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

Abstract

Inflammation and redox imbalance are hallmarks of cancer, diabetes, and other degenerative disorders. Pathophysiological response to these disorders leads to oxidative stress and mitochondrial dysfunction by alterations and reprogramming in cellular signaling and metabolism. Pancreatic beta cells are very sensitive to the inflammatory and altered nutrient signals and hence play a crucial role in diabetes and cancer. In this study, we treated insulin-secreting pancreatic beta cells, Rin-5F, with the bacterial endotoxin, LPS (1 μg/ml) to induce an inflammatory response in vitro and then treated the cells with a known anti-inflammatory, anticancer and antioxidant phytochemical, azadirachtin (AZD, 25 µM for 24 h). Our results demonstrated lipid peroxidation and nitric oxide production causing increased nitro/oxidative stress and alterations in the activities of anti-oxidant enzymes, superoxide dismutase and catalase after LPS treatment. Pro-inflammatory responses caused by translocation of nuclear factor kappa B and release of inflammatory cytokines were also observed. These changes were accompanied by GSH-dependent redox imbalance and alterations in mitochondrial membrane potential and respiratory complexes enzyme activities leading to mitochondrial respiratory dysfunction, reduced ATP synthesis, and intrinsic caspase-9 mediated apoptosis. Caspase-9 was activated due to alterations in Bcl-2 and Bax proteins and release of cytochrome c into the cytosol. The activities of oxidative stress-sensitive mitochondrial matrix enzymes, aconitase, and glutamate dehydrogenase were also inhibited. Treatment with AZD showed beneficial effects on the recovery of antioxidant enzymes, inflammatory responses, and mitochondrial functions. GSH-dependent redox homeostasis also recovered after the treatment with AZD. This study may help in better understanding the etiology and pathogenesis of inflammation-induced disorders in pancreatic beta cells to better manage therapeutic strategies.

Azadirachtin Attenuates Carcinogen Benzo(a) Pyrene-Induced DNA Damage, Cell Cycle Arrest, Apoptosis, Inflammatory, Metabolic, and Oxidative Stress in HepG2 Cells

“…Cells in the control group were treated with the vehicle alone. Cells in the B(a)P group were treated with 25 µM B(a)P for 24 h. In the AZD+B(a)P group, cells were treated with 25 µM AZD for 24 h and 25 µM B(a)P for 24 h. In the AZD group, cells were treated with 25 µM AZD alone for 24 h. The dose and time for AZD and B(a)P treatments were based on the cytotoxicity studies performed as well as our previous studies [20,21]. After treatment with AZD and/or B(a)P, the cells were trypsinized, washed and homogenized in a cold H-medium buffer, pH 7.4, containing 70 mM sucrose, 220 mM mannitol, 2.5 mM HEPES, 2 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride.…”

“…After treatment with AZD and/or B(a)P, the cells were trypsinized, washed and homogenized in a cold H-medium buffer, pH 7.4, containing 70 mM sucrose, 220 mM mannitol, 2.5 mM HEPES, 2 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride. Nuclear, mitochondrial, and post-mitochondrial fractions were isolated via sub-cellular fractionation and protein concentrations were measured as described previously [20,21,29].…”

“…In brief, the cell lysates from control HepG2 and AZD and/or B(a)P-treated cells were incubated with p-nitro aniline-conjugated caspase-specific peptide substrates, DEVD IETD, and LEHD, respectively. The cleavage of the peptide by the respective caspases resulted in the release of the chromophore, which was measured colorimetrically at a wavelength of 405 nm as described before [20,21,29].…”

“…These researchers have shown that different parts of the neem tree, including leaves, seed kernel, seed husk, etc., have been explored to evaluate its potent anti-microbial, anti-pyretic, anti-oxidant, and other medicinal properties. Our previous studies have also shown that AZD treatment could enhance cellular survival under conditions of extreme inflammatory and oxidative stress through an interconnecting network of cell-signaling proteins [20,21]. A recent review by Nagini et al [22] has indicated that neem limonoids including AZD demonstrate anti-carcinogenic effects in different cancer cell lines as well as in animal models by inhibiting cell proliferation, and inducing drug sensitivity, apoptosis, and anti-inflammatory responses.…”

“…Since the liver is the primary target organ for B(a)P-induced toxicities, we chose HepG2 cells as the in vitro model for our present study. In our previous studies, we reported that AZD protects pancreatic β-cells from oxidative and inflammatory stress by regulating cell proliferation and DNA damage, suppressing NF-κB signaling, and restoring redox homeostasis and mitochondrial bioenergetics [20,21]. In the present study, we aim to investigate the defense mechanism of AZD in B(a)P-induced redox and oxidative stress, DNA damage, cell cycle arrest, apoptosis, inflammation, and metabolic and mitochondrial stress, and to identify the regulatory cell signaling markers in HepG2 cells.…”