Protective effect of Moringa oleifera Lam. leaf extract against carbon tetrachloride-induced neuroinammation in a mouse model of hepatic encephalopathy

Hepatic encephalopathy (HE) is a neuropsychiatric disorder associated with acute or chronic liver injury. Carbon tetrachloride (CCl 4 ) is usually used as an experimental model for HE. The present study aimed to assess the neuroprotective impacts of Moringa oleifera Lam. leaf ethanolic extract (MOLE) against neurotoxicity in CCl 4 -induced mouse model of HE. High-performance liquid chromatography (HPLC) analysis was used for the detection of marker compounds; rutin and β-sitosterol. Animals were divided into four groups; vehicle group, CCl 4 treated group, MOLE treated group, and (CCl 4 + MOLE) group treated with MOLE for 14 days before inducing neurotoxicity by CCl 4 .


Results
Pretreatment with MOLE decreased alanine aminotransferase (ALT), aspartate aminotransferase (AST), corticosterone, and ammonia levels in serum as well as it improved the antioxidant status of CCl 4 treated mice in the tissue of hippocampus (HC) and cerebral cortex (CC). It reduced the expression of toll-like receptor (TLR)4, TLR2, myeloid differentiation primary response 88 (MYD88), and nuclear factor kappa B (NF-κB) genes and the protein levels of the pro-in ammatory cytokines in the selected brain regions.
MOLE also exhibited anti-apoptotic effect as revealed by the reduced expression of caspase3, and prevented histological deteriorations caused by CCl 4 treatment. Furthermore, CCl 4 -induced anxiety and depression-like behavioral changes were attenuated by MOLE preadministration.

Conclusions
Taken together, the current results suggest signi cant anxiolytic and antidepressant effects of MOLE via modulation of neuroin ammation, oxidative stress, TLR4/2-MyD88/NF-κB pathway, and apoptosis in HE experimental model.

Background
It is well known that people with liver disease suffer from neuropsychiatric disorders due to alteration of lipid peroxidative and antioxidative mechanisms in the brain along with severe hepatic encephalopathy (HE) propagation 1 . HE is a neuropsychiatric disorder propagated as a result of acute or chronic hepatic failure 2 . Carbon tetrachloride (CCl 4 ) is a toxic substance that is used to induce liver injury with concomitant brain disorders and can be used as an experimental model for HE 3 . In the liver, CCl 4 is metabolized to highly reactive free radicals which oxidize fatty acids in the phospholipids of cell membranes leading to structural and functional changes in these membranes 4 .
Moreover, these free radicals along with CCl 4 itself cause injuries in the endoplasmic reticulum with a consequent effect on protein synthesis and lead to lipid accumulation 4 . Meanwhile, CCl 4 leads to the production of in ammatory mediators from the triggered macrophages in the liver with concomitant systemic in ammation exerting a critical role in aggravating neurological manifestations, possibly through triggering the brain predisposition to the associated hyperammonemia 5 .
Neuroin ammation and oxidative stress have been evidenced to be involved in the development of depression and anxiety 6 . Noteworthy, CCl 4 has been reported to induce neuropsychiatric disorders mimicking what appear in patients with acute or chronic liver damage via targeting brain antioxidative system and in ammatory pathways such as toll-like receptor (TLR)4/nuclear factor kappa B (NF-κB) pathway 1 . Moreover, it has been reported that corticotrophin-releasing factor (CRF) hypersecretion in response to the release of pro-in ammatory cytokines is attributable to the modi cation of hypothalamicpituitary-adrenal (HPA)-axis resulting in an elevated level of plasma corticosterone and depression symptoms' exacerbation 7 .
Plants are well known for having therapeutic effects and have been used in this regard in traditional and modern medications. Moringa oleifera Lam. (MOL), family Moringaceae 8 is widely known as "Miracle tree" as a result of its medicinal outcomes. Leaves, the most common and suitable part of the plant for medicinal commercial mass production 9 can be consumed in different ways and can also be dried and stored for months without losing nutritional bene ts 10 . The following compounds were recorded in our previous study 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 chromatography-mass spectrometry (GC-MS) analysis 11 . Moreover, the total phenolic and the total avonoid contents in MOLE were also measured 11 . Furthermore, it has been reported that MOL possesses pain relief, anti-depression, anti-in ammatory, immunomodulatory, and neuroprotective activities 12 .
The current study is the rst-ever to evaluate the possible role of MOLE as a protective adjuvant against brain manifestations induced by CCl 4 in HE mouse model.The alleviation potentials of MOLE were assessed by tracking its effect on TLR4/2-MyD88/NF-κB pathway, neuroin ammation, 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 was purchased from Sigma (St. Louis, MO, USA). All other chemicals and reagents used were of the highest analytical grade.

