Cytoprotective effects and antioxidant activities of acteoside and various extracts of Clerodendrum cyrtophyllum Turcz leaves against t-BHP induced oxidative damage

Abstract

This study evaluates the antioxidant potential and cytoprotective effects of ethanolic crude extract from Clerodendrum cyrtophyllum leaves (ECE) and five derived fractions (namely, petroleum ether fraction (PEF), dichloromethane fraction (DMF), ethyl acetate fraction (EAF), n-butyl alcohol fraction (BAF) and the remaining fraction (RF)), as well as acteoside (Ac, a major phenolic component in EAF) on oxidative damage caused by tert-butyl hydroperoxide (t-BHP) in HepG2 cells. MTT assay results showed that ECE, EAF, BAF, RF and Ac increased the viability of t-BHP-damaged cells in a dose-dependent manner, while EAF significantly promoted cell viability. EAF, BAF, RF, or Ac reduced the levels of lactate dehydrogenase (LDH) leakage, malondialdehyde (MDA), and reactive oxygen species (ROS). Additionally, glutathione (GSH) levels and the activities of superoxide dismutase (SOD) and catalase (CAT) increased. Western blot analysis further indicated that EAF, BAF, RF, or Ac up-regulated pro-caspase-3 and reduced cleaved caspase-3 during t-BHP-induced oxidative stress. Flow cytometry analysis and fluorescence micrographs showed that Ac could inhibit apoptosis.

Figure 1

Structure of acteoside (Ac) from C. cyrtophyllum leaves.

Figure 2

HepG2 cells viability. (A) Cells were treated with t-BHP at the indicated concentrations (0–1.2 mmol/L). *p < 0.05 compared with no t-BHP treatment. (B) Viability of HepG2 cells after treatment with different concentrations of ECE, PEF, DMF, EAF, BAF, RF. NS indicates that there is no significant difference between control and all sample groups (p < 0.05). (C) Cytoprotective effects of ECE, PEF, DMF, EAF, BAF, RF. (D) Viability of HepG2 cells after treatment with different concentrations of Ac. *p < 0.05 versus no Ac treatment. (E) Cytoprotective effects of Ac. Different letters above bars indicate significant difference (by ANOVA, p < 0.05). Result are expressed as mean ± SD (n ≥ 3). + , treated; −, untreated.

Figure 3

Effects of extracts on the indicators of HepG2 cellular antioxidant defense. (A) Effects of extracts on intracellular ROS. (B) Effects of extracts on extracellular lactate dehydrogenase activity. (C) Effects of extracts on extracellular malondialdehyde level. (D) Effects of extracts on GSH level. (E) Effects of extracts on SOD activity. (F) Effects of extracts on CAT activity. Different letters above bars indicate a significant difference (by ANOVA, p < 0.05). Results are expressed as mean ± SD (n ≥ 3). + , treated; −, untreated.

Figure 4

Effects of extracts on the expression of caspase-3 level in HepG2 cells. HepG2 cells were treated with 30 μg/mL samples for 6 h before being exposed to t-BHP (700 μmol/L) for 3 h. (A) The expressions of pro-caspase-3, cleaved caspase-3 and β-actin were used for normalization and verification of protein loading; (B) Quantitative pro-caspase-3 and cleaved caspase-3 expression after normalization to β-actin. (a) Control; (b) 700 μmol/L t-BHP treated; (c) 30 μg/mL PEF + 700 μmol/L t-BHP; (d) 30 μg/mL DMF + 700 μmol/L t-BHP; (e) 30 μg/mL EAF + 700 μmol/L t-BHP; (f) 30 μg/mL BAF + 700 μmol/L t-BHP; (g) 30 μg/mL RF + 700 μmol/L t-BHP. Different letters above bars indicate significant difference (by ANOVA, p < 0.05). Result are expressed as mean ± SD (n ≥ 3). + , treated; −, untreated.

Figure 5

Effects of Ac, BHT, and VE on the indicators of HepG2 cellular antioxidant defense. (A) Effects of Ac, BHT, and VE on intracellular ROS. (B) Effects of Ac, BHT, and VE on extracellular lactate dehydrogenase activity. (C) Effects of Ac, BHT, and VE on extracellular malondialdehyde level. (D) Effects of Ac, BHT, and VE on GSH level. (E) Effects of Ac, BHT, and VE on SOD activity. (F) Effects of Ac, BHT, and VE on CAT activity. HepG2 cells were treated with 30 μg/mL samples for 6 h before being exposed to TBHP (700 μmol/L) for 3 h. ROS level, LDH activity, MDA level, GSH level, SOD activity, and CAT activity in HepG2 cells were detected through the kit. Different letters above bars indicate significant difference (by ANOVA, p < 0.05). Results are expressed as mean ± SD (n ≥ 3). Ac, acteoside; BHT, butylated hydroxytoluene; VE, vitamin E; + , treated; −, untreated.

Figure 6

Effect of Ac, BHT, or VE on the expression of caspase-3 level in HepG2 cells. (A) The expressions of pro-caspase-3, cleaved caspase-3 and β-actin were used for normalization and verification of protein loading; (B) Quantitative pro-caspase-3 and cleaved caspase-3 expression after normalization to β-actin. (a) Control; (b) 700 μmol/L t-BHP treated; (c) 10 μmol/L Ac + 700 μmol/L t-BHP; (d) 100 μmol/L Ac + 700 μmol/L t-BHP; (e) 200 μmol/L Ac + 700 μmol/L t-BHP; (f) 200 μmol/L BHT + 700 μmol/L t-BHP; (g) 200 μmol/L VE + 700 μmol/L t-BHP. Different letters above bars indicate significant difference (by ANOVA, p < 0.05). Results are expressed as mean ± SD (n ≥ 3). + , treated; −, untreated.

Figure 7

Effect of Ac, BHT and VE on apoptosis. (A) Representative dot pot of apoptosis evaluated by flow cytometric analysis after Annexin-V/PI double staining. Cells were gated based on size and granularity using FSC-A vs SSC-A to eliminate debris and clumped cells. The living cells and apoptotic cells were defined by the four-quarter gate setting tool in the software. (B) Quantitation results of apoptosis evaluated by flow cytometric analysis. (C) Fluorescence micrographs to assess Annexin-V/PI double staining of HepG2 cells. Results are expressed as mean ± SD (n ≥ 3). #p < 0.05 versus the same state of control, *p < 0.05 versus TBHP treatment. + , treated; −, untreated.