Inhibition of hepatitis B virus via selective apoptosis modulation by Chinese patent medicine Liuweiwuling Tablet

Figure 1 Effects of Liuweiwuling Tablet on hepatitis B virus DNA/covalently closed circular DNA/RNA and antigen in cell models. A: The safe concentration of Liuweiwuling Tablet (LWWL), defined as maintaining ≥ 95% cell viability compared with the untreated controls, was evaluated in HepG2.1403F cells; B: The optimal effective time for anti-hepatitis B virus (HBV) activity of LWWL, determined as the time-point demonstrating the strongest suppression of HBV DNA during a 5-d observation period, was evaluated in HepG2.1403F cells; C: The inhibitory effects of LWWL on HBV DNA and supernatant hepatitis B surface antigen (HBsAg) were evaluated in HepG2.1403F cells; D: The inhibitory effects of tenofovir disoproxil fumarate (TDF) on HBV DNA and supernatant HBsAg were assessed in HepG2.1403F cells; E: The inhibitory effects of both LWWL and TDF on pregenomic RNA (pgRNA) were measured in HepG2.1403F cells; F: The inhibitory effects of both LWWL and TDF on covalently closed circular DNA (cccDNA) were measured in HepG2.1403F cells; G: The inhibitory effects of both LWWL and TDF on pgRNA/cccDNA were measured in HepG2.1403F cells; H: The inhibitory effects of LWWL and TDF on cccDNA were evaluated in HepG2.2.15 cells; I: The inhibitory effects of LWWL and TDF on pgRNA/cccDNA were evaluated in HepG2.2.15 cells; J: The inhibitory effects of LWWL and TDF on cccDNA were investigated in HepG2.A64 cells; K: The inhibitory effects of LWWL and TDF on pgRNA/cccDNA were investigated in HepG2.A64 cells. ^aP < 0.05 vs control.

Figure 2 Quality control analysis of serum metabolomics in pAAV-HBV1. 2 replication mice treated with Liuweiwuling Tablet. A: Ion base peak diagram; B: Distribution of metabolic sensitivity; C: Principal component analysis plot; D: Orthogonal partial least squares discriminant analysis plot. LWWL: Liuweiwuling Tablet; HG: High-dose LWWL group; LG: Low-dose LWWL group; CG: Control group; TG: Tenofovir disoproxil fumarate group. OPLS-DA: Orthogonal partial least squares discriminant analysis; PCA: Principal component analysis.

Figure 3 Differential expression metabolites in serum metabolomics of pAAV-HBV1. 2 replication mice treated with Liuweiwuling Tablet. A: Volcanogram of differentially expressed metabolites (DEMs); B: Heat map of DEMs between High-dose Liuweiwuling Tablet group (HG)/control group (CG); C: Kyoto Encyclopedia of Genes and Genomes pathway enrichment for the DEMs between HG/CG; D: Receiver operating characteristic curve validation of the DEMs between HG/CG; E: Heat map of eight key DEMs between HG/CG; F: Potential apoptosis-related metabolic pathways of Liuweiwuling Tablet against hepatitis B virus. LWWL: Liuweiwuling Tablet; HG: High-dose LWWL group; LG: Low-dose LWWL group; CG: Control group; TG: Tenofovir disoproxil fumarate group.

Figure 4 Apoptotic effect of Liuweiwuling Tablet in hepatitis B virus cell models. A: Apoptotic effect of Liuweiwuling Tablet (LWWL) in HepG2.2.15 cells; B: Apoptotic evaluation of control group in HepG2.2.15 cells; C: Statistical analysis of apoptosis in HepG2.2.15 cells; D: Apoptotic effect of LWWL in HepG2.A64 cells; E: Apoptotic evaluation of control group in HepG2.A64 cells; F: Statistical analysis of apoptosis in HepG2.A64 cells; G: Apoptotic effect of LWWL in HepG2.1403F cells; H: Apoptotic evaluation of control group in HepG2.1403F cells; I: Statistical analysis of apoptosis in HepG2.1403F cells. ^aP < 0.05 vs control. LWWL: Liuweiwuling Tablet.

Figure 5 Apoptotic effect of Liuweiwuling Tablet in non- hepatitis B virus cell models. A: Apoptotic effect of Liuweiwuling Tablet (LWWL) in HepG2 cells; B: Apoptosis in the control group is presented in HepG2 cells; C: Statistical analysis of apoptosis is provided in HepG2 cells; D: Apoptotic effect of Liuweiwuling Tablet in L02 cells; E: Apoptosis in the control group is presented in L02 cells; F: Statistical analysis of apoptosis is provided in L02 cells; G: Apoptotic effect of LWWL in LX2 cells; H: Apoptosis in the control group is presented in LX2 cells; I: Statistical analysis of apoptosis is provided in LX2 cells. LWWL: Liuweiwuling Tablet.

Figure 6 Apoptotic effect of Liuweiwuling Tablet in the liver of hepatitis B virus (HBV) and non-HBV-replicating mouse models. A-C: Liver apoptosis in the control group for the non- hepatitis B virus (HBV)-replicating mouse model; D-F: Liver apoptosis in the Liuweiwuling Tablet (LWWL)-treated group in the non-HBV-replicating mouse model; G-I: Liver apoptosis in the CG for the HBV-replicating mouse model; J-L: Liver apoptosis in the LWWL-treated group in the HBV-replicating mouse model. LWWL: Liuweiwuling Tablet; HBV: Hepatitis B virus.

Figure 7 Possible selective apoptosis pathways of Liuweiwuling Tablet against hepatitis B virus. HBV: Hepatitis B virus.