Study on the activation and related mechanism of hepatitis B virus X protein on NLRP3 inflammasome

Abstract

Objective To investigate the influence of hepatitis B virus X (HBx) protein on the activity of NLRP3 inflammasome as well as the mechanism of accelerating HBV-related hepatocellular carcinoma (HCC). Methods The HepG2 cell strains were divided into the 5 groups: blank control group (without plasmid transfection), empty vector group [transfected with pE green fluorescent protein (GFP)-N1 vector plasmid], full-length HBx protein group (transfected with pEGFP-N1-X plasmid), HBx1-127 group (transfected with pEGFP-N1-X1-127 plasmid), HBx1-101 group (transfected with pEGFP-N1-X1-101 plasmid). (1) The expressions of HBx protein and NLRP3 inflammasome were detected by Western blot [Lipopolysaccharide (LPS)+ ATP intervention was performed in the blank control group]. (2) The HepG2 cells in the full-length HBx protein group were respectively intervened by glibenclamide and ammonium pyrrolidinedithiocarbamate (APDC), and the expressions of IL-1β and IL-18 were detected by enzyme-linked immunosorbent assay (ELISA). (3) The expressions of reactive oxygen were detected by flow cytometry. The measurement data with normal distribution were presented by ±s. The one-way ANOVA was adopted in the comparison among groups while the t test was used in the pairwise comparison. Results (1) The results of Western blot showed: ① the relative expressions of HBx recombinant plasmid fusion protein inside the HepG2 cells in the blank control group, empty vector group, full-length HBx protein group, HBx1-127 group and HBx1-101 group were 0.07±0.03, 0.92±0.13, 0.84±0.11, 0.30±0.06 and 0.29±0.05, respectively. The expressions in the HBx1-127 group and the HBx1-101 group represented the expressions of HBx1-127 protein and HBx1-101 protein. There were statistically significant differences among the 5 groups (F=61.790, P 0.05) and between the HBx1-127group and HBx1-101 group (t=0.146, P>0.05). ② The relative expression of NLRP3 inflammasome protein inside the HepG2 cells in the blank control group, full-length HBx protein group, HBx1-127 group, HBx1-101 group and LPS+ ATP group were 0.29±0.06, 0.83±0.14, 0.27±0.06, 0.27±0.05 and 0.90±0.16, respectively, with a statistically significant difference among the 5 groups (F=29.550, P 0.05). (2) The results of ELISA showed: ① the expression of IL-1β inside the HepG2 cells in the blank control group, full-length HBx protein group, HBx1-127 group, HBx1-101 group and LPS+ ATP group was (87±9)pg/mL, (587±56)pg/mL, (125±12)pg/mL, (113±13)pg/mL and (677±74)pg/mL, respectively, with a statistically significant difference among the 5 groups (F=139.010, P 0.05). The expression of IL-18 in the blank control group, full-length HBx protein group, HBx1-127 group, HBx1-101 group and LPS+ ATP group was (43±8)pg/mL, (252±38)pg/mL, (70±13)pg/mL, (63±10)pg/mL and (263±48)pg/mL, respectively, with a statistically significant difference among the 5 groups (F=44.010, P 0.05). ② The expressions of IL-1β and IL-18 in the HepG2 cells of the full-length HBx protein were (587±91)pg/mL and (243±22)pg/mL before the addition of glibenclamide, (115±17)pg/mL and (90±12)pg/mL after the addition of glibenclamide, respectively, with statistically significant differences before and after the addition of glibenclamide (t=8.800, 10.566, P 0.05). Conclusion HBx protein may play an important role in the occurrence and development of HBV-related HCC by activating NLRP3 inflammasome through inducing reactive oxygen generation in the HepG2 cells. Key words: Liver neoplasms; Hepatitis B virus X protein; NLRP3 inflammasome; Reactive oxygen