?M?on THP-1 monocytes was detected by CCK-8 and cytotoxicity assay.The death of THP-1 monocytes treated with DHA?0,10,200?M?was analyzed by PI staining,and observed by microscope.The PI staining positive cells was counted with ImageJ software,and then calculated positive rate;5)CCK-8 test was used to detect the cell viability of peripheral blood mononuclear cells?PBMCs?isolated from healthy human venous blood with different concentrations of DHA.PI staining and flow cytometry were used to further analyze the toxic effects of DHA at different concentrations on PBMCs,and the differences of 50?M DHA between PBMCs and monocyte-macrophages tumor cells THP-1 monocytes cells was compared;Results:1)In anti-inflammatory substances,DHA and SFN regulated the canonical pyroptosis signal pathway in THP-1 monocytes and THP-1 macrophages,by down-regulateing the expression of NLRP3 and Caspase-1?the difference was statistically significant?,cutting GSDMD into p43 fragments?GSDMD-C?,and up-regulating the expression of Cleaved Caspase-3.The expression of NLRP3 and Caspase-1 decreased with the increase of the concentration of DHA,especially at200?M.When the concentration of DHA was 50?M,the expression of NLRP3 and Caspase-1 decreased with the prolongation of DHA processing time,while the content of GSDMD-C fragment increased significantly?the difference was statistically significant?;2)In THP-1 monocytes and THP-1 macrophages,DHA at a certain concentration?10-100?M?could inhibit the shear of Caspase-1,GSDMD and IL-1?induced by LPS-Nigericin;3)Observing under a microscope,it revealed that when the concentration of DHA is more than or equal to 50?M,THP-1 monocytes appear swelling,ruptured and dead,which were typical features of cell pyroptosis.When the concentration of DHA was more than or equal to 25?M,the positive ratio of trypan blue staining for THP-1 monocytes increased significantly with the increase concentration of DHA;4)CCK-8 assay results showed that when the concentration of DHA was more than or equal to 25?M,the OD₄₅₀ value of THP-1 monocytes decreased with the increase of DHA concentration?the difference was statistically significant?.Cytotoxicity assay showed that cytotoxicity of THP-1 monocytes increased significantly with the increase of DHA concentration?the difference was statistically significant?.Observing under a microscope,it showed that when the concentration of DHA is 200?M,the positive rate of PI cell staining is more than 90%;5)In human PBMCs,when the concentration of DHA at 50?M or more,CCK-8 assay revealed that OD₄₅₀ value of cells decreased with the increase of DHA concentration?the difference is statistically significant?,and flow cytometry results indicated that the PI staining positive cells ratio increased?the difference is statistically significant?;when DHA concentration is 50?M,the rate of human PBMCs PI positive cells decreased significantly compared with THP-1 monocytes?the difference is statistically significant?.Conclusions:1)We showed that the treatment of DHA and SFN at the concentration of 50?M could block the NLRP3/Caspase-1/GSDMD canonical pyroptosis pathway in THP-1 monocytes and THP-1 macrophages,both could down-regulate the expression of NLRP3 and Caspase-1 to reduce the formation of inflammasomes in the upstream of the pyroptosis pathway,and cleaved GSDMD into non-toxic p43fragments to block the activation of effector molecules in the downstream of the pathway;2)DHA have anti-pyroptosis and pro-pyroptosis functions in a certain concentration level:when DHA at a certain concentration?10-50?M?,DHA could inhibitor pyroptosis induced by LPS-Nigericin in THP-1 monocytes and THP-1macrophages,and reduced the expression and release of inflammatory factor IL-1?to protect the cells from death.However,DHA caused THP-1 monocytes exhibited pyroptosis-related phenomena when concentration of DHA was at 50,100 and 200?M;3)High concentration