Effects And Mechanisms Of SARS-CoV-2 Envelope Protein On Monocytes Pyroptosis

Background and Objective:Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is a highly infectious and pathogenic coronavirus that emerged in late of 2019.Similar to SARSCoV and MERS-CoV,the new coronavirus can invade the human respiratory system and induce a highly inflammatory response,further triggering symptoms of viral pneumonia.The typical clinical features of SARS-CoV-2 infection include diffuse alveolar hemorrhage and exudative pneumonia,lymphopenia,further development of acute respiratory distress syndrome(ARDS),systemic inflammatory response syndrome(SIRS),thrombosis and embolism,shock,and ultimately multi-organ dysfunction including lung injury and diffuse intravascular coagulation syndrome(DIC).Numerous studies have shown that the development of acute respiratory distress syndrome,systemic inflammatory response syndrome,and multi-organ failure in patients with neo colonies is not directly caused by the invasion and replication of SARS-CoV-2,but is driven by cell death and immune-inflammatory responses induced by multiple factors.Many researchers have proposed that the factors which induced severe cases of COVID-19 might dysregulate cell death and uncontrolled release of inflammatory cytokines.But the role of SARS-CoV-2 structural proteins and nonstructural proteins in the process still remains largely unknown.The E protein is a structural protein that makes up the coronavirus particle and is localized to the outer shell of the viral particle.Studies have shown that the SARS-CoV E protein triggered apoptosis in an in vitro model and it could induce an inflammatory response and lung injury in the mouse models.Gene sequence alignment suggested that the envelope protein is highly conserved between SARS-CoV-2 and SARS-CoV,indicating that they might also have some similarities in functions.Therefore,this project was proposed to investigate the role as well as the mechanism of SARS-CoV-2 E protein on monocyte cell death and multi-organ damage using in vivo and ex vivo models to provide new insights and ideas for potential target screening and clinical drug development of COVID-19.Methods:1.SARS-CoV-2 E protein induced cell death and multi-organ damage: 1)animal pathological models were prepared by tail vein injection of E protein(20 μg/kg)in mice,then serum and tissue samples were collected for serum lactate dehydrogenase(LDH)assay and histopathological analysis;2)human peripheral blood mononuclear cells(THP-1),human umbilical vein endothelial cells(HUVECs)and human bronchial epithelial cells(BEAS-2B)were cultured in vitro,and the toxic effects of E protein on cells were detected by CCK-8 and LDH leakage using different concentrations of E protein acting on the above cells,or using the same concentration of E protein acting on cells of different periods;3)THP-1 cells were treated with different structural and non-structural proteins of SARS-CoV-2 respectively to compare with their toxic effects of E protein on cells by CCK-8 and LDH leakage.2.Analysis of the role and mechanism of SARS-CoV-2 E protein-induced THP-1 pyroptosis:1)pretreatment of THP-1 with different death inhibitors,CCK-8and LDH leakage were used to detect cytotoxicity,Annexin V/PI staining and light microscopy observation were used to evaluate the morphological changes of cells,and analysis of possible forms of E protein-induced cell death;2)detection of NLRP3/GSDMD classical pyroptosis pathway and activation of NF-κB and MAPK pro-inflammatory signaling pathways in THP-1 cells after E protein stimulation by Western blot;3)real-time quantitative PCR(QPCR)analysis of pro-inflammatory cytokine expression;4)pretreatment of THP-1 with NF-κB inhibitor and TLR2 inhibitor,CCK-8 and LDH leakage were used to detect and confirm that E protein induced monocyte pyroptosis through TLR2/NF-κB signaling.3.Analysis of the protective effects and mechanisms of Ruscogenin on SARSCoV-2 E protein-induced THP-1 pyroptosis: 1)screening of small molecule compounds with protective effect on E protein-induced cell death by CCK-8 and LDH leak assays;2)pretreatment of THP-1 with different concentrations of Ruscogenin,then the protective effects of Ruscogenin on E protein-induced monocyte pyroptosis were analyzed by CCK-8,LDH leakage and morphological observation;3)the effects of Ruscogenin on the activation of NLRP3/GSDMD and pro-inflammatory signaling pathway were detected by Western blot.Results:1.Intravenous administration of SARS-CoV-2 E protein resulted in a significant increase in serum LDH levels and significant pathological damage and inflammatory infiltration in lung and liver tissues of mice.Exogenous stimulation of SARS-CoV-2 E protein induced cell death in THP-1,HUVECs and BEAS-2B in a time-and dosedependent manner.In THP-1 cells,the S,N,NSP1,NSP3,NSP5,NSP7,NSP8 proteins of SARS-CoV-2 could not induce cell death,and only the E protein could specifically induce cell death in THP-1 cells.2.SARS-CoV-2 E protein treatment induced pyroptosis-like death with cell swelling in THP-1 and triggered time-and dose-dependent elevation of NLRP3 expression and activation of the pyroptosis-associated protein GSDMD;E protein induced activation of NF-κB,p38 and JNK pro-inflammatory signals and increased NLRP3 and TNF-α,IL-1α,IL-1 β,IL-6,CCL-5 and other pro-inflammatory cytokines expression in m RNA level;pan-caspase,TLR2 and NF-κB inhibitors could partially inhibit E protein-induced THP-1 death.3.The natural compound Ruscogenin was found to attenuate E protein-induced THP-1 pyroptosis in a dose-dependent manner,reducing NLRP3 expression and downstream caspase-1 and GSDMD activation,inhibiting the activation of proinflammatory NF-κB signaling without affecting p38 and JNK signaling as well.Conclusions:1.SARS-CoV-2 E protein induced tissue injury and inflammatory infiltration in mice and cell death in multiple cultured cells,suggesting that E protein may act as a virulence factor involved in tissue injury during SARS-CoV-2 infection;2.SARS-CoV-2 E protein induced monocyte pyroptosis by activating the NLRP3/GSDMD pathway,partly through the TLR2/NF-κB,p38 and JNK signaling pathways.3.Ruscogenin attenuated SARS-CoV-2 E protein-induced monocyte pyroptosis by inhibiting NLRP3/GSDMD pathway and preventing NF-κB signaling activation.