Moringa oleifera Lam. leaf extract mitigates carbon tetrachloride-mediated hepatic inflammation and apoptosis via targeting oxidative stress and toll-like receptor 4/nuclear factor kappa B pathway in mice

Samah M. Fathy, Mohammed S. M. Mohammed

Zoology Department, Faculty of Science, Fayoum University, Fayoum, Egypt

Keywords:

CCl4

inflammation

Moringa oleifera Lam.

Oxidative stress

TLR4/NF-κB

ABSTRACT

Carbon tetrachloride (CCl4) is a hepatotoxin that triggers liver damage. This study aimed to evaluate the protective effect of phytochemicals detected in Moringa oleifera Lam. leaf extract (MOLE) on CCl4-induced hepatotoxicity in mice. Phytochemicals, total phenolics, and total flavonoids were detected in MOLE. MOLE markedly decreased the elevation of serum alanine aminotransferase (ALT) and aspartate aminotransferase(AST) in consistence with the ameliorating effect on CCl4-induced histopathological abnormalities. Moreover,MOLE significantly alleviated the decrease in the antioxidant defense mechanism induced by CCl4. The suppressing effect of MOLE on the boosted inflammatory pathway triggered by CCl4 was detected by measuring the protein levels of nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB-p65),toll-like receptor 4 (TLR4), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β, and IL-8 as well as the relative expressions of nuclear factor kappa B (NF-κB), TNF-α, IL-1β, and TLR4 genes. Apoptosis and genotoxicity induced by CCl4 were significantly alleviated by MOLE. MOLE co-administration modulated TLR4/NF-κB pathway as presented by the suppressed gene expression of TLR4 and NF-κB as well as by the reduced protein expression of TLR4 and NF-κB-p65. In conclusion, MOLE has a multifarious protective role against hepatotoxicity through control of oxidative stress and modulation of TLR4/NF-κB.

1. Introduction

The liver plays a pivotal role in many biochemical processes including metabolism and clearance of various toxicants [1].However, it could also be damaged by these toxicants [2].

Carbon tetrachloride (CCl4) is a hepatotoxin widely used in experimental studies to promote hepatotoxicity in animals. It is metabolized by the cytochrome P450 producing reactive free radicals with subsequent lipid peroxidation [3]. CCl4also activates inflammatory cells and inflammatory mediators leading to hepatic necrosis or apoptosis [4]. The significance of inflammation in CCl4-induced liver injury is obvious as pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 contribute to hepatic fibrosis [5].

Toll-like receptor 4 (TLR4) was reported to activate inflammation in CCl4-induced liver injury through nuclear factor kappa B (NF-κB)provocation with subsequently increased secretion of pro-inflammatory cytokines [4] and apoptosis due to the stimulated inflammatory response [6]. Consequently, reagents could be used to ameliorate CCl4induced hepatotoxicity via targeting TLR4/NF-κB pathway.

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 is a droughtresistant tree widely distributed in tropical and subtropical countries of Asia and Africa. It is known as the “miracle tree” in these areas due to its health benefit.

The leaves of MOL are rich in many nutrients [7]. They have been widely used as an antifungal, antibacterial, antipyretic, antioxidant,anticonvulsant, anti-inflammatory, antiulcer, antihypertensive,anti-tumor reagent, and also as a treatment for hematological and cardiovascular disorders [8-10]. They were also used to ameliorate various diseases [11,12] and they were indicated to have a hepatoprotective effect [13].

However, up to our knowledge, the present experiment is the first ever to hypothesize the possible role of MOL leaf extract (MOLE)phytochemicals against CCl4induced hepatic inflammation, apoptosis,and genotoxicity through modulating TLR4/NF-κB pathway.

2. Materials and methods

2.1 Chemicals

CCl4was purchased from Sigma (St. Louis, MO, USA). Standard silymarin powder was purchased from Sedico, Egypt. All other chemicals and reagents used were of the highest analytical grade.

2.2 Plant material

Leaves of MOL were collected from Jazan City, KSA in November. Degrees minutes seconds (DMS) coordinates of the city are latitude 16° 53’ 12.59” N and longitude 42° 33’ 23.99” E. The plant material was authenticated by comparing it with the herbarium specimens found at Jazan University Herbarium (JAZUH), KSA.

