Protocatechuic acid protects hepatocytes against hydrogen peroxide-induced oxidative stress

Abstract

Oxidative stress is a main cause of tissue damage and highly associated with incidence of human chronic diseases. Among the major target organs attacked by reactive oxygen species (ROS) is the liver. Protocatechuic acid (PCA) is a phenolic compound found in green tea, acai oil and some mushroom species that possesses strong antioxidative and anti-inflammatory activity and may have benefits as a natural phytochemical for prevention of human diseases. However, the protective effect of PCA on hydrogen peroxide (H₂O₂)-induced oxidative stress specifically in the liver has not yet been investigated. The current study aims to observe if PCA possesses protective activity against H₂O₂-induced oxidative stress in HepG2 human liver cancer cells. Relative to untreated control cells, treatment of HepG2 cells with PCA reduced H₂O₂-induced cell death and mitigated H₂O₂-induced production of ROS; furthermore, it mitigated the H₂O₂-induced increase of caspase-3/7 enzyme activity, expression of cleaved poly(ADP-ribose) polymerase (PARP), expression of endoplasmic reticulum (ER) stress genes including activating transcription factor 4 (ATF4), serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1 α (IRE1α) and phosphorylation of p38 mitogen-activated protein kinases (MAPK). These findings indicate that PCA effectively protects hepatic cells from H₂O₂-induced oxidative stress and cell death.

Keywords: Apoptosis; HepG2; Hepatocytes; Oxidative stress; Protocatechuic acid.

Graphical abstract

Fig. 1

Protocatechuic acid (PCA) restored hydrogen peroxide (H₂O₂)-induced suppression of cell viability. A protective effect of PCA against oxidative stress was observed in HepG2 cells co-treated with H₂O₂ (600, 800, and 1000 μM) and PCA (0, 25, and 50 μM). Different letters indicate significant difference at p < 0.05.

Fig. 2

PCA mitigated H₂O₂-induced generation of reactive oxygen species (ROS). Emission from ROS (A) and intensity of fluorescent green color (B) were measured in HepG2 cells co-treated with H₂O₂ (800 μM) and PCA (0, 25, and 50 μM). Different letters indicate significant difference at p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

PCA mitigated H₂O₂-induced increase of caspase 3/7 enzyme activity. Activity of caspase-3/7 enzyme per unit protein (A) and expression of cleaved PARP (cPARP) (B) were measured in HepG2 cells co-treated with H₂O₂ (800 μM) and PCA (0, 25, and 50 μM). Different letters indicate significant difference at p < 0.05.

Fig. 4

PCA mitigated H₂O₂-induced expression of endoplasmic reticulum (ER) stress genes. Western blotting was performed to measure expression of ATF4 (A), IRE1α (B) and phosphorylation of p38 (C) in HepG2 cells co-treated with H₂O₂ (800 μM) and PCA (0, 25, and 50 μM). Different letters indicate significant difference at p < 0.05.