Figures & data
Figure 1. Determination of HBx expression in hepatocytes. HBx-encoding plasmids were transfected into L-02 hepatocytes for 48 h. Western blot analysis revealed the overexpression of HBx in normal human L-02 hepatocytes.
![Figure 1. Determination of HBx expression in hepatocytes. HBx-encoding plasmids were transfected into L-02 hepatocytes for 48 h. Western blot analysis revealed the overexpression of HBx in normal human L-02 hepatocytes.](/cms/asset/c8da0e69-5828-47d3-9b9a-52e7278a30b4/ianb_a_1614014_f0001_b.jpg)
Figure 2. HBx reduces the expression of orexin-1 receptor (OX1R) in L-02 hepatocytes. The HBx-encoding plasmids were transfected into L-02 hepatocytes for 48 h. (A) Real-time PCR analysis revealed that overexpression of HBx decreased the expression of OX1R at the mRNA level; (B) Western blot analysis revealed that overexpression of HBx reduced the expression of OX1R at the protein level (*p < .01 vs vehicle group).
![Figure 2. HBx reduces the expression of orexin-1 receptor (OX1R) in L-02 hepatocytes. The HBx-encoding plasmids were transfected into L-02 hepatocytes for 48 h. (A) Real-time PCR analysis revealed that overexpression of HBx decreased the expression of OX1R at the mRNA level; (B) Western blot analysis revealed that overexpression of HBx reduced the expression of OX1R at the protein level (*p < .01 vs vehicle group).](/cms/asset/65f9e49b-f674-4622-97d7-b2783a77b190/ianb_a_1614014_f0002_b.jpg)
Figure 3. Orexin A mitigates HBx-induced oxidative stress in L-02 hepatocytes. L-02 hepatocytes were transfected with the HBx-encoding plasmid for 24 h, followed by treatment with orexin A at a concentration of 100 or 300 nM for another 24 h. (A) 4-HNE expression was measured by the immunostaining; (B) ROS was measured by the DCFH-DA assay (*, #, $, p < .01 vs previous column group).
![Figure 3. Orexin A mitigates HBx-induced oxidative stress in L-02 hepatocytes. L-02 hepatocytes were transfected with the HBx-encoding plasmid for 24 h, followed by treatment with orexin A at a concentration of 100 or 300 nM for another 24 h. (A) 4-HNE expression was measured by the immunostaining; (B) ROS was measured by the DCFH-DA assay (*, #, $, p < .01 vs previous column group).](/cms/asset/40dfe2c2-d01e-4691-9639-62dda4aec839/ianb_a_1614014_f0003_c.jpg)
Figure 4. Orexin A ameliorates HBx-induced expression of NOX-4 in normal human L-02 hepatocytes. L-02 hepatocytes were transfected with the HBx-encoding plasmid for 24 h, followed by treatment with orexin A at a concentration of 100 or 300 nM for another 24 h. (A) Expression of NOX-4 at the mRNA levels determined by the real time analysis; (B). Expression of NOX-4 at the protein levels determined by the western blot analysis (*, #, $, p < .01 vs previous column group).
![Figure 4. Orexin A ameliorates HBx-induced expression of NOX-4 in normal human L-02 hepatocytes. L-02 hepatocytes were transfected with the HBx-encoding plasmid for 24 h, followed by treatment with orexin A at a concentration of 100 or 300 nM for another 24 h. (A) Expression of NOX-4 at the mRNA levels determined by the real time analysis; (B). Expression of NOX-4 at the protein levels determined by the western blot analysis (*, #, $, p < .01 vs previous column group).](/cms/asset/0abd6392-f257-46fc-b489-ef3db8624fa3/ianb_a_1614014_f0004_b.jpg)
Figure 5. Orexin A ameliorates HBx-induced mitochondrial dysfunction in normal human L-02 hepatocytes. L-02 hepatocytes were transfected with the HBx-encoding plasmid for 24 h, followed by treatment with orexin A at a concentration of 100 or 300 nM for another 24 h. (A) Mitochondrial membrane potential (MMP) was measured by TMRM; (B) Intracellular ATP levels (*, #, $, p < .01 vs previous column group).
![Figure 5. Orexin A ameliorates HBx-induced mitochondrial dysfunction in normal human L-02 hepatocytes. L-02 hepatocytes were transfected with the HBx-encoding plasmid for 24 h, followed by treatment with orexin A at a concentration of 100 or 300 nM for another 24 h. (A) Mitochondrial membrane potential (MMP) was measured by TMRM; (B) Intracellular ATP levels (*, #, $, p < .01 vs previous column group).](/cms/asset/e77cdf7f-241f-4b6a-9523-5b1640637a29/ianb_a_1614014_f0005_c.jpg)
Figure 6. Orexin A inhibits HBx-induced expression and secretion of pro-inflammatory cytokines in normal human L-02 hepatocytes. L-02 hepatocytes were transfected with the HBx-encoding plasmid for 24 h, followed by treatment with orexin A at a concentration of 100 or 300 nM for another 24 h. (A) Expression of IL-8, TNF-α and CXCL2 at the mRNA levels was determined by real time PCR analysis; (B) Expression of IL-8, TNF-α and CXCL2 at the protein levels was determined by ELISA assay (*, #, $, p < .01 vs previous column group).