MOL source and identi cation
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 nally ground. 96% ethanol was mixed with the ground leaves and the mixture was kept in the shaking incubator for 24 h at 37 °C. The obtained extract was then ltered and put in the rotary evaporator at 40 °C until complete evaporation of ethanol. Finally, a semi-solid extract was produced and stored at 4 °C until use.
High-performance liquid chromatography (HPLC) analysis MOLE was analyzed using HPLC method for a qualitative analysis of two marker compounds. About 50 mg of the extract were 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 ow rate of 1 mL/min. The mobile phase consisted of acetonitrile: water/0.1 formic acid with gradient increased from 5% to 95% over 15 min. For β-sitosterol, a waters symmetry shield C18 column (150 x 4.6, 5 µm) and wavelength 210 nm was used. The mobile phase consisted of methanol: acetonitrile with the ratio 30:70 (v/v), with a ow rate of 1.0 mL/min.

Experimental design
Adult healthy BALB/c male albino mice weighting 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 conditions of temperature, humidity, and light/dark cycle. Animals had free access to the standard food and drink ad libitum. Animal experimentation protocols were carried out following the National Institutes of Health (NIH) guidelines for animal experimentation and approved by Cairo University Institutional Animal Care and Use Committee (CU-IACUC), Egypt, (permission number: The experiment lasted for 15 days following 1-week acclimatization. Animals were haphazardly divided into 4 groups with 8 mice in each group; Control group (group 1), CCl 4 -treated group (group 2), MOLEtreated group (group 3), and CCl 4 +MOLE-treated group (group 4). The rst two groups received distilled water orally by gavage on daily basis for consecutive 14 days. The last two groups received MOLE (400 mg/kg body weight) 13 orally by gavage daily for consecutive 14 days. Then on day 15, group 2 and group 4 were administered a single dose of CCl 4 (1 mL/kg body weight) prepared by dilution in olive oil; 1,14 , while other groups (groups 1 & 3) received olive oil, (i.p). 24 hours later, behavioral tests were carried out in separate animal groups 1 . Euthanasia was conducted by decapitation under xylazine/ketamine anesthesia 15 .
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) 16 . Initially, each mouse was placed into water at a depth of 20 cm and a temperature of 23 ± 2 °C inside a transparent cylinder. Afterward, the mice individually were 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) 17 . For 6 min, each mouse was hung about 1 cm from the tip of the tail by using sticky tape on the edge of a rod at a height of 50 cm above the oor. The duration of immobility time was considered by recording the time during which 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 of the blood at 2000 x 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 rst portion of each region was homogenized, centrifuged at 5000 x g, and the protein concentration was evaluated in the tissue supernatant according to the Bradford method by using Biorad assay kit 18 . The analysis of oxidative stress parameters and pro-in ammatory cytokines in the supernatant was followed. The second portion from each brain region was collected in RNA lysis buffer for measuring gene expression of the in ammatory and apoptotic mediators. For the histopathological investigation, brains were kept in 10% neutral buffered formalin.

