Ursolic acid alleviates heat stress-induced lung injury by regulating endoplasmic reticulum stress signaling in mice

Abstract

Acute lung injury has been reported to be associated with heat stress in various animals. Ursolic acid is a natural pentacyclic triterpenoid compound with multiple bioactivities. However, it remains unknown whether ursolic acid supplementation alleviates heat stress-induced lung injury. In the present study, male Institute of Cancer Research mice were left untreated under a normal temperature condition (23±1°C), receiving orally administrated with vehicle (phosphate buffered saline) or ursolic acid (40 mg/kg BW⁻¹·d⁻¹ for 2 d), and then were subjected to high temperature (41±1°C) for 2 h. Histological alterations, activities of antioxidative enzymes, apoptosis, generation of reactive oxygen species, abundance of inflammatory cytokines, and endoplasmic reticulum stress-related proteins were analyzed. Compared with the controls, heat stress treatment led to enhanced apoptosis, increased H₂O₂ production, and upregulated protein levels of inflammatory cytokines in the serum, including tumor necrosis factor alpha, interleukin-6, and interleukin-1 beta. Activities of malondialdehyde, lactate dehydrogenase, and myeloperoxidase were increased, while the activities for superoxide dismutase and catalase were reduced in lung tissues of mice. All these alterations were significantly prevented by ursolic acid administration. Further study showed that heat stress led to activation of protein kinase-like ER kinase eukaryotic initiation factor 2 alpha -the transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP) signaling, which was attenuated by ursolic acid supplementation. These findings indicated that ursolic acid pretreatment protected lung tissues against heat stress-induced injury by regulating inflammatory cytokines and unfolded protein response in mice. Ursolic acid supplementation might be a therapeutic strategy to alleviate high temperature-induced lung injury in humans and animals.

Introduction

Lung injury is commonly observed in patients affected by respiratory diseases [1–4]. Various factors, such as bacterial infection, toxin exposure, hypoxia, and stress, have been reported to be implicated in lung injury [2], [3]–4]. Despite difference in the pathogens of lung disorder, it is generally accepted that reactive oxygen species (ROS)-induced lipid peroxidation and oxidative damage is involved in and contributed to the lung injury and pathogenesis [4]. Consistently, toxic substance exposure, or bacterial lipopolysaccharide challenge has been reported to be associated with increased malondialdehyde (MDA) level, decreased activity of catalase, and reduced glutathione (GSH) in the lung tissues of animals [5], indicating an imbalance between intracellular antioxidant and pro-oxidant, and its contribution to impairment of lung functions. Clinical data show that patients severely affected by heat stress are associated with dysfunction of multiple organs, including the heart and lung [6]. Also, several lines of studies have shown that heat stress exposure leads to markedly lung injury and inflammatory response as shown by increased level of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and interleukin-6, as well as enhanced apoptosis of epithelium in the lung tissues of heat-stressed rodents [7], [8], [9]–10]. However, underlying mechanisms responsible for heat stress-induced lung injury, as well as therapeutic strategies to ameliorate the deleterious effects of heat stress are not available yet.

The endoplasmic reticulum (ER) is a major site for the synthesis, folding, modification, and maturation of both secretory and membrane proteins and therefore playing essential roles in regulating multiple cellular processes [11,12]. Multiple conditions, such as hypoxia, pathogen infection, ROS accumulation, can disturb the ER homeostasis, therefore resulting in ER stress [13,14]. The eukaryotic cells have evolved a conserved signaling cascade termed the unfolded proteins response (UPR) to restore an intracellular homeostasis. UPR is initiated by three essential sensor proteins, including inositol-requiring transmembrane kinase and endonuclease 1α, activation of transcription factor (ATF)6, and the protein kinase-like ER kinase (PERK). Activation of these proteins is transduced to downstream targets, leading to enhanced capability to reduce unfolded or misfolded proteins, and de novo protein synthesis [11]. A prolonged or severe ER stress can activate pro-apoptotic proteins and result in cell death. It has been reported that ER stress is associated with numerous human diseases, including lung fibrosis, intestinal disease, and metabolic syndrome [11,12,15]. Also, activation of the UPR signaling has been described to be associated with lung injury in animals [5,16,17]. These data indicate that small molecules or compounds with abilities to modulate UPR signaling might be potentially therapeutic strategies to ameliorate heat stress-induced lung injury.

Naturally bioactive compounds have gained more attention due to their ability to regulate expression of genes implicated in ROS scavenge and inflammatory response. Ursolic acid is a pentacyclic triterpenoid compound widely distributed in natural plants, including vegetables, fruits, and Chinese medicinal herbs [18,19]. Studies with various animal models have reported that ursolic acid has antioxidative, anti-inflammatory, and antitumor effects [20], [21], [22], [23]–24]. Despite an increasing research on biological activities of ursolic acid and its derivatives in both in vivo and in vitro studies, molecular mechanisms responsible for the beneficial effects remain largely unknown. We have shown that ursolic acid supplementation alleviates heat stress-induced cell death of cardiomyocytes [25], indicating a beneficial effect of ursolic acid on cellular survival. In the present study, we tested the hypothesis that ursolic acid administration abolished heat stress-induced lung injury by repressing the UPR signaling in the lung tissues of mice.