The effect of dietary supplementation of Nigella sativa (black seeds) on cell mediated immunological function of male Wistar rats

This experiment aimed to investigate the effect of dietary Nigella sativa on cell mediated immune response. A total of eighteen male Wistar rats were divided equally into control and black seeds at 30 and 50 g/kg/diet (Sa30 and Sa50), respectively for 30 days. Weight gain, feed intake, feed conversion ratio (FCR), cell mediated immune response was monitored after injection of 0.1 mL of 10% phytohemagglutinin (PHA). Intumesce Index, serum total antioxidant capacity (TAC), Catalase (CAT), Interleukin-12 (IL-12), gamma interferon (γ-IF) and tumor necrosis factor alpha (TNF-α) were determined. Histopathological examination and immunohistochemistry of splenic caspase-3 and CD 8 were done. Nigella sativa signicantly (p<0.05) improved weight gain and FCR in Sa30 and Sa50 groups. Signicant increase for Intumesce Index in Sa50 group was observed. Total TAC, CAT, IL-12, γ-IF and TNF-αincreased signicantly (P<0.05) in Sa30 and Sa50 groups. Histological examination of PHA stimulated foot pads showed more leukocytes inltration and edema in a dose dependent pattern. Splenic caspase-3 and CD 8 showed signicant (P<0.05) decrease and increase, respectively in Sa30 and Sa50 groups. Nigella sativa seeds had immunostimulatory ability through their antioxidant potential, cytokines induction, CD 8 promotion and reducing splenic apoptosis. Present in with Gholamnezhad et al. and Titiek et al. These explained the cell mediated immunostimulatory effect of sativa where IL-12 enhanced IF-γ that acted in autocrine and paracrine manner to increase CD8 cells activity. Both γ-IF and TNF-α could stimulate macrophages activity to eliminate infectious


Introduction
The immunostimulants enhance cell-mediated immune response by the activation of antigen-speci c cytotoxic T-lymphocytes, phagocytes and the discharge of several cytokines toward antigen 1 to achieve therapeutics 2 . Immunostimulant ropes to overwhelm the immunosuppressive effects of infectious agents and stress that interface and/or harm the immune cells function 3 . Diverse substances have been displayed immunostimulatory effects which are; plants or animal derivatives, microbial products, hormones, synthetic chemical and vitamins 4 . Herb, plant extracts and animal originated products are widely used because they are not expensive, can be easily obtained and act versus a wide-ranging spectrum of pathogens 5 . Oral administration of plant or herbal extracts as immunostimulants is considered the most superlative method of immunostimulation 6 .
Herbal medicines extracted from plants or plant extracts itself have historically been used to enhance health. Recently, scientists have been keen on recognizing their key ingredients and comprehending their mechanisms of action [7][8][9][10] . One of them is black seed, or Nigella sativa belonging to the family Ranunculacea that has a rich religious and historical backgrounds 11 . It has been grown and used in various parts of the world as a food additive, spice and remedy for a large variety of diseases, like headache, bronchial asthma, nasal congestion, toothache, allergies, back pain, hypertension, obesity, gastrointestinal troubles and numerous types of cancer 12 . Also Nigella sativa seeds can minimize fatigue 13 and depression 14 beside increasing the strength of the body 15 . Moreover, Nigella sativa was found to possess immunostimulatory effects, due to its documented components, in various in ammatory and immunologic diseases 16 such as experimental allergic encephalomyelitis, colitis, arthritis 17 , sensitized animals 18 , asthma sufferers patients 19 and chemical war victims 20 .
The current study aimed to explore the effect of dietary Nigella sativa seeds supplementation on growth performance parameters, FCR, Intumesce Index as an indicator for immune function in Wistar rats. This was achieved by investigating the parameters of total antioxidant and in ammatory cytokines, such as total antioxidant capacity (TAC), catalase activity, interferon gamma (IF-γ), interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α) in rat model. Also, histopathologic changes in the popliteal lymph node and spleen were also examined; in addition to the immunohistochemical expression of caspase 3 and CD8 in the spleen.

