Protective effect of Moringa oleifera Lam. leaf extract against oxidative stress, inflammation, depression, and apoptosis in a mouse model of hepatic encephalopathy

The present study aimed to assess the antioxidative, anti-inflammatory, antiapoptotic, and anti-depression impacts of Moringa oleifera Lam. leaf ethanolic extract (MOLE) in the hippocampus and cerebral cortex of CCl4-induced hepatic encephalopathy mouse model. High-performance liquid chromatography was used to detect marker compounds: rutin and β-sitosterol. Animals were divided into four groups: vehicle group, CCl4-treated group, MOLE-treated group, and (CCl4 + MOLE) group treated with MOLE for 14 days before CCl4-induced neurotoxicity. MOLE decreased alanine aminotransferase, aspartate aminotransferase, corticosterone, and ammonia levels in serum and improved the antioxidant status of CCl4-treated mice in the hippocampus and cerebral cortex. It reduced the expression of toll-like receptor 4 (TLR4), TLR2, myeloid differentiation primary response 88 (MYD88), and nuclear factor-kappa B (NF-κB) genes and the protein levels of the pro-inflammatory cytokines. MOLE also attenuated apoptosis, as revealed by the reduced expression of caspase3, and prevented histological deterioration. Furthermore, MOLE attenuated CCl4-induced anxiety and depression-like behavioral changes. Collectively, MOLE modulates neuroinflammation, oxidative stress, TLR4/2-MyD88/NF-κB signaling, and apoptosis in the hippocampus and cerebral cortex of the hepatic encephalopathy experimental model.


Introduction
Chlorinated volatile compounds (Cl-VOCs) have been reported to affect several organs such as the liver, the kidney, the heart, and the central nervous system (Genc et al. 2012;Teschke 2018;Webb et al. 2018). Furthermore, direct exposure to Cl-VOCs can cause esophageal cancer, cervical cancer, and liver cancer (Lang and Beier 2018). For these devastating effects, some of them including carbon tetrachloride (CCl 4 ) and chloroform (CHCl 3 ) were considered priority pollutants by the United States Environmental Protection Agency (EPA) (Halawy et al. 2022). The γ-Al2O3 nanocomposite was suggested in a recent study as a cost-effective method to adsorb and remove CCl 4 (Halawy et al. 2022).
According to their review of hepatic encephalopathy animal models, the International Society for Hepatic Encephalopathy and Nitrogen Metabolism (ISHEN), CCL 4 can be used to induce hepatic encephalopathy (HE) in rodents by disrupting the hepatocytes leading to hyperammonemia, which subsequently results in neuroinflammation and decrease in neurogenesis and memory accusation (DeMorrow et al. 2021). CCl 4 is a neurotoxic substance used to mimic brain manifestations of people suffering from acute or chronic hepatic damage by triggering lipid peroxidation and alterations in the antioxidative mechanisms in the brain (Vairappan et al. 2019). The symptoms in patients include cognitive decline, motor, psychiatric disorders, and HE  (Vairappan et al. 2019). HE is a neuropsychiatric disorder propagated as a result of acute or chronic hepatic failure (El-Marasy et al. 2019).
In the liver, CCl 4 is metabolized into highly reactive free radicals which oxidize fatty acids in the phospholipids of cell membranes leading to structural and functional changes in these membranes (Yue et al. 2020). Moreover, these free radicals and CCl 4 cause injuries in the endoplasmic reticulum with a consequential effect on protein synthesis leading to lipid accumulation (Yue et al. 2020). Meanwhile, CCl 4 leads to producing inflammatory mediators from the triggered macrophages in the liver with accompanying systemic inflammation exerting a critical role in aggravating neurological manifestations, possibly by activating the predisposition of the brain to the associated hyperammonemia (Aldridge et al. 2015).
Neuroinflammation and oxidative stress are involved in depression and anxiety development (Mello et al. 2013). Noteworthy, CCl 4 induces neuropsychiatric disorders mimicking what appears in patients with acute or chronic liver damage by targeting the brain antioxidative system and inflammatory pathways such as the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) pathway (Shal et al. 2020). Moreover, it has been reported that corticotropin-releasing factor (CRF) hypersecretion in response to the release of proinflammatory cytokines is attributable to modifying the hypothalamic-pituitary-adrenal (HPA) axis resulting in increased plasma corticosterone levels and exacerbation of depression symptoms (Furtado and Katzman 2015).
Plants are well known for having therapeutic effects and have been used in traditional and modern medications. Moringa oleifera Lam. (MOL), family Moringaceae (Fakurazi et al. 2012) is widely known as the "Miracle Tree" due to its medicinal outcomes. Its leaves, the most common and suitable part of the plant for medicinal commercial mass production (Mahdi et al. 2018), can be consumed in different ways and then dried and stored for months without losing nutritional benefits (Tesfay et al. 2011). Our previous study recorded the following compounds in Moringa oleifera Lam. leaf ethanolic extract (MOLE): quercetagetin-7-O-glucoside, quercetin 3,5,7,3′,4′-pentamethyl ether, and β-sitosterol along with other phytochemicals using gas chromatographymass spectrometry (GC-MS) analysis (Fathy and Mahmoud 2021). Moreover, the total phenolic and the total flavonoid contents in MOLE were also measured (Fathy and Mahmoud 2021). Furthermore, MOL possesses pain relief, anti-depression, anti-inflammatory, immunomodulatory, and neuroprotective activities (Kou et al. 2018).
The current study aimed to assess the antioxidative, antiinflammatory, antiapoptotic, and anti-depression impacts of MOLE in the HC and CC of CCl 4 -induced HE mouse model. The alleviation potentials of MOLE were assessed by tracking its effect on the TLR4/2-MyD88/NF-κB pathway, neuroinflammation, apoptosis, oxidative stress, anxiety and depression-like behavior, and histopathological changes in the hippocampus (HC) and cerebral cortex (CC) regions of the mouse brain.

