Thymol mitigates monosodium glutamate-induced neurotoxic cerebral and hippocampal injury in rats via overexpression of nuclear erythroid 2-related factor 2 signaling pathway as well as altering nuclear factor- kappa β and glial brillary acidic protein expression

Monosodium glutamate (MSG) is commonly used in various food industries as a avor enhancer. MSG is reported to cause many toxic effects, including increased neurotoxicity. The study investigates the molecular mechanisms underlying thymol's neuroprotective effect against MSG-induced neurotoxic cerebral and hippocampal injury in rats. Brain injury was induced by MSG (2 g/kg; i.p.) for 15 days. Thymol (400 & 800 mg/kg/day; p.o.) was administered along with MSG. MSG control rats showed a signicant reduction in behavioral activity, elevated brain tissue oxidative stress, inammatory parameters, Nrf2 gene up-regulation, overexpression of nuclear factor-kappa β (NF-kβ), glial brillary acidic protein (GFAP) as well as neuronal damage in the cerebral cortex & hippocampus. Thymol ameliorated MSG-induced brain injury via overexpression of the Nrf2 gene, thus increasing the cellular defense and organizing antioxidant & anti-inammatory effects. Thymol improved behavioral activity and brain tissue GSH content. Thymol also decreased brain contents of MDA, NO, TNF-α & IL-6. Moreover, Thymol improved NF-kβ & GFAP immunohistochemical expression and histopathological picture in the cerebral cortex & hippocampus compared to MSG control rats. The aforementioned results suggest that thymol exhibits promising neuroprotective effects. The study elucidates the molecular mechanisms linking Nrf2 pathway signaling to oxidative stress, inammation & NF-kβ expression underlying thymol’s protection against MSG-induced neurotoxicity. The study also highlights the role of GFAP expression in MSG-induced astrocyte injury of cerebrum & hippocampus of rats and the promising protective effects of thymol in ameliorating astrocyte injury. of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and nuclear factor-kappa β (NF-kβ) in brain tissues. The study also demonstrates the possible protective effects of thymol on the expression of glial brillary acidic proteins involved in neuronal damage of brain tissue. mainly attributable to thymol’s antioxidant and anti-inammatory properties. These properties might be attributable to the up-regulation of the Nrf2 signaling pathway. The study elucidates the molecular mechanisms linking oxidative stress, inammation, NF-kβ expression and Nrf2 pathway signaling underlying thymol’s protection against MSG-induced neurotoxicity. The study also highlights GFAP expression's role in MSG-induced astrocyte injury of the cerebral cortex & hippocampus of rats and the promising protective effects of thymol in ameliorating astrocyte injury via reduction of elevated GFAP expression.


Introduction
Monosodium glutamate (MSG), a salt form of L-glutamic acid, is commonly used in various food industries as a avor enhancer (Ikeda 2002). MSG is abundant in high concentrations in commercially produced restaurant and household food products (Onaolapo et al. 2016). Despite its overspread use, MSG is always suspected to cause many toxic effects, collectively described as "The Chinese restaurant syndrome" (Walker and Lupien 2000). However, many countries pose no strict limitations on the permissible MSG concentrations in food. MSG is described to produce severe metabolic changes leading to a diverse arena of symptoms including ushing, sweating, weakness, dizziness, headache, and increased oxidative stress in the brain (Rotimi et al. 2012).
MSG dissociates in the body to yield sodium and glutamate ions (Quines et al. 2014). Ingested glutamate is metabolized in the gastrointestinal tract. As more glutamate is ingested, it gets absorbed via the portal vein (Reeds et al. 2000). Glutamate is the primary excitatory neurotransmitter in the human brain that plays a crucial role in brain development, synaptic plasticity, and learning (Maragakis and Rothstein 2001). Elevated glutamate concentrations produce abundant neurotoxicity via over-activation of excitatory amino acid receptors. This dilemma increases intracellular calcium, eventually resulting in cellular damage and severe brain excitotoxicity (Nagakannan et al. 2012).
MSG administration damages the hypothalamic neurons and results in a modi cation in lipid peroxidation and oxidative stress in various areas of the brain, leading eventually to hyperexcitability and motor as well as behavioral changes (López-Pérez et al. 2010;Zan rescu et al. 2019).
Therefore, MSG administration can be considered a reproducible neuro-toxic animal model, for testing drug's effects on oxidative stess and neurotoxic cerebral and hippocampal brain injury (Shivasharan et al. 2013).
Moreover, many studies proved that thymol improves memory (Amiri 2012), possesses anti-cancer (Deb et al. 2011), anti-in ammatory (Liang et al. 2014) as well as immunomodulatory properties (Hashemipour et al. 2013). Most of these effects can be attributed to its antioxidant properties (Javed et al. 2019;Youdim and Deans 2000). However, the neuroprotective effects of thymol in neuro-toxic cerebral and hippocampal injury have not been well studied. Therefore, the current study aims at testing the potential molecular mechanisms of the antioxidant and anti-in ammatory effects of thymol against MSG-induced neurotoxic cerebral and hippocampal injury in rats. The study outlines the possible involvement of nuclear erythroid 2-related factor 2 (Nrf2) signaling pathway as well as expression of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and nuclear factor-kappa β (NF-kβ) in brain tissues. The study also demonstrates the possible protective effects of thymol on the expression of glial brillary acidic proteins involved in neuronal damage of brain tissue.

