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 HBVrelated 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］, fulllength HBx protein group (transfected with pEGFP-N1-X plasmid), HBx^1-127 group (transfected with pEGFP-N1-X^1-127 plasmid), HBx^1-101 group (transfected with pEGFP-N1-X^1-101 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 fulllength HBx protein group were respectively intervened by glibenclamide and ammonium pyrrolidinedithiocarbamate (APDC), and the expressions of IL1β and IL18 were detected by enzymelinked 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 oneway 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, fulllength HBx protein group, HBx^1-127 group and HBx^1-101 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 HBx^1-127 group and the HBx^1-101 group represented the expressions of HBx^1-127 protein and HBx^1-101 protein. There were statistically significant differences among the 5 groups (F=61.790, P<0.05). The relative expression of fulllength HBx protein group was significantly different from that of blank control group, HBx^1-127 group and HBx^1-101 group (t=12.070, 7.465, 7.801, P<0.05). There was no statistically significant difference between fulllength HBx protein group and empty vector group (t=0.867, P>0.05) and between the HBx^1-127group and HBx^1-101 group (t=0.146, P>0.05). ② The relative expression of NLRP3 inflammasome protein inside the HepG2 cells in the blank control group, fulllength HBx protein group, HBx^1-127 group, HBx^1-101 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). The relative expression of NLRP3 inflammasome protein of LPS+ATP group was significantly different from that of blank control group, HBx^1-127 group and HBx^1-101 group, respectively (t=6.310, 6.565, 6.741, P<0.05). There were statistically significant differences between the fulllength HBx group and the HBx^1-127 group or HBx^1-101 group (t=6.381, 6.584, P<0.05) and no statistically significant difference between LPS+ATP group and fulllength HBx protein group (t=0.580, P>0.05). (2) The results of ELISA showed: ① the expression of IL1β inside the HepG2 cells in the blank control group, fulllength HBx protein group, HBx^1-127 group, HBx^1-101 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 IL1β of LPS+ATP group was significantly different from that of blank control group, HBx^1-127 group and HBx^1-101 group (t=13.691, 12.752, 13.001, P<0.05). The expression of IL1β of fulllength HBx group was significantly different from that of the HBx^1-127 group and the HBx^1-101 group (t=14.051, 14.283, P<0.05). There was no statistically significant difference between the LPS+ATP group and the fulllength HBx protein group (t=1.691, P>0.05). The expression of IL18 in the blank control group, fulllength HBx protein group, HBx^1-127 group, HBx^1-101 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). The expression of IL18 of LPS+ATP group was significantly different from that of blank control group, HBx^1-127 group and HBx^1-101 group, respectively (t=7.848, 6.722, 7.065, P<0.05). The expression of IL18 of fulllength HBx group was significantly different from that of HBx^1-127 group and HBx^1-101 group (t=7.882, 8.331, P<0.05). There was no statistically significant difference between LPS+ATP group and fulllength HBx group (t=0.326, P>0.05). ② The expressions of IL1β and IL18 in the HepG2 cells of the fulllength 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). The expressions of IL1β and IL18 in the HepG2 cells of the fulllength HBx protein were (573±89)pg/mL and (252±24)pg/mL before the addition of APDC, (124± 21)pg/mL and (116±15)pg/mL after the addition of APDC, respectively, with statistically significant differences before and after the addition of APDC (t=8.516, 8.269, P<0.05). (3) The results of flow cytometry showed that the relative expression of reactive oxygen in the HepG2 cells in blank control group, fulllength HBx protein group and LPS+ATP group were 66±14, 275±54 and 388±88, with statistically significant differences among the 3 groups (F=22.130, P<0.05) and between the fulllength HBx protein group or LPS+ATP group and blank control group (t=6.489, 6.256, P<0.05). There was no statistically significant difference between fulllength HBx protein group and LPS+ATP group (t=1.887, P>0.05).
Conclusion:HBx protein may play an important role in the occurrence and development of HBVrelated HCC by activating NLRP3 inflammasome through inducing reactive oxygen generation in the HepG2 cells.