Effect of Hedyotis diffusa water extract on protecting human hepatocyte cells (LO₂) from H₂O₂-induced cytotoxicity

Figures & data

Figure 1. DPPH free radical scavenging capacity of WEHDW and vitamin C.

Figure 2. Examination of the cytotoxicity of H₂O₂ and the protective effect of WEHDW against H₂O₂-induced damage on LO₂ cells. (A) Cells were treated with different concentrations (50, 100, 150, 200, 250, 300, 400, or 500 μM) of H₂O₂ for 6 h. Data were expressed as mean ± SEM of three separate experiments; ###p < 0.001 versus control. (B) LO₂ cells were pretreated with WEHDW (0.3, 0.5, 1, 3, 5, or 10 mg/mL, 2 h) before challenging with H₂O₂ (200 μM, 6 h). Data were expressed as the means ± SEM of three separate experiments; ###p < 0.001 versus control and ***p < 0.001 versus the H₂O₂ alone group.

Figure 3. LO₂ cells, treated with different agents, under the view of 400 × fluorescence microscope. LO₂ cells treated with WEHDW (1 mg/mL) could attenuate apoptotic cell death mediated by H₂O₂ (200 μM, 6 h).

Figure 4. WEHDW attenuated the phosphorylation of MEK and ERK caused by H₂O₂. (A) PD98059 prevented H₂O₂-induced cell death in a concentration-dependent manner. After pre-treatment with PD98059 at different concentrations as indicated, LO₂ cells were exposed to 200 μM H₂O₂ for 2 h. Cell viability was measured at 6 h after the H₂O₂ challenge by MTT assay. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments, ^###p < 0.001 versus control and **p < 0.01 versus the H₂O₂ alone group. (B) H₂O₂ time-dependently increased the levels of phospho-MEK and phospho-ERK in the first 15 min. LO₂ cells were incubated with 200 μM H₂O₂ at the indicated time points, and total proteins were detected with the use of specific antibodies. WEHDW attenuated the increase of phospho-MEK and phospho-ERK caused by H₂O₂. LO₂ cells were pre-treated with 1 mg/mL WEHDW for 2 h and then exposed to 200 μM H₂O₂ for 15 min.

Figure 5. WEHDW activated the phosphorylation of P13-K/AKT/GSK3β pathway. (A) SB415286 prevented H₂O₂-induced apoptosis in a concentration-dependent manner. LO₂ cells were exposed to 200 μM H₂O₂ at 2 h after pre-treatment with SB415286 at different concentrations as indicated. Cell viability was measured at 6 h after exposure to H₂O₂ by MTT assay. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments, ###p < 0.001 versus control and ***p < 0.001 versus the H₂O₂ alone group. (B) Specific P13-K inhibitor abrogated the hepatoprotective effects of WEHDW on H₂O₂-induced apoptosis. After pre-treatment with 1 mg/mL WEHDW, LO₂ cells were incubated with 30 μM or 50 μM LY294002 for 30 min. Then, they were exposed to 200 μM H₂O₂ at 2 h. Cell viability was measured at 6 h after exposure to H₂O₂ by MTT assay. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments, ###p < 0.001 versus control and *p < 0.05, **p < 0.01, and ***p < 0.001 versus the H₂O₂ alone group. (C) H₂O₂ time-dependently decreased the levels of phospho-AKT and phospho-GSK3β peaked at 30 min without affecting its expression levels. LO₂ cells were incubated with 200 μM H₂O₂ at the indicated time points, and the total proteins were detected with the use of specific antibodies. WEHDW reversed the reduction of phospho-AKT and phospho-GSK3β caused by H₂O₂. LO₂ cells were pre-treated with 1 mg/mL WEHDW for 2 h and then exposed to 200 μM H₂O₂ for 30 min.