Animals
Forty adult male Albino rats weighing 180-200 g were used in the current study. Standard food pellets and tap water were supplied ad libitum. Animals and food pellets were obtained from the animal house colony of the National Research Center (NRC, Egypt). The study was conducted in accordance with the National Research Centre-Medical Research Ethics Committee (NRC-MREC) for the use of animal subjects and following the recommendations of the National Institutes of Health Guide for Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 2011).
Drugs and chemicals MSG was purchased from Sigma (St. Louis, MO, USA). Thymol uid extract (Thymotal ® syrup; each 5 ml contains 1.66 gram standardized to contain 0.03% phenolic compounds calculated as thymol; ATOS PHARMA, Egypt) was used throughout the study. All other chemicals were of the highest analytical grade available.

Experimental design and treatment protocol
Animals were randomly allocated into four groups (10 rats each). The 1st group's rats received intraperitoneal saline along with distilled water orally for 15 days and served as a normal control group.
Brain injury was induced in the remaining 3 groups by intraperitoneal injection of monosodium glutamate (2 g/kg/day) for 15 days. Group 2 received only distilled water orally for 15 days along with MSG and served as MSG control group. Groups 3 & 4 received thymol (400 & 800 mg/kg/day; p.o.) respectively for 15 days. All animals were sacri ced 48 h after the last drug administration under sodium pentobarbital anesthesia.

Spontaneous locomotor activity
The locomotor activity was recorded by using an actophotometer (Ugo Basile, Milan, Italy). Actophotometer is a clear acrylic plastic box (45 × 45 × 45 cm) with a removable plastic lid perforated with ventilation holes. The device contains photocell beams and detectors that are mounted on opposite walls. Breaks of the photocell beams detect spontaneous locomotor activity. Before the locomotor task, animals were placed individually in the activity meter cage for 2 minutes for habituation. After that, locomotor activity was recorded as the counts within 5 minutes (Kulkarni 1987).

Rotarod performance test
Animals were trained to walk on the accelerating rotarod apparatus 2 days before recording the results (Ugo Basile 47600, Milan, Italy). The apparatus consists of a cylinder on which 5 animals can run simultaneously, separated by su cient size panels to prevent the animals from detecting each other visually. The rod's speed was increased from 4 rpm to 40 rpm during 300s, then the animals were returned to their cages. When the animal could not maintain its balance and fell off the device, it triggered a sensor and the time was recorded (van den Berg et al. 2016).
Real-time polymerase chain reaction (PCR) quanti cation of nuclear erythroid 2-related factor 2 (Nrf2) The mRNA expression level of the Nrf2 gene was assessed using real-time PCR standardized by coampli cation with the housekeeping GAPDH gene as an internal control. Nrf2 RNA was extracted from brain tissue using Trizol reagent. RNA was reverse-transcribed using M-MLV reverse transcriptase

