| 1.Background and objectivesProgrammed cell death is a fundamental process in both normal physiology and pathological situation.Pyroptosis is a caspase-1(Cysteine-requiring Aspartate Protease-1,caspase-1)dependent pro-inflammatory form of programmed cell death,mediated by the activation of inflammasomes such as NLRP3(NACHT,LRR and PYD domains-containing protein 3,NLRP3)inflammasome and triggered by many pathological factors such as stroke,cancer,heart attack.NLRP3 inflammasome increases the inflammatory response and then mediates further damage during many inflammatory diseases especially during atherosclerosis.Many studies have linked caspase-1 depend cell death to atherosclerotic plaque destabilization and sub-endothelial retention of low density lipoprotein(LDL)and its oxidative modification represent the initial event in atherogenesis,following by infiltration and activation of blood inflammatory cells.Moreover,demonstrated in-vivo and ex-vivo analyses revealed that MCC950 is a highly potent specific NLRP3 inhibitor used for the treatment of inflammatory diseases.Through his NLRP3 inhibitor property in vivo,this small molecule has been shown to attenuate EAE(Experimental autoimmune encephalomyelitis,EAE)severity in mice model.MCC950 is also efficacious in a mouse model of CAPS(Cryopyrin-Associated Auto-inflammatory,CAPS)syndrome and in cells from MWS(Muckle-Wells syndrome,MWS)subjects.EAE is the most commonly used experimental model for the human inflammatory demyelinating disease,multiple sclerosis and CAPS is a group of rare,inherited,auto-inflammatory diseases and that have three subtypes: Familial Cold Auto-inflammatory Syndrome(FCAS),Muckle-Wells Syndrome(MWS),and Neonatal-Onset Multisystem Inflammatory Disease(NOMID).EAE and CAPS are two different types of human inflammatory diseases as well as atherosclerosis.Also,MCC950 specifically inhibit IL-1β secretion,blocks NLRP3-induced ASC(apoptosis-associated speck protein containing)oligomerization and LPS(lipopolysaccharide)-dependent TNF(Tumor Necrosis Factor)-α secretion unimpaired.Hypothesis has been made that MCC 950,through its numerous chemical property,may also contribute to attenuate atherosclerosis as it does for the two diseases cited above.The first step of this work was to demonstrate that Ox-LDL,a key factor of atherogenesis,has the property to induce pyroptosis in endothelial cells and following this,and in a second hand to prove the anti-pyroptotic effects of MCC950 on these cells.2.MethodFor this study,HUVEC(Human Umbilical Vein Endothelial Cells)obtained from ATCC company has been cultured following the ATCC protocol.The study includes the analysis of the effect of Ox-LDL on these cells,the effect of the NLRP3 inflammasome inhibitor MCC950 on the HUVEC treated by Ox-LDL.Therefore,Lactate deshydrogenase release was firstly evaluated in cells supernatants as indicator of their toxicity.Cell DNA structure was assessed with Terminal deoxynucleotidyl transferase-mediated dUTP Nick End-Labeling(TUNEL)staining method and Hoechst33342/ Propidium iodide staining was performed to evaluate their viability.Both procaspase-1 and activated caspase-1 activities were finally evaluated.All statistical analysis was performed using SPSS 21.0.3.ResultsThe cytotoxicity on cells treated with Ox-LDL,on cells treated with Ox-LDL and MCC 950 and cells treated with Ox-LDL and VX765 has been evaluated.The cytotoxicity level of cell treated with Ox-LDL alone was very higher than cytotoxicity of cells treated with Ox-LDL+MCC950 and those treated with Ox-LDL+VX765.Tunnel staining(DNA fragmentation)has been used to assess DNA damage on 4 groups of cells(cells cultured alone,cells cultured with Ox-LDL,cells treated with OxLDL+MCC950 and cells treated with Ox-LDL + VX765).As result,DNA damage was very high on cells treated with Ox-LDL,slightly high on those treated with OxLDL+MCC950 and those treated with Ox-LDL+VX765,and very small on cells treated alone.Hoechst33342/ Propidium iodide staining has been used to measure cells viability and cell-cycle phase-specific cell death on 4 groups of cells(cells cultured alone,cells cultured with Ox-LDL,cells treated with Ox-LDL+MCC950 and cells treated with OxLDL+VX765).We noticed that dead cells amount were very high when they were treated with Ox-LDL but very small in the others groups.Finally,western blot analysis was performed to evaluate caspase-1 activity when the cells are treated with ox-LDL+MCC950,cells treated with ox-LDL.As result,caspase-1 activity was great on cells treated with ox-LDL alone but small when cells are treated with MCC950 as same as in control group(cells treated alone with DMSO: Dimethyl sulfoxide).4.ConclusionThe anti-pyroptotic compounds are an emergent class of drugs.Our findings indicate that MCC950 belong to this class.Together with previous data,we are hoping that our results might contribute to unveil the enigmatic pharmacological basis behind the therapeutic effects of MCC950. |
