Orally administered Thymoquinone mitigates cypermethrin-induced dentate gyrus oxidative stress, preventing GABAergic interneuron degeneration and memory impairment in rats via the Nrf2/ARE pathway.

Orally administered Thymoquinone mitigates cypermethrin-induced dentate gyrus oxidative stress, preventing GABAergic interneuron degeneration and memory impairment in rats via the Nrf2/ARE pathway. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Orally administered Thymoquinone mitigates cypermethrin-induced dentate gyrus oxidative stress, preventing GABAergic interneuron degeneration and memory impairment in rats via the Nrf2/ARE pathway. Abubakar Lekan Imam, Akeem Ayodeji Okesina, Fatimo Ajoke Sulamon, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4130260/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Sep, 2024 Read the published version in BMC Neuroscience → Version 1 posted 12 You are reading this latest preprint version Abstract Background Exposure to chemical toxins, including insecticides, has harmful effects on body organs such as the brain. This study examined the neuroprotective of thymoquinone on the cypermethrin's harmful effects on the histoarchitecture of the dentate gyrus as well as motor deficit. Methods Forty adult male rats (180-200g) were randomly divided into 5 groups (n = 8 per group). Groups I, II, III, and IV received oral administration of 0.5 ml of phosphate buffered saline, 20 mg/kg of cypermethrin, 10 mg/kg of thymoquinone, 20 mg/kg cypermethrin plus 5 mg/kg of thymoquinone, and 20 mg/kg of cypermethrin plus 10 mg/kg of thymoquinone for 14 days respectively. The novel Object recognition test assesses intermediate-term memory at days 14 and 21 of the experiment. At the end of these treatments, the animals were euthanized and taken for cytoarchitectural analysis and immunohistochemical studies. Result The study shows that thymoquinone at 5 and 10 mg/kg improved Novelty preference and discrimination index. Thymoquinone enhanced Nissl body integrity, increased GABBAergic interneuron expression, nuclear factor erythroid 2-derived factor 2, and enhanced Bcl-2 expression in the dentate gyrus. It also improved the concentration of nuclear factor erythroid 2-derived factor 2, increased the activities of superoxide dismutase and glutathione, and decreased the concentration of malondialdehyde level against cypermethrin-induced neurotoxicity. Conclusion thymoquinone could be a therapeutic agent against cypermethrin poisoning. Cypermethrin thymoquinone cresyl fast violet GABBAergic interneuron dentate gyrus Figures Figure 1 Figure 2 Figure 3 Background Oxidative stress arises from an imbalance between reactive oxygen species (ROS) and the antioxidant defense system. ROS can lead to covalent oxidative modifications, such as ribonucleic acid (RNA) oxidation, and induce mutations in mitochondrial DNA (mtDNA), thereby destabilizing nucleic acids [ 1 , 2 ]. These modifications may result in cellular dysfunction and apoptosis. The mitochondrial-dependent caspase pathway is crucial in apoptosis [ 3 ]. Stimulation of this cascade releases proapoptotic factors, including cytochrome c (Cyc), activating caspase-9 and caspase-3, ultimately triggering cellular apoptosis [ 4 , 5 ]. Hence, antioxidant pathways that mitigate oxidative damage may exhibit neuroprotective effects [ 6 ]. Persistent exposure to pesticides, such as pyrethroids, adversely affects various physiological functions [ 7 ]. For example; long-term exposure can disrupt the functioning of different organs, posing serious health risks [ 8 , 9 ]. Nigeria has witnessed numerous cases of food poisoning due to pesticides, resulting in significant fatalities and economic losses [ 10 ]. Pyrethroids, widely used in agriculture and household insect control, have been detected in a large portion of the population [ 11 ]. Despite their broad application, pyrethroids exhibit adverse effects, including neurobehavioral effects and disruption of critical molecular targets in the nervous system [ 12 , 13 ]. Cypermethrin, a common pyrethroid, crosses the blood-brain barrier, inducing oxidative stress and apoptotic cell death [ 14 , 15 ]. Medicinal plants, due to their diverse chemical constituents, offer a sustainable therapeutic approach against chemical toxins. Thymoquinone (TQ), derived from Nigella sativa L., possesses antioxidant and anti-inflammatory properties [ 16 , 17 ]. TQ has shown neuroprotective effects in various models of brain injury and neurodegenerative diseases by inhibiting lipid peroxidation and apoptosis [ 18 , 19 ]. Its antioxidant effects are mediated through the nuclear factor erythroid 2-related factor 2 (NRF2) pathway, which regulates cellular defense mechanisms against oxidative stress [ 20 , 21 ]. Activation of NRF2 induces the expression of antioxidative and detoxifying enzymes, crucial for cellular function [ 22 ]. Dysregulation of NRF2/ARE signaling is implicated in neurodegenerative disorders [ 23 ]. Given the increasing incidence of pesticide-induced food poisoning, there is a need for effective antidotes with shared mechanisms of action. This study aims to evaluate the efficacy of thymoquinone against cypermethrin-induced neurotoxicity, focusing on GABAergic interneuron disruption, dentate gyrus cytoarchitectural disorganization, and oxidative stress-induced cell damage. METHODS Experimental Design The experimental design involved the use of 40 adult male Wistar rats (180–200 g). Thymoquinone was obtained from MedChemEpress (MCE) USA (Cat No: HY-d0803) Cypermethrin 10% EC product was sourced from Yubaili Agrotec (ACEC20L068) and NAFDAC No: A5-0108 was obtained from Ibukun Oluwa Agrochemical Distop. Ilorin, Nigeria. The rats were housed in the animal holding facility of the Faculty of Basic Medical Sciences, College of Health Sciences, University of Ilorin, under natural day-night cycles, with standard chow diet and water ad libitum. The rats were randomly divided into 5 groups (n = 8 per group). Groups I, II, III, and IV received oral administration of 0.5 ml of phosphate buffered saline, 20 mg/kg of cypermethrin, 10 mg/kg of thymoquinone, 20 mg/kg cypermethrin plus 5 mg/kg of thymoquinone, and 20 mg/kg of cypermethrin plus 10 mg/kg of thymoquinone for 14 days respectively. Motor behavior was assessed on the 14th day of the experiment. Behavioural Evaluation Intermediate memory recognition of the experimental rats following exposure to cypermethrin and thymoquinone was assessed using the novel object recognition (NOR) paradigm. The test apparatus is made from plywood measuring 100 cm by 100 cm with walls that are 50 cm high. Novelty preference and Discriminatory index were evaluated [ 24 ]. Twenty-four hours after the behavioral study, the animals were euthanized using 20 mg/kg bw ketamine intramuscularly, and brain tissue was excised and processed for histological, immunohistological, and biochemical analysis. Tissue collection After perfusion has been completed, the whole brain tissues were excised and were post-fixed in 4% paraformaldehyde overnight. The whole hippocampal CA regions were excised and equilibrated in 30% sucrose solution, before histological and immunohistochemical analyses. The sections were taken at 2 µm on paraffin wax embedded tissue blocks and mounted on a glass slide Histological Analysis and Immunohistochemistry The hematoxylin and eosin (H and E) staining technique was used to demonstrate the general histo-architecture of the cells; and to show the location of the normal or abnormal nucleus of the hippocampal cells. Cresyl violet: This technique was used to demonstrate Nissl bodies (endoplasmic reticulum and ribosomes) in the cells; and to show normal or abnormal protein synthesis in the cytoplasm of the hippocampus. For immunochemistry: Nuclear factor erythroid 2-related factor 2 (Nrf2), Parvalbumin, and B-cell lymphoma 2 (Bcl2) (human monoclonal; Elisa and microarray) were used to understand their roles in oxidative stress response, neuronal survival, and apoptosis regulation within dentate gyrus. The avidin-biotin complex method was used. The antibody dilution factor used was 1:100 for all the antibody markers. The processed tissues were sectioned at two microns on the rotary microtome and placed on a hotplate at 90°C for at least 40 minutes. Image J software cell counter was used for counting the immunopositive cells for Nrf2, Parvalbumin, and Bcl2 in the dentate gyrus. Biochemical Investigation After the tissues were collected and homogenized, the homogenates were collected in a 5ml plain bottle and centrifuged for 10 minutes at 5000rpm using a centrifuge. The supernatant was carefully decanted and stored at -4°C for enzymatic assays of superoxide dismutase (SOD) activity [ 25 ], glutathione (GSH) concentration [ 26 ], malondialdehyde (MDA) [ 25 ] concentration and nuclear factor erythroid 2-derived factor 2 (NrF2) a product of Elabscience Biotechnology Inc.USA (E-EL-R0673) method and absorbance was read using microplate reader. A four-parameter logistic curve (4PL-curve) was plotted and values for the samples were extrapolated using GraphPad Prism 8.0. Statistical Analysis Data from the behavioural, biochemical assays and