Histopathological examination
After washing the tissue samples, a series of diluted alcohol was used for dehydration, followed by clearance in xylene, in ltration in para n wax, and embedding in para n wax blocks. For the histopathological investigation, 5μm thickness coronal sections were obtained and stained with Ehrlich's hematoxylin and eosin (H&E) as demonstrated by Bancroft and Gamble (2008) 19 . The thickness of dentate gyrus (DG) in HC was measured in different groups using ImageJ software.
Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay Total RNA was isolated from the HC and CC tissues using SV Total RNA Isolation System (Promega Corporation, Madison, WI, USA) as previously described 20 . RNA concentration and purity were analyzed using NanoDrop™ 2000/2000c Spectrophotometer (ThermoScienti c, Lo, UK). Complementary DNA (cDNA) was then produced using SuperScript III First-Strand Synthesis System according to the manufacturer's instructions (Fermentas, Waltham, MA, USA). The cDNA yield was then used to detect the relative expression levels of TLR2, TLR4, myeloid differentiation primary response 88 (MYD88), and NF-κB genes. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene was used for data normalization. Table 1 shows the primers' sequences of mice's genes that have been used in the present study.

Determination of proin ammatory cytokines in HC and CC
The protein level of tumor necrosis factor (TNF)-α and interleukin (IL)-6 was detected in HC and CC supernatant by enzyme-linked immunosorbent assay (ELISA) kits particularly for mice (Merck Millipore, San Francisco, California, USA) 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 checked by measuring ALT as described by Hafkenscheid and Dijt (1979) 21 and AST according to Sampson et al. (1980) 22 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) 23 . The reaction of Nessler's reagent is the key to detect ammonia production using ammonium chloride as a standard. The spectrophotometer was used at 425 nm and the results were presented in percentage.

Evaluation of corticosterone level in serum
Serum corticosterone concentration was determined in serum by using ELISA kits (ThermoScienti c, Lo, UK) as per the manufacturer's instructions.
Determination of malondialdehyde (MDA) level in the HC and CC Measurement of lipid peroxidation (LPO) in the homogenates' supernatant of each brain region was carried out based on thiobarbituric acid (TBA) reaction with MDA 24 . The principle for the reaction is the formation of a product due to 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' level in HC and CC OxiSelect Superoxide dismutase (SOD) kit (CellBiolabs, Inc., CA, USA) was used for the detection of the activity of SOD as described by the producer's protocol following the method reported by (Valentine and Hart, 2003) 25 . The absorbance was recorded spectrophotometrically at 540 nm. Reduced glutathione (GSH) level was measured using the method modi ed by Jollow et al. (1974) 26 . The basis for the assay depends on the formation of yellow color as a result of the reaction between 5, 5dithiobis-2 nitro benzoic acid (DTNB) and free thiol groups of GSH. The absorbance was assessed spectrophotometrically at 412 nm.

Statistical methods
Statistical analyses were performed using GraphPad PRISM (version 8.4.3 (686); Graph Pad Software, USA). Data were represented as mean ± SD. analyses were done using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Differences were considered signi cant at P < 0.05.

Results
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 peaks' retention times, rutin and β-sitosterol were identi ed 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 hours remarkably increased ALT (P < 0.01) and AST (P < 0.001). These increases were markedly prevented in response to MOLE pretreatment at signi cances of (P < 0.01) and (P < 0.001) for ALT and AST, respectively (Fig. 2 A, B).

(C).
Serum corticosterone level was measured to investigate the potential role of MOLE against the alteration in HPA-axis induced by CCl 4 treatment. A signi cant 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. 2 (D).

