P2Y11R regulates cytotoxicity of HBV X protein (HBx) in human normal hepatocytes

Abstract

Hepatitis B infection is a major global health problem and a primary cause of hepatocellular carcinoma (HCC). While various antiviral treatments have been explored, there is not yet a reliable method for preventing the progression of chronic hepatitis B infection into HCC. Hepatitis B virus X protein (HBx) plays a major role in viral replication, chronic inflammation and the pathogenicity of chronic liver disease. Modulation of purinergic receptors using their specific agonists has become a popular new strategy for modifying disease processes. In the present study, we investigated the involvement of the P2Y₁₁ receptor using its specific antagonist NF157 in some key aspects of HBx-induced liver disease in human MIHA hepatocytes, including mitochondrial dysfunction due to compromised mitochondrial membrane potential (MMP), oxidative stress resulting from overproduction of reactive oxygen species (ROS) and decreased antioxidant glutathione (GSH), production of proinflammatory cytokines and chemokines such as interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, and chemokine (C-X-C motif) ligand 2 (CXCL2), as well as activation of cellular signaling pathways including the p38/mitogen-activated protein kinase (p38/MAPK) and nuclear factor-κB (NF-κB) pathways. Our findings present a novel new strategy for the treatment and prevention of chronic liver infection and subsequent morbidities induced by HBx via specific antagonism of the P2Y₁₁ purinergic receptor.

Keywords: HBx; Hepatitis B virus (HBV); NF157; P2Y11; hepatocellular carcinoma; liver disease; purinergic receptors.

Figure 1

HBV X protein (HBx) increased the expression of P2Y₁₁R in normal human MIHA hepatocytes. Normal human MIHA hepatocytes were transfected with the HBx-encoding plasmids. A. At 48 h post-transfection, western blot analysis revealed the successful overexpression of HBx in normal human MIHA hepatocytes; B. Real time PCR analysis revealed that overexpression of HBx increased the expression of P2Y₁₁R at the gene level; C. Western blot analysis revealed that overexpression of HBx increased the expression of P2Y₁₁R at the protein level (*, P < 0.01 vs. vehicle group, n=6).

Figure 2

Inhibition of P2Y₁₁R with its specific antagonist NF157 ameliorated HBx-induced mitochondrial dysfunction in normal human MIHA hepatocytes. Normal human MIHA hepatocytes were transfected with HBx-encoding plasmid. At 24 h post-transfection, cells were treated with NF157 at the concentrations of 25 and 50 μM for 24 h. A. Intracellular levels of MMP were determined by TMRM; Scale bar, 50 μm; B. Cytochrome C oxidase activity (*, #, $, P < 0.01 vs. previous column group, n=5-6).

Figure 3

Inhibition of P2Y₁₁R with its specific antagonist NF157 ameliorated HBx-induced oxidative stress in normal human MIHA hepatocytes. Normal human MIHA hepatocytes were transfected with HBx-encoding plasmid. At 24 h post-transfection, cells were treated with NF157 at the concentrations of 25 and 50 μM for 24 h. A. Intracellular ROS was determined by the DCFH-DA assay; B. Reduced GSH (*, #, $, P < 0.01 vs. previous column group, n=5-6).

Figure 4

Blockage of P2Y₁₁R with its specific antagonist NF157 suppressed HBx-induced expression of IL-6, MCP-1, and CXCL2. Normal human MIHA hepatocytes were transfected with HBx-encoding plasmid. At 24 h post-transfection, cells were treated with NF157 at the concentrations of 25 and 50 μM for 24 h. A. Expression of IL-6, MCP-1, and CXCL2 at the gene level was determined by real time PCR analysis; B. Expression of IL-6, MCP-1, and CXCL2 at the protein level was determined by ELISA assay (*, #, $, P < 0.01 vs. previous column group, n=5-6).

Figure 5

Blockage of P2Y₁₁R with its specific antagonist NF157 prevented HBx-induced secretion of high mobility group box 1 (HMGB1). Normal human MIHA hepatocytes were transfected with HBx-encoding plasmid. At 24 h post-transfection, cells were treated with NF157 at the concentrations of 25 and 50 μM for 24 h. Secretion of HMGB1 was determined by ELISA (*, #, $, P < 0.01 vs. previous column group, n=6).

Figure 6

Antagonism of P2Y₁₁R with its specific antagonist NF157 prevented HBx-induced activation of p38. Normal human MIHA hepatocytes were transfected with HBx-encoding plasmid. At 24 h post-transfection, cells were treated with NF157 at the concentrations of 25 and 50 μM for 2 h. Phosphorylated and total levels of p38 were determined by western blot analysis (*, #, $, P < 0.01 vs. previous column group, n=5-6).

Figure 7

Antagonism of P2Y₁₁R with its specific antagonist NF157 suppressed HBx-induced degradation of IκBα. Normal human MIHA hepatocytes were transfected with HBx-encoding plasmid. At 24 h post-transfection, cells were treated with NF157 at the concentrations of 25 and 50 μM for 6 h. Phosphorylated and total levels of IκBα were determined by western blot analysis (*, #, $, P < 0.01 vs. previous column group, n=5-6).

Figure 8

Antagonism of P2Y₁₁R with its specific antagonist NF157 suppressed HBx-induced activation of NF-κB. Normal human MIHA hepatocytes were transfected with HBx-encoding plasmid. At 24 h post-transfection, cells were treated with NF157 at the concentrations of 25 and 50 μM for 24 h. A. Nuclear translocation of p65; B. Luciferase activity (*, #, $, P < 0.01 vs. previous column group, n=5-6).