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

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.

Alterations in Inflammatory Cytokines and Redox Homeostasis in LPS-Induced Pancreatic Beta-Cell Toxicity and Mitochondrial Stress: Protection by Azadirachtin

“…In our previous study on Rin-5F cells (John and Raza, 2021), we also showed that LPS treatment caused a marked increase in ROS production, which leads to DNA damage and apoptosis. Our present study has further confirmed the consequences of high ROS production by measuring LPO and oxidative stress-sensitive enzymes, SOD, and catalase.…”

“…Our recent study on Rin-5F cells ( John and Raza, 2021 ) showed LPS-induced caspase-3/8-dependent apoptosis followed by increased DNA fragmentation in LPS treated cells and AZD caused attenuation of the apoptotic damages by enhancing the autophagic repair process. Our present study has further demonstrated that intrinsic caspase-9, which is the initiator caspase of the apoptotic pathway in the mitochondria, was also activated by LPS treatment and AZD protected the cells against LPS–induced apoptosis ( Figure 1 ).…”

“…In addition, some of the cells were incubated with azadirachtin (AZD) (25 µM for 24 h), either alone or with LPS. Concentrations and time points for LPS and AZD used in the present study were based on literature search as well as our previous studies (Raza et al, 2014(Raza et al, , 2016John and Raza, 2021). After treatment, cells were harvested and homogenized in cold H-medium buffer (70 mM sucrose, 220 mM mannitol, 2.5 mM HEPES, 2 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride, pH7.4).…”

“…In our previous study, we observed LPS-induced caspase-mediated apoptosis in Rin-5F cells via the caspase-8 extrinsic pathway ( John and Raza, 2021 ). To further elucidate the involvement of the intrinsic pathway, Rin-5F cells were treated with LPS and/or AZD and caspase-9 activity was measured as per the manufacturer’s protocol.…”

“…Our recent study on LPS-induced insulin-secreting pancreatic Rin-5F cells showed increased oxidative stress and apoptosis. AZD treatment attenuated the ROS-dependent DNA damage, oxidative stress, and apoptosis, and enhanced the autophagy signaling ( John and Raza, 2021 ). Our present study further investigated the protection of LPS-induced redox stress, inflammatory responses, and mitochondrial dysfunction in Rin-5F cells by AZD.…”