Because the brain requires high oxygen consumption, low antioxidant activity, and rich polyunsaturated fatty acids, it is very vulnerable to oxidative damage (9). A number of biological studies have shown that oxidative stress is an important risk factor affecting the CNS and causing cognitive impairment (10). Under normal physiological conditions, cellular reactive oxygen species (ROS) levels and the antioxidant system are stable in dynamic equilibrium (11). When the herbicide glyphosate (GLY) and the fungicide Ziram enter the organism and cannot be fully metabolized, they cause lipid peroxidation reactions, leading to an imbalance of oxidants and antioxidants in brain tissue, triggering an excessive accumulation of ROS and further damage to the structure of biological macromolecules such as DNA, lipids, and proteins. It is manifested by an increase in malondialdehyde (MDA) (12) and a decrease in non-enzymatic antioxidants such as the endogenous antioxidant glutathione (GSH) (13). Enzymes associated with oxidative stress, such as superoxide dismutase (SOD) (14), glutathione peroxidase (GSH-Px), catalase (CAT), peroxidase (POD), and heme oxygenase 1 (HO-1), undergo negative changes (15, 16). In addition, changes in GSH-Px expression affect the GSH/oxidized glutathione (GSSG) ratio. ROS can also activate many kinds of immune cells, such as microglia and astrocytes, to release a large number of inflammatory factors, which are associated with neuroinflammatory (17) and induce cholinergic and glutamate toxicity to aggravate oxidative stress (18, 19). These changes cause severe damage to the CNS and are important factors for cognitive impairment.

The structural integrity of mitochondria is very important for the normal operation of energy metabolism, nerve cell energy synthesis (ATP), and the regulation of calcium and ROS homeostasis, which is the basis of the normal CNS (20). Mitochondria contain the main sites of ROS production, which are also important targets of ROS damage, so oxidative stress damage is a major factor in mitochondrial dysfunction (21). Under normal circumstances, mitochondria also play a role in controlling the dynamic balance of Ca²⁺ concentration. When pesticides enter the organism, ROS accumulates in the brain tissue, which reduces mitochondrial membrane potential, resulting in membrane integrity damage and mitochondrial DNA (mtDNA) release (22). The accumulation of mtDNA mutations also increases the level of ROS, which affects the function of mitochondria. ROS excess also causes mitochondrial Ca²⁺ overload. Once the Ca²⁺ concentration exceeds the physiological threshold, ATP supplied by mitochondria is insufficient, and the mitochondrial function is disordered. This will lead to delayed mitochondrial energy metabolism, as well as potential instability and interruption of neural signal transmission, resulting in severe cell damage. Peroxidation further contributes to the decreased affinity of oxidatively modified cardiolipin for the pro-apoptotic factor Cytochrome C (Cyt-C), prompting the dissociation of Cyt-C from the inner mitochondrial membrane and thus its release into the cytoplasm. For example, the pesticide MCP significantly upregulated ROS, Cyt-C, and lipid peroxide in PC12 cells (23). Cyt-C binds to apoptotic protease activator 1, stimulates the formation of apoptotic complex apoptotic vesicles, and participates in the apoptotic process of activating caspases (24), upregulating the protein and gene expression levels of caspase-3 and caspase-9, and inducing apoptosis and severe neurodegenerative changes. This apoptotic pathway is also regulated by the Bcl family and other related pathways, such as Bcl-2, Bcl-W, Mcl-1, and Bax.

Various pesticides have been shown to elevate inflammatory factors levels, leading to neuroinflammation, neuronal cell death, and cognitive impairment (25). Microglia and astrocytes are the main immune cells in the brain, which are more prone to inflammation (26). Under normal physiological conditions, microglia and astrocytes are in a resting state, maintaining the dynamic balance of the internal environment of the CNS and ensuring the smooth progress of synaptic activities. When the CNS is stimulated by the herbicide paraquat and the pyrethroid bifenthrin (BF) (27, 28), microglia and astrocytes rapidly activate and proliferate, and a tissue repair process is initiated in response to this stimulus (29, 30). Activated microglia, astrocytes, and oxidized mtDNA can all stimulate the NLRP3 inflammasome (31), which is the core of inflammation and an important component of innate immunity. NLRP3 triggers the activation of caspase-1, which can induce apoptosis. Simultaneous activation of NF-κb and other pathways leads to the maturation and secretion of a large number of inflammatory factors such as interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2) (32, 33). These inflammatory factors will again lead to the dysfunction of mitochondria, produce ROS, increase the levels of MDA and nitric oxide (NO), and decrease the levels of GSH, CAT, SOD, and GSH-Px (28).

