Roles of pregnane X receptor in sodium arsenite-induced oxidative stress and inflammatory injury in human normal hepatocytes

Abstract

BACKGROUND: As the primary organ for arsenic metabolism in the body, the liver has become a focal point for research on the mechanisms of arsenic toxicity. OBJECTIVE: To investigate the role of pregnane X receptor in sodium arsenite-induced oxidative stress and inflammatory injury in human normal hepatocytes. METHODS: Human normal hepatocyte MIHA cells were exposed to 0 (control), 10, 20, 30 μmol/L sodium arsenite for 48 hours. Changes in cell morphology were observed. Cell viability was measured via the cell counting kit-8 assay. Intracellular reactive oxygen species levels were detected using fluorescence probe staining combined with a microplate reader. Malondialdehyde levels were measured by thiobarbituric acid method. Glutathione reductase activity was detected by NADPH method. Total superoxide dismutase activity was measured by WST-8 method. Levels of interleukin-6, interleukin-1β, and tumor necrosis factor-α in cell supernatant were detected by ELISA. mRNA expression of pregnane X receptor and cytochrome P450 3A4 enzyme was detected by qRT-PCR. Protein expression of pregnane X receptor, cytochrome P450 3A4, nuclear factor-κB p65, nuclear factor-κB p-p65, proliferating cell nuclear antigen, interleukin-6, interleukin-1β, tumor necrosis factor-α, nuclear factor-κB inhibitor protein α, cyclooxygenase-2, p-nuclear factor-κB inhibitor protein α, nuclear factor erythroid 2-related factor 2, Keap1, and p-nuclear factor erythroid 2-related factor 2 was detected by western blot. RESULTS AND CONCLUSION: Compared with the control group, cells in all sodium arsenite groups showed unclear cell membrane boundaries, reduced cytoplasm, decreased cell fusion rate, and widened intercellular spaces. Compared with the control group, intracellular reactive oxygen species and malondialdehyde levels were increased (P < 0.05), levels of interleukin-6, interleukin-1β, and tumor necrosis factor-α in cell supernatant were increased (P < 0.05), protein expression of p-nuclear factor-κB inhibitor protein α, nuclear factor-κB p-p65, nuclear factor-κB p65, tumor necrosis factor-α, and interleukin-1β were increased (P < 0.05), cell viability was decreased (P < 0.05), protein expression of proliferating cell nuclear antigen, nuclear factor erythroid 2-related factor 2, p-nuclear factor erythroid 2-related factor 2 and total superoxide dismutase activity were decreased (P < 0.05), and mRNA and protein expression of pregnane X receptor and cytochrome P450 3A4 enzyme were decreased (P < 0.05). Compared with the control group, glutathione reductase activity was decreased in 20 and 30 μmol/L sodium arsenite groups (P < 0.05), and protein expression of Keap1, interleukin-6, and cyclooxygenase-2 was increased (P < 0.05). These results indicate that sodium arsenite may induce oxidative stress and inflammatory injury in hepatocytes by downregulating pregnane X receptor expression, inhibiting the nuclear factor erythroid 2-related factor 2 antioxidant pathway, and activating the nuclear factor-κB inflammatory pathway, while also inhibiting the expression of the drug-metabolizing enzyme cytochrome P450 3A4.

1. Introduction

Arsenic is a metalloid widely distributed in water, soil, and atmospheric environment. When the human body is exposed to high arsenic environment, arsenic and its compounds can enter the body through respiratory tract, digestive tract, and skin, causing acute and chronic arsenic poisoning. In 2017, the International Agency for Research on Cancer (IARC) classified inorganic arsenic and its compounds as Group I carcinogens [1-2]. Arsenic-contaminated areas have been found in more than 20 countries [3], among which China, India, and Bangladesh are the most severely affected [4]. Arsenic poisoning is a systemic disease characterized by multi-organ and multi-system damage. The liver, as the main site for arsenic metabolism and methylation detoxification, is also one of the main target organs of arsenic toxicity [5]. Long-term chronic arsenic exposure can lead to arsenic accumulation in the liver, resulting in hepatomegaly, liver fibrosis, cirrhosis, and even liver cancer, seriously endangering human health [6]. At present, the pathogenesis of arsenic-induced liver disease has not been fully elucidated. Therefore, exploring the mechanism of arsenic-induced liver injury and finding new therapeutic targets is of great significance.

Pregnane X receptor (PXR) is a ligand-activated transcription factor mainly expressed in the liver. It regulates downstream gene expression by directly or indirectly binding to specific gene regions or crosstalking with other transcription factors, thereby participating in the regulation of drug clearance, inflammation, cell proliferation and differentiation, oxidative stress, and lipid metabolism [7-9]. Studies have found that when PXR is deficient, the expression of nuclear factor-κB (NF-κB) gene is enhanced, and the NF-κB signaling pathway is activated [10]; while the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) is downregulated, and the Nrf2 signaling pathway is inhibited, leading to increased oxidative stress and inflammatory response, and aggravated cell damage, indicating that PXR may regulate NF-κB and Nrf2 pathways to control the occurrence and development of diseases [11]. In view of its pleiotropic regulatory functions, PXR has become a research hotspot as a contributing factor in disease pathophysiology and a new therapeutic target. Our previous research found that sodium arsenite (NaAsO2) can activate the p53 signaling pathway to downregulate the expression of hepatocyte nuclear factor 4α, causing lipid metabolism disorders in hepatocytes [12-13]. Hepatocyte nuclear factor 4α