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.