Background: Gall (Galla Rhois [GR]) is known to have antibacterial, anti-inflammatory, antimetastatic, and anti-invasion activities and exert hepatoprotective effects. However, the hepatoprotective effects of gallotannin-enriched GR (GEGR) and their mechanisms have not yet been investigated.
Objective: The potential protective effect of GEGR against hepatotoxicity induced by hydrogen peroxide (H2O2) was investigated.
Materials and methods: Changes in cell viability, apoptosis protein expression, and reactive oxygen species (ROS) generation were determined in HepG2 cells that were pretreated with four different concentrations of GEGR (6.25-50 μg/ml) for 24 h before H2O2 exposure.
Results: GEGR consisted of gallotannin (69.2%), gallic acid (26.6%), and methyl gallate (4.2%) and showed remarkable 2,2-diphenyl-1-picrylhydrazyl scavenging activity (inhibitory concentration 50% = 0.212 μg/ml). The lethal dose 50% and effective dose 50% values for the response of HepG2 cells to GEGR were determined to be 178 and 6.85 μg/ml, respectively. Significant reductions in the immunofluorescence intensity indicating apoptosis were also detected in the nuclei of HepG2 cells stained with 4',6-diamidino-2-phenylindole and Annexin V after GEGR treatment. The Bax/Bcl-2 ratio and active caspase-3 level were higher in H2O2 + vehicle-treated cells. However, these levels gradually decreased to those of the No-treated group in the GEGR pretreated group even though little or no decrease was observed in response to low GEGR concentrations. Furthermore, the GEGR pretreated group showed a reduced level of 2',7'-dichlorofluorescein diacetate stained cells, indicating ROS generation relative to the H2O2 + vehicle-treated group.
Conclusion: The results of this study provide strong evidence that GEGR can prevent cell death induced by H2O2 in HepG2 cells through the induction of antioxidant conditions.
Summary: The gallotannin (69.2%), gallic acid (26.6%), and methyl gallate (4.2%) are the main constituents of water extracts of GRGEGR was more potent in DPPH scavenging, and gallotannin contributes to this extract activityGEGR significantly reduced the increase of apoptosis, Bax/Bcl-2 ratio, and active caspase-3 level after H2O2 treatmentGEGR pretreatment showed protection against H2O2-induced ROS production in DCFH-DA staining analysis. Abbreviations used: COX: Cyclooxygenase; DAPI: 4',6-diamidino-2-phenylindole; DMSO: Dimethyl sulfoxide; DPPH: 2,2-diphenyl-1-picrylhydrazyl; GEGR: Gallotannin-enriched Galla Rhois; GR: Galla Rhois; HPLC: High-performance liquid chromatography; H2O2: Hydrogen peroxide; MMP: Metallopeptidase; MTT: 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; ROS: Reactive oxygen species; UV-Vis: Ultraviolet-visible.
Keywords: Antioxidant; Caspase-3; cell death; hepatotoxicity; reactive oxygen species.