immunopositiv cell count were analysed using one-way analysis of variance (ANOVA) and subjected to post hoc Bonferroni’s multiple comparison test. The results were expressed as mean ± SEM. Statistical analyses were performed using Graphpad Prism software (version 8.0.2). Values of p ≤ 0.05 were considered statistically significant. RESULTS Thymoquinne Restore Inter-mediate related behaviours following cypermethrin exposure The novelty preference of Wistar rats in CYM group for new object in novel object recognition test was significantly low (32.70 ± 3.93) compared to PBS control group at p < 0.05, compared to CYM group, the TQ group and the CYM-LHQ group showed higher preference for new object which was significant at p < 0.05. However, the CYM-10mgTHQ group showed higher preference than CYM group with no significant at p < 0.05 Fig. 1B. The discrimination index was significantly reduced in CYM group (-0.34 ± 0.08) compared to PBS group (0.47 ± 0.10) and THQ group (0.81 ± 0.11) at p < 0.05. Groups CYM-LTHQ and CYM-HHQ showed higher discrimination index compared to CYM group but not significant at p < 0.05 (Table 1 ). Table 1 Novelty preference and Discrimination Index of rats following cypermethrin and thymoquinone exposure Groups N = 5 Novelty Preference (%) Discrimination index PBS 77.60 ± 3.82 0.28 ± 0.24 CYM 39.30 ± 7.00* -0.20 ± 0.15 TQ 90.30 ± 5.41** 0.81 ± 0.11** CYM-LTQ 65.00 ± 11.20 0.44 ± 0.09 CYM-HTQ 46.00 ± 12.00 0.23 ± 0.06 PBS = phosphate buffered saline, CYM = cypermethrin, TQ = thymoquinone, CYM-LTQ = cypermethrin followed by low dose thymoquinone and CYM-HTQ = cypermethrin followed by High dose thymoquinone. singleasterisk (*) indicates significant (p < 0.05) compared to PBS, Doubleasterisk (**) indicates significant (p < 0.05) compared to CYM Oxidative Stress Biomarker The concentration of Nrf2 in the CYM group was significantly reduced p < 0.05 when compared to both the PBS control and the other experimental groups (Fig. 1A). Moreover, a significant reduction p < 0.05 in the SOD activity was observed in the CYM-exposed group, relative to the PBS control group, while all the other experimental groups showed a significant increase in the SOD activities p < 0.05 when compared to the CYM group (Fig. 1B). However, SOD activities in the TQ, CYM-LTQ and CYM-HTQ groups were not significantly lower compared to the PBS control (Fig. 1B). In addition, the GSH concentration was significantly reduced (p < 0.05) in the CYM group relative to the PBS group (Fig. 1C). Subsequent treatment with LTQ and HTQ led to a significant increase in the GSH levels (p < 0.05) when compared to the CYM-only group (Fig. 1C). Regarding the lipid peroxidation marker MDA, its level was notably higher (p < 0.05) in the CYM group compared to the PBS group (Fig. 1D). While MDA levels were lower in other experimental groups, significance (p < 0.05) was only observed in the CYM-LTQ group compared to the CYM group (Fig. 1D). Histochemical examination of the dentate gyrus revealed chromatolytic-like alterations in rats exposed to CYM. This was characterized by the disrupted integrity of Nissl granules, along with distortions in the shape and organization of granule cells. Additionally, numerous pyknotic and vacuolated cells were observed as a consequence of CYM exposure. Thymoquinone demonstrated a mitigating effect against CYM-induced toxicity, this was observed as a result of a reduction in the extent of neurodegenerative-like changes. Specifically, there was an improvement in Nissl body integrity, as well as the enhancements observed in the cellular shape and arrangement in the rats exposed to thymoquinone following CYM neurotoxicity (Fig. 2 ). The immunohistochemical assessment of the dentate gyrus utilizing anti-Nrf2, anti-Parvalbumin (Parv), and anti-Bcl2 antibodies revealed diminished expression of Nrf2, Parv, and Bcl2 positive cells in the dentate gyrus of rats exposed to CYM (Fig. 3 ). Conversely, post-treatment with thymoquinone exhibited a notable enhancement in the expression of Nrf2, Parv, and Bcl2 immunopositive cells in the dentate gyrus (Fig. 3 ). Quantification of immuno-positive cell counts using ImageJ software demonstrated a significantly higher (p < 0.05) Nrf2 cell count in the dentate gyrus of PBS control rats compared to those exposed to CYM (Fig. 3A1 & 2). Thymoquinone administration led to a significant increase (p < 0.05) in Nrf2 immunopositive cells in the CYM-LTQ and CYM-HTQ groups compared to CYM-exposed rats (Fig. 3A1 & 2). Similarly, there was a significantly higher (p < 0.05) count of Parvalbumin positive cells in the dentate gyrus of PBS control rats compared to CYM-exposed rats (Fig. 3B1 & 2). Thymoquinone significantly (p < 0.05) increased the number of Parvalbumin cells in the CYM-LTQ and CYM-HTQ groups compared to CYM-exposed rats (Fig. 3B1 & 2). Moreover, the cell count of the anti-apoptotic protein Bcl2 was higher in the PBS control group than in CYM-exposed rats (Fig. 3C1 & 2). Administration of thymoquinone resulted in a higher Bcl2 count in the CYM-LTQ and CYM-HTQ groups compared to CYM-exposed rats (Fig. 3C1 & 2). DISCUSSION Continuous application of pesticides and other agrochemicals, driven by the need to increase food production and prevent pest and insect-induced crop damages, has led to increased exposure to the harmful effects of these chemicals, including pyrethroid insecticides, due to their residual accumulation in crops, fruits, and vegetables. When humans and other animals are exposed to these chemicals, they can induce toxicity through mechanisms involving mitochondrial dysfunction, oxidative stress, and inflammation. This toxicity can manifest as movement disorders, loss of cognition, or a combination of both. This study demonstrates that thymoquinone increases the activities of antioxidant enzymes, akin to its parent molecule Nigella sativa oil [ 27 , 28 ], thereby preventing lipid peroxidation and preserving dentate gyrus architecture, ultimately enhancing memory function against CYM toxicity. In this study, cypermethrin caused a reduction in the expression of the Nuclear factor erythroid 2-related factor 2 (Nrf2), a regulatory protein responsible for initiating and expressing the antioxidant system. The reduced concentration and low expression of Nrf2 cells in the dentate gyrus of CYM-exposed rats are undoubtedly responsible for the reduction in the activities of the antioxidant enzymes SOD and GSH, leading to oxidative stress as indicated by the high level of MDA in CYM-exposed rats. Due to the presence of high unsaturated fatty acids, the brain is especially susceptible to oxidative stress, which causes membrane lipid peroxidation and disrupts the normal organizational structure of brain cells, as observed in the dentate gyrus of CYM-exposed rats. Cypermethrin exposure caused neuronal damage, impaired Nissl body integrity, and induced chromatolytic-like changes in the dentate gyrus due to its oxidative stress. It was observed that continuous CYM exposure not only disrupted neuronal shapes in the dentate gyrus but also induced Nrf2 expression. Since appropriate activation of Nrf2 and its nuclear translocation establishes the Nrf2/ARE complex and subsequently boosts the expression and synthesis of antioxidant enzymes, the decreased level of Nrf2 observed contributes to the lower activity of antioxidant enzymes SOD and GSH, which encourages further oxidative stress damage and raises the level of MDA. Cypermethrin decreased the level and activity of antioxidant enzymes like SOD, GSH, and catalase (CAT) in cypermethrin-induced toxicity in the Wistar rat model of Parkinson's disease and peripheral blood. The findings of this study are indeed strengthened by earlier studies by [ 29 – 31 ], which reported an excessive increase in the level of MDA and reduced antioxidant capacity of SOD, CAT, GSH, and GPx, leading to increased lipid peroxidation in the peripheral blood and in the nigrostriatum of cypermethrin-exposed rats. Intervention with thymoquinone was observed to reactivate Nrf2, as shown by the high expression of Nrf2 immunopositive cells. This increased nuclear availability of Nrf2 leads to the Nrf2/ARE complex, thereby stimulating the production and expression of antioxidant enzymes and resulting in high SOD and GSH activity, as reported in this study, and reduced MDA levels, indicating a low level of lipid peroxidation. This finding is consistent with an earlier study by [ 27 ], who found that the parent plant of thymoquinone, black seed oil, increased total antioxidant capacity and GSH while decreasing total ROS levels in rats exposed to Dichlorvos. The findings of this study are also strengthened by the study of Kanter, who reported enhancements in hepatic and pancreatic antioxidant capacities of catalase and GSH following Nigella sativa against STZ-induced diabetes in rats [ 32 ]. Apoptosis is characterized by morphological changes in cells such as nuclear pyknosis, DNA fragmentation, and chromatin condensation, cytoskeleton destruction, membrane blebbing, and eventually the formation of membrane apoptotic bodies that are phagocytosed by macrophages and other cells without inducing an inflammatory response [ 33 ]. Continuous exposure to environmental toxins frequently causes apoptosis in cells [ 34 ] Anti-Bcl-2-stained dentate gyrus had a low expression of Bcl-2 positive cells due to cypermethrin exposure. Cypermethrin, like other pyrethroids, caused apoptosis in the rat brain by producing ROS and cytotoxins. Cypermethrin also induced apoptosis via mitochondrial damage, cytochrome c release, and activation of caspases 3 and 9, which are involved in both extrinsic and intrinsic apoptosis pathways [ 35 – 37 ]. When Bcl-2 and other anti-apoptotic proteins are cleaved by caspases following the initiation of apoptosis, their anti-apoptotic action is frequently converted to pro-apoptotic action [ 33 ]. The findings of this study are similar to the report of the previous study where a type 2 pyrethroid, deltamethrin, following its exposure in rats, induced apoptosis by increasing the level of Bax, caspase-3, cytochrome c, and decreasing the expression of Bcl-2 pro-survival proteins [ 38 , 39 ]. Thymoquinone exhibits anti-apoptotic effects, as administration of thymoquinone brings about a marked increase in the expression of Bcl-2 immunopositive cells in the hippocampal dentate gyrus of the experimental rats. Bcl-2, as a pro-survival protein, has a hydrophilic carboxyl-terminal domain that is linked to the mitochondria outer membrane and helps preserve mitochondrial integrity, preventing unnecessary cytochrome c release and caspase activation [ 33 ]. Bcl-2 prevents Bax and other pro-apoptotic genes from oligomerizing, which stimulates the release of apoptogenic molecules from the mitochondria. Apart from inhibiting Bax oligomerization, Bcl-2 directly binds and inactivates Bax, blocks cytochrome c release, and thus inhibits adaptor molecule APAF-1 and caspase-9 activation, thereby preventing caspase cascade activation [ 33 , 40 ]. In accordance with the findings of this study, [ 41 ] showed that thymoquinone, in concentrations of 10 M and 20 M, prevented arsenic-induced neurotoxicity, apoptosis, and cytotoxicity by either decreasing the levels of Bax or increasing the level of Bcl-2. Also, in agreement with the data of this study, a previous study revealed that thymoquinone administration decreased p53 and Bcl-2 gene expression but increased BAD gene expression in MCF-7 cells; however, it increased the expression of Bcl-2 gene and p53 gene but decreased Bax/BAD gene expression in non-cancer HEK293 cells [ 42 ] In the dentate gyrus, basket cells constitute the GABAergic neurons in the granule layer with the receptors localized in the molecular layer. Reduced levels of parvalbumin-positive cells in the dentate gyrus of cypermethrin-exposed rats indicate that CYM inhibits GABAergic interneurons. GABAergic interneurons constitute the inhibitory neurons in the CNS that are vital for modulating various physiological activities [ 43 ]. Reduced GABAergic interneuron expression due to CYM exposure interferes with the activity of GABAergic interneurons and disrupts excitatory and inhibitory balance in the brain. Previous studies have shown that CYM hinders the opening of the voltage-gated chloride channels and inhibits the GABA-dependent uptake of chloride ions, resulting in hyper-excitation of neuronal cells and leading to changes in the delayed rectifier voltage-dependent potassium channel, which regulates neuronal excitability [ 15 , 44 , 45 ] Thymoquinone enhances parvalbumin-positive cell expression against cypermethrin toxicity. The improvement in motor functions observed in this study, which is one of the crucial functions controlled by GABAergic interneurons, complements the increased expression of the Parvalbumin-positive cells [ 15 ] As a result of thymoquinone's activation of GABA receptors, which results in hyperpolarization and inhibits neuronal activity, the N-methyl-D-aspartate NMDA receptor's enhanced glutamate functions produce prolonged neuronal stimulation [ 46 ]. According to earlier research by [ 47 , 48 ], TQ increased GABA receptor activation after prilocaine-induced cardiotoxicity, epileptiform activity, and seizures in rats as well as seizures brought on by pentylenetetrazole. CONCLUSION Thymoquinone improves motor function by activating Nrf2, reducing the level of NF-қB, increasing the activities of SOD and GSH, and decreasing the concentration of MDA against cypermethrin neurotoxicity. It also enhances the expression of parvalbumin-positive cells as well as Bcl-2 positive cells. Therefore, thymoquinone can be employed in the management of pyrethroid and other insecticide poisoning." List of Abbrevations ROS- reactive oxygen species RNA - ribonucleic acid mtDNA- mutations in mitochondrial DNA Cyc - cytochrome c TQ - Thymoquinone NRF2- nuclear factor erythroid 2-related factor 2 NOR - novel object recognition Nrf2- Nuclear factor erythroid 2-related factor 2 Bcl2- B-cell lymphoma 2 MCE - MedChemEpress NAFDAC- national authority for food and drugs administration control SOD - superoxide dismutase GSH - glutathione MDA - malondialdehyde E-EL - Elabscience Biotechnology 4PL-curve - four-parameter logistic curve ANOVA - analysis of variance SEM – standard error of mean CYM- cypermethrin PBS- phosphate buffered saline CYM-LTQ - cypermethrin followed by low dose thymoquinone CYM-HTQ- cypermethrin followed by High dose thymoquinone H and E - hematoxylin and eosin CYM-LTQ - cypermethrin followed by low dose thymoquinone CYM-HTQ - cypermethrin followed by High dose thymoquinone. NMDA - N-methyl-D-aspartate * - singleasterisk ** - Doubleasterisk Declarations Ethics approval and consent to participate This study was approved by the University of Ilorin ethical review committee (UERC\ASN\2021\2137). Consent for publication Not applicable. Availability of Data and Materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare no competing interest Funding Not Applicable Author contributions ALI: Conception, design, data collection data analysis, and interpretation, animal breeding, animal treatments, and writing of the manuscript AAO: Conception, design, histochemical analysis and interpretation, critical revision and writing of the manuscript FAS: Conception, design, histochemical analysis and interpretation, and critical revision AI: Conception, design, histochemical analysis and interpretation, and critical revision RYI: Conception, design, histochemical analysis and interpretation, and critical revision LAO: Conception, design, data collection, biochemical analysis and interpretation SAB: Conception, design, data collection, biochemical analysis and interpretation SM: Conception, design, data collection, biochemical analysis and interpretation AOA: Conception, design, histochemical analysis and interpretation, and critical revision OOO: Critical revision and referencing SMA: Conception, design, and project supervision. 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Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 2016;1863:2977–92. https://doi.org/10.1016/j.bbamcr.2016.09.012. Martinez MM, Reif RD, Pappas D. Detection of apoptosis: A review of conventional and novel techniques. Anal Methods 2010;2:996–1004. https://doi.org/10.1039/C0AY00247J. Wong RS. Apoptosis in cancer: from pathogenesis to treatment. J Exp Clin Cancer Res 2011;30:87. https://doi.org/10.1186/1756-9966-30-87. Ko J, Park JH, Park YS, Koh HC. PPAR-γ activation attenuates deltamethrin-induced apoptosis by regulating cytosolic PINK1 and inhibiting mitochondrial dysfunction. Toxicology Letters 2016;260:8–17. https://doi.org/10.1016/j.toxlet.2016.08.016. Gasmi S, Rouabhi R, Kebieche M, Boussekine S, Salmi A, Toualbia N, et al. Effects of Deltamethrin on striatum and hippocampus mitochondrial integrity and the protective role of Quercetin in rats. Environ Sci Pollut Res 2017;24:16440–57. https://doi.org/10.1007/s11356-017-9218-8. Khalatbary AR, Ghaffari E, Mohammadnegad B. Protective Role of Oleuropein against Acute Deltamethrin-Induced Neurotoxicity in Rat Brain. Iran Biomed J 2015;19:247–53. https://doi.org/10.7508/ibj.2015.04.009. Stewart CR, Stuart LM, Wilkinson K, Van Gils JM, Deng J, Halle A, et al. CD36 ligands promote sterile inflammation through assembly of a Toll-like receptor 4 and 6 heterodimer. Nature Immunology 2010;11:155–61. Firdaus F, Zafeer MohdF, Anis E, Ahmad F, Hossain MM, Ali A, et al. Evaluation of phyto-medicinal efficacy of thymoquinone against Arsenic induced mitochondrial dysfunction and cytotoxicity in SH-SY5Y cells. Phytomedicine 2019;54:224–30. https://doi.org/10.1016/j.phymed.2018.09.197. Yıldırım İH, Azzawri AA, Duran T. Thymoquinone induces apoptosis via targeting the Bax/BAD and Bcl-2 pathway in breast cancer cells. Dicle Tıp Dergisi 2019:411–7. https://doi.org/10.5798/dicletip.620329. Xu M, Wong AHC. GABAergic inhibitory neurons as therapeutic targets for cognitive impairment in schizophrenia. Acta Pharmacol Sin 2018;39:733–53. https://doi.org/10.1038/aps.2017.172. Ullah R, Rehman A, Zafeer MF, Rehman L, Khan YA, Khan MAH, et al. Anthelmintic Potential of Thymoquinone and Curcumin on Fasciola gigantica. PLoS ONE 2017;12:e0171267. https://doi.org/10.1371/journal.pone.0171267. Yu-Tao T, Zhao-Wei L, Yang Y, Zhuo Y, Tao Z. Effect of alpha-cypermethrin