Evaluating the Effect of Thymoquinone on Keap-1/Nrf-2 Signaling, Oxidative Stress, and Behavior in Rats

Summary

This protocol demonstrates how to assess the effects of thymoquinone on the Keap-1/Nrf-2 pathway, oxidative stress biomarkers, and behavioral outcomes in a rat model of subchronic deltamethrin exposure.

Abstract

Recent developments in pesticide exposure research have increased interest in antioxidant-based protective strategies. Thymoquinone (TQ), a plant-derived bioactive compound, has been reported to exert protective effects against various toxic agents. This study describes a protocol to evaluate the effects of TQ on oxidative stress parameters, the Keap-1/Nrf-2 signaling pathway, and behavioral outcomes in rats exposed to low-dose Deltamethrin (DTM). Here, 24 adult male Wistar Albino rats (250 ± 20 g) were randomly assigned to four groups (n = 6/group): control, TQ, DTM, and DTM+TQ. DTM (1.28 mg/kg) and TQ (10 mg/kg) were administered intragastrically for 30 days. Body weight was monitored throughout the study. Locomotor activity and anxiety-like behavior were assessed using the open field test on day 31, and depression-like behavior was evaluated using the forced swim test on day 32. At the end of the experimental period, plasma and brain tissues were collected for biochemical, histopathological, and molecular analyses. Oxidative stress markers, including malondialdehyde, total nitric oxide, glutathione, and sulfhydryl group levels, were measured in plasma and cerebral cortex samples. Kelch-like ECH-associated protein 1 (Keap-1) and Nuclear factor erythroid 2–related factor 2 (Nrf-2) expression levels were analyzed using Western blotting and immunohistochemistry. This protocol provides a comprehensive and reproducible approach for investigating pesticide-induced neurotoxicity and the modulatory effects of antioxidant compounds.

Introduction

Insecticides are widely used in agriculture and domestic environments to control pests; however, chronic exposure to low doses can result in environmental contamination and adverse health effects in both humans and animals. Such exposure, particularly through contaminated food and water, has been associated with an increased risk of neurological disorders and other systemic toxicities¹.

Pyrethroids have largely replaced organophosphorus insecticides due to stricter regulations on the latter and their relatively lower mammalian toxicity. DTM, a type II pyrethroid containing an α-cyano group, is among the most com....

Protocol

The Institutional Animal Experimentation Local Ethics Committee approved the experimental protocol under registration number 68429034/35. The total duration of the study was 32 days. Prior to the experiments, animals were acclimatized to standard laboratory conditions. All experimental procedures were conducted in strict accordance with institutional and international animal ethics guidelines. The experimental materials used in this protocol are listed in the Table of Materials.

NOTE: Chemical and biological wastes were disposed of in accordance with the institution’s laboratory safety and waste management guidelines.....

Results

Body weight measurements
Body weight was recorded on days 1, 10, 20, and 30. Rats in the C and TQ groups showed a progressive increase in body weight over the experimental period, with no significant difference between these groups on day 30. In contrast, rats exposed to DTM exhibited a significant reduction in body weight compared with controls. Co-administration of TQ attenuated DTM-associated weight loss. No mortality was observed during the study period (Figure 1).

Discussion

This study demonstrates that subchronic oral exposure to DTM induces consistent behavioral, biochemical, molecular, and histopathological alterations in rats and that TQ effectively mitigates these effects when administered concomitantly. By integrating standardized behavioral tests with oxidative stress profiling, pathway-level analysis of Keap-1/Nrf-2 signaling, and histopathological evaluation, the protocol provides a robust and reproducible framework for assessing pesticide-induced neurotoxicity and antioxidant-based.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5,5'-Dithiobis(2-nitrobenzoic acid)	Thermo Scientific	69-78-3
Benchtop centrifuge NF048	Nüve	43560
Beta_Actin	ThermoFisher Scientific, United States	7D2C10
BSA solution	Proteintech, United States)	66201-1-Ig
Butylated hydroxytoluene	Sigma Aldrich	128-37-0
Dijital Homojenizatör	Daihan Scientific	https://www.artlaboratuv arcihazlari.com/urun/dai han-hg-15d-dijital-homoje nizator-set-a?srsltid=Afm BOorx_az06ulcHpSzKZs uP8-47Za4BkvLOM22Xh p00tgwWhJ-86Vc
DTM	Bayer	TR01426058O
Hematoxylin–eosin	Bio Optica	W01030708
Hydrochloric acid	Merck	7647-01-0
Image J software program	NIH; Washington, U.S.A.	https://imagej.net/ij/
Immunohistochemical staining kit	Lab VisionTM UltraVisionTM Large Volume Detection System: anti-polyvalent	HRP, TA-125-HL
Leica Autocut 14051956472	Germany	https://www.leicabiosystems .com/histology-equipment/mi crotomes/histocore-autocut/
Light microscope	Nikon, Eclipse	Ni-U, 940728
N-(1-Naphthyl)ethylenediamine	Thermo Scientific	1465-25-4
Nano Microplate Reader	BMG LABTECH	SPECTROstar Nano
phosphate-buffered saline	Merck	P4417
Primary antibodies Keap-1	Proteintech	Cat No. 10503-2-AP, 1:200
Primary antibodies Nrf-2	Proteintech	Cat No. 16396-1-AP, 1/100
Qubit Protein BR Assay Kit	ThermoFisher Scientific-INVITROGEN	Q33211
sodium citrate	Merck	03-04-6132
Sodium Dodecyl Sulfate	Thermo Scientific	151-21-3
sodium hydroxide	Merck	1310-73-2
Sodium nitrate	Merck	7631-99-4
Streptavidin Peroxidase	Thermo Scientific	SHRP248-B
sulfanilamide	Thermo Scientific	63-74-1
thiobarbituric acid	Merck	504-17-6
TQ	CAYMAN	490-91-5
Tricarballylic acid, 99%	Thermo Scientific Chemicals	139360500
Trichloroacetic acid	Thermo Scientific Chemicals	76-03-9
Tris-HCl	Thermo Scientific	1185-53-1
Vanadium(III) chloride	Sigma Aldrich	7718-98-1
West Pico PLUS Chemiluminescent Substrate and the iBright 750	Thermo Fisher Scientific system	https://www.clinxsci.com/product /mini_chemiluminescence_imagi ng_system?gad_source=1&gad _campaignid=16224184202&gbr aid=0AAAAAoN6UOOtoB8UD0d eumBAMD8Xi-1l-&gclid=CjwKCAj wgeLHBhBuEiwAL5gNEXtK2CNS LS1GqllMuNgkrFYDaW7yK585njQ jFja485Fkwj9lRUEKDBoCgLoQA vD_BwE
Zinc sulfate solution	Merck	7733-02-0