Synergetic effect of Probiotic and Nigella sativa to control enteric and respiratory infections in an animal model.
The medicinal plants have been used for curing diseases and are promising for the treatment of various diseases such as diabetes, cancer, inflammation, and rheumatoid. [1-3] Nigella sativa (Family Ranunculaceae; NS) is a miracle herb as many researchers discovered its broad spectrum of pharmacological potential. NS is commonly identified as the black seed.  NS has been extensively studied for its biological and beneficial effect and shown to have a wide spectrum of activities as diuretic, antidiabetic, antihypertensive, anticancer and immunomodulatory, analgesic, and antimicrobial. [2,3] The antibacterial effect of NS was studied in disc diffusion method. A clear inhibition zone of growth of Staphylococcus aureus was observed, this inhibition may be attributed to the main active ingredients of NS.  The most valuable extracts were the crude alkaloid and water extracts of NS against Gram-negative bacterial isolates were affected more than the Gram-positive ones.  The antibacterial spectrum of NS against clinical isolates of methicillin-resistant S. aureus (MRSA) was investigated. All examined strains of MRSA were sensitive to the ethanolic extract of NS at a concentration of 4 mg/disc with an MIC range of 0.2-0.5 mg/mL.  The immunomodulatory properties of NS on splenocytes proliferation, natural killer cells, and macrophages activity on BLAB/c were investigated. The results clarified that the aqueous extract of NS significantly enhances splenocyte proliferation in a dose-dependent manner. Moreover, it favors the Th-2 versus Th-1cytokines as the secretion of interleukin-6 (IL-6), tumor necrosis factor-a, and nitric oxide, known pro-inflammatory mediators, is modulated by NS. 
Gum arabic (GA) is considered as an edible, dehydrated, sticky exudates excreted from the stems and branches of Acacia Senegal and Acacia seyal.  GA is prebiotic that enhances the growth of intestinal flora,  it is similar to breast milk and inulin in its importance. [10,11] It plays an important role in protection against pathogenic microorganisms, growth, and homeostasis of immune cells, digestion of polysaccharides that is indigestible by human enzymes and fat metabolism.  Microbiological studies showed that GA has antibacterial activities that attributed to second metabolites in its contents as tannins, carbohydrates, phenols, flavonoids, and glycosides. [13,14]
This study was designed to investigate the ejects of ethanolic extract of NS and GA (natural powder) as natural probiotics on the antibacterial and immune-modulator factors on rats after infection by either Gram-negative or Gram-positive bacteria. The gene expression of immune-modulator cytokines was examined after challenge in rats and possible protection by NS and GA were outlined compared to Lactobacillus (LA) species bacteria.
MATERIALS AND METHODS
MRSA and Escherichia coli were kindly provided from the (Animal Reproduction Research Institute, Department of Udder and Neonates, Egypt). S. aureus was grown in nutrient broth for 24 h. The culture was spin at 15,000 x g for 20 min, and the pellet was washed using sterile phosphate buffer saline (PBS). The viable bacterial count was adjusted to approximately 2 x [10.sup.9] colony forming units (CFU)/mL in PBS.  E. coli was grown in broth, the suspension was spin at 15,000 x g for 20 min, the supernatant was discarded, and then the bacteria were diluted in sterile saline. The viable bacterial count was adjusted to approximately 5 x [10.sup.10] CFU/mL in PBS. 
Preparation of NS and GA for Administration
NS seeds were purchased from a local herb store at Taif area, Saudi Arabia. The plant was identified by botanists in the herbarium of Taif University, and the specimen number of the plant is 293-0303-1. The hydro-ethanolic extract was prepared using a maceration method.  GA powder was a generous gift from Dar Savanna Ltd., Khartoum, Sudan (www.ssgums.com). It is a 100% natural extract powder produced mechanically from the wildly 85 grown A. Senegal tree with a particle size <210 [micro]m. GA (10% wt/wt) was added to the drinking water, which has previously been shown to be effective. [18,19] Lacteol Fort probiotic composed of LA corresponding to Lactobacillus delbrueckii and Lactobacillus fermentum 10 billion (one sachet of lacteal fort added to 500 ml of water) daily in a free intake during all period of the experiment. 