2.3 Preparation of MOLE

Leaves were washed, dried, and about 70 g were ground. 700 mL of ethanol (96%) was added to the powder and was left in the shaking incubator for 24 h at 37 °C. Following the filtration of the soluble extract, a rotary evaporator at 40 °C was used, and the semi-solid extract was obtained and kept at 4 °C until use.

2.4 Gas chromatography-mass spectrometry (GC-MS)analysis

The GC-MS technique was performed according to Fathy and Drees [14], using a GC system (Agilent Technologies 7890A) fitted out with a mass-selective detector (MSD, Agilent 7000 Triple Quad) and an apolar Agilent HP-5 ms (5%-phenyl methyl poly siloxane) capillary column (30 m × 0.25 mm i. d.and 0.25 μm film thickness). The recorded components were interpreted by comparing their mass spectra and retention time with those of known compounds catered by the National Institute of Standards and Technology (NIST) and WILEY library as well as by contrasting the fragmentation pattern of the mass spectral information with those documented before [7,15].

2.5 Measurement of total phenolic content

The total phenolic content of MOLE was determined by using gallic acid as a standard following the method described by Folin-Ciocalteu according to Vázquez-León et al. [16].

2.6 Measurement of total flavonoid content

The total flavonoid content of MOLE was measured by using aluminum chloride (AlCl3) colorimetric assay following Martono et al. [17]. Results were expressed as mg catechin equivalents (CE)/100 g dry weight (DW).

2.7 Experimental design

Thirty healthy adult male mice weighting 20-25 g (8 weeks old) were preserved under standard conditions of ventilation and temperature (25 ± 2) °C. Mice were subjected to 12 h light/dark cycle,provided with water ad libitum, and fed with the standard laboratory diet. 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: CU/I/F/38/20). The time for the experiment was 21 days and animals were randomized into 5 groups with 6 mice in each group. Group (I): vehicle group in which mice received olive oil,intraperitoneally (i.p.). Group (II): mice received CCl4(0.5 mL/kg body weight) in olive oil (10%), i.p, 3 times a week for 3 weeks [18].Group (III): mice received CCl4(10% in olive oil, i.p.) 3 times a week and silymarin (100 mg/kg·day, orally by gavage) [19] simultaneously.Group (IV): mice received CCl4(10% in olive oil, i.p.) 3 times a week and MOLE (200 mg/kg·day by gavage) [11] concurrently. Group (V):mice received CCl4(10% in olive oil, i.p.) 3 times a week and MOLE(400 mg/kg·day by gavage) [11] concurrently.

2.8 Blood and tissue sample collection

All mice were anesthetized by i.p. injection of xylazine/ketamine(10/110, mg/kg) anesthesia [20] and euthanized by drainage of blood through the right ventricle puncture. Fasting blood samples were collected in plain tables and serum was isolated by centrifuging the blood at 2 000 × g for 15 min at 4 °C. The separated serum was used for Alanine aminotransferase (ALT) and Aspartate aminotransferase(AST) colorimetric assay.

The liver was dissected and divided into 3 parts. The first part was kept in a 10% neutral buffered formalin for liver histopathological assessment. The second part was subjected to homogenization,centrifugation at 5 000 × g, and the amount of protein was estimated in the tissue supernatant by using Biorad assay kit according to the Bradford method [21]. The collected supernatant was used for further analysis of oxidative stress parameters, pro-inflammatory cytokines, apoptotic protein levels, and the degree of genotoxicity.The remaining liver tissue was collected in RNA lysis buffer for measuring gene expression of the inflammatory biomarkers.

2.9 Histopathological examination

Tissue samples were washed, dehydrated using ascending series of alcohol, cleared in xylene, and embedded in paraffin blocks using the standard technique. Sections of 5 μm thickness were obtained and stained with Ehrlich’s hematoxylin and eosin (H&E) for histopathological examination as described by Bancroft and Gamble [22].

