Upregulation and stabilization of senescence marker protein-30 by epigallocatechin gallate against tert-butyl hydroperoxide-induced liver injury in vitro and in vivo

Abstract

Senescence marker protein-30 (SMP30), a novel ageing marker, suppresses oxidative stress in the liver. However, studies on phytochemical-mediated regulation of SMP30 expression are lacking. Here, we showed that epigallocatechin gallate (EGCg), a polyphenol abundant in green tea, positively regulates SMP30 expression in the rat hepatoma-derived Fao cells. EGCg maintained SMP30 expression even in the presence of cycloheximide, a protein synthesis inhibitor. Furthermore, treatment of cells with tert-butyl hydroperoxide (tert-BHP), an oxidative promoter, decreased SMP30 expression and ERK1/2 phosphorylation, while EGCg treatment inhibited these effects. Male mice (7-week-old) were divided into 4 groups-Control (saline), tert-BHP (1.5 mmol/kg tert-BHP), EGCg + tert-BHP (30 mg/kg/day of EGCg and 1.5 mmol/kg tert-BHP), and EGCg (30 mg/kg/day). After oral EGCg administration for 6 consecutive days, EGCg + tert-BHP group mice were administered tert-BHP. The tert-BHP-administered mice showed decreased SMP30 expression in the liver and increased aspartate aminotransferase and alanine transaminase (hepatic injury marker enzymes) activities; however, EGCg treatment attenuated these changes. Thus, EGCg-induced SMP30 upregulation may alleviate tert-BHP-induced liver injury. The findings of this study offer new perspectives of the anti-ageing properties of EGCg.

Keywords: epigallocatechin gallate; senescence marker protein-30; tert-BHP-induced liver injury.

Fig. 1

Effects of EGCg on cell toxicity and SMP30 expression. (A) Cell viability of EGCg: Fao cells (1.0 × 10⁵ cells/well) were cultured in a 96-well plate before treating with various EGCg concentrations for 24 h. Cell viability was assessed using the CCK-8 assay. Cell viability was expressed as a percentage of the values obtained for EGCg-untreated cells. (B) Protein expression of SMP30: Fao cells were treated with EGCg (0–100 µM) for 24 h, and the cell lysate was immunoblotted with each indicated antibody. Densitometry quantification of SMP30 was performed using ImageJ. SMP30 levels were normalized to β-actin. Data are shown as the means ± SE from at least three independent experiments. Unmatching letters denote significantly different, p<0.05.

Fig. 2

Effects of EGCg on SMP30 up-regulation and stabilization in Fao cells. (A) Fao cells were pre-treated with or without CHX (10 mg/ml) for 0.5 h before stimulating with or without 30 µM EGCg for 24 h. (B) Densitometry quantification was performed using ImageJ. SMP30 levels were normalized to β-actin. Data are shown as the means ± SE from at least three independent experiments. Unmatching letters denote significantly different, p<0.05.

Fig. 3

Effects of EGCg on LDH release with tert-BHP-induced oxidative stress in Fao cells. Fao cells were pre-treated with 30 µM EGCg for 8 h. After incubating with EGCg, cells were treated with 100 µM tert-BHP with or without 30 µM EGCg for 24 h. Quantitative LDH level analyses were performed in the cell culture supernatant. Data are shown as the means ± SE from at least three independent experiments. Unmatching letters denote significantly different, p<0.05.

Fig. 4

Effects of EGCg on SMP30 expression and ERK1/2 phosphorylation with tert-BHP-treated Fao cells. (A) Cells were pre-treated with 30 µM EGCg for 8 h. After incubating with EGCg, cells were treated with 100 µM tert-BHP with or without 30 µM EGCg for 24 h. Cells were homogenized using RIPA buffer, and the supernatants were separated to measure SMP30, phosphorylated ERK1/2 (activation form), and ERK1/2 expression. (B) Densitometry quantification was performed using ImageJ. SMP30 levels were normalized to β-actin. p-ERK1/2 levels (Thr202/Tyr204) were normalized to ERK1/2. (C) Cells were treated with 100 µM tert-BHP with/without 0.5 mM NAC for 24 h. The supernatants were separated for SMP30 expression measurement. (D) Densitometry quantification was performed using ImageJ. SMP30 levels were normalized to β-actin. Data are shown as the means ± SE from at least three independent experiments. Unmatching letters denote significantly different, p<0.05.

Fig. 5

Effects of EGCg on SMP30 expression and hepatic toxicity in tert-BHP-injected mice. (A) All groups were fed an AIN93G diet as a basal diet and given tap water for 7 days. EGCg (30 mg/kg body weight) was orally administered to the EGCg and EGCg + tert-BHP groups for 6 days. After 6 days, the tert-BHP and EGCg + tert-BHP groups were intraperitoneally injected with tert-BHP for 24 h. (B) Livers were homogenized using RIPA buffer, and the supernatants were separated for SMP30 expression measurement. Densitometry quantification was performed using ImageJ. SMP30 levels were normalized to β-actin. Data are shown as the means ± SE. (C) Blood was collected and serum separated to determine serum AST and (D) ALT levels. Unmatching letters denote significantly different, p<0.05.