Animals and Experimental Design
Male Wistar rats (66 rats), aged 3 months and weighing 200-250 g, were purchased from the King Fahd Institute for Scientific Research, King AbdulAziz University, Jeddah, Saudi Arabia). Rats were subjected to 12 h/12 h daylight with free access to food and water. The rats were divided into 11 groups (6 rats per group). The control group was fed normal diet and water, E. coli group was injected intraperitoneally (IP) with virulent strain of E. coli in a dose of 2 x [10.sup.10] CFU/ml per rat, and S. aureus group was injected IP with virulent strain of S. aureus in a dose of 2 x [10.sup.9] CFU/mL per rat. The remaining 8 groups were as following: E. coli plus Luria broth (LB) (one sachet of lacteal fort added to 500 ml of water daily) group; S. aureus plus LB; E. coli plus NS (150 mg/kg bw daily); E. coli plus GA (10% wt/wt daily), S. aureus group plus NS; S. aureus group plus GA; E. coli group plus Ns plus GA; S. aureus plus NS plus GA. All animals were kept under observation for 1 week after infection. Animals received NS and GA for 2 weeks before infection by E. coli and S. aureus then continued for 1 week later. At the end of the experimental schedule, the rats were overnight fasted, and inhaled diethyl ether then decapitated. Organs were used for pathogens isolation under aseptic conditions. Kidney, lung, spleen, and intestine tissues were taken a weighted and checked for bacterial isolation and count (CFU/gram tissues for S. aureus and E. coli), and for RNA extraction for some cytokines.
Total Bacterial Counts
The collected organs were weighted under aseptic condition then minced into small pieces and incubated in broth at 37[degrees]C for 24 h. Serial dilution was performed from the broth; each sample has been diluted in six tubes (10x dilution), then cultured on specific media for the pathogen.
RNA Extraction, cDNA Synthesis, and Cytokines Expression in Tissues
Total RNA was extracted from the tissue as previously discussed.  2 [micro]g of total RNA and 0.5 ng oligoDT primer (Qiagen Valencia, CA, USA) were used for cDNA synthesis. For semi-quantitative gene expression and polymerase chain reaction (PCR) expression analysis, primers for the examined genes (Table 1) were designed using Oligo-4 computer program (Version 7.0; Molecular Biology Insights, Cascade, CO, USA) according to the nucleotide sequences published in GenBank (http://www.ncbi.nlm.nih.gov/ genbank/; Table 1) and were synthesized by Macrogen Korea (Seoul, South Korea). PCR was conducted in a final volume of 25 [micro]l consisting of 1 [micro]l cDNA, 1 [micro]l of 10 [micro]M of each primer (forward and reverse), and 12.5 [micro]l PCR master mix (Promega Corporation, Madison, WI, USA), the volume was adjusted to 25 [micro]l using sterilized, deionized water. PCR was carried out using Bio-Rad T100TM Thermal cycle machine (Bio-Rad Laboratories, Inc.) with the cycle sequence at 94[degrees]C for 5 min one cycle, followed by 26 cycles, each consists of denaturation at 94[degrees]C for 1 min, annealing at the specific temperature corresponding to each primer (Table 1) and extension at 72[degrees]C for 1 min with an additional final extension at 72[degrees]C for 7 min. The expression of glyceraldehyde-3-phosphate dehydrogenase mRNA was used as a reference. PCR products were run in 1.5% agarose (Bio Basic, Markham, ON, Canada) gel stained with ethidium bromide in Tris-Borate-EDTA buffer and visualized under UV light gel using an In Genius 3.0 gel documentation system (Syngene, Frederick, MD, USA). Band intensities from the various rats from each group were quantified densitometrically using Image Software Version 1.47 (http://www.imagej. en.softonic.com/).
Data are represented as means [+ or -] standard error of means for 3 different independent experiments. Data were analyzed using analysis of variance (ANOVA) (one-way ANOVA) by SPSS software version 11.5 for Windows (SPSS, IBM, Chicago, IL, USA). Values with P < 0.05 are considered statistically significant.