Histopathological examination
Following scari cation, the brains were removed and xed in 10% neutral formalin for 48h. Then, the brain tissues were coronally sectioned, washed, dehydrated in alcohol, and embedded in para n wax. The sections were cut into 4μm thickness and stained with H&E for histopathological examination (Bancroft et al. 1996). For neuronal damage assessment, the number of degenerated and/or necrotic neurons was counted in ve random high microscopic power elds (HPF) in both the cerebral cortex and hippocampus of normal and treated groups.
All the immunohistochemical procedures for the demonstration of NF-kβ and GFAP expression were performed according to the method of Khalil et al. (2019) (Khalil et al. 2019). Brie y, the para nembedded brain sections were depara nized and rehydrated. Then, the sections were incubated with rabbit polyclonal anti-NF-kβ (ab16502) and anti-GFAP (ab7260) (Abcam) as primary antibodies. Finally, diaminobenzidine (DAB) (Sigma, USA) was used to visualize the immune reaction. Relying on the percentage of positive cells per HPF, the immune reactivity was semi-quantitatively assessed in ten random HPF, according to the method of Hassan et al. (2019), in which; 0=no staining,1=positive staining in < 30% of cells per HPF, 2=positive staining in 30-70% of cells per HPF, or 3= positive staining in > 70% of cells per HPF ).

Statistical analysis
All the values are presented as means ± standard error of the means (SEM). Comparisons between different groups were carried out using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison post hoc test. The difference was considered signi cant when p 0.05. GraphPad prism ® software (version 6 for Windows, San Diego, California, USA) was used to carry out these statistical tests.

Results
Effects of thymol on spontaneous locomotor activity and rotarod rotation time in MSG-induced neurotoxic brain injury in rats.
MSG (2 g/kg; i.p.) resulted in acute brain injury in rats, as evidenced by a signi cant reduction in animal's movement (measured as counts/5 min) to 52% compared to normal control animals.
The treatment of rats with thymol (400 & 800 mg/kg/day; p.o) signi cantly improved the decreased spontaneous locomotor (measured as counts/5 min) to 152% & 177% as respectively compared to the MSG control group.
MSG resulted in a signi cant reduction in animals' rotarod rotation time (seconds) to 28% compared to normal control animals.
While treatment of rats with thymol (400 & 800 mg/kg/day; p.o) signi cantly improved the decreased rotarod rotation time (seconds) to 166% & 226% respectively as compared to the MSG control group (Table 1). Effects of thymol on brain tissue oxidative stress parameters in MSG-induced neurotoxic brain injury in rats.
MSG resulted in acute brain injury in rats as evidenced by the signi cant elevation of brain tissue  Effects of thymol on brain tissue concentration of TNF-α and IL-6 in MSG-induced neurotoxic brain injury in rats. Acute brain injury in rats induced by MSG resulted in a signi cant elevation of brain tissue concentration of TNF-α & IL-6 to 393% & 432%, respectively, as compared to the normal control group.
Quantitative RT-PCR for Nrf2 gene expression in brain tissue The expression response of the Nrf2 gene in rat brains following monosodium glutamate-induced oxidative stress and neuro-toxic brain injury was analyzed using qRT-PCR. Nrf2 was up-regulated in the MSG-control group to reach 1.15-fold as compared to the normal control group.

Histopathological examination of cerebral cortex and hippocampus
The result of the histopathological assessment of neuronal damage is illustrated in table 3. Brains of normal rats showed typical histological structure with normal rounded cerebral cortical and hippocampal neurons (Figs. 3a &3b, respectively). Meanwhile, apparent neuronal damage was demonstrated in the brains of MSG-treated rats. This damage was manifested by the presence of shrunken eosinophilic necrotic neurons associated with neuronophagia and diffuse proliferation of microglia cells and astrocytes that are characteristically demonstrated in the cerebral cortex (Fig. 3c) and hippocampus (Fig. 3d) of this group. Treatment with thymol remarkably ameliorates these histopathological alterations in a dose-dependent manner. A decreased number of degenerated and/or necrotic neurons and moderate gliosis were observed in the cerebral cortex and hippocampus of the MSG-Thymol (400 mg/kg) group ( Fig. 3e &3f, respectively). On the other side, minimal neuronal damage and gliosis were demonstrated in the MSG-Thymol (800 mg/kg) group. Most of the cerebral cortical and hippocampal neurons appeared normal with scant degenerated ones ( Fig. 3g &3 h, respectively).