Chemicals
CCl 4 and L-glutathione reduced were purchased from Sigma (St. Louis, MO, US). All other chemicals and reagents used were of the highest analytical grade.

MOL source and identification
The source for the plant leaves was from Jazan City, KSA, with latitude 16° 53′ 12.59″ N and longitude 42° 33′ 23.99″ E coordinates according to degrees minutes seconds (DMS). The authentication of the plant was carried out by taking the herbarium specimens found at Jazan University Herbarium (JAZUH), KSA, as a reference.

Preparation of MOLE
MOL leaves were washed, dried, and finally ground. Ninetysix percent ethanol was mixed with the ground leaves, and the mixture was kept in the shaking incubator at 37 °C for 24 h. The obtained extract was then filtered and put in the rotary evaporator at 40 °C until the ethanol was completely evaporated. Finally, a semi-solid extract was produced and stored at 4 °C until use.

High-performance liquid chromatography (HPLC) analysis
MOLE was analyzed using the HPLC method for a qualitative analysis of two marker compounds. About 50 mg of the extract was dissolved in 25 mL methanol and injected into an HPLC (Agilent 1200 series, UV detector). For rutin, Agilent Eclipse XDB-C18 (150 × 4.6 mm, 5 μm), wavelength 254 nm, and 1 mL/min flow rate. The mobile phase consisted of acetonitrile: water/0.1 formic acid with a gradient increased from 5 to 95% over 15 min. For β-sitosterol, a waters symmetry shield C18 column (150 × 4.6, 5 µm) and wavelength 210 nm was used. The mobile phase consisted of methanol: acetonitrile with a ratio of 30:70 (v/v), with 1.0 mL/min flow rate.

Experimental design
Adult healthy BALB/c male albino mice weighing 20-25 g (8 weeks old) were brought from the National Cancer Institute (NCI). Throughout the experiment, animals were kept in conventional cages at the standard temperature, humidity, and light/dark cycle conditions. Animals had free access to the standard food and drink ad libitum.
The experiment lasted 15 days following a week of acclimatization. Animals were randomly divided into four groups with eight mice in each group; vehicle group (group 1), CCl 4 -treated group (group 2), MOLE-treated group (group 3), and CCl 4 + MOLE-treated group (group 4). The first two groups received distilled water orally by gavage daily for consecutive 14 days. The last two groups received MOLE (400 mg/kg body weight) (Singh et al. 2014) orally by gavage daily for consecutive 14 days. On day 15, group 2 and group 4 were then given a single dose of CCl 4 (1 mL/kg body weight) prepared by dilution in olive oil; 1:1 (v/v), intraperitoneally (i.p.) (Makni et al. 2012;Shal et al. 2020), while the other groups (groups 1 and 3) received olive oil (i.p). Behavioral tests were carried out on separate animal groups 24 h later (Shal et al. 2020). For quantitative reverse transcription-polymerase chain reaction (RT-qPCR), the enzyme-linked immunosorbent assay (ELISA), and the evaluation of biochemical parameters, the animals were euthanized by decapitation under i.p. injection with pentobarbital (30 mg/kg) anesthesia.