2.10 Western blotting analysis for apoptosis regulatory proteins and inflammatory mediators

The Western blotting technique was followed to detect the levels of the apoptotic regulator proteins; B-cell lymphoma 2 (Bcl-2) and Bcl-2 Associated X (Bax) as well as the levels of inflammatory mediators; TLR4 and nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB-p65). Equal amounts of total proteins were processed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 12% gel percentage. The fractionated sample was transferred onto polyvinylidene difluoride(PVDF) membranes (Millipore Corp., Bedford, MA, USA) and blocked with 5% nonfat milk in Tris-buffered saline with Tween-20 for 1 h at room temperature. The membranes were incubated with 1:1 000 dilution of the specific primary antibodies (Millipore Ltd.,Oxen, UK) at 4 °C overnight. Further incubation with secondary antibodies (1:5 000, Thermo Scientific, LO, UK) was followed for 1 h at room temperature. The bands were detected by using the enhanced chemiluminescence detection reagents (Millipore, CA, USA) and were captured using a chemiluminescence system (New Life Science Products, Boston, MA, USA).

2.11 Gene expression analysis of pro-inflammatory cytokines and inflammatory mediators by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay

Total RNA isolation from the liver tissue was performed using SV Total RNA Isolation System (Promega Corporation, Madison, WI,USA) according to Fathy and Said [23]. The RNA concentrations and purity were measured with NanoDrop? 2000/2000c Spectrophotometer(ThermoScientific, Lo, UK). The complementary DNA (cDNA) was produced from RNA by using SuperScript III First-Strand Synthesis System as referred to in the instructions of the manufacturer (Fermentas,Waltham, MA, USA). The cDNA yield was then used to detect the relative expression levels ofNF-κB,TNF-α,IL-1β, andTLR4genes.Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene was used for normalization of the data. Mice primer sequences used in the current experiment were presented in Table 1.

Table 1Sequence of primers used for RT-qPCR.

2.12 Determination of pro-inflammatory cytokine levels using enzyme-linked immunosorbent assay (ELISA)

The protein levels of TNF-α, IL-1β, IL-6, and IL-8, were detected in the hepatic supernatant by ELISA kits specific for mice(Merck Millipore, San Francisco, California, USA) following the manufacturer’s protocol. Absorbance at 450 nm was used to detect the protein levels of the above mentioned cytokines by microplate ELISA reader.

2.13 Estimation of biochemical parameters

2.13.1 Measurement of ALT and AST levels in serum

Biochemical parameters in the serum were estimated to assess liver functions in all groups of mice. ALT and AST were measured by the enzymatic methods according to Hosen et al. [24].

2.13.2 Antioxidant enzyme activities in liver

Catalase (CAT) activity was measured using a CAT assay kit(CAT Activity Colorimetric/Fluorometric Assay Kit; BioVision Incorporated., CA, USA) according to the manufacturer’s instructions based on the method previously explained [25].

The activity of superoxide dismutase (SOD) was detected using the OxiSelect SOD kit (CellBiolabs, Inc., CA, USA) according to the producer’s instructions depending on the method in the text [26].

The activity of glutathione peroxidase (GPx) was assessed using Abnova kit (Taipei, Taiwan) according to the instructions from the manufacturer based on the described method [27].

2.14 Measurement of genotoxicity using comet assay

Analysis of DNA damage and genotoxicity was conducted using comet assay (alkaline single cell gel electrophoresis) as previously described [28]. The analysis was conducted using Komet 5 image analysis software (Kinetics Imaging Ltd., Liverpool, UK). Tail moment, the product of multiplying the percent of DNA in the tail and tail length (μm), was used to compare DNA damage in different groups.

2.15 Statistical analysis

Statistical analyses were carried out using GraphPad PRISM(version 6.01; Graph Pad Software, USA). Data were expressed as the mean ± SD and analyzed using one-way repeated measures analysis of variance (ANOVA) followed by Tukey’s test. Differences were considered significant atP＜ 0.05.

3. Results

3.1 GC-MS analysis of MOLE

Various polyphenols were recorded in MOLE including phenolic acids (1-12), flavonols (13, 14), chalcones (15, 16), isoflavonoids(17), and flavones (18-31). Other phenolic compounds were recorded(32-40). Other phytochemicals were also detected such as irbesartan,oleic acid, heptadecanoic acid, andβ-sitosterol (Table 2).

Table 2GC-MS analysis of the phytochemical compounds in MOLE.

3.2 Total phenolic and total flavonoid contents in MOLE

Total phenolic content in MOLE was (275.25 ± 8.77) mg of gallic acid equivalents/100 g DW whereas total flavonoid content was(38.50 ± 3.42) mg of CE/100 g DW.

