IL-6 and NFE2L2: A putative role for the hepatoprotective effect of N. Sativa, P. Ginseng and C. Sempervirens in AFB-1 induced hepatocellular carcinoma in rats

Graphical abstract


Introduction
Hepatocellular carcinoma (HCC) is considered to be a primary cause of cancer related deaths globally [1][2][3]. There may rise of the incidence of HCC in Egypt due to several risk factors e.g. hepatitis C infection [4,5]. With the highest prevalence of HCV infection worldwide, finding alternative as well as preventive therapy for HCC is of a great concern to researchers and oncologists both in Egypt and globally [3,6,7]. Herbal medicine poses a potential complementary and alternative medicine for many diseases including HCC [8,9].
Nigella Sativa (N. Sativa) also known as black or Blessings seed is a common food condiment in the middle east and has been widely used by several cultures as a treatment of various diseases [10]. N. Sativa was reported to have anti-inflammatory and anti-oxidant effect [10][11][12]. Its anti-cancer and chemotherapeutic actions both in vitro and in vivo animal cancer models have also been reported [13,14]. Not only that, but N. Sativa has also been shown to have protective effects against chemotherapeutic agent's side effects e.g. cardio-and nephrotoxicity [15][16][17].
Panax Ginseng (P. Ginseng) is a Chinese herb that has been used commonly in medicine. Studies have shown Ginseng family members of herbs have potential roles as anticancer agents [18,19]. An anti-angiogenic saponin; Ginsenoside Rg3 extracted from ginseng was used for the treatment of HCC tumors in rats via reducing metastasis and promoting more survival [20]. Another study has shown that fermented Ginseng (FG) had hepatoprotective effect in liver cancer rat model [21]. In patients of epithelial ovarian cancer (EOC), who received chemotherapy, red ginseng reduced genotoxicity and improved quality of life [19]. Another study illustrated the mechanism of action of ginsenosides in Alzheimer's disease and the effect of different factors on the yield of extractable materials [22,23].{Razgonova, 2019 #62}{Razgonova, 2019 #62}{Razgonova, 2019 #62}{Razgonova, 2019 #62} Cupressus Sempervirens (C. Sempervirens) commonly known as American yellow jasmine was reported to have potential anticancer effects both in vitro and in vivo [24,25]. It is a native herb in north Africa that possesses several therapeutic actions [26]. Its extract was shown to inhibit cancer cell proliferation in mouse skin carcinoma model [27].
Studies have shown that inflammation and oxidative stress are key components in the pathophysiology of HCC [28,29]. In fact inflammatory condition and pro-oxidative state may synergistically act to enhance tumor progression [30]. Inflammatory cytokines e.g. IL-6 plays a central role in the pathogenesis of HCC [31]. Targeting antioxidant enzymes and inflammatory pathways, pharmacologically, poses a logical potential strategy to control tumor-genesis in HCC models [30,32].
Nuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or Nrf2 plays an important role in activating antioxidant response in the cell for its protection from exogenous and endogenous insults; moreover, it is thought to be a cytoprotective transcription factor and the main regulator of the survival of the cell [33].
The anti-tumor mechanism of action of these three aforementioned herbs is an active area of research and yet to be elaborated. In this study, we investigated possible hepato-protective effects of N. Sativa, P. Ginseng, and C. Sempervirens in Aflatoxin B1 (AFB-1) induced hepatocellular carcinoma rat model. We hypothesized that the mechanism of this hepato-protective action of these herbs act is putatively mediated via anti-oxidant and anti-inflammatory effects.

Animals
Fifty-four male albino rats, 200 ± 20 grams (gm) at the start of the experiments were used. Animals were randomly assigned to experimental groups. Four rats (maximum) were housed per cage (size 26 × 41 cm) and placed in the experimental room for acclimatization 24 hours before any procedure performed. Animals were fed with standard laboratory diet, tap water ad libitum, and kept in an airconditioned animal room at 23 ± 1°C with a 12 h light/dark cycle.
Animal care and handling was performed in conformance with approved protocols of MSA University and Egyptian Community guidelines for animal care. MSA Faculty of Pharmacy Research Ethics Committee approved the study

Experimental groups
Experimental doses of all herbs were selected based on previous studies and within non-toxic range [26,37,38]. Rats were randomly allocated into nine groups of six animals each.

Preparation of blood samples
At the end of the study, rats were fasted overnight, anesthetized with thiopental sodium (50 mg/kg) [39] and blood samples were collected (5 ml per rat). Blood samples were centrifuged at 3000 rpm for 15 min after 30 minutes of collection and stored at -80 0 C until analyzed. Serum Alpha-fetoprotein (AFP), Interleukin-6 (IL-6) and high sensitive C-reactive protein (hs-CRP) were determined using the rat enzyme immunoassay kits.