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 markedly elevated 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. 3 (A, B).
To evaluate the potential antioxidant power of MOLE against CCl 4 neurotoxicity, SOD and GSH levels were measured in the HC and CC brain tissues. CCl 4 markedly enhanced the oxidative stress as evidenced by the signi cant decrease in SOD and GSH levels in the HC (P < 0.05) and CC (P < 0.01). However, MOLE administration before CCl 4 treatment signi cantly attenuated these decreases in both tissues (P < 0.05), To evaluate the role of MOLE pretreatment on the TLR4/2-MyD88/NF-κB pathway, RT-qPCR was used to measure the expression of TLR2, TLR4, MyD88, and NF-κB genes in the HC and CC brain tissues. CCl 4 markedly raised the gene expression of TLR2 (P < 0.001), TLR4 (P < 0.001) and MyD88 (P < 0.0001) in both brain regions. NF-κB gene expression also upregulated signi cantly in response to CCl 4 toxicity in the HC (P < 0.01) and CC (P < 0.05). Most of these increases were signi cantly alleviated in response to MOLE pretreatment. TLR2 gene expression was signi cantly decreased in both HC (P < 0.01) and CC (P < 0.001). TLR4 was signi cantly decreased in both tissues as well (P < 0.001). Marked restoration of MyD88 gene expression was also noticed (P < 0.0001). Although a decrease in NF-κB gene expression was noticed in both tissues, it was only signi cant in the HC (P < 0.01), (Table 2).

MOLE pretreatment reverses alterations in TNF-α and IL-6 levels induced by CCl 4
The effect of MOLE pretreatment on the in ammatory 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 signi cantly elevated 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).
FST and TST were performed to investigate the protective role of MOLE pretreatment against depressionlike behaviors manifested by CCl 4 -treated mice. Depression-like behaviors represented by mice immobility in seconds after FST and TST were recorded. It was found that CCl 4 markedly increased depression-like behaviors 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).
MOLE pretreatment protects against histopathological changes induced in brain regions by CCl 4 Coronal sections in the HC and CC were stained with H&E to assess the neuroprotective effect of MOLE against histopathological alterations induced by CCl 4 . CCl 4 induced histopathological changes including thinning of the DG region in the HC (Fig. 5) and neuron degeneration (Nd) in the CC (Fig. 6). However, MOLE pretreatment alleviates these changes signi cantly.