Synapses are the key site for information transmission between neurons, and neurotransmitters are crucial for maintaining brain function, as important information transmission substances between nerve cells. Abnormal release and transmission of neurotransmitters can affect the formation of the neurological basis of learning and memory long-term potentiation (LTP) (34) and lead to signal transduction disorders in the brain and ultimately to cognitive impairment.

The neurotransmitters affected by pesticide-induced brain injury are mainly divided into choline, free amino acids, and monoamines. (1) Choline substances, such as acetylcholine (ACh) and acetylcholinesterase (AChE), together constitute the central acetylcholinergic neuron system (35). Normally, in the presynaptic membrane, ACh is synthesized by the reversible transfer of acetyl groups from acetyl-CoA to choline (Ch) catalyzed by the enzyme choline acetyltransferase (ChAT). ACh relies on the acetylcholine transporter (VAChT) for transport to the synaptic vesicle and then released into the synaptic gap. Then binds to the cholinergic receptor of the postsynaptic membrane to form acetylase (HAT) and rapidly sheds acetyl groups automatically to form free AChE, while excess ACh in the synaptic gap is hydrolyzed by AChE to Ch and acetate (36). After monocrotophos (MCP) and other pesticides enter the organism (37), the positively charged part of the pesticides structure binds to the negative moment part of AChE. While the electrophilic phosphoryl group covalently binds to the serine hydroxyl group of the esterolysis site of AChE, inhibiting the activity of AChE. It is difficult to restore to free AChE. As a result of the loss of ability to hydrolyze ACh, ACh released from cholinergic nerve endings accumulated in large quantities. This will adapt changes in the cholinergic receptor, muscarinic receptor (mAChR), and nicotinic receptor (nAChR) (38, 39), resulting in CNS toxicity, disturbances in neural signaling, and cognitive impairment. (2) Free amino acids in the brain also play an important role as neurotransmitters in the cerebral cortex and hippocampus, participate in synaptic excitation transmission, and promote learning and memory formation. Free amino acids can be divided into excitatory amino acids (EAAs) such as glutamic acid (Glu) and N-methyl-D-aspartic acid (NMDA) and inhibitory amino acids (IAAs) such as γ-aminobutyric acid (GABA) and Gly. Under normal circumstances, these neurotransmitters are in a state of dynamic balance to ensure the normal transmission of nerve signals. For example, as an important inhibitory neurotransmitter in the CNS, GABA can antagonize the toxicity of Glu. When the herbicide atrazine and organophosphorus insecticide chlorpyrifos (CPF) enter the organism, the levels of these amino acids fluctuate abnormally, and nerve signal transduction is disordered, affecting the synthesis and regulation of related learning and memory proteins and resulting in brain function impair and cognitive impairment (40, 41). (3) Monoamine neurotransmitters include dopamine (DA), norepinephrine (NE), and 5-hydroxytryptamine (5-HT). They play an important role in the signal transmission of the CNS and have the functions of regulating movement, emotion, sleep, cognition, and memory. Pesticides such as Amitraz, Paclobutrazol, and Profenofos can reduce the expression of monoamine neurotransmitters in the brain tissue (42, 43).

