Azadirachtin Attenuates Lipopolysaccharide-Induced ROS Production, DNA Damage, and Apoptosis by Regulating JNK/Akt and AMPK/mTOR-Dependent Pathways in Rin-5F Pancreatic Beta Cells.

Annie John,Haider Raza

doi:10.3390/biomedicines9121943

Abstract

Pancreatic inflammation and the resulting cellular responses have been implicated in pancreatitis, diabetes, and pancreatic cancer. Inflammatory responses due to the bacterial endotoxin, lipopolysaccharide (LPS), have been demonstrated to alter cellular metabolism, autophagy, apoptosis, and cell proliferation in different cell populations, and hence increases the risks for organ toxicity including cancer. The exact molecular mechanism is however not clear. In the present study, we investigated the role and mechanism of an antioxidant, azadirachtin (AZD), a limonoid extracted from the neem tree (Azadirachta indica), against LPS-induced oxidative stress in the pancreatic β-cell line, Rin-5F. We demonstrated that cells treated with LPS (1 µg/mL for 24 h) showed increased reactive oxygen species (ROS) production, DNA damage, cell cycle arrest, and apoptosis. Our results also showed that LPS induced alterations in the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathways, suppressing autophagy and augmenting apoptosis. Treatment with Azadirachtin (25 µM for 24 h), on the other hand, rendered some degree of protection to the pancreatic cells from apoptosis by inducing the autophagy signals required for cell survival. These results may have significance in elucidating the mechanisms of pancreatic β-cell survival and death by balancing the molecular communication between autophagy and apoptosis under inflammatory and pathological conditions.

Highlights

Lipopolysaccharide (LPS), a bacterial endotoxin, induces septic shock, which may result in multiple organ dysfunction
Cells treated with 1 μg/mL LPS exhibited around 15% late apoptotic or necrotic cell death, whereas treatment with 25 μM AZD for 24 h alone or in combination with LPS resulted in less than 10% late apoptosis. These results suggest some degree of protection by AZD on LPS-induced cell death
To confirm the cell cycle arrest observed by flow cytometry, we investigated the exTo confirm the cell cycle arrest observed by flow cytometry, we investigated the pression of p53 protein, known to suppress cell cycle progression and cause apoptosis and expression of p53 protein, known to suppress cell cycle progression and cause apoptosis cell death

Summary

Introduction

Lipopolysaccharide (LPS), a bacterial endotoxin, induces septic shock, which may result in multiple organ dysfunction. The release of inflammatory mediators, such as tumor necrosis factor (TNF-α) and interleukins are considered to be the main triggers inducing cellular response and organ dysfunction [1]. These pro-inflammatory mediators are responsible for initiating the cascade of secondary factors and signaling of autophagy, apoptosis, or cellular proliferation [2,3,4,5,6]. A high-fat diet can cause alterations in gut microbiota and induce up to three-fold lipopolysaccharide production, contributing to increased oxidative and ER stress, glucotoxicity, and lipotoxicity-associated complications, resulting in decreased pancreatic functions and triggering type 2 diabetes (T2D) development [9,10,11]. LPS-induced pancreatitis and β-cell mass destruction have been reported to be due to increased reactive nitrogen species and inflammatory signaling [12,13,14]

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