and theta-cypermethrin on delayed rectifier potassium currents in rat hippocampal neurons. NeuroToxicology 2009;30:269–73. https://doi.org/10.1016/j.neuro.2009.01.001. Pottoo FH, Ibrahim AM, Alammar A, Alsinan R, Aleid M, Alshehhi A, et al. Thymoquinone: Review of Its Potential in the Treatment of Neurological Diseases. Pharmaceuticals 2022;15:408. https://doi.org/10.3390/ph15040408. Akgül B, Aycan İÖ, Hidişoğlu E, Afşar E, Yıldırım S, Tanrıöver G, et al. Alleviation of prilocaine-induced epileptiform activity and cardiotoxicity by thymoquinone. DARU J Pharm Sci 2021;29:85–99. https://doi.org/10.1007/s40199-020-00385-2. Seghatoleslam M, Alipour F, Shafieian R, Hassanzadeh Z, Edalatmanesh MA, Sadeghnia HR, et al. The effects of Nigella sativa on neural damage after pentylenetetrazole induced seizures in rats. Journal of Traditional and Complementary Medicine 2016;6:262–8. https://doi.org/10.1016/j.jtcme.2015.06.003. Additional Declarations No competing interests reported. Supplementary Files MANUSCRIPImamDATASET.xlsx Cite Share Download PDF Status: Published Journal Publication published 27 Sep, 2024 Read the published version in BMC Neuroscience → Version 1 posted Editorial decision: Revision requested 29 May, 2024 Reviews received at journal 17 May, 2024 Reviews received at journal 14 May, 2024 Reviewers agreed at journal 12 May, 2024 Reviewers agreed at journal 12 May, 2024 Reviewers agreed at journal 25 Apr, 2024 Reviewers agreed at journal 23 Apr, 2024 Reviewers invited by journal 23 Apr, 2024 Editor invited by journal 25 Mar, 2024 Submission checks completed at journal 21 Mar, 2024 Editor assigned by journal 21 Mar, 2024 First submitted to journal 19 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4130260","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":281366902,"identity":"e88f8007-253c-486b-bc2f-0acb4589ea45","order_by":0,"name":"Abubakar Lekan Imam","email":"","orcid":"","institution":"University of Ilorin","correspondingAuthor":false,"prefix":"","firstName":"Abubakar","middleName":"Lekan","lastName":"Imam","suffix":""},{"id":281366903,"identity":"1d80c973-3de0-413c-ac10-be694f47f1c1","order_by":1,"name":"Akeem Ayodeji Okesina","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYDACHjCZwMDADKQ+ADEbOylaGGeAtDATrQUImMEcQlr4ew4ffHSjIk1Ovp334GebX9vk+ZgZGD98zMGtReJsW7JxzpkcY4PDfMnSuX23DduYGZglZ27DY815HjPp3LaKxA3MPAbSuT23GYFa2Jh58WiRP8///TdQS/38Zh7j35Y9t+0JajE428PGnNuWk8BwGGgdw4/biQS1GJ45ZiydcybNcANQi2Vvw+3kNmbGZrx+kTuT/PBzTkWyvHz/GeMbP/7ctp3f3nzww0d83kcBjG1gsoFY9SDwhxTFo2AUjIJRMFIAAEvbTS4CsUL3AAAAAElFTkSuQmCC","orcid":"","institution":"University of Rwanda","correspondingAuthor":true,"prefix":"","firstName":"Akeem","middleName":"Ayodeji","lastName":"Okesina","suffix":""},{"id":281366904,"identity":"2fb350fd-4e7d-4f29-9312-c55503d0a2b6","order_by":2,"name":"Fatimo Ajoke Sulamon","email":"","orcid":"","institution":"University of Ilorin","correspondingAuthor":false,"prefix":"","firstName":"Fatimo","middleName":"Ajoke","lastName":"Sulamon","suffix":""},{"id":281366905,"identity":"79b00d29-0b99-43ab-977f-fdf7fd2a1214","order_by":3,"name":"Aminu Imam","email":"","orcid":"","institution":"University of Ilorin","correspondingAuthor":false,"prefix":"","firstName":"Aminu","middleName":"","lastName":"Imam","suffix":""},{"id":281366906,"identity":"cb732bb4-f747-4ded-bad8-08f9ec836481","order_by":4,"name":"Ruqayyah Yetunde Ibiyeye","email":"","orcid":"","institution":"Kwara State University","correspondingAuthor":false,"prefix":"","firstName":"Ruqayyah","middleName":"Yetunde","lastName":"Ibiyeye","suffix":""},{"id":281366907,"identity":"2bc5ca9b-a212-474f-b444-73c6bf8e03ef","order_by":5,"name":"Lukuman Aboyeji Oyewole","email":"","orcid":"","institution":"University of Ilorin","correspondingAuthor":false,"prefix":"","firstName":"Lukuman","middleName":"Aboyeji","lastName":"Oyewole","suffix":""},{"id":281366908,"identity":"6942287d-b463-4b23-b1ed-655692c31cb3","order_by":6,"name":"Sikiru Abayomi Biliaminu","email":"","orcid":"","institution":"University of Ilorin","correspondingAuthor":false,"prefix":"","firstName":"Sikiru","middleName":"Abayomi","lastName":"Biliaminu","suffix":""},{"id":281366909,"identity":"e077e5c5-709e-4fa5-8721-22724ab91421","order_by":7,"name":"Monsur Shehu","email":"","orcid":"","institution":"University of Ilorin","correspondingAuthor":false,"prefix":"","firstName":"Monsur","middleName":"","lastName":"Shehu","suffix":""},{"id":281366910,"identity":"8d9fe921-bf2e-4ca9-818d-a84d25e99020","order_by":8,"name":"Alli Oluwatomi Abdulhameed","email":"","orcid":"","institution":"University of Ilorin","correspondingAuthor":false,"prefix":"","firstName":"Alli","middleName":"Oluwatomi","lastName":"Abdulhameed","suffix":""},{"id":281366911,"identity":"d4b646f6-c266-4d49-a348-9aaa58b49997","order_by":9,"name":"Oluwatosin Olasheu Omoola","email":"","orcid":"","institution":"Kampala International University","correspondingAuthor":false,"prefix":"","firstName":"Oluwatosin","middleName":"Olasheu","lastName":"Omoola","suffix":""},{"id":281366912,"identity":"7100935d-29ff-42da-b4b1-f321f4a515ee","order_by":10,"name":"Salihu Moyosore Ajao","email":"","orcid":"","institution":"University of Ilorin","correspondingAuthor":false,"prefix":"","firstName":"Salihu","middleName":"Moyosore","lastName":"Ajao","suffix":""}],"badges":[],"createdAt":"2024-03-19 12:29:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4130260/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4130260/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12868-024-00896-7","type":"published","date":"2024-09-27T15:58:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53087162,"identity":"5a707919-8f10-4a31-b6ac-1435eb698639","added_by":"auto","created_at":"2024-03-20 12:08:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":405178,"visible":true,"origin":"","legend":"\u003cp\u003eHippocampal Nrf2, SOD activity, GSH activity, and MDA concentrations; level of rats exposed to Cypermethrin and thymoquinone. PBS= phosphate buffered saline, CYM= cypermethrin, TQ= thymoquinone, CYM-LTQ= cypermethrin followed by low dose thymoquinone and CYM-HTQ= cypermethrin followed by High dose thymoquinone. singleasterisk (*) indicates significant (p \u0026lt; 0.05) compared to PBS, Doubleasterisk (**) indicates significant (p \u0026lt; 0.05) compared to CYM.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4130260/v1/8fa3b6ec1537436dfa540aa0.png"},{"id":53086133,"identity":"14877961-b20c-4bdb-9c77-a24d1a6e73c5","added_by":"auto","created_at":"2024-03-20 12:00:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":408845,"visible":true,"origin":"","legend":"\u003cp\u003eThe Dentate gyrus of rats exposed to cypermethrin and thymoquinone. CFV x40 100µ. PBS= phosphate buffered saline, CYM= cypermethrin, TQ= thymoquinone, CYM-LTQ= cypermethrin followed by low dose thymoquinone and CYM-HTQ= cypermethrin followed by High dose thymoquinone.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4130260/v1/134c8ed8e385e55f756b432a.png"},{"id":53086125,"identity":"a96a02d3-5e61-4f54-b65d-5ec8c2f545b9","added_by":"auto","created_at":"2024-03-20 12:00:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4073335,"visible":true,"origin":"","legend":"\u003cp\u003eDentate gyrus of rats exposed to cypermethrin and thymoquinone. \u003cstrong\u003e(A1)\u003c/strong\u003e Anti-Nrf2 antibody; \u003cstrong\u003e(B1)\u003c/strong\u003eAnti-Parv antibody; \u003cstrong\u003e(C1)\u003c/strong\u003e Anti-Bcl2 antibody x10 100µ. \u003cstrong\u003e(A2) \u003c/strong\u003eNrF2 immuno positive; \u003cstrong\u003e(B2) \u003c/strong\u003eParvalbumin immunopositive cell count; \u003cstrong\u003e(C2)\u003c/strong\u003e Bcl2 immunopositive cell count.\u003cstrong\u003e \u003c/strong\u003ePBS= phosphate buffered saline, CYM= cypermethrin, TQ= thymoquinone, CYM-LTQ= cypermethrin followed by low dose thymoquinone and CYM-HTQ= cypermethrin followed by High dose thymoquinone.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4130260/v1/c0f7be67c5e51957786de999.png"},{"id":65627396,"identity":"797c47cd-a59d-4879-9d33-b368d7c3f78e","added_by":"auto","created_at":"2024-09-30 16:15:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6537615,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4130260/v1/62bc8e47-5460-4e47-b311-04aa98d6028b.pdf"},{"id":53086121,"identity":"204e2a4f-40d1-4764-b039-824d845c6110","added_by":"auto","created_at":"2024-03-20 12:00:20","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11520,"visible":true,"origin":"","legend":"","description":"","filename":"MANUSCRIPImamDATASET.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4130260/v1/46c949d1578733e1b6d9833c.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eOrally administered Thymoquinone mitigates cypermethrin-induced dentate gyrus oxidative stress, preventing GABAergic interneuron degeneration and memory impairment in rats via the Nrf2/ARE pathway.