Preparation of liver samples
Animals were euthanized by cervical dislocation at 24 hours of AFB1 injection, and then liver was rapidly removed from each rat. Each liver was divided into two parts. The first part was fixed in formalinsaline for 48 hours for histopathological study. The second part was homogenized, using glass homogenizer (Universal Lab. Aid MPW-309, mechanika precyzyjna, Poland), with 5 mL phosphate buffer saline (PBS) then centrifuged using cooling ultra-centrifuge. The homogenate was divided into three aliquots for measuring total protein, malondialdehyde (MDA) and superoxide dismutase (SOD). Another part of the liver was homogenized with 5 mL phosphate buffer saline (PBS), using glass homogenizer and was subjected to nuclear extraction protocol according to methodology of LSBio TM DNA-Binding ELISA Kit contains the necessary buffers and inhibitors for nuclear extraction from cells for measuring Nuclear Factor Erythroid Derived 2 Like 2 Protein (NFE2L2).

Biochemical analysis
Serum analysis was performed to measure AFP, IL-6 and hs-CRP levels using rat respective enzyme immunoassay kits (Wuhan Fine Biological Technology Co., Ltd, China, Immuno-Biological Laboratories, Inc. (IBL-America), Wuhan EIAAB science Co, Ltd, China). Liver MDA and SOD were measured using colorimetric method according to manufacturer instructions using commercial kits purchased from Biodiagnostic, Egypt, while Liver NFE2L2 was measured using rat immunoassay kits purchased from Cloud Clone Corp, USA.

Histopathological Examination
At the end of the study, livers were harvested, as mentioned previously. Specimens were fixed in 10% formalin and then liver tissues were decalcified in nitric oxide for 4 days, routinely processed and embedded in paraffin. Five microns sections were cut and stained with hematoxylin and Eosin (H&E).

Statistical analyses
All data were expressed as mean ± SEM and analyzed using Prism ® program version 6. For all parameters, comparisons among groups were carried out using one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparisons test. All P values reported are twotailed and P < 0.05 were considered significance.

Effect of C. Sempervirens, P. Ginseng and N. Sativa extracts on liver enzymes level
Mean serum level of liver enzymes were significantly increased in AFB1 induced HCC group compared to the control group; P value was < 0.0001. The mean serum levels of ALT & AST were significantly reduced in C. Sempervirens 300, C. Sempervirens 600, P. Ginseng 250, P. Ginseng 500, N. Sativa 500, N. Sativa 1000 and silymarin groups compared to AFB1 induced HCC group (P value < 0.0001). Non-significant difference was found between 3 herbs in reducing liver enzymes (Table 1).