3.3 MOLE alleviates CCl4-induced liver injury

Mice injection with CCl4induced a highly significant increase in the activity of aminotransferase enzymes; ALT and AST as indicated by serum levels (P＞ 0.001). Treatment with MOLE conspicuously reversed this increase irrespective of the dose used (P＞ 0.01) (Fig. 1).There was no significant difference between the protective effect of MOLE at either dose and that of the standard drug (silymarin).

Fig. 1 Effect of MOLE on serum levels of aminotransferases, ALT and AST in CCl4-treated mice. Data were expressed as mean ± SD, n = 6. *** P ＜ 0.001,compared with vehicle group; ## P ＜ 0.01 and ### P ＜ 0.001 compared with CCl4 group.

Fig. 2 Photomicrographs of liver sections stained with hematoxylin and eosin (H & E) (× 200) representing different experimental groups. I: vehicle group,showing normal structure of hepatocytes with preserved cytoplasm and prominent nuclei, hepatic cords, central vein and sinusoids; II: group injected with CCl4,showing many histopathological lesions such as necrosis, pyknosis, steatosis, and inflammatory cell in filtration; III: group injected with CCl4 and treated with silymarin, showing a high restoration of liver architecture and some binucleated cells, but also showing some pyknotic nuclei; IV: group injected with CCl4 and treated with MOLE (200 mg/kg), showed mild restoration of normal liver histology with some pyknotic nuclei; V: group injected with CCl4 and treated with MOLE (400 mg/kg), showing mostly the same appearance as vehicle group sections besides many binucleated cells. CV. central vein, S. sinusoid, H. hepatocyte, N.nucleus, K. Kupffer cell, Ne. necrosis, Pk. pyknosis, St. steatosis, B. blood, LI. leukocyte in filtration, BN. binucleated cell, L. leukocyte. Scale bar = 20 μm.

3.4 Ameliorating effect of MOLE against histopathological lesions induced by CCl4

The liver of the vehicle group showed normal central veins,sinusoids, and hepatic strands, and hepatocytes with preserved cytoplasm and prominent nuclei. CCl4administration showed many histopathological lesions including hepatocyte necrosis, pyknosis,steatosis, and inflammatory cell infiltration. MOLE lower dose showed mild restoration of normal liver histology while the higher dose showed mostly the same histological features noticed in the vehicle group (Fig. 2).

3.5 MOLE reverses the reduction in antioxidant enzyme activities induced by CCl4

The activity of antioxidant enzymes; SOD, CAT, and GPx, was significantly reduced in the group injected with CCl4as compared with the vehicle group (P ＞ 0.001). The group treated with CCl4along with MOLE at the lower dose manifested noticeable alleviation of the reduction in SOD, CAT, and GPx (P ＞ 0.05, P ＜ 0.01, and P＜ 0.01, respectively) when compared with the CCl4group.Moreover, the group treated with CCl4along with MOLE at the higher dose manifested a highly substantial alleviation (P ＜ 0.01) in the reduced activity of these enzymes. There was no significant difference between the protective role of MOLE and that of silymarin, except for CAT enzyme (P ＜ 0.05) (Fig. 3).

3.6 MOLE suppresses inflammatory response triggered by CCl4

mRNA levels of TLR4, NF-κB, TNF-α, and IL-1β as well as the protein levels of hepatic TLR4, NF-κB-p65, TNF-α, IL-1β, IL-6, and IL-8 were measured in the liver.

There were evident elevations in mRNA levels of proinflammatory cytokines (P ＜ 0.001 and P ＜ 0.000 1 for TNF-α and IL-1β, respectively) and inflammatory mediators (P ＜ 0.01 and P ＜ 0.001 for TLR4 and NF-κB, respectively) in CCl4-induced mice when compared with the vehicle group. Nevertheless, treatment with MOLE at either dose markedly suppressed these elevations (P ＜ 0.01 for TNF-α,TLR4, and NF-κB and P ＜ 0.001 for IL-1β) as compared to CCl4-treated animals. Moreover, most of the results showed no significant difference between the protective role of silymarin and that of MOLE. The only exceptions were for NF-κB (P ＜ 0.05), and IL-1β (P ＜ 0.01) (Fig. 4).

