HBx Promotes Liver Cell Inflammation And Pyroptosis Under Oxidative Stress Through NLRP3 Inflammasome Activation

Background and Aim:Hepatitis B virus X protein?HBx?is a pivotal factor of hepatitis B virus?HBV?induced hepatitis;however,the precise mechanisms involved remain poorly understood.Our previous studies found that HBx can interact with mitochondrial cytochrome c oxidase subunit??COX??and facilitate oxidative stress-induced mitochondrial damage and reactive oxygen species?ROS?production.NOD-like receptor family,pyrin domain containing 3?NLRP3?inflammasome has recently garnered considerable attention regarding its role in liver inflammation and fibrosis.The mitochondrial ROS?mitoROS?model is the classical activation mechanisms of NLRP3 inflammasome.Based on these findings,we aimed to investigate HBx mediated hepatitis and pyroptosis under oxidative stress through mitoROS/NLRP3inflammasome pathway.Methods:Western blot was used to verify the expression of plasmids pcDNA3.1?pNC?,pcDNA3.1-HBx?pHBx?,pGEM-4Z?pGEM?,pGEM-HBV-?HBx?pHBV-HBx?and pGEM-HBV?pHBV?.The optimal concentration for hydrogen peroxide?H₂O₂?intervention was determined by cell counting kit-8?CCK-8?to induce of oxidative stress.The effect of HBx on the expression of inflammatory factors apoptosis-associated speck-like protein containing a caspase recruitment domain?ASC?,interleukin-1??IL-1??,interleukin-18?IL-18?and high mobility group box 1?HMGB1?under oxidative stress in normal human hepatic HL7702cells was detected by enzyme-linked immunosorbent assay?ELISA?.Then,qPCR and Western blot were used to detect the expression and activation of NLRP3inflammasome.PI and Hoechst 3342 staining,and transmission electron microscopy were used to observe the formation of hepatocyte membrane pores.L-lactate dehydrogenase?LDH?content of cell supernatant was detected to further verify the membrane pores formation.Furthermore,mitochondrial protein extraction combined with Western blot was performed to detect mitochondrial localization of HBx,and mitoSOX staining combined with flow cytometry to detect mitoROS levels.ROS inhibitor further verified the above changes.Finally,immunohistochemical?IHC?staining was used to evaluate the expression of NLRP3,ASC and IL-1?in liver tissues of patients with different HBV DNA loading,and the correlation between HBV DNA content and the above indicators was analyzed.ELISA was used to detect serum ASC level in patients with or without HBV infected.Results:In H₂O₂-suppressed HL7702 cells,HBx caused the release of pro-inflammatory mediators ASC,IL-1?,IL-18,and HMGB1.HBx was then shown to induce NLRP3 activation and pro-inflammatory cell death?pyroptosis?in hepatocytes and elevate the production of pro-inflammatory factors mentioned.HBx localized to the mitochondria and induced mitochondrial damage and production of mitoROS.Treatment of HL7702 cells with a ROS inhibitor attenuated HBx-induced NLRP3 activation and pyroptosis.Finally,the expression levels of NLRP3,ASC,and IL-1?in liver tissues from patients were positively correlated with the amount of HBV DNA,and the serum level of ASC was higher in HBV patients than in HBV-negative patients.Conclusions:These results suggested that the NLRP3 inflammasome was activated by the elevation of mitoROS levels and mediated HBx-induced liver inflammation and hepatocellular pyroptosis under H₂O₂-stress conditions.Our data offer novel insights into the chronic inflammation mechanism induced by HBx and provide new targets and ideas for the treatment of chronic hepatitis.