Study On The Mechanism Of Hepatocyte Injury By SARS-CoV-2 ORF3a Protein

Objective:To investigate the role of SARS-Co V-2 ORF3 a protein on hepatocytes and the mechanism of injury,and to provide new ideas for the treatment of liver injury associated with COVID-19.Methods:(1)Constructed eukaryotic expression plasmid pc DNA3.1(+)-ORF3a-myc-His A expressing ORF3 a protein,and transfected L02 cells with it after successful enzymatic digestion and sequencing.(2)The morphological and ultrastructural changes of L02 cells expressing ORF3 a protein were observed by optical microscope and transmission electron microscopy.(3)Flow cytometry was applied to investigate the level of apoptosis induced by ORF3 a protein in L02 cells.(4)The changes of lactate dehydrogenase(LDH)activities in the culture supernatant were measured by a fully automated biochemical analyzer to assess the cellular damage of L02 cells expressing ORF3 a protein.(5)The secretion of cytokines IL-1β in the cell culture supernatant was detected by ELISA.(6)The secretion of cytokines IL-6 and TNF-α in the cell culture supernatant was detected by chemiluminescence.(7)The secretion of ORF3 a protein and various cell death proteins in the cell lysate was detected by Western Blotting.ORF3 a protein and various cell death pathway proteins were detected by western blotting.(8)Phosphorylated mixed-lineage kinase region-like protein(p MLKL)pore-forming activity inhibitor Necrosulfonamide(NSA)and Caspase-8specific inhibitor Z-IETD-FMK were applied to treat L02 cells to assess the effects of necroptosis and Caspase-8 on ORF3 a protein-expressing L02 cells to assess the role of necroptosis and caspase-8 in the damage process of L02 cells after ORF3 a protein treatment.Results:(1)The plasmid pc DNA3.1(+)-ORF3a-myc-His A expressing ORF3 a protein was successfully constructed.Enzyme digestion,sequencing and database comparison confirmed that the ORF3 a gene fragment in the eukaryotic expression plasmids was consistent with the standard sequence in the NCBI database and could be successfully expressed after transient transfection of L02 cells.(2)The plasmid was compared with pc DNA3.1(+)-myc-His A myc-His A Empty Vector transfected group(EV group),L02 cells in the pc DNA3.1(+)-ORF3a-myc-His A transfected group(ORF3a group)showed apoptotic and inflammatory cell death morphological alterations.Apoptotic-like ultrastructural alterations were observed in L02 cells overexpressing ORF3 a protein under transmission electron microscopy.(3)Early apoptosis was increased of L02 cells in the ORF3 a group by flow cytometry analysis.(4)LDH and IL-1β release levels were increased in L02 cells in the ORF3 a group compared with the EV group,the secretion of IL-6 and TNF-α was not detected.(5)Caspase-8 and p MLKL protein expression levels were increased in L02 cells in the ORF3 a group compared with the EV group.(6)The release of both IL-1β and LDH induced by ORF3 a protein was significantly reduced by the application of NSA.(7)ORF3a protein-induced apoptosis in L02 cells was inhibited and the secretion level of IL-1β was decreased by the application of Z-IETD-FMK.Conclusions:(1)ORF3a protein induces apoptosis in L02 cells via Caspase-8-dependent extrinsic pathway.(2)ORF3a protein induces IL-1βsecretion from L02 cells dependent on Caspase-8 activation and necroptosis pathway.