Fig. 3 Effect of MOLE on the activity of antioxidant enzymes in liver of CCl4-injected mice. SOD (A), CAT (B), and GPx (C); Data were expressed as mean ± SD, n = 6. * P ＜ 0.05 and *** P ＜ 0.001 compared with vehicle group; # P ＜ 0.05, ## P ＜ 0.01, and ### P ＜ 0.001 compared with CCl4 group; Δ P ＜ 0.05 compared with (CCl4 + silymarin) group.

Fig. 4 Effect of MOLE on relative expression of pro-inflammatory cytokines and inflammatory mediators in the liver of CCl4 induced mice. mRNA levels of TNF-α (A), IL-1β (B), TLR4 (C), and NF-кB (D); Data were expressed as mean ± SD, n = 6. * P ＜ 0.05, ** P ＜ 0.01, *** P ＜ 0.001, and **** P ＜ 0.000 1 compared with vehicle group; ## P ＜ 0.01, ### P ＜ 0.001 and #### P ＜ 0.000 1 compared with CCl4 group; Δ P ＜ 0.05 and ΔΔ P ＜ 0.01 compared with(CCl4 + silymarin) group.

Fig. 5 Effect of MOLE on protein levels of hepatic pro-inflammatory cytokines and inflammatory mediators in CCl4 induced mice. Protein expression of TLR4 (A)and NF-κB-p65 (B) was assessed by western blotting while, protein content of TNF-α (C), IL-1β (D), IL-6 (E), and IL-8 (F) was assessed by ELISA; Data were expressed as mean ± SD, n = 6. * P ＜ 0.05, ** P ＜ 0.01, *** P ＜ 0.001, and **** P ＜ 0.0001 compared with vehicle group; # P ＜ 0.05, ## P ＜ 0.01, ### P ＜ 0.001 and#### P ＜ 0.000 1 compared with CCl4 group; Δ P ＜ 0.05 and ΔΔ P ＜ 0.01 compared with (CCl4 + silymarin) group.

Fig. 6 Impact of MOLE supplementation on hepatic apoptotic proteins expressions in CCl4-injected mice. Pro-apoptotic protein Bax (A) and apoptosis suppressing protein Bcl-2 (B); Data were expressed as mean ± SD, n = 6. * P ＜ 0.05, ** P ＜ 0.01, and **** P ＜ 0.000 1 compared with vehicle group; # P ＜ 0.05, ## P＜ 0.01, ### P ＜ 0.001 and ####: P ＜ 0.000 1 compared with CCl4 group; ΔΔ P ＜ 0.01 compared with (CCl4 + silymarin) group.

Significant increases in the protein expressions of the proinflammatory cytokines (P ＜ 0.01, P ＜ 0.000 1, P ＜ 0.001, and P ＜ 0.01 for TNF-α, IL-1β, IL-6, and IL-8, respectively) as well as the inflammatory mediators, TLR4 and NF-κB-p65, (P ＜ 0.001) were noticed following CCl4-induced hepatotoxicity when compared with the vehicle group. MOLE substantially reduced the above mentioned cytokine levels as compared with the CCl4-treated group (P ＜ 0.05,P ＜ 0.001, and P ＜ 0.05 for TNF-α, IL-1β, and IL-8, respectively at both doses, and P ＜ 0.01 and P ＜ 0.001 for IL-6 at the lower dose and higher dose, respectively). MOLE, at both doses, markedly reduced the protein levels of TLR4 and NF-κB-p65 as well (P ＜ 0.01).Significant differences between the protective role of silymarin and that of MOLE were only noticed for IL-1β (P ＜ 0.01 for both doses of MOLE), IL-6 (P ＜ 0.05, only for the lower dose of MOLE), and NF-κB-p65 (P ＜ 0.05 for both doses of MOLE) (Fig. 5).

3.7 MOLE protects against apoptosis induced in liver cells by CCl4

The elevation of pro-apoptotic protein Bax (P ＞ 0.000 1) and the reduction of apoptosis suppressing protein Bcl-2 (P ＞ 0.01) observed in CCl4model group were reversed in the presence of MOLE at both doses (P ＞ 0.01, P ＞ 0.001 for Bax and P ＞ 0.05 for Bcl-2). However,there was a significant difference between the protective role of silymarin and that of MOLE for Bax (P ＞ 0.01) (Fig. 6).