Effect of NS and GA on E. coli and S. aureus Counts in Wistar Rats
The total bacterial count for E. coli showed that NS strongly and significantly (P < 0.05) inhibited bacterial growth. In contrast, without explanation GA increased alone or in combination E. coli growth. Of note, LB increased also the pathogens growth (Table 2). For S. aureus growth, the total bacterial count was decreased significantly (P < 0.01) when NS administered 2 weeks before S. aureus infection. In parallel, same results with less significance (P < 0.05) for GA were obtained. When both co-administered together, additive inhibition for the bacterial count (P < 0.01) was reported as seen in Table 3. LB induced more inhibition for S. aureus count.
Protective Effect of NS and GA on Superoxide Dismutase (SOD) Activity on Ileum and Spleen after Infection by E. coli and S. aureus in Wistar Rats
Induction of pathogenicity by E. coli in ileum and S. aureus in spleen downregulated mRNA expression of SOD as seen in Figure 1a. Administration of NS alone normalized and upregulated SOD mRNA expression (Figure 1a). GA alone did not normalize the downregulation in SOD expression and combination with NS did not induce any effect. The normalized effect seen in Figure 1a is due to NS action. Administration of LA to E. coli infected rats upregulated SOD expression compared to E. coli administered rats. In Figure 1b, SOD mRNA expression was examined after infection of rats with S. aureus. The same pattern of SOD mRNA expression reported for E. coli in ileum is the same for SOD in S. aureus in spleen (Figure 1b) as NS was stimulatory while GA is inhibitory when both co-administered the effect is due to NS action.
Protective Effect of NS and GA on Glutathione (GHS) Peroxidase (GPx) Activity on Ileum and Spleen after Infection by E. coli and S. aureus in Wistar Rats
In ileum, infection of rats by E. coli induced downregulation in mRNA expression of GPX (Figure 2a). Administration of NS to infected rats normalized the downregulation in GPX mRNA while GA did not show any alteration in GPX expression and remained downregulated. When NS co-administered with GA, NS normalized the inhibitory effect of both E. coli and GA. As seen LB normalized GPX mRNA expression that is downregulated in E. coli. Staph aureus induced partial (15%) downregulation in GPX expression (Figure 2b). NS administration normalized and upregulated mRNA expression of examined GPX (Figure 2b). GA induced additive downregulation in GPX mRNA expression (Figure 2b). Co-administration of NS with GA did not show any additive or suppressive effect to their effect alone on GPX mRNA expression. However, the prominent effect is the suppressive action of GA (Figure 2b) on the stimulatory effect of NS.
Effect of NS and GA on IL-4 and Interferon-gamma (INF-y) on Ileum after Infection by E. coli in Wistar Rats
As seen in Figure 3, E. coli significantly upregulated IL-4 expression; this suppression was inhibited by NS as it induced downregulation in IL-4 mRNA. GA induced more inhibition for E. coli suppressive effect on IL-4 mRNA (P < 0.05). Co-administration of NS plus GA showed inhibition of IL-4 mRNA (Figure 3a). LB showed upregulation of IL-4 expression. INF expression in Figure 3b showed upregulation by E. coli and administration of NS to E. coli infected rats showed additive stimulatory effect (P < 0.05). GA showed inhibition for upregulated expression induced by E. coli. Moreover, GA inhibited the additive stimulatory effect of both E. coli and NS (Figure 3b).
Effect of NS and GA on IL-12 Spleen after Infection by S. aureus in Wistar Rats
Finally, we examined the expression of IL-12 in the spleen of rats after S. aureus challenge. As seen in Figure 4, S. aureus infection downregulated mRNA expression of IL-12. In contrast, NS administration into infected rats normalized and upregulated IL-12 expression in examined groups. GA induced downregulation of IL-12 mRNA and co-administration of GA with NS inhibited NS induced upregulation of IL-12 mRNA. IL-12 expression was downregulated in spleen after administration of LB to S. aureus infected rats.
GA has been widely used as an emulsifier and stabilizer agent basically in food industry.  In Falk medicine, GA is used for management of inflammatory bowel diseases. Some recent reports explained that GA has antioxidant and nephron protected property.  NS has been employed for many years as potential medicinal properties; the main constituent of seeds includes thymoquinone, thymol, dithymoquinone, carvacrol, nigellicine, and alpha-hedrin.  Immunomodulatory and therapeutic properties of NS as antibacterial, antifungal, and cytotic effect have been reported.