![Figure 6. Orexin A inhibits HBx-induced expression and secretion of pro-inflammatory cytokines in normal human L-02 hepatocytes. L-02 hepatocytes were transfected with the HBx-encoding plasmid for 24 h, followed by treatment with orexin A at a concentration of 100 or 300 nM for another 24 h. (A) Expression of IL-8, TNF-α and CXCL2 at the mRNA levels was determined by real time PCR analysis; (B) Expression of IL-8, TNF-α and CXCL2 at the protein levels was determined by ELISA assay (*, #, $, p < .01 vs previous column group).](/cms/asset/fb92bdb7-a597-45ff-9347-f46f4e4f4b56/ianb_a_1614014_f0006_b.jpg)
Figure 7. Orexin A attenuates HBx-induced cytotoxicity. L-02 hepatocytes were transfected with the HBx-encoding plasmid for 24 h, followed by treatment with orexin A at a concentration of 100 or 300 nM for another 24 h. Release of LDH was assayed (*, #, $, p < .01 vs previous column group).
![Figure 7. Orexin A attenuates HBx-induced cytotoxicity. L-02 hepatocytes were transfected with the HBx-encoding plasmid for 24 h, followed by treatment with orexin A at a concentration of 100 or 300 nM for another 24 h. Release of LDH was assayed (*, #, $, p < .01 vs previous column group).](/cms/asset/a6265f62-ccf5-4a5c-8401-f8b9b8515106/ianb_a_1614014_f0007_b.jpg)
Figure 8. Orexin A suppresses HBx-induced phosphorylation of JNK in L-02 hepatocytes. L-02 normal hepatocytes were transfected with the HBx-encoding plasmid. After 24 h, cells were treated with orexin A at the concentrations of 100 and 300 nM for another 2 h. Phosphorylated and total levels of JNK were determined using western blot analysis (*, #, $, p < .01 vs previous column group).
![Figure 8. Orexin A suppresses HBx-induced phosphorylation of JNK in L-02 hepatocytes. L-02 normal hepatocytes were transfected with the HBx-encoding plasmid. After 24 h, cells were treated with orexin A at the concentrations of 100 and 300 nM for another 2 h. Phosphorylated and total levels of JNK were determined using western blot analysis (*, #, $, p < .01 vs previous column group).](/cms/asset/17ed4003-a4ec-47c3-97eb-66f0c3cdc70b/ianb_a_1614014_f0008_b.jpg)
Figure 9. Orexin A treatment inhibited HBx-induced activation of NF-κB in L-02 hepatocytes. L-02 normal hepatocytes were transfected with HBx-encoding plasmids. After 24 h, cells were treated with orexin A at the concentrations of 100 and 300 nM for another 24 h. (A) Nuclear translocation of p65; (B) Luciferase activity of NF-κB (*, #, $, p < .01 vs previous column group).
![Figure 9. Orexin A treatment inhibited HBx-induced activation of NF-κB in L-02 hepatocytes. L-02 normal hepatocytes were transfected with HBx-encoding plasmids. After 24 h, cells were treated with orexin A at the concentrations of 100 and 300 nM for another 24 h. (A) Nuclear translocation of p65; (B) Luciferase activity of NF-κB (*, #, $, p < .01 vs previous column group).](/cms/asset/c109ce18-a7d9-41cb-9da5-d3eb48abedb8/ianb_a_1614014_f0009_b.jpg)
Figure 10. The protective effects of orexin A against HBx-induced cytotoxicity in human MIHA hepatocytes. Human MIHA hepatocytes were transfected with the HBx-encoding plasmid for 24 h, followed by treatment with orexin A at a concentration of 100 or 300 nM for another 24 h. (A) ROS was measured by DCFH-DA assay; (B). Secretions of IL-8, TNF-α and CXCL2 were measured by ELISA analysis; (C). Luciferase activity of NF-κB (*, #, $, p < 0.01 vs previous column group).
![Figure 10. The protective effects of orexin A against HBx-induced cytotoxicity in human MIHA hepatocytes. Human MIHA hepatocytes were transfected with the HBx-encoding plasmid for 24 h, followed by treatment with orexin A at a concentration of 100 or 300 nM for another 24 h. (A) ROS was measured by DCFH-DA assay; (B). Secretions of IL-8, TNF-α and CXCL2 were measured by ELISA analysis; (C). Luciferase activity of NF-κB (*, #, $, p < 0.01 vs previous column group).](/cms/asset/15c54897-e247-475f-a8c5-532e52436e2a/ianb_a_1614014_f0010_b.jpg)