3.8 Ameliorating effect of MOLE on CCl4-induced genotoxicity

Genotoxicity induced in CCl4treated group was significantly different (P ＜ 0.05) compared with that in the vehicle group. This genotoxicity was attenuated in the case of co-administration with MOLE at both doses as evidenced by the lack of significant difference between these experimental groups and the vehicle group (Fig. 7).

Fig. 7 Ameliorating Effect of MOLE on CCl4-induced DNA damage in hepatocytes of mice. DNA damage in different groups analyzed by comet assay; Data were expressed as mean ± SD, n = 6. * P ＜ 0.05 compared with vehicle group.

4. Discussion

The results of the current study revealed that CCl4induced hepatotoxicity while MOLE protected liver cells against CCl4-induced injury. The previous outcome was proved by the reduced serum levels of ALT and AST as well as by the ameliorated histopathological lesions in MOLE treated groups. This was represented by restoring the normal features of liver histology, reinstating the normal appearance of the nuclei, and the presence of many binucleated cells indicating hepatocyte regeneration [29]. Similar results have been observed against CCl4-induced hepatotoxicity in rats [30] and against pendimethalin-induced hepatotoxicity in fish [7]. Exposure to toxic insults affects the antioxidant defense system with disturbance of the balance between reactive oxygen species (ROS) release and removal by the antioxidative mechanisms. This imbalance may lead to liver damage after CCl4treatment with altered activities of the antioxidant enzymes such as SOD, CAT, and GPx. The present experiment revealed that CCl4treatment significantly reduced the above mentioned enzyme activities. Our findings were in line with Singh et al. [11] who attributed the defective liver function in rats to the decline of antioxidant enzyme activity as a result of CCl4treatment. Reduced enzymes’ activities were recorded to be a result of ROS accumulation [14]. MOLE administration showed antioxidative effect and enhanced the levels of the antioxidant enzymes in CCl4intoxicated mice. The previous outcome is consistent with Singh et al. [11] who attributed the antioxidant influence of MOL leaves to their total phenolic and flavonoids as well as their phytochemicals components with a hydroxyl group. It has been reported that phenolics and flavonoid constituents possess ROS scavenging activity and protect against oxidative stress associated diseases [31].

inflammation of the liver as a result of a hepatotoxic agent was reported to be induced by different pro-inflammatory cytokines through ROS production and imbalance of antioxidative stress mechanism with liver damage [32]. The present study showed increased expression and/or release of pro-inflammatory cytokines(TNF-α, IL-1β, IL-6, and IL-8) in CCl4-treated mice. It has been reported that TNF-α, IL-1, IL-6, IL-8, and ROS are released by Kupffer cells in response to hepatotoxic agent induction with subsequent propagation of hepatic inflammation [19]. Despite IL-6 was suggested to exhibit paradoxical, pro- or anti-inflammatory,impacts [33], elevated IL-6 level was associated with the propagation of various diseases [34]. Supplementation of MOLE in the present study alleviated hepatic inflammation as it reduced the expression and/or the release of TNF-α, IL-1β, IL-6, and IL-8. The previous outcome may associate with the antioxidant properties of MOLE which underlie its anti-inflammatory impact [34,35].

The stress effect of CCl4was accompanied by TNF-α mediated activation of different transcriptional factors including NF-κB which activates inflammation and liver fibrosis [19]. NF-κB is an important transcriptional factor during liver-induced inflammation that controls the expression and the release of different inflammatory mediators such as TNF-α, IL-8, IL-6, and IL-1β [36,37]. NF-κB is recognized as a homo- or hetero-dimer. p50:p65 is the heterodimer that is extensively linked with cell survival and induced inflammation [34].Upon inflammation, NF-κB dissociates from inhibitory protein I-κBα following its phosphorylation rendering NF-κB free to translocate into the nucleus and associates with the κB binding sites in the promoter part of the target genes [34]. Noteworthy, upregulated transcription of pro-inflammatory mediators was reported upon this association [34].

Consequently, the hepatoprotective effect of MOLE may be attributed to the inhibiting action on the activation of NF-κB pathway as revealed by the downregulated expression of NF-κB gene and the suppressed protein expression of NF-κB-p65 with a further reduction of hepatic CCL4-induced inflammation as a result of the detected polyphenolic compounds [38] in the extract. Our result was in line with Fard et al. [34] who ascribed the anti-inflammatory effect of MOLE to its role in NF-κB pathway inhibition in lipopolysaccharide(LPS) activated macrophages.