Effect of N. Sativa, P. Ginseng and C. Sempervirens extracts on serum Alpha-fetoprotein (AFP) level
As shown in Fig. 1, the mean serum level of AFP was significantly increased in AFB1 induced HCC group compared to the control group, P value was < 0.05. The mean serum level of AFP was significantly reduced in C. Sempervirens 600, P. Ginseng 250, 500, N. Sativa 1000 and silymarin groups compared to AFB1 induced HCC group (P value < 0.05).
3.3. Effect of N. Sativa, P. Ginseng and C. Sempervirens extracts on serum Interleukin-6 (IL-6) and high sensitive C-reactive protein (hs-CRP) Fig. 2 shows that, the mean serum level of IL-6 was significantly increased in AFB1 induced HCC group compared to the control group, P value was < 0.0001, the mean serum level of IL-6 was significantly reduced in C. Sempervirens 300, 600, P. Ginseng 250, 500, N. Sativa 1000 and silymarin groups compared to AFB1 induced HCC group (P value < 0.0001), P. Ginseng 500 reduced IL-6 level compared to C. Sempervirens 600, N. Sativa 1000 and Silymarin groups (P value was < 0.0001) also, non-significant difference was found between control and P. Ginseng 500 group in serum level of IL-6 Fig. 3 shows that the mean serum level of hs-CRP was significantly increased in AFB1 induced HCC group compared to the control group, P value was < 0.01. The mean serum level of hs-CRP was significantly reduced in C. Sempervirens 300, 600, P. Ginseng 250, 500, N. Sativa 500, 1000 and silymarin groups compared to AFB1 induced HCC group (P value < 0.02). C. Sempervirens 600 and P. Ginseng 500 showed a better result compared to N. Sativa 1000 in reducing serum hs-CRP levels. Non-significant difference was found between silymarin group and C. Sempervirens 600 and P. Ginseng 500 in the serum level of hs-CRP The mean tissue level of MDA was significantly increased in AFB1 induced HCC group compared to the control group, P value was < 0.0001, the mean tissue level of MDA was significantly reduced in C. Sempervirens 300, 600, P. Ginseng 250, 500, N. Sativa 500, 1000 and silymarin groups compared to AFB1 induced HCC group (P value < 0.0001), C. Sempervirens 600 reduced MDA level compared to P. Ginseng 500, N. Sativa 1000 groups even to control group (P value was < 0.03) also, Silymarin showed a good response when compared to C. Sempervirens 600 and P. Ginseng 500 groups (Fig. 4) The mean tissue level of SOD was significantly decreased in AFB1 induced HCC group compared to the control group, P value was < 0.0001, the mean tissue level of SOD was significantly increased in C. Sempervirens 300, 600, P. Ginseng 250, 500, N. Sativa 500, 1000 and silymarin groups compared to AFB1 induced HCC group (P value < 0.0001). Non-significant difference between C. Sempervirens 600 and P. Ginseng 500 groups in tissue level of SOD (Fig. 5), on the other hand C. Sempervirens 600 and P. Ginseng 500 raised the SOD level compared to N. Sativa 1000 group, P value was < 0.0001 3.5. Effect of N. Sativa, P. Ginseng and C. Sempervirens extracts on tissue Rat Nuclear Factor Erythroid Derived 2 Like 2 Protein (NFE2L2) The mean tissue level of NFE2L2 was significantly increased in AFB1 induced HCC group compared to the control group, P value Results were expressed as mean ± SEM and analyzed using one-way ANOVA followed by Bonferroni's post hoc test, a: significant from control at P < 0.0001, b: significant from AFB1 induced HCC group at P < 0.0001 Fig. 1. Serum level of Alpha-fetoprotein (ng/ mL) in the experimental groups. N. Sativa, P. Ginseng and C. Sempervirens extracts reduced serum level of AFP in the liver cancer rats after 1 month of prophylaxis. Results were expressed as mean ± SEM and analyzed using one-way ANOVA followed by Bonferroni's post hoc test, a: significant from control at P < 0.05, b: significant from AFB1 induced HCC group at P < 0.05 Fig. 2. Serum level of IL-6 (Pg/mL) in the experimental groups. N. Sativa, P. Ginseng and C. Sempervirens extracts reduced serum level of IL-6 in the liver cancer rats after 1 month of prophylaxis. Results were expressed as mean ± SEM and analyzed using one-way ANOVA followed by Bonferroni's post hoc test, a: significant from control at P < 0.0001, b: significant from AFB1 induced HCC group at P < 0.0001, c: significant from C. Sempervirens 600 at P < 0.0001, d: significant from P. Ginseng 500 at P < 0.0001 Fig. 3. Serum level of hs-CRP (ng/mL) in the experimental groups. N. Sativa, P. Ginseng and C. Sempervirens extracts reduced serum level of hs-CRP in the liver cancer rats after 1 month of prophylaxis. Results were expressed as mean ± SEM and analyzed using one-way ANOVA followed by Bonferroni's post hoc test, a: significant from control at P < 0.01, b: significant from AFB1 induced HCC group at P < 0.001, c: significant from C. Sempervirens 600 at P < 0.05, d: significant from P. Ginseng 500 at P < 0.05. Fig. 4. Tissue level of MDA (nmole/mL/1gm total protein) in the experimental groups. N. Sativa, P. Ginseng and C. Sempervirens extracts reduced tissue level of MDA in the liver cancer rats after 1 month of prophylaxis. Results were expressed as mean ± SEM and analyzed using one-way ANOVA followed by Bonferroni's post hoc test, a: significant from control at P < 0.0001, b: significant from AFB1 induced HCC group at P < 0.0001, c: significant from C. Sempervirens 600 at P < 0.05, d: significant from P. Ginseng 500 at P < 0.05.
was < 0.0001, the mean tissue level of NFE2L2 was significantly raised in C. Sempervirens 300, 600, P. Ginseng 250, 500, N. Sativa 500, 1000 and silymarin groups compared to AFB1 induced HCC group (P value < 0.0001), C. Sempervirens 600 and N. Sativa 1000 increased NFE2L2 level compared to P. Ginseng 500 group (P value was < 0.01) also, Silymarin showed a good response when compared to C. Sempervirens 600 and P. Ginseng 500 groups. Significant difference was found between the N. sativa 500 and 1000 groups (P value was < 0.01) (Fig. 6) 3.6. Histopathological effects of N. Sativa, P. Ginseng and C. Sempervirens extracts Histological examination of the liver tissue of all experimental groups using Hematoxylin and Eosin (H&E) staining revealed that normal hepatocytes and preserved hepatic lobular architecture, with regular central veins (C) and unremarkable portal tracts (P) of the liver of the control group. Sections show wide neoplastic transformation of the hepatocytes, with increased N/C ratio, nuclear hyperchromasia and irregular nuclear membranes in Aflatoxin group. In Silymarin treated group sections showed preservation of the lobular architecture, subserosal hepatocyte dysplastic changes is focally evident with no frank neoplastic transformation. Examination N. Sativa group P. Ginseng and C. Sempervirens treated sections showed evident preservation of the lobular architecture, minimal hepatocyte dysplastic changes are focally evident with no neoplastic transformation (Fig. 7).