In this study, the antioxidant effect of GA and NS in Wistar rats infected with E. coli and S. aureus either in ilium or spleen tissue. E. coli and S. aureus infection downregulated the expression of SOD and GPX. NS administration alone normalized and upregulated mRNA of SOD and GPX compared to control. While GA alone did not normalize the downregulation in case of E. coli infection and somewhat more than S. aureus but less than normal and in combination with NS induce changes related to the co-administrated effect of NS. The combination of NS and GA in E. coli infected rats gives more expression than S. aureus in GPX. In contrast, LB upregulated SOD mRNA. These results agree as reported that GA in a dose of 2.5%, 5%, and 10% in drinking water did not significantly alter either the concentration of free radicals scavenger, reduced GPX, GHS, ascorbic acid, and SOD and there is no evidence that GA has strongly antioxidant effects.  The antioxidant potential of NS which indicating a higher free radical scavenger activities. 
Many researchers reported the immunomodulatory effect of NS on the immune system and the immunoprotective role against tumors.  In our study, cytokines expression were ameliorated by NS administration and are in parallel with other reports  as the aqueous extract of NS favors the secretion of pro-inflammatory cytokines and increase the secretion of INF-y as protective effect.  The co-administration of NS and GA reduced each other that may be attributed to the interaction of the two plants ingredients. The two plants (GA and NS) increased INF-[gamma] more than IL-4. It has been shown that NS favors the secretion of Th-2 versus Th-1. On the same time, cytokines profile of rats infected with S. aureus regulated the secretion of IL-12. IL-12 play a role in Th-1 response, especially on macrophages function and regulate Th-1 response by regulating the secretion of IL-4, 10, and IL-13. 
The antibacterial effect of NS components (thymoquinone, thymol, dithymoquinone) found to possess an inhibitory response against both Gram-positive and Gram-negative bacteria.  In the current study, the higher antibacterial effect (lowest bacterial growth) reported was found in the NS-treated group (p < 0.01) followed by the LA-treated group, followed by GA group then GA plus NS combination. On the other hand, E. coli infection showed lower bacterial count in NS-treated group compare with the non-treated group, followed by GA group, GA plus NS group, and LA group. It has been confirmed that GA suppresses the macrophages activities of rats to produce superoxide anion and capable for almost blocking completely macrophages function, especially in chronic liver diseases. 
From the data obtained in this study, we concluded that GA has weak antioxidant activities as it downregulates the two genes expressed (SOD and GPX). Moreover, GA increases both bacterial counts of each Gram-positive and Gram-negative bacterium in the spleen, lung tissues; on the contrary, NS very strong antioxidant, reduce bacterial count, and modulate the immune response through activation of Th-1 cytokines. The combination between NS and GA is not recommended and GA not suitable prebiotic in the case of bacterial infection.
This study was supported by a grant in aid of the Deans of Scientific Research Affairs, Taif University, Saudi Arabia (# 4344-1436-1).
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Source of Support: Nil, Conflict of Interest: None declared.