Estimation of depression-like behavior by forced swimming test (FST) and tail suspension test (TST)
FST was carried out as described by Porsolt et al. (1977). Initially, each mouse was placed inside a transparent cylinder in the water at a depth of 20 cm and a temperature of 23 °C ± 2 °C. Afterwards, the mice were individually forced to swim for 6 min. The time of immobility was recorded by considering the halt of escape-oriented behavior during the last 5 min.
TST was also executed as reported by Steru et al. (1985). For 6 min, each mouse was hung about 1 cm from the tip of its tail with adhesive tape on the edge of a rod at a height of 50 cm above the floor. The duration of immobility time was considered by recording the time each mouse was suspended without any activity or any motion in the last 5 min.

Collection of blood and tissue samples
The blood was collected and the serum was isolated by centrifugation at 2000 × g for 15 min at 4 °C for biochemical analysis.
Brains were dissected from the skull. For the histopathological investigation, one side of each brain was kept in 10% neutral buffered formalin for later use. HC and CC were excised from the other side and assigned into two portions. The first portion of each region was homogenized in 1.5% KCl, centrifuged at 5000 × g, and the protein concentration in the tissue supernatant was evaluated following the Bradford method using the Bio-Rad assay kit (Bradford 1976). The analysis of oxidative stress parameters and proinflammatory cytokines in the supernatant was followed. The second part from each brain region was collected in RNA lysis buffer to measure the gene expression of the inflammatory and apoptotic mediators.

Histopathological examination
After washing the tissue samples, a series of diluted alcohol was used for dehydration, followed by clearance in xylene, infiltration in paraffin wax, and embedding in paraffin wax blocks. For the histopathological examination, 5-μm thickness coronal sections were obtained and stained with Ehrlich's hematoxylin and eosin (H&E) as demonstrated by Bancroft and Gamble (2008). The thickness of dentate gyrus (DG) in HC was measured in different groups using ImageJ software.

RT-qPCR assay
Total RNA was isolated from the HC and CC tissues using SV Total RNA Isolation System (Promega Corporation, Madison, WI, US) as previously described (Fathy and Said 2019). RNA concentration and purity were analyzed using the NanoDrop™ 2000/2000c Spectrophotometer (Thermo-Scientific, Lo, UK). Complementary DNA (cDNA) was then prepared using SuperScript III First-Strand Synthesis System following the manufacturer's instructions (Fermentas, Waltham, MA, US). The cDNA yield was then used to detect the relative expression levels of TLR2, TLR4, myeloid differentiation primary response 88 (MYD88), caspase 3, and NF-κB genes. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene was used for data normalization. Table 1 shows the primer sequences of mice genes used in the present study.

Determination of proinflammatory cytokines in HC and CC
The protein levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 were detected in HC and CC supernatant by ELISA kits particularly for mice (Thermo Fisher Scientific, BMS607-3 for TNF-α and CBMS603-2 for Il-6) following the producer's protocol. Protein levels were measured using the microplate ELISA reader at 450 nm.

Evaluation of biochemical parameters
Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in serum Liver functions were determined by measuring ALT (Abcam, ab105134) as described by Hafkenscheid and Dijt (1979) and AST (Abcam, ab105135) according to Sampson et al. (1980) in serum using the enzymatic methods.

Detection of ammonia level in serum
Ammonia assay was used to assess the level of ammonia in serum, as described by Gutiérrez-de-Juan et al. (2017). The reaction of Nessler's reagent is key to detecting ammonia production using ammonium chloride as a standard. The spectrophotometer was used at 425 nm, and the results were presented in percentages.

Evaluation of corticosterone level in serum
Serum corticosterone concentration was determined in serum by using ELISA kits (ThermoScientific, Lo, UK) as per the manufacturer's instructions. The levels were measured using the microplate ELISA reader at 450 nm.

Determination of malondialdehyde (MDA) level in the HC and CC
The lipid peroxidation (LPO) was measured in the homogenates' supernatant of each brain region based on thiobarbituric acid (TBA) reaction with MDA (Ohkawa et al. 1979). The principle for the reaction is the formation of a product due to the LPO of the membranes. After incubation, the spectrophotometer was used to record the absorbance at 532 nm (MDA Colorimetric/Fluorometric Assay kit, Biovision Inc., CA, USA).