of DHA has toxic effect on human PBMCs;DHA was more likely to induce THP-1 monocytes to pyroptosis compared with normal cells?PBMCs?.Part ? DHA treatment induces Caspase-3/GSDME pyroptosis pathway in THP-1 monocytesObjective: To explore the clear mechanism of DHA induced pyroptosis in THP-1 monocytes.Methods: 1)Western blot was used to verify the effect of DHA on Caspase-3/-7/-8 and GSDME in THP-1 monocytes and THP-1 macrophages after treating with DHA at different concentration?0,10,25 and 50 ?M?and times?0,2,4,8,16 and 24 hours?,respectively;2)PI staining and flow cytometry were used to analyze the cell death after THP-1 monocytes were pretreated with Z-VAD-FMK?Pan Caspase inhibitor?and Z-DEVD-FMK?Caspase-3 inhibitor?and then treated with 50 ?M DHA for 16 hours;western blot was applied to detect the expression of GSDME,caspase-3/-7/-8/-9 and PARP;3)Western blot was used to detect the expression of NLRP3,GSDMD,GSDME and Caspase-3 after THP-1 monocytes and THP-1 macrophages treating with EPA,AA,OA,GLA,PA and MA for 16 hours;4)THP-1 monocytes were priming with five oxidase inhibitors participating in DHA metabolism pathway,including NDGA,Baicalein,COX 2 V FK3311,MK886?the inhibitors of 5-lipoxygenase,12-lipoxygenase,15-lipoxygenase,cyclooxygenase-2 and pan lipoxygenase?for 1 hour,and then treated with DHA for 16 hours.Western blot was applied to detect the expression of NLRP3,caspase-1,GSDMD,GSDME and apoptosis factor Caspase-3;5)THP-1 monocytes were treated with three final metabolites?Maresin 1,Resolvin D1 and Protectin D1?of DHA at various concentrations?50,100 and 200 n M?;and the expression of NLRP3,Caspase-1,GSDMD,GSDME and Caspase-3 in THP-1 monocytes were analyzed by western blot.Results: 1)In THP-1 monocytes,the fragments of Cleaved Caspase-7/-8/-3 and GSDME-N were detected when the concentration of DHA was 10 ?M or more,and the expression of those fragments increased significantly when DHA was at the concentration of 50 ?M?the difference was statistically significant?.In addition,when the concentration of DHA was 50 ?M,Caspase-7/-8 started to be activated after treated with DHA for 2 hours,but Caspase-3 and GSDME started to be activated after DHA treatment for 4 hours,and the expression of activated fragments increased significantly when treated with DHA for 16 and 24 hours?the difference was statistically significant?.Nevertheless,in THP-1 macrophages,GSDME started to be activated when the concentration of 10 ?M,and GSDME-N fragment were detected after 2 hours with 50 ?M DHA treatment,and increased with the elevation of dose and time?the difference was statistically significant?;2)Both Z-VAD-FMK?Pan Caspase inhibitor?and Z-DEVD-FMK?Caspase-3 inhibitor?could decline the PI positive cells ratio and inhibit the cleavage of GSDME in THP-1 monocytes?the difference was statistically significant?.Z-VAD-FMK and Z-DEVD-FMK could also inhibit the decrease of PARP,caspase-9/-8/-7/-3 in THP-1 monocytes with treating DHA;3)EPA,AA,OA,GLA,PA and MA could not reduce the expression of NLRP3 and Caspase-1,and could not induce the activated cleavage of GSDMD,GSDME and Caspase-3 either;4)THP-1 monocytes pretreated with the oxidases inhibitors involved in DHA metabolism pathway including NDGA,Baicalein,COX-2 V FK3311,MK886?the inhibitors of 5-lipoxygenase,12-lipoxygenase,15 lipoxygenase,cyclooxygenase-2 and pan lipoxygenase?did not have effect on the expression of NLRP3 and Caspase-1 in THP-1 monocytes and THP-1 macrophage,and could not inhibit the cleavage of GSDME and Caspase-3;5)In THP-1 monocytes and THP-1 macrophage,the treatment of the final metabolites of DHA,including Maresin 1,Resolvin D1 and Protectin D1 that at various doses?50,100,and 200 n M?,could not block NLRP3/caspase-1/GSDMD in canonical pyroptosis pathway and either activate Caspase-3/GSDME-induced the secondary pyroptosis pathway.Conclusions: 1)We firstly proved that DHA could induce