乙型肝炎病毒X蛋白对核苷酸结合寡聚化结构域样受体蛋白3炎性小体的激活及机制研究

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

目的：探讨乙型肝炎病毒X蛋白（HBx蛋白）对核苷酸结合寡聚化结构域样受体蛋白3 （NLRP3）炎性小体活性的影响及其促进HBV相关性肝细胞肝癌（HCC）的作用机制。 方法：采用实验研究方法。将HepG2细胞株分为5组：空白对照组（未转染质粒），空载质粒组［转染pE绿色荧光蛋白（GFP）N1空载质粒］，全长HBx蛋白组（转染pEGFP-N1-X质粒），HBx 1-127组（转染pEGFP-N1-X1-127质粒），HBx1-101组（转染pEGFP-N1-X1-101质粒）。（1）采用Western blot检测HBx蛋白和NLRP3炎性小体蛋白［采用脂多糖＋ATP干预空白对照组HepG2细胞］的表达。（2）分别采用格列本脲、吡咯烷二硫代甲酸铵盐（APDC）干预全长HBx蛋白HepG2细胞，采用ELISA检测IL-1β和IL-18的表达。（3）采用流式细胞仪检测活性氧的表达。正态分布的计量资料以±s表示，多组间比较采用单因素方差分析，两两比较采用t检验。 结果：（1）Western blot检测结果显示：①空白对照组、空载质粒组、全长HBx蛋白组、HBx1-127组、HBx1-101组HepG2细胞中HBx重组质粒融合蛋白的相对表达量分别为0.07±0.03、0.92±0.13、0.84±0.11、0.30±0.06、0.29±0.05，其中HBx1-127组和HBx1-101组HepG2细胞中分别为HBx1-127蛋白和HBx1-101蛋白的表达。5组比较，差异有统计学意义（F=61.790，P〈0.05）。其中全长HBx蛋白组分别与空白对照组、HBx1-127组、HBx1-101组比较，差异均有统计学意义（t=12.070，7.465，7.801，P〈0.05）；全长HBx蛋白组与空载质粒组比较，差异无统计学意义（t=0.867，P〉0.05）；HBx1-127组和HBx1-101组比较，差异无统计学意义（t=0.146，P〉0.05）。②空白对照组、全长HBx蛋白组、HBx1-127组、HBx1-101组、脂多糖＋ATP组HepG2细胞中NLRP3炎性小体蛋白的相对表达量分别为0.29±0.06、0.83±0.14、0.27±0.06、0.27±0.05、0.90±0.16，5组比较，差异有统计学意义（F=29.550，P〈0.05）。其中脂多糖＋ATP组分别与空白对照组、HBx1-127组、HBx1-101组比较，差异均有统计学意义（t=6.310，6.565，6.741，P〈0.05）；全长HBx蛋白组分别与HBx1-127组、HBx1-101组比较，差异均有统计学意义（t=6.381，6.584，P〈0.05）；脂多糖＋ATP组与全长HBx蛋白组比较，差异无统计学意义（t=0.580，P〉0.05）。（2）ELISA检测结果显示：①空白对照组、全长HBx蛋白组、HBx1-127组、HBx1-101组、脂多糖＋ATP组HepG2细胞中IL-1β的表达量分别为（87±9）pg/mL、 （587±56）pg/mL、（125±12）pg/mL、（113±13）pg/mL、（677±74）pg/mL，IL-18的表达量分别为（43±8）pg/mL、（252±38）pg/mL、（70±13）pg/mL、（63±10）pg/mL、（263±48）pg/mL。HepG2细胞中IL-1β的表达量 5组比较，差异有统计学意义（F=139.010，P〈0.05）；其中脂多糖＋ATP组分别与空白对照组、HBx1-127组、HBx1-101组比较，差异均有统计学意义（t=13.691，12.752，13.001，P〈0.05）；全长HBx蛋白组分别与HBx1-127组、HBx1-101组比较，差异均有统计学意义（t=14.051，14.283，P〈0.05）；脂多糖＋ATP组与全长HBx蛋白组比较，差异无统计学意义 （t=1.691，P〉0.05）。HepG2细胞中IL-18的表达量5组比较，差异有统计学意义（F=44.010，P〈0.05）；其中脂多糖＋ATP组分别与空白对照组、HBx1-127组、HBx1-101组比较，差异均有统计学意义（t=7.848，6.722，7.065，P〈0.05）；全长HBx蛋白组分别与HBx1-127组、HBx1-101组比较，差异均有统计学意义 （t=7.882，8.331，P〈0.05）；脂多糖＋ATP组与全长HBx蛋白组比较，差异无统计学意义（t=0.326，P〉0.05）。②加格列本脲前、后全长HBx蛋白HepG2细胞中IL-1β的表达量分别为（587±91）pg/mL、（115±17）pg/mL，两者比较，差异有统计学意义（t=8.800，P〈0.05）。加格列本脲前、后全长HBx蛋白HepG2细胞中IL-18的表达量分别为（243±22）pg/mL、（90±12）pg/mL，两者比较，差异有统计学意义（t=10.566， P〈0.05）。加APDC前、后全长HBx蛋白HepG2细胞中IL-1β的表达量分别为（573±89）pg/mL、（124±21）pg/mL，两者比较，差异有统计学意义（t=8.516，P〈0.05）。加APDC前、后全长HBx蛋白HepG2细胞中IL-18的表达量分别为（252±24）pg/mL、（116±15）pg/mL，两者比较，差异有统计学意义（t=8.269，P〈0.05）。（3）流式细胞仪检测结果显示：空白对照组、全长HBx蛋白组、脂多糖＋ATP组HepG2细胞中活性氧的相对表达量分别为66±14、275±54、388±88，3组比较，差异有统计学意义（F=22.130，P〈0.05）；其中全长HBx蛋白组和脂多糖＋ATP组分别与空白对照组比较，差异均有统计学意义（t=6.489，6.256，P〈0.05）；全长HBx蛋白组和脂多糖＋ATP组比较，差异无统计学意义（t=1.887，P〉0.05）。 结论：HBx蛋白通过诱导HepG2细胞内活性氧的产生，进而激活NLRP3炎性小体，可能在HBV 相关性HCC的发生发展过程中发挥重要作用。

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）, 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 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, 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 fulllength 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 fulllength 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, fulllength 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 fulllength 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 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, 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 IL1β 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 IL1β of fulllength 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 fulllength HBx protein group （t=1.691, P〉0.05）. The expression of IL18 in the blank control group, fulllength 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 IL18 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 IL18 of fulllength 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 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.