Enzymatic and non-enzymatic antioxidants' levels in HC and CC
OxiSelect Superoxide dismutase (SOD) kit (CellBiolabs, Inc., CA, US) was used for detecting the activity of SOD as described by the producer's protocol following the method reported by Valentine and Hart (2003). The absorbance was recorded spectrophotometrically at 540 nm. Reduced glutathione (GSH) level was measured using the method modified by Jollow et al. (1974), and L-glutathione reduced was used to prepare the standard serial dilutions. The basis for the assay depends on the formation of a yellow color due to the reaction between 5,5-dithiobis-2-nitrobenzoic acid (DTNB) and free thiol groups of GSH. The absorbance was determined spectrophotometrically at 412 nm.

Rutin and β-sitosterol detection by HPLC
For HPLC analysis, the authentic reference standards of rutin and β-sitosterol were analyzed using an identical chromatographic method. By comparing the peak retention times, rutin and β-sitosterol were identified in MOLE (Fig. 1).

MOLE pretreatment reverses the increase in serum aminotransferases, ammonia, and corticosterone levels induced by CCl 4
CCl 4 treatment for 24 h remarkably increased ALT (P < 0.01) and AST (P < 0.001). These increases were markedly prevented in response to MOLE pretreatment at significances of (P < 0.01) and (P < 0.001) for ALT and AST, respectively (Fig. 2a, b). An apparent increase in serum ammonia levels was manifested in CCl 4 -treated mice (P < 0.0001). However, MOLE pretreatment remarkably reversed this change (P < 0.0001) (Fig. 2c).
Serum corticosterone levels were measured to investigate the potential role of MOLE against the alteration in the HPA-axis induced by CCl 4 treatment. A significant increase in serum corticosterone level was found in CCl 4 -treated mice (P < 0.01). However, this increase was markedly prevented upon MOLE pretreatment (P < 0.01) (Fig. 2d).

MOLE pretreatment alleviates oxidative stress induced by CCl 4
The role of MOLE pretreatment against CCl 4 LPO was investigated by measuring MDA in the HC and CC brain tissues. CCl 4 significantly increased LPO in both HC and CC as evidenced by the striking increase in MDA (P < 0.0001). This effect was remarkably attenuated in both tissues in the presence of MOLE pretreatment (P < 0.001) (Fig. 3a, b).
SOD and GSH levels were measured in the HC and CC brain tissues to evaluate the potential antioxidant power of MOLE against CCl 4 neurotoxicity. CCl 4 markedly enhanced the oxidative stress as evidenced by the significant decrease in SOD and GSH levels in the HC (P < 0.05) and CC (P < 0.01). However, MOLE administration before CCl 4 treatment attenuated these decreases significantly in both tissues (P < 0.05) (Fig. 3c-f).

MOLE pretreatment reverses alterations in TNF-α and IL-6 levels induced by CCl 4
The effect of MOLE pretreatment on the inflammatory mediators activated by CCl 4 was evaluated by measuring TNF-α and IL-6 protein levels in the HC and CC brain tissues in different groups. CCl 4 significantly increased the protein levels of TNF-α and IL-6 in both HC and CC (P < 0.01). Nevertheless, pretreatment with MOLE remarkably prevented these elevations in the HC (P < 0.05) and CC (P < 0.01) ( Table 2).

MOLE exhibits an antiapoptotic effect against CCl 4 neurotoxicity
Caspase 3 gene expression was measured in the HC and CC brain tissues to assess the antiapoptotic effect of MOLE against CCl 4 neurotoxicity. A remarkable increase in caspase 3 gene expression was manifested in both brain tissues in response to CCl 4 toxicity (P < 0.0001). However, MOLE was found to have an antiapoptotic role against this effect as evidenced by the striking decrease in caspase 3 gene expression in both tissues (P < 0.0001), (Table 2).

MOLE protects against depression-like behavior induced by CCl 4
FST and TST were performed to investigate the protective role of MOLE pretreatment against depression-like behavior manifested in CCl 4 -treated mice. Depression-like behavior was represented by mice immobility in seconds after FST and TST were recorded. CCl 4 was found to significantly increase depression-like behavior based on both FST (P < 0.01) and TST (P < 0.0001). Nevertheless, pretreatment with MOLE had a marked anxiolytic effect as evidenced by FST (P < 0.01) and TST (P < 0.001) (Fig. 4).