Discussion
This study clearly shows and confirms a hepatoprotective role for N. Sativa, P. Ginseng and C. Sempervirens extracts in AFB1 induced HCC rat model evidenced by reduction of liver enzymes (AST and ALT), tumor marker (AFP) and histopathological observations. This hepatoprotective effect is possibly mediated via antioxidant and anti-inflammatory effects as N. Sativa, P. Ginseng and C. Sempervirens extracts, orally administered for about a month, clearly improved inflammatory (IL-6 and Fig. 5. Tissue level of SOD (U/mL/1 gm total protein) in the experimental groups. N. Sativa, P. Ginseng and C. Sempervirens extracts reduced tissue level of SOD in the liver cancer rats after 1 month of prophylaxis. Results were expressed as mean ± SEM and analyzed using one-way ANOVA followed by Bonferroni's post hoc test, a: significant from control at P < 0.0001, b: significant from AFB1 induced HCC group at P < 0.0001, c: significant from C. Sempervirens 600 at P < 0.0001, d: significant from P. Ginseng 500 at P < 0.0001 Fig. 6. Tissue level of NFE2L2 (U/mL/1gm total protein) in the experimental groups. N. Sativa, P. Ginseng and C. Sempervirens extracts raised tissue level of NFE2L2 in the liver cancer rats after 1 month of prophylaxis. Results were expressed as mean ± SEM and analyzed using one-way ANOVA followed by Bonferroni's post hoc test, a: significant from control at P < 0.0001, b: significant from AFB1 induced HCC group at P < 0.0001, d: significant from P. Ginseng 500 at P < 0.01 hs-CRP) as well as antioxidant (MDA and SOD) biochemical markers. These effects were comparable to the effects of Silymarin, a known hepatoprotective agent.
N. Sativa extract and some of its components as thymoquinone, dithymoquinone and alpha-hederin have been shown to possess antitumor activities against cancer cell lines, as well as in a diverse animal models of cancer e.g. lung, pancreatic and multiple myeloma [10,40].
Our results confirm such anti-cancer effect for N. Sativa in HCC rat Fig. 7. Histological examination of the liver tissue of all experimental groups using Hematoxylin and Eosin (H&E) staining: a) illustrates control group sections that shows normal hepatocytes and preserved hepatic lobular architecture, with regular central veins (C) and unremarkable portal tracts (P). b) Illustrates Aflatoxin group section that shows wide neoplastic transformation of the hepatocytes, with increased N/C ratio, nuclear hyperchromasia and irregular nuclear membranes. c) Illustrates Silymarin treated group section that shows preservation of the lobular architecture, subserosal hepatocyte dysplastic changes is focally evident, however with no frank neoplastic transformation. d) Illustrates N. Sativa group sections that shows evident preservation of the lobular architecture, minimal hepatocyte dysplastic changes is focally evident (thick arrow), however with no neoplastic transformation. e) and f) illustrates C. Sempervirens and P. Ginseng group sections that shows evident preservation of the lobular architecture with unremarkable hepatocyte dysplastic changes and no neoplastic transformation. Magnification 10 × .
model. Although the exact mechanism(s) for the anti-tumor action of N. Sativa is not fully understood, it has been postulated that its active components inhibit cell proliferation and arrest cell cycle as well as antioxidant effects [10,41]. Our results clearly show an anti-inflammatory effect of N. Sativa as it significantly reduced IL-6 and hs-CRP in our HCC rat model. These results come in agreement with other studies that demonstrated that Thymoquinone, the major N. Sativa active component, inhibited the production of IL-6 in human rheumatoid arthritis synovial fibroblast cell culture [42,43]. Another study showed that blockage of IL-6 inhibited mice fibroblast growth factor 19 (FGF19) induced hepatocarcinogenesis [44]. Similarly, activation of IL-6-JAK-STAT3 (signal transducer and activator of transcription 3) signaling pathway accelerated HCC growth in rat model [45]. Since the majority of human HCC is associated with in vivo inflammatory environment [31] N. Sativa mediated inhibition/reduction of IL-6 production may contribute to its hepatoprotective effect on HCC. Furthermore, N. Sativa reduction of hs-CRP reduction comes in agreement with, Alizadeh et al., 2016 who found that N. Sativa reduced hs-CRP in women with rheumatoid arthritis [46]. Along with reduction of IL-6 our results confirm anti-inflammatory effect for N. Sativa in HCC as well as the potential role for hs-CRP as an early diagnostic tool for HCC [47].