Immunohistochemical analysis of NF-kβ expression in cerebral cortex and hippocampus
The results of immunohistochemical analysis of NF-kβ expression in the cerebral cortex and hippocampus are illustrated in table 4. Brains of normal rats showed normal cytoplasmic staining of neurons of both the cerebral cortex ( Figure 4a) and hippocampus ( Figure 4b) with no nuclear translocation evidence. On the contrary, the signi cant increase of NF-kβ expression was demonstrated mainly in the nuclei of cerebral cortical neurons ( Figure 4c) and pyramidal cells of the hippocampus (Figure 4d). Conversely, the percentage of NF-kβ-positively stained cells was signi cantly reduced in the cerebral cortex ( Figure 4e) and hippocampus (Figure 4f) of the MSG-Thymol (400mg/kg) group, in which most neurons showed cytoplasmic staining. Pronounced reduction of NF-kβ expression, with minimal nuclear staining, was demonstrated in the cerebral cortex ( Figure 4g) and hippocampus (Figure 4h) of MSG-Thymol (800mg/kg) group.

Immunohistochemical analysis of GFAP expression in cerebral cortex and hippocampus
The results of immunohistochemical analysis of GFAP expression in the cerebral cortex and hippocampus are illustrated in table 5. Few weakly stained astrocytes with small cell bodies and short cytoplasmic processes were demonstrated in the cerebral cortex ( Figure 5a) and hippocampus ( Figure  5b) of normal rats. Whereas the signi cant increase in the percentage of GFAP+ astrocytes with large hypertrophied cell bodies and thicker as well as longer cytoplasmic processes recorded in the cerebral cortex ( Figure 5c) and hippocampus (Figure 5d) of MSG-treated rats. On the other hand, the percentage of GFAP+ astrocytes was signi cantly decreased in thyme-treated groups in a dose-dependent manner. Few GFAP+ astrocytes with hypertrophied cell bodies were demonstrated in the cerebral cortex of the MSG-Thymol (400mg/kg) group (Figure 5e). But these cells were much hypertrophied in the hippocampus of this group (Figure 5f). Signi cant reduction of GFAP+ astrocytes, which appeared in the focal aggregate, was demonstrated in the cerebral cortex and hippocampus of the MSG-Thymol (800mg/kg) group ( Figures 5g & 3h, respectively).