Discussion
HE is ascribed with hyperammonemia in the bloodstream that can pass via the blood-brain barrier (BBB) and causes damage to the brain tissue through oxidative stress, inflammatory response, and dysfunction of energy metabolism (de Souza Machado et al. 2015). It is well known that CCl 4 treatment can successfully stimulate liver injury and HE-associated brain tissue damage (de Souza Machado et al. 2015).
For confirmation, the current experiment revealed that serum levels of liver enzymes, ALT and AST, were increased significantly in CCl 4 -induced mice compared to the vehicle group. Pretreatment with MOLE halts the increase of ALT and AST levels in CCl 4 -treated mice.
In the present study, CCl 4 induced oxidative stress whereas MOLE pretreatment protected brain tissue from oxidative stress damage. In the current study, an increase in MDA and a decrease in antioxidants were observed in HC and CC following CCl4 injection. CYP450-mediated bioactivation of CCl 4 into reactive free radicals, trichloromethyl (CCl 3 ) and trichloromethyl peroxy radical (CCl 3 OO), initiates hepatocyte damage. This initiation activates the release of reactive oxygen species (ROS) leading to LPO (Risal et al. 2012). The severity of LPO from cell membranes can be monitored by assessing the MDA formed in the brain tissue (Lıu and Tıan 2016). Moreover, the decline in GSH and SOD levels was reportedly due to ROS release in the CCl 4 mouse model of encephalopathy (Ogaly et al. 2018). The dysregulated antioxidant mechanism in the current model was consistent with previous reports (Han et al. 2019;Shal et al. 2020), where an elevated MDA and reduced antioxidant mechanism in different brain regions were observed after CCl 4 intoxication. MOLE pretreatment successfully restored the antioxidative power in HC and CC by reducing MDA and restoring antioxidative mechanisms, consistent with Idoga et al. (2018). The reversed alterations of antioxidant power in MOLE/CCl 4 -treated mice demonstrate the neuroprotective effect of MOLE and its prominent antioxidant capacity protecting against LPO.
It was recorded that inflammation is a crucial inducer of HE (Lu et al. 2020). Liver damage was associated with peripheral inflammation and cytokine storming that can cross BBB with ascribed neuroinflammation disorders (Lu et al. 2020). To evaluate the protective effects of MOLE against neuroinflammation consequences in HC and CC regions of CCl 4 -injected mice, relative expressions of TLR2, TLR4, MyD88, and NF-κB genes and the protein levels of TNF-α and IL-6 were measured.
Toll-like receptors (TLRs) play major roles in inflammatory responses (Kong and Le 2011). TLR2 and TLR4 are considered neuroinflammatory receptors residential in neurons, astrocytes, and microglia (Mao et al. 2012). Almost all TLRs adaptor protein is MyD88. It links the receptors and the downstream signaling components with subsequent activation of transcription and inflammatory responses (Mao et al. 2012). The current experiment detected upregulation of TLR4, TLR2, and MyD88 gene expressions in CCl 4 -treated mice. Our findings were consistent with previous studies (Lu et al. 2020;Zhou et al. 2020) where upregulated TLR4, TLR2, and MyD88 gene expressions were noticed in HE rat model. MOLE interferes with this cascade as evidenced by the downregulation of TLR4, TLR2, and MyD88 gene expressions manifested in the CCl 4 /MOLE-treated group.
Gene expression of NF-κB was also elevated in the present CCl 4 -treated mice and was downregulated by MOLE pretreatment. In addition, activating the TLR4/2-MyD88dependent signaling pathway has been reported to lead to NF-κB transcription (Zhou et al. 2020). Consequently, the MOLE pretreatment protects against the exhibition of depression-like behaviors induced by CCl 4 based on forced swimming test (FST) (a) and tail suspension test (TST) (b). All data are represented as mean ± SD, n = 8. **: P < 0.01, ***: P < 0.001, and ****: P < 0.0001 compared to vehicle group. ##: P < 0.01, and ###: P < 0.001 compared to CCl 4 group denote different regions of cornu ammonis (CA). DG denotes dentate gyrus. Graph (e) shows that MOLE pretreatment protects against the reduction in DG thickness induced by CCl 4 . All data are represented as mean ± SD, n = 8. **: P < 0.01 compared to vehicle group TLR4/2-MyD88/NF-κB signaling pathway might be targeted by MOLE to mitigate CCl 4 toxicity.
