Study On The Mechanism Of Pyroptosis Induced By Zika Virus In Infected HUVECs And SH-SY5Y Cells

Background and ObjectsZika virus is among the most widely transmitted arboviruses in the world.The virus was first discovered in the Zika Forest in Uganda in 1947 and isolated from primates.By the end of 2016,Zika virus cause different scales of outbreaks in the Africa,the South Pacific region and the Americas successively,which has become a serious public health problem in the world.Approximately 80%of Zika virus infections are asymptomatic,and the most common symptoms include fever,arthralgia,rash,myalgia,edema,vomiting,and non-purulent conjunctivitis.It’s neurotropism is associated with encephalitis,fetal microcephaly,and Guillain-Barre syndrome closely.Zika virus can be detected in the amniotic fluid of pregnant women and the brain tissue of microcephaly fetuses,indicating that Zika virus can infect fetuses through the placental barrier.Zika virus can also infects skin fibroblasts,neurons,neurons stem cells,human umbilical vein endothelial cells persistently,causing cell death.The mechanism of cell death caused by Zika virus remains unclear.The way of cell death includes apoptosis,necrosis and pyroptosis.Apoptosis is a well-studied process of programmed cell death,which is characterized by activation of Cysteinyl aspartate specific proteinase-3(Caspase-3).Pyroptosis is a newly discovered programmed cell death process,which has attracted much attention in recent years.It is characterized by the activation of Caspase-1.Caspase-1 could promote the maturation and secretion of interleukin-1β(IL-1β)and inter-leukin-18(IL-18),which are released into the extracellular environment,causing severe inflammatory response of cells.Studies have shown that Zika virus activated the Nod-like receptor protein 3(NLRP3)inflammasome in infected immune cells,glial cells and mononuclear cells,which in turn activated Caspase-1,promoted the secretion of IL-1β.NLRP3 is currently the most extensively investigated inflammasome,many pathogens can activate the NLRP3,form NLRP3 inflammasome,trigger pyroptosis signal pathway.Influenza viruses,encephalitis viruses,human immunodeficiency virus I and enterovirus 71 can activate the NLRP3 and trigger an inflammatory response.In addition,there was significantly increased production of various cytokines such as interleukin-6(IL-6),inter-leukin-8(IL-8),Tumor Necrosis Factor α(TNFα),Granulocyte macrophage colony-stimulating factor(GM-CSF),chemokine ligand 2(CCL-2),chemokine ligand 5(CCL-5)in infected cells,which is closely related to the inflammatory death of cells and cytokine storm caused by Zika virus.Methods1.The detection of cytokines.The concentration of ten cytokines in cell culture supernatant was detected by Luminex xMAP technology.The relative expression of cytokines at mRNA level was determined by quantitative real-time PCR(qPCR)technology.2.The detection of indicators of apoptosisCell activity was detected by cell counting kit-8(CCK-8),lactate dehydrogenase(LDH)release assay was used to evaluate the integrity of cell membrane.qPCR technology was used to detect the relative expression of pyroptosis-related genes at mRNA level.Western-blot was used to detect the expression of pyroptosis genes at protein level.Annexin V-FITC/PI staining was performed by flow cytometry,Caspase1 and NLRP3 inhibitors were added or synthesize a small interfering RNA(siRNA)specific to NLRP3 to verify that Zika virus causes pyroptosis by activating Caspase-1 dependent on NLRP3 inflammasome.Results1.There was significantly up-regulated of GM-CSF,IL-6,IL-8,TNFa and CCL-2 in HUVECs infected with Zika virus in comparison with mock-infected group and the concertation of these cytokines increased in supernatants.There was significantly upregulated of GM-CSF,IL-6,IL-8,TNFα,and CCL-5 in SH-SY5Y cells infected with Zika virus in comparison with mock-infected group and the concertation of these cytokines increased in supernatants.2.The cell viability decreased by 50%72 h after Zika virus infection.LDH release detection and flow cytometry results showed that cell membrane integrity was lost,the expression of pyroptosis-related genes at mRNA levels increased 72 h after Zika virus infected HUVECs and SH-SY5Y cells.3.The activity of Caspase-1 and IL-1β was inhibited after transfected siRNA-NLRP3 or treated with NLRP3 inhibitor MCC950 and Caspase-1 inhibitor VX-765 before infected with Zika virus.Conclusion1.The expression of GM-CSF,IL-6,IL-8,TNFα,CCL-2 or CCL-5 were up-regulated in HUVECs and SH-S Y5 Y cells infected with Zika virus,and the concertation of these cytokines increased in supernatant.These cytokines may be related to cytokines storm caused by Zika virus.2.Zika virus induces pyroptosis by activating Caspase-1 in infected HUVECs and SHSY5Y cells.3.Zika virus induces pyroptosis by activating Caspase-1 partly dependent on the NLRP3 inflammasome.