Inhibition of Protease Activated Receptor 2 Attenuates HBx-Induced Inflammation and Mitochondria Oxidative Stress

Figures & data

Figure 1 Overexpression of HBx elevates PAR2 protein level in LO2 cells. (A) The protein level of HBx in LO₂ cells transfected with pcDNA3.1-HBx was measured by Western blotting. (B) The protein level of PAR2 in LO₂ cells transfected with pcDNA3.1-HBx was measured by Western blotting. **P < 0.01 vs the vector group. The experiments were performed in triplicate.

Figure 2 Inhibition of PAR2 reverses the influences of HBx overexpression on the proliferation and apoptosis of LO₂ cells. (A) The proliferation of LO₂ cells under different treatments was measured by EdU assay. (B) The apoptosis of LO₂ cells under different treatments was analysed using a flow cytometer. *P < 0.05 vs the HBx(-) + FSL(-) group. ^#P < 0.05 vs the HBx(+) + FSL(-) group. The experiments were performed in triplicate.

Figure 3 Suppression of PAR2 attenuates HBx-induced mitochondria oxidative stress in LO₂ cells. (A) ROS generation assay was performed using flow cytometry analysis. (B) The level of GSH in LO₂ cells under different treatments. (C) The level of SOD in LO₂ cells under different treatments was measured by a commercial kit. (D) The level of MDA in LO₂ cells under different treatments was measured by a commercial kit. (E) MMP value in LO₂ cells under different treatments. (F) ATP value in LO₂ cells under different treatments. (G) H₂O₂ concentration in LO₂ cells under different treatments. *P < 0.05 vs the HBx(-) + FSL(-) group. ^#P < 0.05 vs the HBx(+) + FSL(-) group. The experiments were performed in triplicate.

Figure 4 Decreased PAR2 represses inflammation responses in HBx overexpressed LO₂ cells. (A) The mRNA expression of TNF-α, IL-6, and CXCL-2 in LO₂ cells under different treatments was detected by qRT-PCR. (B) The protein level of TNF-α, IL-6, and CXCL-2 in LO₂ cells under different treatments was measured by ELISA. *P < 0.05 vs the HBx(-) + FSL(-) group. ^#P < 0.05 vs the HBx(+) + FSL(-) group. The experiments were performed in triplicate.

Figure 5 PAR2 knockdown alleviates HBx-induced liver injuries in a mouse model (n = 8 in each group). (A) The protein levels of HBx and PAR2 in PAR2-KO mice after injection of pcDNA3.1-HBx. (B) The levels of AST and ALT in PAR2-KO mice after injection of pcDNA3.1-HBx were measured by the corresponding commercial kits. *P < 0.05 vs the control mice group. ^#P < 0.05 vs the WT mice + pcDNA3.1-HBx group. (C) The HE staining and (D) immunohistochemistry assay of PAR2-KO mice after injection of pcDNA3.1-HBx.

Figure 6 Inflammation caused by HBx is improved in PAR2-KO mice (n = 8 in each group). (A) The mRNA expression of TNF-α, IL-6, and CXCL-2 in liver tissues of PAR2-KO mice was detected by qRT-PCR. (B) The protein level of TNF-α, IL-6, and CXCL-2 in liver tissues of PAR2-KO mice was measured by ELISA. *P < 0.05 vs the control mice group. ^#P < 0.05 vs the WT mice + pcDNA3.1-HBx group.

Figure 7 Mitochondria oxidative stress caused by HBx is alleviated in PAR2-KO mice (n = 8 in each group). (A) The level of GSH in liver tissues of PAR2-KO mice. (B) The level of SOD in liver tissues of PAR2-KO mice was measured by a commercial kit. (C) The level of MDA in liver tissues of PAR2-KO mice was measured by a commercial kit. (D) MMP value in liver tissues of PAR2-KO mice. (E) ATP value in liver tissues of PAR2-KO mice. (F) H₂O₂ concentration in liver tissues of PAR2-KO mice. *P < 0.05 vs the control mice group. ^#P < 0.05 vs the WT mice + pcDNA3.1-HBx group.