Herbal plants
Black seeds (100% organic Nigella sativa seeds) were bought from a local market, kept with a voucher number 54782: 228/056/572 in Prophetic Medicine Foundation, Ismailia, Egypt. The whole seeds were daily shriveled in a blender and mingled well with basal diet just before administration to rats.
Quantitative phytochemical constituent's analysis of Nigella sativa seeds were done using previously reported protocols as follow: preparation of seeds extract for quantitative phytochemical constituent's analysis as previously mentioned by Silahtaroglu, et al. 49 .
Phytochemical analysis of Nigella sativa seeds and antioxidant activity Phytochemical screening of phenolic compounds was done according to Oladeji, et al. 50 . Total phenolic content was estimated in Nigella sativa seeds according to Khattak and Simpson 51 . The total avonoid content was estimated in Nigella sativa seeds extract based on the method of Nergiz and Ötleş 52 . The antioxidant activity of the seeds was assessed by the method described by Mariod, et al. 53 .

Experimental animals
Blood and tissue collection The rats were anesthetized with tetrahydrofuran inhalation anesthesia after overnight fasting. Blood samples were drawn, at the end of experiment, from orbital venous plexus under the effect of the latter anesthesia in plain tubes. Sera were alienated and kept at -70 °C until analysis of antioxidants (catalase and TAC), IF-γ, IL-12 and TNF-α.
Rats were scari ed and the spleen was excised, microscopically examined and weighed in relation to body weight to obtain the relative weight. Also, popliteal lymph node of PHA injected paw and PBS injected one were excised. Both spleen and popliteal lymph nodes were xed in a 10% neutral formalin solution for histopathological examination and immunohistochemical detection.

Catalase activity and TAC
Catalase activity and TAC were measured in the sera by colorimetric method and following kits (K773 and K274) instruction that were purchased from BioVision Inc., Milpitas, CA, USA.
IF-γ, IL-12 and TNF-α levels IF-γ, IL-12 and TNF-α levels were assayed via rat enzyme linked immunosorbent assay sandwich ELISA kit (Thermo Fisher Scienti c, USA) according to producing company directions. IF-γ (BMS629) with detection limit 11.0 pg/mL; Serum IL-12 (KRC0121) with detection limit <2.5 pg/mL and TNF-α levels (BMS621) with detection limit 9.9 pg/mL. They were carefully checked for their sensitivity, specify and reliability. Spectrophotometer and a microplate reader (Biotech, USA) were used to measure the absorbance.

Histopathology and immunohistochemistry examination
Formalin xed spleen and foot paws were progressively dehydrated, cleared, then submerged in para n wax. Numerous 5μm sections were obtained and stained with Haematoxylin and Eosin (H&E) according to Bancroft and Gamble 59 for histopathological examination.
Immunohistochemistry of splenic caspase 3 was done using a primary antibody for caspase 3 (#PAI29157, Thermo Scienti c Co., USA) and CD8 (Cat. No. 6A242, Santa Cruz, CA, USA) at dilution rates 1:1000 and 1:200, respectively. The procedures were performed secondary polyvalent Biotinylated antibody according to the methodology of Zhao et al. and Elgawish et al. 60,61 , respectively. The percentages of the IHC-stained area (IHC area %) were acquired via ImageJ software as described by Elgawish and Abdelrazek 61 . Tissues were examined blindly to which group the samples belonged.

Statistical analysis
Data were tested for normality and they found to follow normal distribution. The results were presented as the mean ± standard error of mean (SEM). The differences between groups were calculated using one-way analysis of variance (ANOVA) followed by Duncan post hoc multiple comparison tests (SPSS software, version 16.0; SPSS Inc., Chicago, IL, USA) with signi cance at P < 0.05.

Phytochemical constituent analysis of Nigella sativa seeds
The standard laboratory procedures for phytochemical screening demonstrated the presence of phenolic compounds (p-hydroxybenzoic; Catechin; Chlorogenic; Ferulic; Sinapic; p-coumaric; Kaempferol) Table (1). The results indicated that the highest amount of phenolics was p-hydroxybenzoic (69.685 μg/g) while the lowest amount was Kaempferol (1.277 μg/g). As shown in Table (2), the contents of Nigella sativa seeds from total phenol compound were 2.077 mg equivalents to Gallic acid , total avonoid contents is 0.565 mg equivalents to catechin and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities of black seeds was 1.367 g equivalents to Trolox.

Relative weight of the spleen
There was no noteworthy nding in the spleen external appearance. The relative weight of the spleen in the sa50 rats signi cantly (P< 0.05) improved than those of the control group. Relative weight of spleen did not differ in 30 g/ kg black seeds fed group than control rats (Table 3).