It is found that the intestinal microbiota is closely related to the physiological function and disease of the CNS. The intestinal microbiota is linked to the brain through the nervous system, immune system, and endocrine system by neurotransmitters (such as 5-HT, DA, and GABA), immune factors (such as 1L-1β and TNF-α), and endocrine hormones (such as cortisol and corticotropin), thereby directly or indirectly affecting the brain function (44, 45). The accumulation of pesticides can change the composition of intestinal microbiota, promoting the downregulation of beneficial bacteria and overgrowing pathogenic bacteria, which can cause damage to intestinal mucosal and increase intestinal permeability, and allow intestinal bacteria and metabolites to enter the systemic circulation and be recognized by the brain through the nerve, immune, and endocrine pathways, affecting the CNS and inducing nerve injury. Endotoxin lipopolysaccharide (LPS), an important harmful metabolite, is released into the intestinal cavity during proliferation or lysis, activating macrophages and glial cells, increasing NF-κB activity (46), releasing inflammatory factors such as TNF-α and 1L-1β, and inducing inflammation and oxidative stress. However, the levels of beneficial metabolite short-chain fatty acids (SCFAs) decrease (47), affecting brain development and behavior. SCFAs are involved in the regulation of blood–brain barrier permeability and human glial cell homeostasis, and they maintain homeostasis in the CNS (48).

It is found that intestinal microbiota is, indeed, related to brain cognitive function. Aitbali et al. (49) found that the number of the beneficial intestinal microbiota of Actinobacteria (Corynebacterium), Firmicutes (Lactobacillus), and Bacteroidetes in the gut was decreased after acute (one administration of 0.3 mL), sub-chronic, and chronic exposure (250 or 500 mg/kg/d for 6 and 12 weeks) to GLY-based herbicides. The elevated plus maze and tail suspension test showed that the mouse showed anxiety and depression-like symptoms, indicating that intestinal ecological disorders may lead to neurobehavioral changes. By establishing a mice model of chronic Rotenone-induced Parkinson’s disease (PD), Zhao et al. (50) found that the insecticide rotenone (30 mg/kg/d BW by gavage) could induce intestinal microbiota imbalance and lead to intestinal dysfunction and behavioral disorder. Akkermansia and Desulfovibrio in feces were increased by 16S rRNA sequencing. Rotenone decreased the expression of tight junction proteins (claudin-1, occludin, and ZO-1), and transmission electron microscopy showed that the intestinal barrier and blood–brain barrier were damaged. The destruction of the intestinal microbiota induced the increase of its metabolite LPS in colon, serum and substantia nigra. LPS promoted the activation of the TLR4/MyD88/NF-κB signaling pathway and enhanced the expression of inflammatory factors (TNF-α, IL-6, IL-1β, iNOS and COX-2), thereby causing damage to neurons. The study showed that rotenone-induced microbiota imbalance may mediate inflammation caused by LPS-TLR4 signaling pathways, thus participating in the occurrence of PD through the microbiota–gut–brain axis. Based on these studies, it can be speculated that intestinal dysfunction may be the key to cognitive impairment caused by pesticides, but there are few related studies at present. Therefore, we think it is necessary to deeply explore the specific mechanism of gut microbiota affecting brain function through information exchange between the gut–brain axis, which may rewrite the definition of a healthy diet and life, and human beings will find new microecological methods to prevent and improve cognitive impairment in pesticides poisoning.

In summary, pesticides do not play a toxic role through a single mechanism, and there are multiple links between the mechanisms (Figure 1). A large amount of ROS is accumulated due to oxidative stress, which leads to the increase of mitochondrial mtDNA and Ca²⁺, and triggers mitochondrial dysfunction. Oxidized mtDNA promotes the activation of NLRP3 inflammasomes, releases a large number of inflammatory factors, and causes neuroinflammatory reactions, forming three feedback loops. Excessive free radicals in oxidative stress can also attack the structure and function of cholinergic neurotransmitter receptors, change cholinesterase activity, and damage the cholinergic system. Oxidative stress affects the expression of Gly; Gly is a constituent amino acid of the endogenous antioxidant reduced Glu in free amino acids. Acetyl-CoA is the raw material for the synthesis of ACh in the cholinergic system, which needs to be synthesized in mitochondria, and Glu is formed by multiple steps in the mitochondria of astrocytes (51). When the supply of ATP from mitochondria is insufficient, it will affect the synthesis of ACh, depolarize the cell membrane, and release a large amount of Ca²⁺-dependent Glu, resulting in cytotoxicity. Immune cells are involved in the expression of Ch acetyltransferase, AChE direct synthetase, and other corresponding cholinergic components. ACh released by immune cells or cholinergic neurons regulates immune function by acting on their receptors in an autocrine/paracrine manner, which can be called the “cholinergic anti-inflammatory pathway” mechanism (52). Therefore, the mechanisms are closely related and inseparable.