\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eOxidative stress arises from an imbalance between reactive oxygen species (ROS) and the antioxidant defense system. ROS can lead to covalent oxidative modifications, such as ribonucleic acid (RNA) oxidation, and induce mutations in mitochondrial DNA (mtDNA), thereby destabilizing nucleic acids [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These modifications may result in cellular dysfunction and apoptosis. The mitochondrial-dependent caspase pathway is crucial in apoptosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Stimulation of this cascade releases proapoptotic factors, including cytochrome c (Cyc), activating caspase-9 and caspase-3, ultimately triggering cellular apoptosis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Hence, antioxidant pathways that mitigate oxidative damage may exhibit neuroprotective effects [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePersistent exposure to pesticides, such as pyrethroids, adversely affects various physiological functions [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. For example; long-term exposure can disrupt the functioning of different organs, posing serious health risks [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Nigeria has witnessed numerous cases of food poisoning due to pesticides, resulting in significant fatalities and economic losses [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Pyrethroids, widely used in agriculture and household insect control, have been detected in a large portion of the population [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Despite their broad application, pyrethroids exhibit adverse effects, including neurobehavioral effects and disruption of critical molecular targets in the nervous system [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Cypermethrin, a common pyrethroid, crosses the blood-brain barrier, inducing oxidative stress and apoptotic cell death [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMedicinal plants, due to their diverse chemical constituents, offer a sustainable therapeutic approach against chemical toxins. Thymoquinone (TQ), derived from Nigella sativa L., possesses antioxidant and anti-inflammatory properties [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. TQ has shown neuroprotective effects in various models of brain injury and neurodegenerative diseases by inhibiting lipid peroxidation and apoptosis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Its antioxidant effects are mediated through the nuclear factor erythroid 2-related factor 2 (NRF2) pathway, which regulates cellular defense mechanisms against oxidative stress [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Activation of NRF2 induces the expression of antioxidative and detoxifying enzymes, crucial for cellular function [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Dysregulation of NRF2/ARE signaling is implicated in neurodegenerative disorders [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the increasing incidence of pesticide-induced food poisoning, there is a need for effective antidotes with shared mechanisms of action. This study aims to evaluate the efficacy of thymoquinone against cypermethrin-induced neurotoxicity, focusing on GABAergic interneuron disruption, dentate gyrus cytoarchitectural disorganization, and oxidative stress-induced cell damage.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Design\u003c/h2\u003e \u003cp\u003eThe experimental design involved the use of 40 adult male Wistar rats (180\u0026ndash;200 g). Thymoquinone was obtained from MedChemEpress (MCE) USA (Cat No: HY-d0803) Cypermethrin 10% EC product was sourced from Yubaili Agrotec (ACEC20L068) and NAFDAC No: A5-0108 was obtained from Ibukun Oluwa Agrochemical Distop. Ilorin, Nigeria. The rats were housed in the animal holding facility of the Faculty of Basic Medical Sciences, College of Health Sciences, University of Ilorin, under natural day-night cycles, with standard chow diet and water ad libitum.\u003c/p\u003e \u003cp\u003eThe rats were randomly divided into 5 groups (n\u0026thinsp;=\u0026thinsp;8 per group). Groups I, II, III, and IV received oral administration of 0.5 ml of phosphate buffered saline, 20 mg/kg of cypermethrin, 10 mg/kg of thymoquinone, 20 mg/kg cypermethrin plus 5 mg/kg of thymoquinone, and 20 mg/kg of cypermethrin plus 10 mg/kg of thymoquinone for 14 days respectively. Motor behavior was assessed on the 14th day of the experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBehavioural Evaluation\u003c/h2\u003e \u003cp\u003eIntermediate memory recognition of the experimental rats following exposure to cypermethrin and thymoquinone was assessed using the novel object recognition (NOR) paradigm. The test apparatus is made from plywood measuring 100 cm by 100 cm with walls that are 50 cm high. Novelty preference and Discriminatory index were evaluated [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Twenty-four hours after the behavioral study, the animals were euthanized using 20 mg/kg bw ketamine intramuscularly, and brain tissue was excised and processed for histological, immunohistological, and biochemical analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTissue collection\u003c/h2\u003e \u003cp\u003eAfter perfusion has been completed, the whole brain tissues were excised and were post-fixed in 4% paraformaldehyde overnight. The whole hippocampal CA regions were excised and equilibrated in 30% sucrose solution, before histological and immunohistochemical analyses. The sections were taken at 2 \u0026micro;m on paraffin wax embedded tissue blocks and mounted on a glass slide\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eHistological Analysis and Immunohistochemistry\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe hematoxylin and eosin (H and E) staining technique was used to demonstrate the general histo-architecture of the cells; and to show the location of the normal or abnormal nucleus of the hippocampal cells. Cresyl violet: This technique was used to demonstrate Nissl bodies (endoplasmic reticulum and ribosomes) in the cells; and to show normal or abnormal protein synthesis in the cytoplasm of the hippocampus.\u003c/p\u003e \u003cp\u003eFor immunochemistry: Nuclear factor erythroid 2-related factor 2 (Nrf2), Parvalbumin, and B-cell lymphoma 2 (Bcl2) (human monoclonal; Elisa and microarray) were used to understand their roles in oxidative stress response, neuronal survival, and apoptosis regulation within dentate gyrus. The avidin-biotin complex method was used. The antibody dilution factor used was 1:100 for all the antibody markers. The processed tissues were sectioned at two microns on the rotary microtome and placed on a hotplate at 90\u0026deg;C for at least 40 minutes. Image J software cell counter was used for counting the immunopositive cells for Nrf2, Parvalbumin, and Bcl2 in the dentate gyrus.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical Investigation\u003c/h2\u003e \u003cp\u003eAfter the tissues were collected and homogenized, the homogenates were collected in a 5ml plain bottle and centrifuged for 10 minutes at 5000rpm using a centrifuge. The supernatant was carefully decanted and stored at -4\u0026deg;C for enzymatic assays of superoxide dismutase (SOD) activity [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], glutathione (GSH) concentration [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], malondialdehyde (MDA) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] concentration and nuclear factor erythroid 2-derived factor 2 (NrF2) a product of Elabscience Biotechnology Inc.USA (E-EL-R0673) method and absorbance was read using microplate reader. A four-parameter logistic curve (4PL-curve) was plotted and values for the samples were extrapolated using GraphPad Prism 8.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData from the behavioural, biochemical assays and immunopositiv cell count were analysed using one-way analysis of variance (ANOVA) and subjected to post hoc Bonferroni\u0026rsquo;s multiple comparison test. The results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical analyses were performed using Graphpad Prism software (version 8.0.2). Values of p\u0026thinsp;\u0026le;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003eThymoquinne Restore Inter-mediate related behaviours following cypermethrin exposure\u003c/h2\u003e\n\u003cp\u003eThe novelty preference of Wistar rats in CYM group for new object in novel object recognition test was significantly low (32.70\u0026thinsp;\u0026plusmn;\u0026thinsp;3.93) compared to PBS control group at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, compared to CYM group, the TQ group and the CYM-LHQ group showed higher preference for new object which was significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. However, the CYM-10mgTHQ group showed higher preference than CYM group with no significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 Fig.