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 3 . The mechanism for brain injury associated with HE was reported to be through oxidative stress, in ammatory response, and dysfunction of energy metabolism 3 . It was previously documented that CCl 4 treatment can successfully stimulate liver injury along with HE associated brain tissue damage 3 . For con rmation, the current experiment revealed that serum levels of liver enzymes; ALT and AST, elevated signi cantly in CCl 4 -induced mice when compared with the control 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. An increase of MDA and a decrease of the level of antioxidants were observed in HC and CC following CCl 4 injection in the current study. Hepatocyte damage is initiated by CYP450-mediated bioactivation of CCl 4 into reactive free radicals; trichloromethyl (CCl 3 ) and trichloromethyl peroxy radical (CCl 3 OO). This activates the release of reactive oxygen species (ROS) which leads to LPO 27 . The severity of LPO of cell membranes can be monitored via assessing the formed MDA in brain tissue 28 . Moreover, the decline of GSH and SOD levels was reportedly due to ROS release in the CCl 4 mouse model of encephalopathy 29 . The dysregulated antioxidant mechanism in the current model was in line with previous reports 1, 30 , in which an elevated MDA and reduced antioxidant mechanism in different brain regions were observed following CCl 4 intoxication. It was recorded that In ammation is a crucial inducer for HE 32 . Liver damage was associated with peripheral in ammation with cytokine storming that can cross BBB with ascribed neuroin ammation disorders 32 . To evaluate the protective effects of MOLE against neuroin ammation consequences in HC and CC regions of CCl 4 injected mice, relative expressions of TLR2, TLR4, MyD88, and NF-κB genes as well as the protein levels of TNF-α and IL-6 were measured.
Toll-like receptors (TLRs) play major roles in inflammatory responses 33 . TLR2 and TLR4 are considered as neuroin ammatory receptors that are residential in neurons, astrocytes, and microglia 34 . The adaptor protein of almost all TLRs is MyD88 and acts as a link between the receptors and the downstream signaling components with subsequent activation of transcription and in ammatory responses 34  Gene expression of NF-κB was also elevated in the present CCl 4 treated mice, which was downregulated by MOLE pretreatment. It has been reported that activating TLR4/2-MyD88 dependent signaling pathway leads to NF-κB transcription 35 . Consequently, TLR4/2-MyD88/NF-κB signaling pathway might be targeted by MOLE in the mitigation of CCl 4 toxicity.
TLR4, a member of TLRs, has been evidenced to play a signi cant role in initiating the inflammatory response after brain damage 36 . In the present study, we found an increase in proin ammatory cytokines in the CCl 4 group which was prevented in 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 via the production of cytokines and chemokines 37 . This peripheral intensi cation in inflammatory response promotes the activation of microglia and TLR4.
Microglial activation is ascribed with the secretion of pro-in ammatory cytokines such as IL-6 and TNF-α that were associated with the brain deteriorations observed in CCl 4 treated mice 38 . Whereas, activation of TLR4 exacerbates the inflammatory reactions by inducing NF-κB pathway leading to the generation of proinflammatory factors 39 .
It has been established that oxidative stress and neuroinflammation are associated with neurobehavioral changes 40 . For instance, proin ammatory cytokines such as IL-1β, IL-6, and TNF-α have been demonstrated to be elevated in depression and anxiety, implying immune dysregulation 41 . Moreover, symptoms of depression were proven to be aggravated by pro-in ammatory cytokines which lead to disturbance of HPA-axis as a result of hypersecretion of CRF with elevated corticosterone level in plasma and subsequent induction of depression symptoms 1,7 . Subsequently, the neuroprotective effect of MOLE against CCl 4 -induced anxiety and depression-like 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 1 . However, pretreatment with MOLE signi cantly improved the behavioral status of mice.
This may indicate that MOLE contains anxiolytic and anti-depression phytochemicals. These phytochemicals could be acting on serotonergic, dopaminergic, and/or noradrenergic neurotransmitter systems 42 .
Besides, the elevated level of corticosterone in CCl 4 -treated mice was alleviated by MOLE pretreatment in the current study con rming the previously reported antidepressant effect of MOLE 12 .
An increased level of ammonia in serum was observed in CCl 4 group in agreement with previous ndings 1 . 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 43 . Hyperammonemia has been implicated in neurological disorders through activating brain oxidative stress and neuroin ammation 44 . Hyperammonemia results from liver injury and ammonia can easily pass through BBB causing neurotoxicity 45 .
In the current study, the antiapoptotic effect of MOLE pretreatment against CCl 4 -induced apoptosis was Apoptosis can be triggered via in ammatory response or ROS generated by CCl 4 47 . It can also be explained by the pro-apoptotic effect of corticosteroids on the brain regions especially HC 48 . Consequently, neuroin ammation, oxidative stress, along with apoptosis in the brain cells, can cause anxiety and depression-like behaviors 46 . 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, 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 of the control mice. This agrees with a previous study 46 . Nd was also manifested in the CC of CCl 4 -treated mice in agreement with Shaalan et al. (2017) 49 . However, MOLE evidently attenuated these alterations. Histopathological results were consistent with the biochemical ndings.

Conclusions
The present study demonstrates, for the rst time, the neuroprotective role of MOLE against CCl 4 -induced neurotoxicity through biochemical, molecular, behavioral, and histological examinations. MOLE attenuated neuroin ammation, brain oxidative stress, apoptosis, biochemical alterations, and histopathological changes in HC and CC. Furthermore, MOLE signi cantly improved anxiety and depression-like behaviors. Accordingly, MOLE may be used in the prevention of HE related brain dysfunctions.  and cerebral cortex (CC) of CCl 4 -injected mice.  Protective effect of MOLE pretreatment on serum aminotransferases, ammonia, and corticosterone levels.