Puerarin prevents cadmium-induced endoplasmic reticulum stress via SIRT1-dependent PERK-CHOP pathway in HepG2 cells

Abstract

Cadmium (Cd) is a high-risk heavy metal that induces oxidative stress, endoplasmic reticulum (ER) stress and inflammation, damaging organs such as the liver. Puerarin (PUE) has been shown to treat liver injury and especially prevent Cd-induced hepatic damage via its antioxidant activity. Sirtuin 1 (SIRT1), a histone deacetylase, is a key protector against various stress insults. However, its role in the protection of PUE against Cd-induced liver damage has not been clarified. Thus, this study is designed to elucidate the molecular mechanism in the human hepatoma cell line HepG2. The results first reveal that Cd-induced apoptosis is significantly restored by PUE pretreatment, as confirmed by the CCK-8, flow cytometric, Hoechst 33258 and TUNEL assays. Mechanistically, PUE significantly decreases ROS production and increases SOD levels in Cd-treated HepG2 cells. Moreover, PUE pretreatment alleviates ER stress by inhibiting the PERK-eIF2α-ATF4-CHOP axis and subsequently partially restores ER function as revealed by decreased Ca2+ release from the ER. In addition, further study demonstrates that PUE upregulates SIRT1 expression, which suppresses the PERK signaling cascade and reduces CHOP levels. Collectively, our results first demonstrate that PUE protects HepG2 cells from Cd-induced apoptosis at least partially by inhibiting the PERK-eIF2α-ATF4-CHOP pathway in a SIRT1 expression-dependent manner. Puerarin appears to have great potential as a hepatoprotective agent.

1. Introduction

Cadmium (Cd) is a toxic pollutant in industry and agriculture. The biological half-life of Cd is long, approximately 20–25 years. Thus, contaminated food, the atmosphere and water easily accumulate in and damage various organs, including the liver, spleen, kidney, and ovaries, leading to permanent damage [1–3]. As a primary organ for metabolism and detoxification, the liver is the major site of Cd accumulation. Growing evidence has revealed that Cd accumulation in the liver impacts normal function [4]. Mechanistically, oxidative stress-related cellular apoptosis is a major cause of Cd-induced hepatotoxicity via the production of excessive reactive oxygen species (ROS) and weakening of the antioxidant system [3,5,6].

The endoplasmic reticulum (ER) is a specialized eukaryotic organelle involved in protein synthesis, modification and processing, as well as the storage of cellular calcium. A variety of stimuli, such as environmental toxins, Ca2+ perturbations and oxidative injury, can affect ER function, leading to the accumulation of misfolded and/or unfolded proteins, resulting in ER stress [7,8]. Consequently, ER stress activates the unfolded protein response (UPR) to alleviate mild ER stress, reestablish ER homeostasis and restore cell viability [8,9]. The UPR is performed by three sensors: protein kinase RNA-like ER kinase (PERK), inositol requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6). However, the UPR cannot restore the normal ER when severe ER stress occurs, which ultimately promotes cell apoptosis [8,9]. Since the liver is the major organ for protein production with an elaborate ER system, ER stress is involved in the pathogenesis of various liver disorders. Interestingly, we previously demonstrated that Cd exposure induced hepatoxicity through a ROS-initiated PERK-CHOP pathway, providing a new strategy for the prevention or treatment of Cd-mediated hepatotoxicity [5].

Puerarin (PUE), an isoflavone compound derived from the dried root of Pueraria lobata (Willd.) Ohwi, has been widely used to treat diabetes, liver injury, cancers and neurological disorders [10–13]. PUE possesses diverse pharmacological activities, such as potent anti-oxidant, anti-inflammatory and hepatoprotective effects [13]. Emerging studies have revealed that PUE can play a protective role in various liver disorders by improving oxidative stress, the inflammatory response and lipid metabolism [13,14]. Wang et al. [15] demonstrated that PUE-mediated alleviation of Cd-induced liver injury is associated with the restoration of autophagic flux and the alleviation of autophagy blockade via the restoration of Rab7 expression in the hepatocyte line AML-12. Additionally, Yu et al. [16] revealed that PUE prevents Cd-induced autophagy inhibition and NLRP3 inflammasome activation via the induction of Nrf2 in AML12. Zhou et al. [17] reported that PUE could markedly block Cd-induced apoptosis in hepatocytes. However, whether these beneficial effects induced by PUE are associated with reducing ER stress-induced apoptosis in Cd-treated HepG2 cells remains unclear.