\u0026nbsp;1B. The discrimination index was significantly reduced in CYM group (-0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08) compared to PBS group (0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10) and THQ group (0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11) at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Groups CYM-LTHQ and CYM-HHQ showed higher discrimination index compared to CYM group but not significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eNovelty preference and Discrimination Index of rats following cypermethrin and thymoquinone exposure\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGroups\u003c/p\u003e\n\u003cp\u003eN\u0026thinsp;=\u0026thinsp;5\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eNovelty Preference (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eDiscrimination index\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePBS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e77.60\u0026thinsp;\u0026plusmn;\u0026thinsp;3.82\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCYM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e39.30\u0026thinsp;\u0026plusmn;\u0026thinsp;7.00*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e-0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTQ\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e90.30\u0026thinsp;\u0026plusmn;\u0026thinsp;5.41**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11**\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCYM-LTQ\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e65.00\u0026thinsp;\u0026plusmn;\u0026thinsp;11.20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCYM-HTQ\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e46.00\u0026thinsp;\u0026plusmn;\u0026thinsp;12.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"3\"\u003ePBS\u0026thinsp;=\u0026thinsp;phosphate buffered saline, CYM\u0026thinsp;=\u0026thinsp;cypermethrin, TQ\u0026thinsp;=\u0026thinsp;thymoquinone, CYM-LTQ\u0026thinsp;=\u0026thinsp;cypermethrin followed by low dose thymoquinone and CYM-HTQ\u0026thinsp;=\u0026thinsp;cypermethrin followed by High dose thymoquinone. singleasterisk (*) indicates significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to PBS, Doubleasterisk (**) indicates significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to CYM\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eOxidative Stress Biomarker\u003c/h2\u003e\n\u003cp\u003eThe concentration of Nrf2 in the CYM group was significantly reduced p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 when compared to both the PBS control and the other experimental groups (Fig.\u0026nbsp;1A). Moreover, a significant reduction p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 in the SOD activity was observed in the CYM-exposed group, relative to the PBS control group, while all the other experimental groups showed a significant increase in the SOD activities p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 when compared to the CYM group (Fig.\u0026nbsp;1B). However, SOD activities in the TQ, CYM-LTQ and CYM-HTQ groups were not significantly lower compared to the PBS control (Fig.\u0026nbsp;1B). In addition, the GSH concentration was significantly reduced (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the CYM group relative to the PBS group (Fig.\u0026nbsp;1C). Subsequent treatment with LTQ and HTQ led to a significant increase in the GSH levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) when compared to the CYM-only group (Fig.\u0026nbsp;1C). Regarding the lipid peroxidation marker MDA, its level was notably higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the CYM group compared to the PBS group (Fig.\u0026nbsp;1D). While MDA levels were lower in other experimental groups, significance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was only observed in the CYM-LTQ group compared to the CYM group (Fig.\u0026nbsp;1D).\u003c/p\u003e\n\u003cp\u003eHistochemical examination of the dentate gyrus revealed chromatolytic-like alterations in rats exposed to CYM. This was characterized by the disrupted integrity of Nissl granules, along with distortions in the shape and organization of granule cells. Additionally, numerous pyknotic and vacuolated cells were observed as a consequence of CYM exposure. Thymoquinone demonstrated a mitigating effect against CYM-induced toxicity, this was observed as a result of a reduction in the extent of neurodegenerative-like changes. Specifically, there was an improvement in Nissl body integrity, as well as the enhancements observed in the cellular shape and arrangement in the rats exposed to thymoquinone following CYM neurotoxicity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe immunohistochemical assessment of the dentate gyrus utilizing anti-Nrf2, anti-Parvalbumin (Parv), and anti-Bcl2 antibodies revealed diminished expression of Nrf2, Parv, and Bcl2 positive cells in the dentate gyrus of rats exposed to CYM (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Conversely, post-treatment with thymoquinone exhibited a notable enhancement in the expression of Nrf2, Parv, and Bcl2 immunopositive cells in the dentate gyrus (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eQuantification of immuno-positive cell counts using ImageJ software demonstrated a significantly higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) Nrf2 cell count in the dentate gyrus of PBS control rats compared to those exposed to CYM (Fig.\u0026nbsp;3A1 \u0026amp; 2). Thymoquinone administration led to a significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in Nrf2 immunopositive cells in the CYM-LTQ and CYM-HTQ groups compared to CYM-exposed rats (Fig.\u0026nbsp;3A1 \u0026amp; 2). Similarly, there was a significantly higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) count of Parvalbumin positive cells in the dentate gyrus of PBS control rats compared to CYM-exposed rats (Fig.\u0026nbsp;3B1 \u0026amp; 2). Thymoquinone significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increased the number of Parvalbumin cells in the CYM-LTQ and CYM-HTQ groups compared to CYM-exposed rats (Fig.\u0026nbsp;3B1 \u0026amp; 2).\u003c/p\u003e\n\u003cp\u003eMoreover, the cell count of the anti-apoptotic protein Bcl2 was higher in the PBS control group than in CYM-exposed rats (Fig.\u0026nbsp;3C1 \u0026amp; 2). Administration of thymoquinone resulted in a higher Bcl2 count in the CYM-LTQ and CYM-HTQ groups compared to CYM-exposed rats (Fig.\u0026nbsp;3C1 \u0026amp; 2).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eContinuous application of pesticides and other agrochemicals, driven by the need to increase food production and prevent pest and insect-induced crop damages, has led to increased exposure to the harmful effects of these chemicals, including pyrethroid insecticides, due to their residual accumulation in crops, fruits, and vegetables. When humans and other animals are exposed to these chemicals, they can induce toxicity through mechanisms involving mitochondrial dysfunction, oxidative stress, and inflammation. This toxicity can manifest as movement disorders, loss of cognition, or a combination of both.\u003c/p\u003e \u003cp\u003eThis study demonstrates that thymoquinone increases the activities of antioxidant enzymes, akin to its parent molecule Nigella sativa oil [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], thereby preventing lipid peroxidation and preserving dentate gyrus architecture, ultimately enhancing memory function against CYM toxicity.\u003c/p\u003e \u003cp\u003eIn this study, cypermethrin caused a reduction in the expression of the Nuclear factor erythroid 2-related factor 2 (Nrf2), a regulatory protein responsible for initiating and expressing the antioxidant system. The reduced concentration and low expression of Nrf2 cells in the dentate gyrus of CYM-exposed rats are undoubtedly responsible for the reduction in the activities of the antioxidant enzymes SOD and GSH, leading to oxidative stress as indicated by the high level of MDA in CYM-exposed rats. Due to the presence of high unsaturated fatty acids, the brain is especially susceptible to oxidative stress, which causes membrane lipid peroxidation and disrupts the normal organizational structure of brain cells, as observed in the dentate gyrus of CYM-exposed rats. Cypermethrin exposure caused neuronal damage, impaired Nissl body integrity, and induced chromatolytic-like changes in the dentate gyrus due to its oxidative stress. It was observed that continuous CYM exposure not only disrupted neuronal shapes in the dentate gyrus but also induced Nrf2 expression. Since appropriate activation of Nrf2 and its nuclear translocation establishes the Nrf2/ARE complex and subsequently boosts the expression and synthesis of antioxidant enzymes, the decreased level of Nrf2 observed contributes to the lower activity of antioxidant enzymes SOD and GSH, which encourages further oxidative stress damage and raises the level of MDA. Cypermethrin decreased the level and activity of antioxidant enzymes like SOD, GSH, and catalase (CAT) in cypermethrin-induced toxicity in the Wistar rat model of Parkinson's disease and peripheral blood. The findings of this study are indeed strengthened by earlier studies by [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], which reported an excessive increase in the level of MDA and reduced antioxidant capacity of SOD, CAT, GSH, and GPx, leading to increased lipid peroxidation in the peripheral blood and in the nigrostriatum of cypermethrin-exposed rats.