In addition, N. Sativa demonstrated an antioxidant effect in our rat model of HCC as it significantly reduced oxidative stress markers as MDA and increased SOD levels. Studies have shown that both MDA (a byproduct of lipid peroxidation that is produced under oxidative stress conditions) and SOD (a pivotal superoxide scavenging enzyme) are reliable markers of oxidative stress [48][49][50]. Both MDA and SOD were postulated to be involved in the pathogenesis of HCC [51]. Further, NFE2L2 is a transcription factor that is thought to be involved in hepato-protection against oxidative stress induced HCC mainly by the regulation of many genes involved in glutathione biosynthesis that protect against oxidative damage [52,53]. Together with the aforementioned effect on MDA and SOD, our finding that N. Sativa significantly increased NFE2L2 levels in AFB1 induced HCC rat model further supports the idea that its hepatoprotective effect in HCC can be attributed to its antioxidant effect.
P. Ginseng and C. Sempervirens extract administration to our HCC rat model had similar effect to N. sativa on anti-inflammatory markers measured (IL-6 and hs-CRP) as well as anti-oxidant markers (MDA, SOD, and NFE2L2). P. Ginseng and C. Sempervirens extract also had hepatoprotective effect in our HCC rat model indicated by histopathological examination and liver enzymes reduction. Our P. Ginseng results comes in agreement with other studies that demonstrated hepatoprotective effect for different P. Ginseng constituents and extracts against liver cancer in animals [18][19][20][21]. It is well documented that P. Ginseng extracts and active constituents has anti-inflammatory and antioxidant effects [54,55]. Ginsenoside Rg1, one of P. Ginseng components, exhibited a neuroprotective effect via modulation of IL-6 and MDA in rats [56]. Our results further support that this hepatoprotective effect is possibly mediated via reduction of oxidative stress as well as modulation of inflammatory cytokines as IL-6. Our data supports Lu et al., 2016 conclusion who found that Ginseng had hepatoprotective effects against carbon tetrachloride (CCl 4 ) liver injury possibly via reduction of liver inflammation [57].
On the other hand, other studies have shown that C. Sempervirens extract had hepatoprotective effect against CCl 4 liver injury indicated by liver function tests and liver pathological changes [58,59]. Our results not only concur with these studies but also propose its possible mediation of such hepatoprotective action via anti-inflammatory and anti-oxidant effect. To our knowledge, this is the first demonstration that C. Sempervirens extract could in fact alter IL-6, hs-CRP together with MDA, SOD, and NFE2L2 AFB1 induced HCC rat model. It was previously reported that C. Sempervirens extract administration protected against indomethacin-induced gastric ulcer. Such protection was associated with increased SOD activity [26].
Our data supports other studies that showed that health beneficial effects as antioxidant and anti-inflammatory effects can be achieved using plant-derived flavonoids [60,61]. This can be achieved by modulation of gene expression of certain proteins involved in the inflammatory response and alteration of activity levels of enzymes involved in antioxidant response [26].
In conclusion, this study confirms a beneficial hepatoprotective effect for N. Sativa, P. Ginseng, and C. Sempervirens extracts orally administered in rat model of AFB1 induced HCC. This effect is putatively mediated via modulation of inflammatory cytokines as IL-6 as well as amelioration of oxidative stress.

Ethics approval
Animal care and handling was performed in conformance with approved protocols of MSA University and Egyptian Community guidelines for animal care.

Consent to publish
Not applicable

Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its additional files.

Competing interest
The authors declare no competing interest associated with this article. There is no significant financial support for this work that could affect its outcome.

Author contribution
Dr. Aborehab and Dr. Waly contributed equally to the experimental design, execution of the experiments, data, sample collection the study, conduction of biochemical analyses, interpretation of data. Dr. Waly drafted the manuscript. Dr. Aborehab performed the statistical analyses of this study. Both authors read and approved the final manuscript.

Funding
This research is not supported by any funding agency. The Transparency document associated with this article can be found in the online version.