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. 2020 Jul;69(7):683-696.
doi: 10.1007/s00011-020-01351-z. Epub 2020 Apr 28.

Hepatitis B virus X protein promotes liver cell pyroptosis under oxidative stress through NLRP3 inflammasome activation

Wen-Hui Xie  1 , Jian Ding  2 , Xiao-Xia Xie  3 , Xiao-Huang Yang  1 , Xiao-Fan Wu  3 , Zhi-Xin Chen  1 , Qi-Lan Guo  1 , Wen-Yu Gao  1 , Xiao-Zhong Wang  4 , Dan Li  5
Affiliations

Hepatitis B virus X protein promotes liver cell pyroptosis under oxidative stress through NLRP3 inflammasome activation

"V体育平台登录" Wen-Hui Xie et al. Inflamm Res. 2020 Jul.

Abstract

Objective: Hepatitis B virus X protein (HBx) is a pivotal factor for HBV-induced hepatitis. Herein, we sought to investigate HBx-mediated NLR pyrin domain containing 3 (NLRP3) inflammasome activation and pyroptosis under oxidative stress. VSports手机版.

Methods: The effect of HBx on the NLRP3 inflammasome was analyzed by enzyme-linked immunosorbent assays, quantitative reverse transcription-polymerase chain reaction, western blotting, and immunofluorescence in hepatic HL7702 cells. Pyroptosis was evaluated by western blotting, lactate dehydrogenase release, propidium iodide staining, and transmission electron microscopy V体育安卓版. NLRP3 expression in the inflammasome from liver tissues was assessed by immunohistochemistry. .

Results: In hydrogen peroxide (H2O2)-stimulated HL7702 cells, HBx triggered the release of pro-inflammatory mediators apoptosis-associated speck-like protein containing a CARD (ASC), interleukin (IL)-1β, IL-18, and high-mobility group box 1 (HMGB1); activated NLRP3; and initiated pro-inflammatory cell death (pyroptosis) V体育ios版. HBx localized to the mitochondria, where it induced mitochondrial damage and production of mitochondrial reactive oxygen species (mitoROS). Treatment of HL7702 cells with a mitoROS scavenger attenuated HBx-induced NLRP3 activation and pyroptosis. Expression levels of NLRP3, ASC, and IL-1β in liver tissues from patients were positively correlated with HBV DNA concentration. .

Conclusions: The NLRP3 inflammasome was activated by elevated mitoROS levels and mediated HBx-induced liver inflammation and hepatocellular pyroptosis under H2O2-stress conditions. VSports最新版本.

Keywords: Hepatitis B virus X protein; Liver inflammation; NLRP3 inflammasome; Pyroptosis. V体育平台登录.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Cytotoxicity of H2O2 and verification of recombinant plasmid expression in HL7702 cells. Cells were treated with different concentrations of H2O2, as indicated for 12 h. Cell viability was evaluated using a Cell Counting Kit-8 assay, n = 4 (a). HBV X antigen (HBx) and C antigen (HBc) were transiently expressed in HL7702 cells and protein expression levels determined by western blotting, n = 4 (b, c). Abbreviations: pNC, cells transfected with control vector pcDNA3.1; pHBx, cells transfected with reconstituted pHBx vector; pGEM, cells transfected with control vector pGEM-4Z; pHBV-HBx, cells transfected with pHBV-HBx vector; pHBV, cells transfected with pHBV vector. Data are displayed as mean ± SD. *P < 0.05 and **P < 0.01
Fig. 2
Fig. 2
Impact of hepatitis B virus (HBV) X protein (HBx) on the expression of inflammatory mediators in hepatocytes. Concentrations of ASC, IL-1β, IL-18, and HMGB1 in the culture media of HBx-expressing HL7702 cells with or without H2O2 stimulation (100 μM) were determined by ELISA, n = 4 (a). Concentrations of ASC, IL-1β, IL-18, and HMGB1 from HBV-expressing HL7702 cells were quantified by ELISA, n = 4 (b). Data are presented as mean ± SD. *P < 0.05 and **P < 0.01
Fig. 3
Fig. 3
HBV X protein (HBx) promoted the upregulation and activation of NLRP3 inflammasome expression under H2O2 stress. Quantification of mRNA expression of GAPDH, NLRP3, ASC, caspase-1, IL-1β, IL-18, HMGB1, and GSDMD using real-time PCR, n = 5 (a, b). Data are expressed as mean ± SD. *P < 0.05 and **P < 0.01. Western blot analysis of NLRP3, ASC, pro-caspase-1, Cleaved-caspase-1 (p10), and IL-1β (mature IL-1β), n = 4 (c, d). GAPDH served as a loading control
Fig. 4
Fig. 4
Hepatitis B virus X protein (HBx) induced liver cell pyroptosis under H2O2 stimulation. Western blot analysis of GSDMD and loading control GAPDH, n = 4 (a, b). Representative micrographs of PI staining (Red) and Hoechst 33342 staining (Blue), n = 5 (c–f). Scale bar indicates 200 µm. The pore formation of the cellular membrane was examined by TEM, n = 4 (g, h). Levels of LDH released into the cell culture medium from membrane pores were examined using an LDH Cytotoxicity Detection Kit, n = 5 (i–k). Data are expressed as mean ± SD. *P < 0.05 and **P < 0.01
Fig. 5
Fig. 5
Mitochondrial reactive oxygen species (mitoROS) mediated HBV X protein (HBx)-induced NLRP3 activation and pyroptosis in liver cells. Western blot analysis of HBx localized in the mitochondria. COXIV served as a loading control of mitochondrial protein, n = 4 (a). Morphological changes of mitochondria were observed by TEM, n = 4 (b). mitoROS levels were examined by flow cytometry, n = 4 (c, d). Effects of the ROS scavenger NAC and specific mitoROS inhibitor mito-TEMPO on HBx-induced inflammasome activation analyzed by western blotting, n = 4 (e–h). NAC and mito-TEMPO attenuated HBx-induced LDH release, n = 5 (i, j). NAC and mito-TEMPO inhibited both the translocation of ASC and formation of ASC specks, as detected by immunofluorescence, n = 4 (k). Data are shown as mean ± SD. *P < 0.05, **P < 0.01,#P < 0.05, ##P < 0.01
Fig. 6
Fig. 6
Association between the expression levels of NLRP3 inflammasome components and hepatitis B virus (HBV) DNA load. Analysis by immunohistochemistry of expression levels of NLRP3, ASC, and IL-1β in tumor-adjacent tissues of HCC patients with or without HBV infection. Representative images are shown, and the mean integrated optical density (IOD) of protein expression was statistically analyzed, n = 51 (a–d). Pearson correlation analysis of NLRP3, ASC, and IL-1β expression and HBV DNA copy number in a cohort of 51 patients, n = 51 (e–g). Serum ASC in patients with varied HBV DNA load was detected by ELISA, n = 84 (h). The height of the histogram represents the DNA copy number counts. Data are shown as mean ± SD. *P < 0.05 and **P < 0.01
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