In vitro study on the inhibition of hepatitis D virus replication by bulevirtide based on liver organoids
Objective To construct the liver organoid infected with hepatitis D virus(HDV),and to investigate the role of the sodium taurocholate cotransporting polypeptide(NTCP)receptor inhibitor bulevirtide in inhibiting viral replication.Methods Hepatocyte-like cells(HLC)differentiated from induced pluripotent stem cells(iPSC)were seeded onto inverted colloidal crystal polyethylene glycol scaffolds(ICC)to construct liver organoids.After transfecting human hepatocelluar carcinoma cells(HuH7 cells)with plasmids,HDV particles were harvested from the supernatant,while HBV particles were extracted from the HepG2.2.15 cell supernatant.The liver organoids were infected with both HBV and HDV particles,and the negative control group without HDV infection was set up.The microstructure of the liver organoid units and the expression of hepatitis D antigen(HDAg)and hepatitis B surface antigen(HBsAg)were observed under laser scanning confocal microscope by immunofluorescence method.The protein levels of NTCP and HDAg in the liver organoids were detected by Western blotting.Bulevirtide was added before HDV infection(bulevirtide pre group)and 24 hours after infection(bulevirtide post group),and interferon-alpha(IFN-α)was also added after 24 hours infection(IFN-α group),and a control group without drug treatment was set up.HDV replication was compared among the four groups after drug intervention.Real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)was used to measure the relative mRNA expression levels.of Nanog homeobox(NANOG),sex determining region Y-box(SOX)2,SOX17,forkhead box protein A2(FOXA2),hepatocyte nuclear factor 4 alpha(HNF-4α),albumin(ALB),alpha-fetoprotein(AFP),NTCP during the differentiation of iPSC,and the mRNA expression of HDV after the drug intervention of the four groups.Statistical analysis was performed using two independent sample t tests.Results Within 21 days of the differentiation of iPSC into HLC,the mRNA expression level of NANOG gradually decreased,while the expression levels of SOX17,FOXA2 initially increased then decreased,and the expression levels of the HNF-4α,ALB,AFP and NTCP progressively increased.The protein level of NTCP in iPSC(0.118±0.003)was lower than that in HLC(1.315±0.073),and the difference was statistically significant(t=11.92,P＜0.001).The protein level of HDAg in the liver organoids after HDV infection was higher than that in the negative control group without HDV infection(1.284±0.128 vs 0.157±0.040),and the difference was statistically significant(t=23.27,P＜0.001).Laser scanning confocal microscopy showed three-dimensional spheroid structures and high expressions of HDAg and HBsAg at the 14th day of infection.Compared with the control group(1.000±0.077),the HDV mRNA expressions in both IFN-α group(0.453±0.028)and bulevirtide pre group(0.136±0.012)decreased after three days of drug intervention.The differences were statistically significant(t=19.95 and 33.15,respectively,both P＜0.001).However,there was no significant difference in HDV mRNA expressions between the bulevirtide post group(0.968±0.069)and the control group(t=0.94,P＞0.05).Conclusions The liver organoids constructed from iPSC-derived HLC and ICC can simulate human liver functions and successfully be infected by HDV particles.Early blockade with bulevirtide can effectively reduce the level of viral replication in the HDV-infected liver organoids.
Hepatitis DBulevirtideInduced pluripotent stem cellsLiver organoidsHepatitis D virus infection
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