Intumesce Index
There was a signi cant (P < 0.05) increase in Intumesce index of ankle of rats fed 50 g/kg Nigella sativa for 30 days compared to control groups. However, 30 g/kg black seeds fed group was non-signi cantly differed than control and 50 g/kg black seeds fed groups (Table 3).

Catalase and TAC
Serum catalase activity revealed signi cant (P <0.05) increase in Sa30 and Sa50 groups than control. Also, serum TAC was signi cantly increased (P<0.05) in Sa30 and Sa50 groups than control (Table 4).

Spleen histopathology and histomorphometry
Microscopical examination of control and Nigella Sativa treated spleens revealed no histopathological lesions in normal white and red pulps separated by marginal zones. The white pulp consists of follicle with pale germinal center and peripherally located central arterioles (Fig. 1). Histomorphometric analysis of 30 and 50 g/kg Nigella Sativa spleens showed that area of the white pulp, periarterial lymphoid sheath and the germinal center showed signi cant increase compared to the spleen of control rats (Fig. 1) that appeared to be dose dependent.
The CD8 protein expression appeared as brownish color in splenocytes. Spleen of control group showed very weak expression of CD8 expression. In contrast to spleen of Sa30 and Sa50 groups showed more expression pattern compared to control group (Fig. 2). The percent area of positive Immunohistochemical expression of splenocytes containing CD8 signi cantly (P<0.05) increased in Sa30 and Sa50 groups compared to control groups.
On the other hand, spleen of control group showed a strong expression of caspase 3 expression that was represented by brown coloration. In contrast to spleen of Sa30 and Sa50 groups showed more decrease to the expression pattern of caspase 3 protein compared to control group (Fig. 3). The percent area of positive Immunohistochemical expression of splenocytes containing caspase 3 protein signi cantly (P < 0.05) decreased in rats fed (30 and 50) g/kg Nigella sativa seeds daily for 30 days compared to control groups.
The popliteal lymph node of control group injected with PHA Fig. 4 (A&D) showed a reduction in the width of the cortex. The lymphoid follicle with a pale staining area (germinal center) was poorly demarcated.
Reactive in ammatory hyperplasia of the lymph node showed a slight increase in the number of lymphocytes seen extending to the medulla and dispersed throughout the whole section. Severe degeneration with mild necrotic area and lymphatic sinus ectasia were also observed. The histological inspection of the popliteal lymph nodes of Sa30 Fig. 4 (B&E) and Sa50 Fig. 4 (C&F) groups showed medullary lymphoid hyperplasia with a slight increase in the number of lymphocytes that were seen extending to the medulla and distributed throughout the whole section. Sa50 lymph nodes showed parafollicular hyperplasia.
The footpad 24 hours after PHA injection Fig. 5 showed the control footpad with marked cellular diffuse in ltration in the connective tissue with edema in the dermis. In the other hand, the footpad of male rats fed Nigella sativa seeds showed an increased in the in ammatory response with an increase of lymphocyte in ltration and edema in the dermis when matched with control group.