However, there are still some shortcomings in the research on the pesticide-induced cognitive impairment mechanism: the mechanism of the toxic effect of different pesticides is not fixed and cannot be fully classified, and it is also unknown whether the structure of pesticides is one of the causes of cognitive impairment. Studies have shown that the action mechanism of organophosphorus pesticides is widely considered to inhibit AChE. The atomic energy of P in organophosphorus pesticides combines with AChE to form phosphoryl cholinesterase, which makes the organism lose the ability to balance ACh, thus causing damage to the cholinergic central nervous system (53). Bipyridine heterocycle herbicide paraquat relies on the transfer of high-energy electrons in its planar bicyclic structure from the mitochondrial electron transfer chain to molecular oxygen to produce toxic ROS, which can induce cognitive impairment through oxidative stress (54). The rest of the pesticides according to their structure also can be divided into organic chlorine, carbamate, and pyrethroid (55, 56). There is no detailed report on whether the mechanism causing cognitive impairment is related to their respective special structures. In addition, a kind of pesticide can damage brain tissue through different mechanisms, but the specific reasons still need a lot of research and verification. At present, many pesticides have been banned in different countries, so it is very important to identify the specific mechanism of cognitive impairment caused by pesticides, and the specific effects of the structural components of pesticides on the CNS and human body. The development of new low-toxicity and high-efficiency pesticides has become an important topic for the global pesticide environment and human safety.

N-methyl-D-aspartic acid receptor (NMDAR) is a Glu excitatory nerve receptor, which is mainly distributed in the presynaptic and postsynaptic membranes of the cerebral cortex, hippocampus, thalamus, and striatum. It plays an important role in synaptic plasticity and synaptic transmission (57). At resting potential, NMDAR channels are blocked by Mg²⁺, and the NMDAR function is inhibited. Glu and Gly can activate NMDAR to open its channel. At this time, Mg²⁺ flows out of the channel, Ca²⁺ flows in and conducts signal transduction after synapse, which induces the formation of LTP and promotes synaptic plasticity. When pesticides, such as CPF, enter the organism (58), neurons release a large number of Glu, and NMDAR is overactivated, resulting in a Ca²⁺ influx. Ca²⁺-bound calmodulin (CaM) compensation increases, resulting in calmodulin-dependent protein kinase II (CaMKII) expression (59). It also affects the expression of cascade mitogen-activated protein kinase (MAPK), cAMP response element binding protein (CREB), and brain-derived neurotrophic factor (BDNF). NMDAR also affects the normal expression of proteins related to learning and memory such as PSD-95, NR2A, NR2B, and phosphatidylinositol 3 kinase (PI3K) (60). PSD-95 is a key skeleton protein of NMDAR and neuronal nitric oxide synthase (nNOS), which plays an important role in neural development (61). NR2A and NR2B can regulate Ca²⁺ permeability and Mg²⁺ sensitivity, and induce and maintain LTP.