Discussion
MSG is commonly used in various food industries as a avor enhancer (Ikeda 2002). MSG is reported to produce severe metabolic changes leading to abundant neurotoxicity and motor as well as behavioral changes (Nagakannan et al. 2012). Therefore, the current study aims at scouting the potential underlying molecular mechanisms involved in MSG-induced neurotoxic brain injury in rats. This study is the rst to evaluate the role of the Nrf2 signaling pathway as well as NF-kβ and GFAP expression in MSG-induced brain injury and the potential neuroprotective antioxidant and anti-in ammatory effects of thymol on MSG-induced neurotoxic cerebral and hippocampal injury in rats.
In the current study, MSG administration resulted in a signi cant alteration in motor and behavioral activities in rats as manifested by a reduction of the spontaneous locomotor activity as well as the rotarod rotation time of rats. This data agrees with Shivasharan et al. (2013), who stated that MSG signi cantly decreased the locomotor activity of adult female Wistar rats (Shivasharan et al. 2013). Swamy et al. (2014) also demonstrated that MSG caused a signi cant reduction in spontaneous locomotor activity in Wistar albino rats (Swamy et al. 2013).
MSG administration also resulted in a signi cant elevation of lipid peroxidation products and NO metabolites with a substantial reduction in GSH concentration in rats' brain tissue compared to the normal group; indicating severe oxidative damage to brain tissues. Many studies agree with this data demonstrating alteration in brain tissues antioxidative defense mechanisms post-MSG administration (Onaolapo et al. 2016;Sadek et al. 2016;Shivasharan et al. 2013;Swamy et al. 2013).
In the current work, MSG administration resulted in a signi cant elevation of brain tissue concentration of IL-6 and TNF-α compared to the normal group; indicating signi cant in ammatory damage of brain tissue. Khalil and Khedr (2016) stated that MSG administration resulted in a signi cant in ammatory response to brain tissue as manifested by an elevation of TNF-α in the hippocampus of MSG-treated rats (Khalil and Khedr 2016).
In the current work, Nrf2 was up-regulated in the MSG-control group as compared to the normal control group. In the basal non-stressed condition, Nrf2 is primarily abundant in the cytoplasm bound to the cytoplasmic Kelch-like ECH associated protein 1(Keapl). Binding to Keap1 blocks Nrf2 activation to keep Nrf2 concentration low. In case of occurrence of any endogenous or exogenous cellular events mandating cellular defense mechanisms, abrupt induction of Nrf2 occurs. Oxidative stress and/or in ammation decouple Keap1 from Nrf2 to activate the latter along with a further synthesis of Nrf2. Nrf2 is then transferred into the nucleus to bind with antioxidant response elements (ARE); also known as electrophile response elements (EpRE). This binding further activates Nrf2/ARE pathway. Nrf2 activation eventually results in an organized antioxidant, anti-in ammatory and anti-apoptotic responses in vivo (Vomhof-DeKrey and Picklo Sr 2012). Nrf2 gene over-expression also stimulates protective proteins such as; the brain-derived neurotrophic factor, peroxisome proliferator-activated receptor gamma co-activator 1α and the auto-phagic protein p62. External causes can induce Nrf2 activation, and it has been linked to many endogenous pathophysiological defense mechanisms in numerous systems, including the cardiovascular and the nervous systems (Zhu et al. 2018). It has been con rmed that the Nrf2 pathway plays a crucial role in CNS, in the case of brain in ammation, acute cerebral injuries as well as neurodegenerative disorders (Sandberg et al. 2014). Besides, a signi cant increase of NF-kβ expression, in the current work, was demonstrated in the nuclei of cerebral cortical neurons and pyramidal cells of the hippocampus of the MSG-control group. NF-kβ is found to suppress the transcription of ARE-dependent genes, and thus it acts as a crucial regulator of in ammation. NF-kβ is inactivated in the cytoplasm via binding to an inhibitor of NF-kβ-kinase subunit beta. The molecular mechanisms linking NF-kβ and Nrf2 are not yet well-de ned. However, the Nrf2 gene contains binding sites for NF-kβ in the promoter region which proposes a possible inter-relation between these two in ammatory regulators (Nair et al. 2008). In line with our ndings, many studies reported over-expression of Nrf2 as well as NF-kβ in different experimental animal models of brain injury. Li et al. (2016), Zhang et al. (2017) and Cheng et al. (2016) reported over-expression of Nrf2 signaling in experimental traumatic brain injury (Cheng et al. 2016;Li et al. 2016;Zhang et al. 2017). Wei et al. (2017) reported activation of the Nrf2 signaling pathway postintracerebral hemorrhage (Wei et al. 2017). Du et al. (2020) reported up-regulation of Keap1/Nrf2 pathway in MSG-induced obese cardiomyopathy (Du et al. 2020). Xu et al. (2020) reported overexpression of NF-kβ pathway in neuro-in ammation in rats following subarachnoid hemorrhage (Xu et al. 2020). In line with this data, Nurmasitoh et al. (2018) reported that NF-kβ reduction results in antioxidant and anti-in ammatory effects (Nurmasitoh et al. 2018). The current study showed a signi cant histopathological injury of the brains of MSG-treated rats as manifested by the presence of shrunken necrotic neurons and proliferation of microglia cells and astrocytes in the cerebral cortex and hippocampus. Many studies reported severe alteration in brain tissue histopathological picture following MSG