Discussion
Nigella sativa seeds are one of the most frequently used plants in traditional medicine. They play a signi cant role as anti-in ammatory 62 , antioxidant 63,64 agents and immunological activator 65 . Some researchers have demonstrated that Nigella sativa has in uences on the immune system; it could increase the antibody response 66 and ameliorate in ammation 67,68 as well as immunological attacks 69 . Therefore, the current study investigated the cellular mediated immunomodulatory action of Nigella sativa seeds on male adult Wister rats with their in uence on performance. To the best of our knowledge, current study is the rst study presuming experimental evidence that dietary Nigella sativa has immunomodulatory properties against 10% PHA in rats. Additionally, no studies have described the immune response to the PHA skin test in rat fed Nigella sativa seeds.
Analyses of Nigella sativa seeds showed a strong antioxidant free radical DPPH scavenging action which may be attributed to the high levels of phenolic and avonoid constituents. Similarly, Adetuyi and Ibrahim 70 , Hameed et al. 71 , Hirose et al. 72 and Zhang et al. 73 found the DPPH function associated with the phenolic and avanoid components.
Nigella sativa seeds fed groups showed a signi cant improvement in nal body weight and FCR relative to control group. Nigella sativa seeds greatly improved growth performance. This growth performance promotion could be due to the nutritional value of Nigella sativa key components which contain high fatty acid percentages and essential amino acids [74][75][76] . Moreover, Nigella sativa contributes an enhancing effect to digestive enzymes 77,78 and gastrointestinal motility 79 thus, improving feed utilization and FCR.
The previous results were in harmony with Dollah et al. 80 and Ahmed and El-Sayed 21 .
Spleen represent enlarged lymphatic tissue which responsible for clearance of the damaged old particles of the body and foreign particles from the blood 81 . In the present study, dietary Nigella sativa seeds were found to increase relative splenic weight at dose 50 g/kg. Moreover, the splenic histomorphometry of both Sa30 and Sa50 groups was signi cantly higher than control. These results harmonized with Ghonime et al. 45 as Nigella sativa was proven to have lympho-regenerating effect in lymphoid organs 82 . This illustrates its role in increasing splenic weight.
According to our results, feeding Nigella sativa seeds had resulted in a signi cant increase of catalase activity and TAC rates compared with the control group. These results proposed that feeding of Nigella sativa seeds participated in cellular protection as a source of antioxidant molecules and indirectly as a stimulator to the activity of these enzymes 83,84 . The active ingredients of Nigella sativa seeds such as phydroxybenzoic acid 85 , Chlorogenic acid 86 , Catechin 87 , sinapic acid 88 . ferulic acid 89 , p-coumaric acid 90 and kaempferol 91 were established to have antioxidant in uences beside ROS scavenging potential thus, promoting higher antioxidant enzymes level. The abolishing of oxidative stress has close association with promotion of body weight 92 . Moreover, the later antioxidant potency of Nigella sativa seeds ingredients could be attributed to their immunostimulant effect 17,[93][94][95][96] . This was manifested by increased TNF-α and IF-γ toward PHA stimulation as well as increased splenic lymphoproliferation denoted by the increased histomorphometric parameters in Nigella sativa treated groups.
Phytohaemagglutinin (PHA) is a mitogen derived plant that provokes leucocytes recruitment in both innate and adaptive immune responses at the place of inoculation resulting in a quanti able tissue swelling that could quantify such immune response [97][98][99] . Current results demonstrated signi cant increase in Intumesce index of Sa50 group. These results were parallel to the observed upgrading of cellular in ltration, edema and lymphocytes in ltration at PHA injected paws in Sa30 and Sa50 groups.
The increments of IF-γ, IL-12 and TNF-α in Sa30 and Sa50 groups as cellular immune promoting cytokines were con rmative to Nigella sativa immunostimulatory effect.
Interleukin 12 (IL-12) is a substantial immunomodulatory cytokine which is manufactured by macrophages, dendritic cells and antigen presenting cells. The production of this cytokine during infection adjusts innate immune responses and determines the adaptive immune responses sequence to be elicited. Also, IL-12 can evoke the assembly of IF-γ from T helper type 1 (Th1), activated CD8 T cells and natural killer cells that in turn, aggravates macrophages to destroy intracellular organisms 100 . IF-γ prompts the differentiation of CD4+ T cells into Th1 cells that further produces IF-γ again 101 . Moreover, IL-12 provokes TNF-α production that possesses a pivotal role in scenario of immune regulation through monitoring lymphocyte proliferation, survival and apoptosis via paracrine/autocrine signals [102][103][104] . The later function of TNF-α is concerned with maintenance of immune homeostasis and self-tolerance 105,106 . The crosstalk between innate and adaptive immune system that is arbitrated by IL-12 and IF-γ, contributes a substantial role in infectious agent control. The present study revealed signi cant promotion in the levels of IF-γ, IL-12 and TNF-α in Sa30 and Sa50 groups that seemed to be dose dependent. Present data were in harmony with Gholamnezhad et al. 107 , Aljabre et al. 108 , Gholamnezhad et al. 109 and Titiek et al. 110 . These results explained the cell mediated immunostimulatory effect of Nigella sativa where IL-12 enhanced IF-γ that acted in autocrine and paracrine manner to increase CD8 cells activity. Both γ-IF and TNF-α could stimulate macrophages activity to eliminate infectious agent 111,112 .
