Thymoquinone prevents endoplasmic reticulum stress and mitochondria-induced apoptosis in a rat model of partial hepatic warm ischemia reperfusion

Abstract

This study was undertaken to evaluate the protective effect of thymoquinone (TQ), the bioactive compound of Nigella sativa seeds, against warm ischemia-reperfusion (I/R) injury in liver. Rats were given an oral administration of a vehicle solution (sham group) or TQ at the appropriate dose (10, 20, 30 and 40 mg/kg) for ten days consecutively. Following, they were subjected to 60 min of partial hepatic ischemia followed by 24 h of reperfusion. .Transaminase activities, histopathological changes, TNFα and antioxidant parameters were evaluated. Also, endoplasmic reticulum (ER) stress, mitochondrial damage and apoptosis were studied. In addition, ERK and P38 phosphorylation was determined by Western blot technique. We found that TQ at 30 mg/kg is the effective dose to protect rat liver against I/R injury. Moreover, 30 mg/kg of TQ prevented histological damages, inflammation and oxidative stress. Interestingly, it decreased the expression of ER stress parameters including GRP78, CHOP and caspase-12. In parallel, it improved mitochondrial function and attenuated the expression of apoptotic parameters. Furthermore, TQ significantly enhanced ERK and P38 phosphorylation. In conclusion, we demonstrated the potential of TQ to protect the rat liver against I/R injury through the prevention of ER stress and mitochondrial dysfunction. These effects implicate the prevention of oxidative stress.

Introduction

Hepatic ischemia reperfusion (I/R) is one of the major concerns after liver surgery and transplantation. In the past few decades, different works have explored the molecular pathways involved in liver I/R injury. The pathophysiological mechanisms of I/R-induced damage are multifactorial and a lot of experimental data indicate that reactive oxygen species (ROS) accumulated in the injured tissues are the hallmark of all subsequent lesions [1]. ROS can cause lipid peroxidation, protein oxidation and DNA damage which can lead to organelles malfunction and further cellular disorders.

Oxygen and glucose deprivations induce abnormalities in protein-folding processes and accumulation of unfolded/misfolded proteins into endoplasmic reticulum (ER). This protein misfolding in the ER lumen can initiate ROS production. In addition, ROS could exacerbate protein misfolding in the ER lumen by oxidizing amino acids in folding proteins or modifying chaperone [2]. This situation activates the unfolded protein response (UPR) to resolve this protein-folding defect and so to restore ER homeostasis [3], [4]. However, an excessive and continuous ER stress can activate pro-apoptotic factors, including C/EBP-homologous protein (CHOP) and caspase-12, conducting to cell dysfunction and even cell death [5]. Several studies were interested in developing strategies that limit or avoid the consequences of ER stress in I/R process [6]. Among these strategies, pharmacological pretreatment has been of great interest [7], [8].

Mitochondria produce cellular energy needed for normal cellular function through oxidative phosphorylation [9]. Oxygen deprivation during ischemia triggers events that alter mitochondrial electron transport chain complexes resulting in further oxidative stress increase and mitochondrial bioenergetic decline [10], [11]. Moreover, mitochondrial membrane permeability can be affected by Ca²⁺-accumulation in the mitochondrial matrix during the ischemic period [12]. A continuous damage in mitochondria leads cell to undergo apoptosis. Therefore, remedial strategies targeting ER and mitochondria stress are fundamental to reduce the harmful effects of I/R.

Thymoquinone (TQ) (2-isopropyl-5-methylbenzo-1, 4-quinone), is the main active compound of the volatile oil isolated from the Nigella sativa seeds [13]. TQ was reported to possess strong antioxidant properties [14], [15], [16] and to scavenge ROS [17]. Also, TQ was shown to have anti-inflammatory action [18]. Previous research demonstrated that TQ pretreatment protects from warm I/R injury in a variety of tissues and organs [19], [20], [21], [22], [23]. Although its antioxidant and anti-inflammatory properties has been reported in a number of studies, its effect on ER stress has not been studied yet. Cellular mechanisms involved in TQ protection against I/R injury are still unclear and need more experiments to be clarified. Hence, the present study, by using a partial warm hepatic ischemia rat model, investigates TQ effects on ER stress, mitochondrial damage and apoptosis.