Previous studies recorded that TLR4 is an essential contributor for the inflammatory response in CCl4-induced liver fibrosis with augmentation of pro-inflammatory and profibrogenic cytokine expressions through stimulation of NF-κB [4]. It was also stated that TLR4 activation is accountable for acute liver damage [39]. Our findings have shown upregulated TLR4 gene and protein expression following CCl4injection. TLR4 was proven to be activated in response to different hepatotoxic agents with subsequent enhancement of inflammatory reactions [39]. TLR4 activation induces NF-κB phosphorylation which provokes inflammatory cytokine release [4].The exact mechanism for TLR4/NF-κB activation following hepatic injury was reported to be initiated by TLR4 overexpression which involves the up-regulation of various downstream molecules with enhanced expression of specific genes involved in the inflammatory response through NF-κB induction [4]. Thus, it can be inferred that MOLE prompted an attenuated inflammatory cascade in CCl4-induced liver damage through the down-regulation of TLR4/NF-κB pathway as was demonstrated by their reduced gene and protein expressions with subsequent reduction of pro-inflammatory cytokine secretion. For support, it was reported that various extracts of MOL leaves exhibited a wide variety of protective effects including immunomodulation consequences [40].

Pro-apoptotic Bax gene and anti-apoptotic Bcl-2 gene control cell readiness to undergo apoptosis. Bax, as a consequence of oxidative stress and inflammation, stimulates apoptosis [6], while Bcl-2 suppresses apoptosis [41]. In our study, an increase in Bax expression and a decrease in Bcl-2 expression was observed in the CCl4-treated group confirming previous studies stating that CCl4induces apoptosis [4,42]. CCl4-induced apoptosis in the liver is mostly due to ROS release that leads to necrosis and apoptosis [4].It has been reported that induced apoptosis in liver cells is always ascribed with an inflammatory response with subsequent liver fibrosis[43]. MOLE administration showed down-regulation of Bax, and up-regulation of Bcl-2 protein expressions. The above mentioned mitigation impact may be attributed to the decline of ROS and proinflammatory cytokine release [39]. Similar results supporting MOL anti-apoptotic effect were recently reported in methotrexate-induced liver dysfunction [44].

Treating mice with CCl4significantly damages DNA as revealed by comet assay (single-cell gel electrophoresis). Genotoxicity noticed in the CCl4-treated group can be attributed to the CCl4-mediated oxidative stress causing DNA strand breaks, sugar and base lesions,and missing bases [45]. However, co-administration with MOLE has diminished the genotoxicity to be comparable with that of the vehicle group. This is consistent with the findings of Hamed and El-Sayed [7]who indicated the attenuating effect of MOLE against genotoxicity.

The detected phytochemicals in MOLE in the current study have been reported to intimately exert hepatoprotective impacts against CCl4-induced liver injury via the exhibition of one or more of the antioxidative, anti-inflammatory, immunomodulatory, anti-apoptotic,and anti-genotoxic consequences [36,46,47].

5. Conclusions

In conclusion, MOLE phytochemicals exhibit hepatoprotective effects against CCl4-induced liver damage by reducing inflammation mainly through the restoration of antioxidative power and inhibition of TLR4/NF-κB pathway with a subsequent decline of proinflammatory cytokine expression and/or production. Moreover,MOLE protects from apoptotic cell death via antioxidative and antiinflammatory impacts with consequent attenuation of DNA-induced genotoxicity. Thus, MOLE may be a useful therapeutic agent that protects from liver damage in a multifarious manner.

conflict of interests

The authors declare that they have no competing interests.

Acknowledgment

We are grateful to Prof. Ibrahim Abd-Elkader (Faculty of Science,Cairo University, Egypt), Prof. Ashraf M. Essa (Faculty of Science,Fayoum University, Egypt), Dr. Saida S. Ncibi (Biology Department,Faculty of Science, Jazan University, Jazan), and Dr. Mohammed H. Elgammal (Research head, Regional Centre for food and feed,Agricultural Research Centre, Egypt) for their technical support. The authors also thank Dr. Lamiaa F. Shalabi (Ain Shams University,Egypt) for her help in the identification of the plant.