Nigella sativa seeds treated rats showed a signi cant increase in the CD8 immunoreactivity in splenocytes. Salem et al. 113 had shown similar results where they found that addition of thymoquinone stimulated CD8 and markedly increased IF-γ production. Our ndings indicate that the expression of CD8 may be linked to the activation state of the T cell when injected with PHA. CD8 cells are crucial constituent of the cellular immune response where their frequency is increased during conditions where the immune system is activated by infection 114 or autoimmune disease or after transplantation 115 .
CD8 + T cells produce cytokines like IF-γ and TNF-α. The latter is a proin ammatory cytokine that initiates apoptotic gesturing and inhibits viral replication as well as gene expression 116 . Also, CD8+ T cells could directly attack and induce cytolysis to the infected targets 117,118 .
Nigella sativa seeds treated rats showed a signi cant decrease in the expression of caspase-3 splenic immunoreactivity in a dose dependent pattern. on the same trend, Salem 12 found that spleen of treated with Nigella sativa associated with decreasing rates of apoptosis. Numerous lines of evidence had indicated that Nigella sativa seeds are able to modulate pro-in ammatory cytokines as multiple cell signaling molecules 27 , apoptotic proteins 119 and antioxidants 12 . Reduction of caspase 3 protein expression in Nigella sativa seeds administered groups indicated their anti-apoptotic potential that could be attributed to the antioxidant ingredients in them. This was manifested by the elevated TAC and catalase activities in Nigella sativa treated groups. Moreover, the reduced splenic caspase 3 denoted active dynamic status in such organ toward PHA injection that was augmented by the increased splenic CD8 expression as well as serum IF-γ, IL-12 and TNF-α. This scenario was re ected on the increased edema and in ltrations of in ammatory cells in the PHA stimulated foot pads with increment in Intumesce Index. Moreover, white pulp, periarterial lymphoid sheath and the germinal center were signi cantly increased in Sa30 and Sa50 groups as a re ection of active splenic performance with lower apoptosis.
Histological assessment of lymph nodes is crucial to comprehend the immunologic effects of chemicals 120 . PHA stimulates a characteristic pattern of reaction in lymph nodes. The control group injected with PHA has shown lymphoid follicle size reduction with medullary lymphoid hyperplasia, severe degeneration with mild necrotic area and lymphatic sinus ectasia. The appeared histopathological alteration of lymph node is due to its in ammatory response to the PHA injection which trigger an immunologic response according to O'Dowd et al. 121 .The groups Sa30 and Sa50 lymph nodes showed medullary lymphoid hyperplasia and lymphoid follicle size reduction with less notable distortion in architecture seen in the control sections. Hyperplasia occurs in lymph node is an acute immune response to antigens 122 . Therefore, PHA acts as antigen stimulator. Sa50 lymph nodes showed parafollicular hyperplasia where the follicle pushed to the periphery of the node beneath of the capsule that may be a response to the reactive hyperplasia of the lymph node 123 . Nigella sativa immunologic response is explicit dose dependent trend where the higher dose showed the least damage and more lymphocyte to the popliteal lymph nodes tissues of the experimental rats.
With consideration to all the previous data, it seems that Nigella sativa possesses a favorable cell mediated immune response toward PHA injection through its antioxidant active ingredients that positively in uenced weight gain and FCR. The effect of Nigella sativa seeds was represented by increased IL-12 that promoted CD8 IF-γ production and TNF-α that could effectively face infectious agent.
This active immunomodulatory cell mediated immune response was accompanied with active splenic state of increased CD8 expression and reduced caspase 3 as apoptotic marker. These were manifested by increased lymphoid histomorphometry in spleen with increased chemotaxis and in ammatory reaction at the site of PHA injection. Also, popliteal lymph node of PHA injected leg showed lymphoid hyperplasia.

Conclusion
The existing study established that Nigella sativa seeds are useful to be introduced in foods where the positive effect of these seeds is to boost the overall growth output parameters, FCR and immunological response. Later effects are due to their antioxidant constituents that promoted cell mediated cytokines production, splenic CD8 and reduced splenic caspase-3 expression. From this study Nigella sativa seeds could be useful as a dietary supplement that has a positive modulatory effect to cell mediated immunoresponse to disease. Thus, Nigella sativa dietary supplementation could be bene cial in viral or bacterial infections where cellular mediated immune response plays a pivotal role. Con icts of interest: The authors declare no competing interests.