Study on the activation and related mechanism of hepatitis B virus X protein on NLRP3 inflammasome / 中华消化外科杂志

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 groupsblank 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),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 (x) ± 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 showed① 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).The relative expression of full-length HBx protein group was significantly different from that of blank control group,HBx1-127 group and HBx1-101 group (t =12.070,7.465,7.801,P ＜0.05).There was no statistically significant difference between full-length HBx protein group and empty vector group (t =0.867,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).The relative expression of NLRP3 inflammasome protein of LPS + ATP group was significantly different from that of blank control group,HBx1-127 group and HBx1-101 group,respectively (t =6.310,6.565,6.741,P ＜0.05).There were statistically significant differences between the full-length HBx group and the HBx1-127 group or HBx1-101 group (t =6.381,6.584,P ＜ 0.05) and no statistically significant difference between LPS + ATP group and full-length HBx protein group (t =0.580,P ＞ 0.05).(2) The results of ELISA showed①) 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-1 β of LPS + ATP group was significantly different from that of blank control group,HBx1-127 group and HBx1-101 group (t =13.691,12.752,13.001,P ＜0.05).The expression of IL-1β of full-length HBx group was significantly different from that of the HBx1-127 group and the HBx1-101 group (t =14.051,14.283,P ＜ 0.05).There was no statistically significant difference between the LPS + ATP group and the full-length HBx protein group (t =1.691,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).The expression of IL-18 of LPS + ATP group was significantly different from that of blank control group,HBx1-127 group and HBx1-101 group,respectively (t =7.848,6.722,7.065,P ＜ 0.05).The expression of IL-18 of full-length HBx group was significantly different from that of HBx1-127 group and HBx1-101 group (t =7.882,8.331,P ＜ 0.05).There was no statistically significant difference between LPS + ATP group and full-length HBx group (t =0.326,P ＞ 0.05).②The 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).The expressions of IL-1β and IL-18 in the HepG2 cells of the full-length 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 full-length 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 full-length 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 HBV-related HCC by activating NLRP3 inflammasome through inducing reactive oxygen generation in the HepG2 cells.