\u003c/p\u003e \u003cp\u003eIntervention with thymoquinone was observed to reactivate Nrf2, as shown by the high expression of Nrf2 immunopositive cells. This increased nuclear availability of Nrf2 leads to the Nrf2/ARE complex, thereby stimulating the production and expression of antioxidant enzymes and resulting in high SOD and GSH activity, as reported in this study, and reduced MDA levels, indicating a low level of lipid peroxidation. This finding is consistent with an earlier study by [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], who found that the parent plant of thymoquinone, black seed oil, increased total antioxidant capacity and GSH while decreasing total ROS levels in rats exposed to Dichlorvos. The findings of this study are also strengthened by the study of Kanter, who reported enhancements in hepatic and pancreatic antioxidant capacities of catalase and GSH following Nigella sativa against STZ-induced diabetes in rats [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eApoptosis is characterized by morphological changes in cells such as nuclear pyknosis, DNA fragmentation, and chromatin condensation, cytoskeleton destruction, membrane blebbing, and eventually the formation of membrane apoptotic bodies that are phagocytosed by macrophages and other cells without inducing an inflammatory response [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Continuous exposure to environmental toxins frequently causes apoptosis in cells [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] Anti-Bcl-2-stained dentate gyrus had a low expression of Bcl-2 positive cells due to cypermethrin exposure. Cypermethrin, like other pyrethroids, caused apoptosis in the rat brain by producing ROS and cytotoxins. Cypermethrin also induced apoptosis via mitochondrial damage, cytochrome c release, and activation of caspases 3 and 9, which are involved in both extrinsic and intrinsic apoptosis pathways [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. When Bcl-2 and other anti-apoptotic proteins are cleaved by caspases following the initiation of apoptosis, their anti-apoptotic action is frequently converted to pro-apoptotic action [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The findings of this study are similar to the report of the previous study where a type 2 pyrethroid, deltamethrin, following its exposure in rats, induced apoptosis by increasing the level of Bax, caspase-3, cytochrome c, and decreasing the expression of Bcl-2 pro-survival proteins [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Thymoquinone exhibits anti-apoptotic effects, as administration of thymoquinone brings about a marked increase in the expression of Bcl-2 immunopositive cells in the hippocampal dentate gyrus of the experimental rats. Bcl-2, as a pro-survival protein, has a hydrophilic carboxyl-terminal domain that is linked to the mitochondria outer membrane and helps preserve mitochondrial integrity, preventing unnecessary cytochrome c release and caspase activation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Bcl-2 prevents Bax and other pro-apoptotic genes from oligomerizing, which stimulates the release of apoptogenic molecules from the mitochondria. Apart from inhibiting Bax oligomerization, Bcl-2 directly binds and inactivates Bax, blocks cytochrome c release, and thus inhibits adaptor molecule APAF-1 and caspase-9 activation, thereby preventing caspase cascade activation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In accordance with the findings of this study, [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] showed that thymoquinone, in concentrations of 10 M and 20 M, prevented arsenic-induced neurotoxicity, apoptosis, and cytotoxicity by either decreasing the levels of Bax or increasing the level of Bcl-2. Also, in agreement with the data of this study, a previous study revealed that thymoquinone administration decreased p53 and Bcl-2 gene expression but increased BAD gene expression in MCF-7 cells; however, it increased the expression of Bcl-2 gene and p53 gene but decreased Bax/BAD gene expression in non-cancer HEK293 cells [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIn the dentate gyrus, basket cells constitute the GABAergic neurons in the granule layer with the receptors localized in the molecular layer. Reduced levels of parvalbumin-positive cells in the dentate gyrus of cypermethrin-exposed rats indicate that CYM inhibits GABAergic interneurons. GABAergic interneurons constitute the inhibitory neurons in the CNS that are vital for modulating various physiological activities [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Reduced GABAergic interneuron expression due to CYM exposure interferes with the activity of GABAergic interneurons and disrupts excitatory and inhibitory balance in the brain. Previous studies have shown that CYM hinders the opening of the voltage-gated chloride channels and inhibits the GABA-dependent uptake of chloride ions, resulting in hyper-excitation of neuronal cells and leading to changes in the delayed rectifier voltage-dependent potassium channel, which regulates neuronal excitability [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThymoquinone enhances parvalbumin-positive cell expression against cypermethrin toxicity. The improvement in motor functions observed in this study, which is one of the crucial functions controlled by GABAergic interneurons, complements the increased expression of the Parvalbumin-positive cells [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] As a result of thymoquinone's activation of GABA receptors, which results in hyperpolarization and inhibits neuronal activity, the N-methyl-D-aspartate NMDA receptor's enhanced glutamate functions produce prolonged neuronal stimulation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. According to earlier research by [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], TQ increased GABA receptor activation after prilocaine-induced cardiotoxicity, epileptiform activity, and seizures in rats as well as seizures brought on by pentylenetetrazole.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThymoquinone improves motor function by activating Nrf2, reducing the level of NF-қB, increasing the activities of SOD and GSH, and decreasing the concentration of MDA against cypermethrin neurotoxicity. It also enhances the expression of parvalbumin-positive cells as well as Bcl-2 positive cells. Therefore, thymoquinone can be employed in the management of pyrethroid and other insecticide poisoning.\"\u003c/p\u003e "},{"header":"List of Abbrevations","content":"\u003cp\u003eROS- reactive oxygen species\u003c/p\u003e\n\u003cp\u003eRNA - ribonucleic acid\u003c/p\u003e\n\u003cp\u003emtDNA- mutations in mitochondrial DNA\u003c/p\u003e\n\u003cp\u003eCyc - cytochrome c\u003c/p\u003e\n\u003cp\u003eTQ - Thymoquinone\u003c/p\u003e\n\u003cp\u003eNRF2- nuclear factor erythroid 2-related factor 2\u003c/p\u003e\n\u003cp\u003eNOR - novel object recognition\u003c/p\u003e\n\u003cp\u003eNrf2- Nuclear factor erythroid 2-related factor 2\u003c/p\u003e\n\u003cp\u003eBcl2- B-cell lymphoma 2\u003c/p\u003e\n\u003cp\u003eMCE - MedChemEpress\u003c/p\u003e\n\u003cp\u003eNAFDAC- national authority for food and drugs administration control\u003c/p\u003e\n\u003cp\u003eSOD - superoxide dismutase\u003c/p\u003e\n\u003cp\u003eGSH - glutathione\u003c/p\u003e\n\u003cp\u003eMDA - malondialdehyde\u003c/p\u003e\n\u003cp\u003eE-EL - Elabscience Biotechnology\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4PL-curve - four-parameter logistic curve\u003c/p\u003e\n\u003cp\u003eANOVA - analysis of variance\u003c/p\u003e\n\u003cp\u003eSEM \u0026ndash; standard error of mean\u003c/p\u003e\n\u003cp\u003eCYM- cypermethrin\u003c/p\u003e\n\u003cp\u003ePBS- phosphate buffered saline\u003c/p\u003e\n\u003cp\u003eCYM-LTQ - cypermethrin followed by low dose thymoquinone\u003c/p\u003e\n\u003cp\u003eCYM-HTQ- cypermethrin followed by High dose thymoquinone\u003c/p\u003e\n\u003cp\u003eH and E - hematoxylin and eosin\u003c/p\u003e\n\u003cp\u003eCYM-LTQ - cypermethrin followed by low dose thymoquinone\u003c/p\u003e\n\u003cp\u003eCYM-HTQ - cypermethrin followed by High dose thymoquinone.\u003c/p\u003e\n\u003cp\u003eNMDA - N-methyl-D-aspartate * - singleasterisk\u003c/p\u003e\n\u003cp\u003e** - Doubleasterisk\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the University of Ilorin ethical review committee (UERC\\ASN\\2021\\2137).