A number of studies suggest that PI3K/Akt is an important oxidative signaling pathway and is closely related to learning and memory ability (62). PI3K/Akt signaling pathway is associated with multiple cascade pathways to maintain cellular homeostasis through antioxidant, anti-inflammation, and other mechanisms. When cells are damaged by oxidative stress caused by pesticides, excessive production of ROS over-regulates the level of PI3K (63). PI3K promotes the production of phosphatidylinositol triphosphate (PIP3), which then activates the high expression of Akt activated by PIP3 (64), affecting the normal regulation of downstream signaling molecules such as glycogen synthase kinase-3β (GSK3β), Nrf2, NF-κB, and mammalian rapamycin target protein (mTOR). For example, rotenone mediates the PI3K/Akt/mTOR pathway leading to brain injury and motor dysfunction in mice, and multiple impairments including the degeneration of dopaminergic neurons, decreased DA and GSH, and elevated MDA are also present (65). Mohammadzadeh et al. (66) found that the organophosphorus pesticide malathion induced spatial learning and memory impairment, activated GSK-3β, and inhibited protein phosphatase 2A (PP2A) in rats, while also increasing the levels of TNF-α and IL-6. GSK3β of an appropriate amount widely exists in neurons and glial cells of the brain and plays an important role in the regulation of cognitive function (67). Instead, excess GSK3β inhibits the activity of the antioxidant transcription factor Nrf2, preventing Nrf2 from playing a normal role in regulating cognitive function.

Mitogen-activated protein kinase is a group of serine–threonine protein kinases that can be activated by cytokines, neurotransmitters, hormones, and cell stress, which is an important information transmission chain (68). The main subclasses of MAPKs are extracellular regulated protein kinases (ERK1/2), c-jun terminal kinase (JNK), and p38 kinase (p38/MAPK) (69). After MAPKs activation, ERK1/2 is preferentially activated by growth factors, while p38/MAPK and JNK are particularly sensitive to environmental stress and cytokines (such as TNF-α) (70). They participate in the regulation of several inflammatory transcription factors (such as NF-κB) through phosphorylation. ERK1/2 is mainly responsible for cell growth and differentiation, and its upstream signal is the famous Ras/Raf protein (71). Stimulated by pesticides such as CPF (72), Ras is activated by binding to guanosine triphosphate, thus phosphorylating and activating Raf. Raf reactivates MEK1/2, and phosphorylated MEK1/2 activates ERK1/2. Activated ERK1/2 phosphorylates ribosomal S6 protein kinase (RSK)-like protein kinase substrates and co-enters the nucleus to promote the phosphorylation of important transcription factors such as CREB and regulate the transcriptional expression of Bcl-2, c-fos, c-myc, c-jun, Egrl, and other genes (73). p38/MAPK may be involved in the post-transcriptional regulation of COX-2, iNOS, and TNF-α (74). JNK is similar to p38 in function. The damage to brain tissue by pesticides may be related to three important branch pathways of MAPK, ERK1/2, p38/MAPK, and JNK.

Keap1/Nrf2/ARE signaling pathway plays an important role in cellular resistance to endogenous or exogenous oxidative stress and inflammation (75). Normal cells resist the interference of foreign substances through the antioxidant and anti-inflammation effects produced by the Keap1/Nrf2/ARE signaling pathway and its downstream gene activation. Increased intracellular ROS levels after pesticide intervention can promote the dissociation of the important transcription factor Nrf2 and the specific receptor Keap1 that regulates the anti-oxidative stress response in the body (76). Nrf2 enters the nucleus and forms a heterodimer with the small Maf protein, activating the antioxidant response element (ARE) to regulate the expression of phase II detoxification enzyme genes (NQO1 and GSTs) and antioxidant enzyme genes (SOD, HO-1, and CAT) (77, 78). For example, cypermethrin could inhibit the Nrf2/ARE pathway and decrease the expression of HO-1 and NQO1 (79). Nrf2 also regulates the GSH redox system, mainly regulating the expression of γ-GCS, and γ-GCS is the rate-limiting enzyme of GSH biosynthesis (80). Nrf2 also regulates the regeneration of GSH by controlling GSH-Px expression, GSH can be oxidized to GSSG by GSH-Px, and GR regenerates GSSG generated by GSH-Px to GSH (81). At the same time, this pathway increases the ability of cells to react to ROS and resistance to oxidative stress markers such as lipid peroxidation products, thus having a protective effect on multiple organs.

