Gallic acid attenuates LPS-induced inflammation in Caco-2 cells by suppressing the activation of the NF-κB/MAPK signaling pathway

Abstract

Inflammatory bowel disease (IBD) is a chronic inflammatory disease characterized by intestinal barrier dysfunction, inflammatory synergistic effects and excessive tissue injury. Gallic acid (GA) is renowned for its remarkable biological activity, encompassing anti-inflammatory and antioxidant properties. However, the underlying mechanisms by which GA protects against intestinal inflammation have not been fully elucidated. The aim of this study is to investigate the effect of GA on the inflammation of a lipopolysaccharide (LPS)-stimulated human colon carcinoma cell line (Caco-2) and on the intestinal barrier dysfunction, and explore the underlying molecular mechanism involved. Our findings demonstrate that 5 μg/mL GA restores the downregulation of the mRNA and protein levels of Claudin-1, Occludin, and ZO-1 and decreases the expressions of inflammatory factors such as IL-6, IL-1β and TNF-α induced by LPS. In addition, GA exhibits a protective effect by reducing the LPS-enhanced early and late apoptotic ratios, downregulating the mRNA levels of pro-apoptotic factors (Bax, Bad, Caspase-3, Caspase-8, and Caspase-9), and upregulating the mRNA levels of anti-apoptotic factor Bcl-2 in Caco-2 cells. GA also reduces the levels of reactive oxygen species increased by LPS and restores the activity of antioxidant enzymes, namely, superoxide dismutase and catalase, as well as the level of glutathione. More importantly, GA exerts its anti-inflammatory effects by inhibiting the LPS-induced phosphorylation of key signaling molecules in the NF-κB/MAPK pathway, including p65, IκB-α, p38, JNK, and ERK, in Caco-2 cells. Overall, our findings show that GA increases the expressions of tight junction proteins, reduces cell apoptosis, relieves oxidative stress and suppresses the activation of the NF-κB/MAPK pathway to reduce LPS-induced intestinal inflammation in Caco-2 cells, indicating that GA has potential as a therapeutic agent for intestinal inflammation.

1. Introduction

Inflammatory bowel disease (IBD), encompassing Crohn’s disease (CD) and ulcerative colitis (UC), manifests chronic inflammation and dysplasia of epithelial barrier function [1,2]. An increasing incidence of IBD has been reported worldwide. Due to shifts in diet and lifestyle, the prevalence of IBD has been steadily increasing, especially in developing countries, in recent years [3,4]. The pathogenesis of IBD may be related to the interaction of genetic, immune, infectious and psychiatric factors [5,6]. The damage mediated by inflammation disrupts tight junction (TJ) proteins and increases paracellular permeability to microbes and antigens [7,8]. Long-term damage to TJ proteins may lead to IBD [9,10]. Consequently, remission of IBD necessitates a decrease in inflammatory responses and reinforcement of intestinal barrier integrity [11].

In vivo animal models are too complex to control many gut processes alone. Thus, new treatments for IBD have been developed in a variety of cell models to simulate intestinal inflammation. Caco-2 cells have emerged as valuable tools for studying intestinal diseases [12,13]. Various drugs, such as 5-aminosalicylic acid drugs, steroids and immunosuppressants [14], have been used to treat IBD. However, the clinical treatment efficacy is unsatisfactory, and the side effects of long-term use are serious [14]. Therefore, there is an urgent need for some mild and effective treatment alternatives. Many studies have reported that plant phenols, including sinapic acid, have attracted increasing attention due to their antioxidant and anti-inflammatory properties and minimal harm to human health [15‒19]. These compounds act as antioxidants that inhibit the oxidation of DNA, proteins, lipids and enzymes linked to the production of free radicals [20‒22].

Gallic acid (GA), a plant polyphenol, is a naturally produced secondary metabolite that is present in a variety of fruits, plants, vegetables and nuts, such as strawberries, green tea and oak bark [23,24]. GA has a low molecular weight and triphenolic structure, endowing it with potent anti-inflammatory and antioxidant capabilities [25]. The phenol hydroxyl group of GA can eliminate reactive oxygen species (ROS) and interrupt the cycle of new free radical formation. GA has anti-inflammatory effects by reducing proinflammatory mediators, inhibiting the expressions of nuclear transcription factors and downregulating downstream inflammatory targets [26,27]. In addition to its anti-inflammatory effects, GA has been shown to have pharmacological effects on tumors, diabetes, and obesity [27‒29]. Despite its proven efficacy in treating various inflammation-related diseases, the effects and mechanism of action of GA in IBD remain unexplored. Therefore, in this study we used Caco-2 cells as an in vitro model to explore the effects of GA on the inflammatory response induced by lipopolysaccharide (LPS) and to investigate the possible underlying mechanisms involved.