administration. Shivasharan et al. (2013) reported the appearance of numerous shrunken cells in the hippocampus of MSG-treated rats (Shivasharan et al. 2013  Astrocytes makeup about 50% of the brain tissue and are essential for the normal physiology and survival of the neurons. GFAP is a crucial protein secreted by mature astrocytes. GFAP is an astrocytespeci c marker that controls astrocytes' shape and movement. GFAP expression is an immunohistochemical marker for reactive astrocytes. GFAP is overexpressed in response to neuronal damage, especially synaptic e cacy (Hashem et al. 2012). The current study demonstrates a signi cant increase in the percentage of GFAP astrocytes with large hypertrophied cell bodies and thicker as well as longer cytoplasmic processes recorded in the cerebral cortex and hippocampus of MSG-treated rats. In line with this nding, Zaher (2020) reported signi cant histopathological alterations and severe GFAP immunoreactivity in the hippocampus of MSG-treated rats (Zaher 2020). Hashem et al. (2012) also described histological distortment accompanied by moderate GFAP immunohistochemical expression in the astrocytes of cytoplasm as well as a granular layer of the cerebellar cortex of MSG-treated rats (Hashem et al. 2012).
The current study aimed to test the protective effects of thymol against MSG-induced neuro-toxic cerebral and hippocampal injury in rats, emphasizing the potential molecular mechanisms underlying this neuroprotection.
In the current work, thymol in the selected doses improved behavioral changes caused by MSG administration. This was manifested by an improvement of the decreased spontaneous locomotor and rotarod rotation time compared to the MSG control group.
Thymol has been reported to be a positive activator of the GABAA receptors in humans (Priestley et al. 2003). This may account for its neuroprotective effects, especially in the case of MSG-induced neurotoxicity. MSG dissociates in the body to yield glutamate ions producing abundant neuro-toxicity via over-activation of excitatory amino acid receptors. Sancheti et al. (2014) stated that thymol administration resulted in anti-epileptogenic activity in rats, manifested by improved locomotor activity and seizure score (Sancheti et al. 2014).
According to previous studies, thymol improved oxidative stress parameters by decreasing MDA and NO metabolites and increasing GSH concentration in brain tissues compared to the MSG control group. This can be attributed to thymol's antioxidative properties. Moreover, thymol administration resulted in a signi cant reduction of elevated IL-6 and TNF-α concentration in brain tissues as compared to the MSGtreated group. This can be attributable to thymol's potential anti-in ammatory properties. The antiin ammatory effects of thymol also account for the signi cant reduction of NF-kβ immunohistochemical expression in the cerebral cortex and hippocampus of rats as compared to MSG-treated rats. Interestingly, thymol administration also resulted in considerable overexpression of the Nrf2 gene in brain tissue compared to the MSG control group. This indicates activation of the theNrf2 signaling pathway, which results in organized antioxidant, anti-in ammatory, anti-apoptotic actions. Thymol also improved the overall histopathological pictures of the cerebral cortex and hippocampus of rats. Moreover, thymol signi cantly decreased GFAP expression in the cerebral cortex and hippocampus compared to the MSG group.
In accordance with our results, Javed et al. (2019) stated that thymol administration resulted in signi cant attenuation of oxidative stress, in ammation, and neuronal damage induced by Parkinson's disease in rats. These actions were attributed to inhibition of lipid peroxidation, besides the restoration of GSH, catalase and superoxide dismutase antioxidant activates. Thymol administration signi cantly decreased the pro-in ammatory cytokines, viz. IL-6, IL-1β, and TNF-α and inhibited glial cell activation (Javed et al. 2019). In another study, thymol administration was associated with improved cognitive de cits due to up-regulation of the Nrf2 signaling pathway in mice . Chamanara et al. (2019) reported signi cant NF-kβ signaling inhibition in acetic acid-induced in ammation in colon tissue in rats (Chamanara et al. 2019).
Further investigation is warranted to explore other potential pharmacological interventions to overcome MSG-induced neuro-toxic cerebral and hippocampal injury.

Conclusion
To summarize, from the current study, it can be concluded that thymol exhibited promising protective effects against MSG-induced neurotoxic cerebral and hippocampal injury in rats. These effects are mainly attributable to thymol's antioxidant and anti-in ammatory properties. These properties might be attributable to the up-regulation of the Nrf2 signaling pathway. The study elucidates the molecular mechanisms linking oxidative stress, in ammation, NF-kβ expression and Nrf2 pathway signaling underlying thymol's protection against MSG-induced neurotoxicity. The study also highlights GFAP expression's role in MSG-induced astrocyte injury of the cerebral cortex & hippocampus of rats and the promising protective effects of thymol in ameliorating astrocyte injury via reduction of elevated GFAP expression.

Declarations
Funding: The authors have no relevant nancial or non-nancial interests to disclose.
Con icts of interest/Competing interests: The authors have no con icts of interest to declare that are relevant to the content of this article.
Availability of data and material: All data will be available upon request