NF-κB is a multiple nuclear transcription-inducing factor that exists in almost all animal cells. It participates in the response of cells to external stimuli. It plays a key role in cellular immune response and inflammatory response and is closely related to learning and memory (82). NF-κB protein activity is activated by the phosphorylation of IκB kinase (IKK). In the resting state, NF-κB binds to IκB in the cytoplasm to form an inert complex that stays in the cytoplasm. When cells are stimulated by pesticide-induced inflammation signals, IKK is first activated (83). Then IKK phosphorylates the intracellular IκB protein. The phosphorylated IκB protein is, in turn, degraded by proteases, thus releasing NF-κB (84). The activated NF-κB enters the nucleus, binds to genes with NF-κB binding sites, and thus initiates the transcription process, inducing an increase in the levels of inflammatory factors such as TNF-α, IL-1β, and apoptotic factors such as Bcl-2 to affect learning and memory (85). Michel et al. (86) found that rotenone-induced NF-κB transcription increased the expression of iNOS and COX-2 in the rat’s midbrain and striatum. Rotenone also increased caspase-3 and α-synuclein activity, as well as the Bax/Bcl-2 ratio in the striatum to induce apoptosis.

After sorting out, it is found that each pathway is not independent but has interconnected targets, which establishes a huge pathways system (Figure 2) and together play a regulatory role in the process of pesticide-induced brain tissue injury. It is assumed that under the stimulation of pesticides, a large amount of Glu accumulates, activating that NMDAR pathway, resulting in Ca²⁺ influx and activation of downstream PI3K/Akt and MAPK signaling pathways through CaM and Ras. The γ subtype of PI3K plays a key role in NMDAR receptor-mediated synaptic plasticity and hippocampal-dependent learning and memory ability. PI3K can activate CREB related to the ERK pathway, and its over-activation can reduce the probability of ERK entering the nucleus. As an important injury junction, Akt can attenuate the expression of the Nrf2 nuclear transcription factor in the Keap1/Nrf2/ARE pathway, and reduce the activities of antioxidant enzymes such as SOD. It can also promote the initiation of the NF-κB pathway and release a large number of inflammatory factors such as TNF-α and IL-1β. NF-κB can also initiate the Bcl apoptosis pathway, resulting in cell damage and death. All three branches of MAPK will promote the production of inflammatory factors finally, and it is believed that MAPK and NF-κB pathways are inextricably linked. By constructing the pathway network, it is not difficult to guess that the related signaling pathways in pesticide-induced cognitive impairment are an organic whole and closely related to the action mechanism. These pathways together lead to cognitive impairment caused by pesticide poisoning.

Carnosic acid (CA) has many biological activities, such as inhibiting oxidative stress and inflammation (113, 114), and it has a good protective effect in the experimental model of neurodegenerative diseases. De Oliveira et al. (115) used CA (0.1–2 μm) to intervene with SH-SY5Y cells in the PD model induced by Paraquat (100 μm) and found that CA could be used as a neuroprotective agent to prevent the effects of Paraquat on cell viability and redox parameters, increase the contents of total GSH and mitochondrial GSH in SH-SY5Y cells, reduce the production of ROS, RNS, and the toxicity to mitochondrial function. At the same time, inhibition of the PI3K/Akt pathway or silencing of Nrf2 expression by LY294002 partially blocked the improvement of CA. The results showed that CA could activate Nrf2 by regulating PI3K/Akt pathway, resulting in an increase in the level of antioxidant enzymes and a neuroprotective effect. AlKahtane et al. (116) studied the improving effect of CA (60 mg/kg/d BW by gavage) on mice brain and eye cytotoxicity induced by CPF (12 mg/kg/d BW by gavage). It was found that CA could reduce the contents of MDA and NO in the brain and eye tissue, decrease the levels of IL-1β, IL-6, and TNF-α in serum, and increase the level of AChE in serum and the levels of GSH, GSH-Px, SOD, and CAT in the brain.

Carnosic acid is a natural phenolic diterpene with an MW of 332.434. It is speculated that the two hydroxyl groups on the benzene ring in its molecule are one of the key reasons for its antioxidant activity. However, whether other characteristics of CA structure are related to the improvement of cognitive impairment needs to be verified.