Caspase-3/GSDME-mediated pyroptosis in THP-1 monocytes and THP-1 macrophages;2)In THP-1 monocytes,the oxidative enzymes and the final metabolites of DHA did not induce pyroptosis.Part ? DHA induces pyroptosis in HUVECsObjective: To explore the expression levels of canonical pyroptosis signaling pathway associated molecules in different cell lines?endothelial cells,monocyte-macrophages and non-monocyte-macrophages tumor cells?,and whether DHA can induce pyroptosis in HUVECs.Methods: 1)Western blot was used to analyze the expression levels of four canonical pyroptosis pathway associated molecules NLRP3,Caspase-1,GSDMD,IL-1?,and the GSDMs family members?GSDMA,GSDMB,GSDMC,GSDME,DFNB59?in 5 endothelial cell lines?HUVECs,HCAECs,HAECs,HLMECs,HDMECs?,3 monocyte-macrophages cell lines?THP-1 monocytes,U937,RAW264.7?,and 5 non-monocyte-macrophages tumor cell lines?HEK293T,Hela,A549,3T3,COS-7?;2)Western blot was used to detect the expression of GSDMD in HUVECs after HUVECs were treated with 1 ?g/m L LPS,10ng/m L TNF-? and 25 m M glucose for 0,1,2,4,6,8,16,24 and 32 hours,respectively;3)Western blot was used to screen which could regulate pyroptosis in HUVECs among these anti-inflammatory small molecule chemicals,including Curcumin,DHA,SFN,Atorvastatin,Puerarin,Honokiol,Artemisinin,JTE013,MI-2;4)Western blot was used to detect the expression of GSDMD and Caspase-1 in HUVECs with prolonging the cultured time of DHA;5)CCK-8 was used to detect the cell viability after HUVECs were treated with different doses of DHA?0,10,25,50,100,and 200 ?M?or different times?0,4,8,16 and 24 hours?,respectively.PI staining and fluorescence microscopy were used to observe the dead cells after HUVECs were treated with DHA at 50 ?M.And western blot was used to further verify the expression levels of GSDME.Results: 1)Compared with non-specific expression protein apoptosis factor,including Caspsase-3,NLRP3,Caspase-1,GSDMD,and IL-1?,were molecules related to pyroptosis signaling pathway,and only expressed in certain cell lines.Among the four monocyte-macrophages tumor cells involved in this study,the above four cell pyroptosis pathway related molecules were all expressed.GSDMD was expressed in endothelial cells and monocyte-macrophages tumor cells.However in non-monocyte-macrophages tumor cell lines,GSDMD was only detected in A549 cells.And that,GSDME was expressed in normal endothelial cells,monocyte-macrophages and non-monocyte-macrophages tumor cells;2)Western blot showed that the cleavage of GSDMD fragment was not produced after HUEVCs was treated with LPS,TNF-? and Glucose,respectively;3)Only DHA could cause the abnormal expression of GSDMD in HUVECs among these anti-inflammatory substances;4)HUVECs was treated with 50 ?M DHA,the cleaved fragments of GSDMD-C were detected.And with the prolonging the cultured time of DHA,the content of GSDMD-C was increased gradually?the difference was statistically significant?,but the expression of Caspase-1 was normal.HUVECs treated with DHA were swelling and ruptured,and the PI staining was positive;5)When DHA was treated with the dose of 50 ?M or more,CCK-8 assay revealed that the OD450 value of HUVECs began to decline significantly with the increase of DHA dose,and western blot analysis indicated that the content of GSDME-N fragment increased obviously.In addition,the production of GSDME-N fragment facilitated evidently with the augment of time after HUVECs were treated with 50 ?M DHA for different times.Conclusions: 1)According to the expression levels of NLRP3,Caspase-1 and GSDMD in endothelial cells,monocyte-macrophages tumor cells and non-monocyte-macrophages tumor cells,monocyte-macrophages tumor cells have all the associated molecules of canonical pyroptosis pathway,which is closely related to the functions of anti-pathogen and inflammation;GSDME is expressed in these cells;2)We firstly proposed that DHA could induce GSDME-medicated pyroptosis in HUVECs.