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eALI: Conception, design, data collection data analysis, and interpretation, animal breeding, animal treatments, and writing of the manuscript\u003c/p\u003e\n\u003cp\u003eAAO: Conception, design, histochemical analysis and interpretation, critical revision and writing of the manuscript\u003c/p\u003e\n\u003cp\u003eFAS: Conception, design, histochemical analysis and interpretation, and critical revision\u003c/p\u003e\n\u003cp\u003eAI: Conception, design, histochemical analysis and interpretation, and critical revision\u003c/p\u003e\n\u003cp\u003eRYI: Conception, design, histochemical analysis and interpretation, and critical revision\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLAO: Conception, design, data collection, biochemical analysis and interpretation\u003c/p\u003e\n\u003cp\u003eSAB: Conception, design, data collection, biochemical analysis and interpretation\u003c/p\u003e\n\u003cp\u003eSM: Conception, design, data collection, biochemical analysis and interpretation\u003c/p\u003e\n\u003cp\u003eAOA: Conception, design, histochemical analysis and interpretation, and critical revision OOO: Critical revision and referencing\u003c/p\u003e\n\u003cp\u003eSMA: Conception, design, and project supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the support of Bioresearch Hub director staff, the entire staff of the Departments of; Human anatomy, Physiology, Histopathology and Chemical Pathology, University of Ilorin, Nigeria, for their technical support throughout this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAngelova PR, Abramov AY. 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Archives of Biochemistry and Biophysics 1959;82:70\u0026ndash;7. https://doi.org/10.1016/0003-9861(59)90090-6.\u003c/li\u003e\n\u003cli\u003eImam A, Sulaiman NA, Oyewole AL, Chengetanai S, Williams V, Ajibola MI, et al. Chlorpyrifos- and Dichlorvos-Induced Oxidative and Neurogenic Damage Elicits Neuro-Cognitive Deficits and Increases Anxiety-Like Behavior in Wild-Type Rats. Toxics 2018;6:71. https://doi.org/10.3390/toxics6040071.\u003c/li\u003e\n\u003cli\u003eAminu I, Moyosore Saliu A, Abdulbasit A, Wahab Imam A, Abdulmumin I, Olajide, et al. Canabis-induced Moto-cognitive Dysfunction in Wistar Rats: Ameliorative Efficacy of Nigella Sativa. MJMS 2016;23:17\u0026ndash;28. https://doi.org/10.21315/mjms2016.23.5.3.\u003c/li\u003e\n\u003cli\u003eAgrawal S, Dixit A, Singh A, Tripathi P, Singh D, Patel DK, et al. Cyclosporine A and MnTMPyP Alleviate \u0026alpha;-Synuclein Expression and Aggregation in Cypermethrin-Induced Parkinsonism. Mol Neurobiol 2015;52:1619\u0026ndash;28. https://doi.org/10.1007/s12035-014-8954-8.\u003c/li\u003e\n\u003cli\u003eTripathi P, Singh A, Agrawal S, Prakash O, Singh MP. Cypermethrin alters the status of oxidative stress in the peripheral blood: relevance to Parkinsonism. J Physiol Biochem 2014;70:915\u0026ndash;24. https://doi.org/10.1007/s13105-014-0359-7.\u003c/li\u003e\n\u003cli\u003eAbdelhafidh K, Mhadhbi L, Mezni A, Badreddine S, Beyrem H, Mahmoudi E. Protective effect of \u003cem\u003eZizyphus lotus\u003c/em\u003e jujube fruits against cypermethrin-induced oxidative stress and neurotoxicity in mice. Biomarkers 2018;23:167\u0026ndash;73. https://doi.org/10.1080/1354750X.2017.1390609.\u003c/li\u003e\n\u003cli\u003eKanter M, Coskun O, Korkmaz A, Oter S. Effects of \u003cem\u003eNigella sativa\u003c/em\u003e on oxidative stress and \u0026beta;‐cell damage in streptozotocin‐induced diabetic rats. 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GABAergic inhibitory neurons as therapeutic targets for cognitive impairment in schizophrenia. Acta Pharmacol Sin 2018;39:733\u0026ndash;53. https://doi.org/10.1038/aps.2017.172.\u003c/li\u003e\n\u003cli\u003eUllah R, Rehman A, Zafeer MF, Rehman L, Khan YA, Khan MAH, et al. Anthelmintic Potential of Thymoquinone and Curcumin on Fasciola gigantica. PLoS ONE 2017;12:e0171267. https://doi.org/10.1371/journal.pone.0171267.\u003c/li\u003e\n\u003cli\u003eYu-Tao T, Zhao-Wei L, Yang Y, Zhuo Y, Tao Z. Effect of alpha-cypermethrin and theta-cypermethrin on delayed rectifier potassium currents in rat hippocampal neurons. NeuroToxicology 2009;30:269\u0026ndash;73. https://doi.org/10.1016/j.neuro.2009.01.001.\u003c/li\u003e\n\u003cli\u003ePottoo FH, Ibrahim AM, Alammar A, Alsinan R, Aleid M, Alshehhi A, et al. Thymoquinone: Review of Its Potential in the Treatment of Neurological Diseases. Pharmaceuticals 2022;15:408. https://doi.org/10.3390/ph15040408.\u003c/li\u003e\n\u003cli\u003eAkg\u0026uuml;l B, Aycan İ\u0026Ouml;, Hidişoğlu E, Afşar E, Yıldırım S, Tanrı\u0026ouml;ver G, et al. Alleviation of prilocaine-induced epileptiform activity and cardiotoxicity by thymoquinone. DARU J Pharm Sci 2021;29:85\u0026ndash;99. https://doi.org/10.1007/s40199-020-00385-2.\u003c/li\u003e\n\u003cli\u003eSeghatoleslam M, Alipour F, Shafieian R, Hassanzadeh Z, Edalatmanesh MA, Sadeghnia HR, et al. The effects of Nigella sativa on neural damage after pentylenetetrazole induced seizures in rats. Journal of Traditional and Complementary Medicine 2016;6:262\u0026ndash;8. https://doi.org/10.1016/j.jtcme.2015.06.003.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-neuroscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nros","sideBox":"Learn more about [BMC Neuroscience](http://bmcneurosci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/nros/default.aspx","title":"BMC Neuroscience","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cypermethrin, thymoquinone, cresyl fast violet, GABBAergic interneuron, dentate gyrus","lastPublishedDoi":"10.21203/rs.3.rs-4130260/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4130260/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eExposure to chemical toxins, including insecticides, has harmful effects on body organs such as the brain. This study examined the neuroprotective of thymoquinone on the cypermethrin's harmful effects on the histoarchitecture of the dentate gyrus as well as motor deficit.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eForty adult male rats (180-200g) were randomly divided into 5 groups (n\u0026thinsp;=\u0026thinsp;8 per group). Groups I, II, III, and IV received oral administration of 0.5 ml of phosphate buffered saline, 20 mg/kg of cypermethrin, 10 mg/kg of thymoquinone, 20 mg/kg cypermethrin plus 5 mg/kg of thymoquinone, and 20 mg/kg of cypermethrin plus 10 mg/kg of thymoquinone for 14 days respectively. The novel Object recognition test assesses intermediate-term memory at days 14 and 21 of the experiment. At the end of these treatments, the animals were euthanized and taken for cytoarchitectural analysis and immunohistochemical studies.\u003c/p\u003e\u003ch2\u003eResult\u003c/h2\u003e \u003cp\u003eThe study shows that thymoquinone at 5 and 10 mg/kg improved Novelty preference and discrimination index. Thymoquinone enhanced Nissl body integrity, increased GABBAergic interneuron expression, nuclear factor erythroid 2-derived factor 2, and enhanced Bcl-2 expression in the dentate gyrus. It also improved the concentration of nuclear factor erythroid 2-derived factor 2, increased the activities of superoxide dismutase and glutathione, and decreased the concentration of malondialdehyde level against cypermethrin-induced neurotoxicity.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003ethymoquinone could be a therapeutic agent against cypermethrin poisoning.\u003c/p\u003e","manuscriptTitle":"Orally administered Thymoquinone mitigates cypermethrin-induced dentate gyrus oxidative stress, preventing GABAergic interneuron degeneration and memory impairment in rats via the Nrf2/ARE pathway.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-20 12:00:08","doi":"10.21203/rs.3.rs-4130260/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-29T06:04:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-17T12:49:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-14T11:19:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"196466855742307658653490074461653491292","date":"2024-05-12T08:26:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"293994780819230417024266444202940073400","date":"2024-05-12T04:31:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"b8038080-db17-45de-81d9-58438d14cb16","date":"2024-04-25T21:32:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"a85048be-26ce-4f3c-83b9-f5bce7956345","date":"2024-04-23T22:51:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-23T21:28:01+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-03-25T18:08:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-21T10:12:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-21T10:12:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Neuroscience","date":"2024-03-19T12:26:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-neuroscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nros","sideBox":"Learn more about [BMC Neuroscience](http://bmcneurosci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/nros/default.aspx","title":"BMC Neuroscience","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"00d5e215-fa59-4647-b036-2ed6d2dcf8db","owner":[],"postedDate":"March 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-30T16:06:41+00:00","versionOfRecord":{"articleIdentity":"rs-4130260","link":"https://doi.org/10.1186/s12868-024-00896-7","journal":{"identity":"bmc-neuroscience","isVorOnly":false,"title":"BMC Neuroscience"},"publishedOn":"2024-09-27 15:58:00","publishedOnDateReadable":"September 27th, 2024"},"versionCreatedAt":"2024-03-20 12:00:08","video":"","vorDoi":"10.1186/s12868-024-00896-7","vorDoiUrl":"https://doi.org/10.1186/s12868-024-00896-7","workflowStages":[]},"version":"v1","identity":"rs-4130260","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4130260","identity":"rs-4130260","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}