TLR4, a member of TLRs, has been evidenced to play a significant role in initiating the inflammatory response after brain damage (Brown et al. 2011). The present study found an increase in proinflammatory cytokines in the CCl 4 group, prevented in the CCL 4 + MOLE group. CCl 4 toxicity increases the levels of proinflammatory cytokines produced by Kupffer cells. Consequently, liver stromal cells are recruited to assist in intensifying the inflammatory response by producing cytokines and chemokines (Shim et al. 2010). This peripheral intensification in inflammatory response activates microglia and TLR4. Microglial activation is ascribed to the secretion of proinflammatory cytokines such as IL-6 and TNF-α that were associated with the brain deteriorations observed in CCl 4 -treated mice (Vairappan et al. 2019), whereas TLR4 activation exacerbates the inflammatory reactions by inducing the NF-κB pathway leading to the generation of proinflammatory factors (Yoshimura and Shichita 2012).
Oxidative stress and neuroinflammation are associated with neurobehavioral changes (Kubera et al. 2011). For instance, proinflammatory cytokines such as IL-1β, IL-6, and TNF-α are increased in depression and anxiety, implying immune dysregulation (Dowlati et al. 2010). Moreover, symptoms of depression were proven to be aggravated by proinflammatory cytokines, leading to disturbance of the HPA-axis as a result of hypersecretion of CRF with elevated corticosterone level in plasma and subsequent induction of depression symptoms (Furtado and Katzman 2015;Shal et al. 2020). Subsequently, the neuroprotective effect of MOLE against CCl 4 -induced anxiety and depressionlike behavioral changes via FST and TST was assessed. CCl 4 -induced anxiety and depression-like behavioral changes observed in the current study agree with a previous report (Shal et al. 2020). However, pretreatment with MOLE and CCl 4 + MOLE groups, respectively. Neuron degeneration is manifested in the CCl 4 group but MOLE pretreatment protected the neurons against this effect significantly improved the mice's behavioral status. This improvement may indicate that MOLE contains anxiolytic and anti-depression phytochemicals. These phytochemicals could act on serotonergic, dopaminergic, and/or noradrenergic neurotransmitter systems (Millan 2004). Besides, the elevated corticosterone level in CCl 4 -treated mice was alleviated by MOLE pretreatment in the current study confirming the previously reported antidepressant effect of MOLE (Kou et al. 2018).
An increased ammonia level in serum was observed in the CCl 4 group, consistent with previous findings (Shal et al. 2020). Ammonia exists in biological solutions in two forms, NH 3 and NH 4 + . CCl 4 -induced liver damage results in hyperammonemia represented by increased levels of circulating ammonia (Damink et al. 2002). Hyperammonemia is associated with neurological disorders by activating brain oxidative stress and neuroinflammation (Heidari et al. 2016). Hyperammonemia results from liver injury, and ammonia can easily pass through BBB, causing neurotoxicity (Bosoi and Rose 2009).
The current study assessed the antiapoptotic effect of MOLE pretreatment against CCl4-induced apoptosis by evaluating the relative caspase 3 mRNA level. A decrease in caspase 3 expression in the presence of MOLE confirms the antidepressant effect of MOLE as suggested by Khan et al. (2019). Apoptosis can be triggered by an inflammatory response or by ROS generated by CCl 4 (Liu et al. 2018). It can also be explained by the proapoptotic effect of corticosteroids on the brain regions, especially HC (Kurek et al. 2016). Consequently, neuroinflammation, oxidative stress, and apoptosis in the brain cells can cause anxiety and depression-like behavior (Khan et al. 2019). However, our results proved the anxiolytic and anti-depression properties of MOLE.
To further explore the mechanisms behind the neuroprotective effect of MOLE against CCl 4 -induced neurotoxicity, the histopathology of CC and HC was assessed using H&E. The thickness of the DG cellular layer in HC region in CCl 4 -challenged mice was found to be markedly thinner than that in the vehicle group. This result agrees with a previous study (Khan et al. 2019). Neuron degeneration was also manifested in the CC of CCl 4 -treated mice in agreement with Shaalan et al. (2017). However, MOLE attenuated these alterations. Histopathological results were consistent with the biochemical findings.

Conclusions
The present study demonstrates the neuroprotective role of MOLE against CCl 4 -induced neurotoxicity as indicated by mitigating the neuroinflammation, brain oxidative stress, apoptosis, biochemical alterations, and histopathological changes in HC and CC, and the significant improvement in anxiety and depression-like behavior. This protective effect can be attributed to the antioxidative and anti-inflammatory characteristics of MOLE which prevent the cascade of HE progression. We suggest that TLR4/2-MyD88/NF-κB signaling pathway could be the main target for the ameliorative effect of MOLE.