METHODS: The cytoprotective role of AEIA was measured on mouse hepatocytes by cell viability assay followed by Hoechst staining and flow cytometric assay. The effect on ROS production, lipid peroxidation, protein carbonylation, intracellular redox status were measured after incubating the hepatocytes with Pb-acetate (6.8 μM) along with AEIA (400 μg/ml). The effects on the expressions of apoptotic signal proteins were estimated by western blotting. The protective role of AEIA was measured by in vivo assay in mice. Haematological, serum biochemical, tissue redox status, Pb bioaccumulation and histological parameters were evaluated to estimate the protective role of AEIA (100 mg/kg) against Pb-acetate (5 mg/kg) intoxication.
RESULTS: Pb-acetate treated hepatocytes showed a gradual reduction of cell viability dose-dependently with an IC50 value of 6.8 μM. Pb-acetate treated hepatocytes exhibited significantly enhanced levels (p < 0.01) of ROS production, lipid peroxidation, protein carbonylation with concomitant depletion (p < 0.01) of antioxidant enzymes and GSH. However, AEIA treatment could significantly restore the aforementioned parameters in murine hepatocytes near to normalcy. Besides, AEIA significantly reversed (p < 0.05-0.01) the alterations of transcription levels of apoptotic proteins viz. Bcl 2, Bad, Cyt C, Apaf-1, cleaved caspases [caspase 3, caspase 8 and caspase 9], Fas and Bid. In in vivo bioassay, Pb-acetate treatment caused significantly high intracellular Pb burden and oxidative pressure in the kidney, liver, heart, brain and testes in mice. In addition, the haematological and serum biochemical factors were changed significantly in Pb-acetate-treated animals. AEIA treatment restored significantly the evaluated-parameters to the near-normal position.
CONCLUSION: The extract may offer the protective effect via counteracting with Pb mediated oxidative stress and/or promoting the elimination of Pb by chelating. The presence of substantial quantities of flavonoids, phenolics and saponins would be responsible for the overall protective effect.
OBJECTIVE: The antioxidant, cytotoxic, and protective effects of a series of synthesized 2- trifluoromethylquinazolines (2, 4, and 5) and quinazolinones (6-8) in lipopolysaccharide (LPS)- murine microglia (BV2) and hydrogen peroxide (H2O2)-mouse neuroblastoma-2a (N2a) cells were investigated.
METHOD: The antioxidant activity of synthesized compounds was evaluated with ABTS and DPPH assays. The cytotoxic activities were determined by MTS assay in BV2 and N2a cells. The production of nitric oxide (NO) in LPS-induced BV2 microglia cells was quantified.
RESULTS: The highest ABTS and DPPH scavenging activities were observed for compound 8 with 87.7% of ABTS scavenge percentage and 54.2% DPPH inhibition. All compounds were noncytotoxic in BV2 and N2a cells at 5 and 50 μg/mL. The compounds which showed the highest protective effects in LPS-induced BV2 and H2O2-induced N2a cells were 5 and 7. All tested compounds, except 4, also reduced NO production at concentrations of 50 μg/mL. The quinazolinone series 6-8 exhibited the highest percentage of NO reduction, ranging from 38 to 60%. Compounds 5 and 8 possess balanced antioxidant and protective properties against LPS- and H2O2-induced cell death, thus showing great potential to be developed into anti-inflammatory and neuroprotective agents.
CONCLUSION: Compounds 5 and 7 were able to protect the BV2 and N2a cells against LPS and H2O2 toxicity, respectively, at a low concentration (5 μg/mL). Compounds 6-8 showed potent reduction of NO production in BV2 cells.
Methods: NQC was synthesised and characterised using spectroscopic techniques. The compound was tested for its anti-inflammatory effect using Lipopolysaccharide from Escherichia coli (LPSEc) induced inflammation in mouse macrophages (RAW 264.7 cells). The effect of NQC on inflammatory cytokines was measured using enzyme-linked immune sorbent assay (ELISA). The Nrf2 activity of the compound NQC was determined using 'Keap1:Nrf2 Inhibitor Screening Assay Kit'. To obtain the insights on NQC's activity on Nrf2, molecular docking studies were performed using Schrödinger suite. The metabolic stability of NQC was determined using mouse, rat and human microsomes.
Results: NQC was found to be non-toxic at the dose of 50 µM on RAW 264.7 cells. NQC showed potent anti-inflammatory effect in an in vitro model of LPSEc stimulated murine macrophages (RAW 264.7 cells) with an IC50 value 26.13 ± 1.17 µM. NQC dose-dependently down-regulated the pro-inflammatory cytokines [interleukin (IL)-1β (13.27 ± 2.37 μM), IL-6 (10.13 ± 0.58 μM) and tumor necrosis factor (TNF)-α] (14.41 ± 1.83 μM); and inflammatory mediator, prostaglandin E2 (PGE2) with IC50 values, 15.23 ± 0.91 µM. Molecular docking studies confirmed the favourable binding of NQC at Kelch domain of Keap-1. It disrupts the Nrf2 interaction with kelch domain of keap 1 and its IC50 value was 4.21 ± 0.89 µM. The metabolic stability studies of NQC in human, rat and mouse liver microsomes revealed that it is quite stable with half-life values; 63.30 ± 1.73, 52.23 ± 0.81, 24.55 ± 1.13 min; microsomal intrinsic clearance values; 1.14 ± 0.31, 1.39 ± 0.87 and 2.96 ± 0.34 µL/min/g liver; respectively. It is observed that rat has comparable metabolic profile with human, thus, rat could be used as an in vivo model for prediction of pharmacokinetics and metabolism profiles of NQC in human.
Conclusion: NQC is a new class of NRF2 activator with potent in vitro anti-inflammatory activity and good metabolic stability.