Salama Moustafa Abd-El-Hafez (1,2), Alaa Bassuny Ismael (3,4), Omaima Nasir (5), Mohamed Soliman (6), Essam Hassan Mohamed (7), Ibrahim Khalid Kafaween (1)
(1) Department of Medical Microbiology, Faculty of Applied Medical Sciences, Taif University, Turrabah, Saudi Arabia, (2) Department of Immunobiology and Immunopharmacology, Animal Reproduction Research Institute, Giza, Egypt, (3) Department of Medical Biotechnology, Faculty of Applied Medical Sciences, Taif University, Turrabah, Saudi Arabia, (4) Department of Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44519, Egypt, (5) Department of Medical Laboratories, Faculty of Applied Medical Sciences, TaifUniversity, Turrabah, Saudi Arabia, (6) Department of Biochemistry, Faculty of Veterinary Medicine, Benha University, Benha 13736, Egypt, (7) Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt
Correspondence to: Salama Moustafa Abd-El-Haf, E-mail: email@example.com
Received: April 17, 2016; Accepted: May 03, 2016
Caption: Figure 1: (a and b) Protective effect Nigella sativa and gum arabic (GA) on superoxide dismutase (SOD) mRNA on ileum and spleen after infection by Escherichia coli and Staphylococcus aureus in Wistar rats. RNA was extracted and reverse transcribed (2 mcg) and reverse transcription polymerase chain reaction analysis was carried out for SOD expression as described in materials and methods. Densitometric analysis (down columns) was carried for 3 different experiments. Data are means [+ or -] standard error of the mean for 3 independent experiments. Values are statistically significant at * <0.05 versus control, (#) P<0.05 versus either E. coli or S. aureus and ($) P<0.05 versus GA plus either E. coli or S. aureus
Caption: Figure 2: (a and b) Protective effect of Nigella sativa and gum Arabic (GA) on glutathione peroxidase (GPX) mRNA on ileum and spleen after infection by either Escherichia coli or Staphylococcus aureus in Wistar rats. RNA was extracted and reverse transcribed (2 mcg) and reverse transcription polymerase chain reaction analysis was carried out for GPX expression as described in materials and methods. Densitometric analysis (down columns) was carried for 3 different experiments. Data are means [+ or -] standard error of the mean for 3 independent experiments. Values are statistically significant at * P<0.05 versus control, (#) P<0.05 versus either E. coli or S. aureus and ($) P<0.05 versus GA plus either E. coli or S. aureus
Caption: Figure 3: (a and b) Effect of Nigella sativa and gum Arabic (GA) on interleukin-4 (IL-4) and interferon-gamma (IFN-[gamma]) on ileum after infection by Escherichia coli in Wistar rats. RNA was extracted and reverse transcribed (2 mcg) and reverse transcription polymerase chain reaction analysis was carried out for IL-4 and IFN-[gamma] expression as described in materials and methods. Densitometric analysis (down columns) was carried for 3 different experiments. Data are means [+ or -] standard error of the mean for 3 independent experiments. Values are statistically significant at * P<0.05 versus control, (#) P<0.05 versus E. coli and ($) P< 0.05 versus GA plus E. coli
Caption: Figure 4: Effect of Nigella sativa and gum Arabia (GA) on interleukin-12 (IL-12) on spleen after infection by S. aureus in Wistar rats. RNA was extracted and reverse transcribed (2 mcg) and reverse transcription polymerase chain reaction analysis was carried out for IL-12 expression as described in materials and methods. Densitometric analysis (down columns) was carried for 3 different experiments. Data are means [+ or -] standard error of the mean for 3 independent experiments. Values are statistically significant at * P<0.05 versus control, (#) P<0.05 versus S. aureus and ($) P<0.05 versus GA plus S. aureus
Table 1: Primer sequence of examined genes and PCR conditions Gene Product Annealing Direction Sequence size (bp) ([degrees]C) (5'-3') SOD 410 55 Forward AGGATTAACTGAA GGCGAGCAT Reverse TCTACAGTTAGCA GGCCAGCAG GPX 407 57 Forward AAGGTGCTGCTCA TTGAGAATG Reverse CGTCTGGACCTAC CAGGAACn IL-4 327 57 Forward AGGTCAACACCAC GGAGAAC Reverse AGGACATGGAAGT GCAGGAC IFN- 321 58 Forward AGGAAAGAGCC [gamma] TCCTCTTGG Reverse TCTACCCCAGAAT CAGCACC G3PDH 309 52 Forward AGATCCACAACG GATACATT Reverse TCCCTCAAGATTG TCAGCAA SOD: Superoxide dismutase, PCR: Polymerase chain reaction, GPX: Glutathione peroxidase, IL-4: Interleukin-4, IFN-[gamma]: Interferon-gamma G3PDH: Glyceraldehyde-3-phosphate dehydrogenase Table 2: Effect of different treatments on E. coli count in organs (kidney and spleen) of Wistar rats after E. coli challenge Group Control E. coli E. coli+NS Means 00 76.33x[10.sup.10] 45.83x[10.sup.10] SEM 00 4.19x[10.sup.10] 3.5x[10.sup.10] P- value P<0.05 Group E. coli+gum E. coi;+(NS+gum) Means 170.17x[10.sup.10] 619.0x[10.sup.10] SEM 19.8x[10.sup.10] 88.8x[10.sup.10] P- value P<0.05 P<0.05 Group E. coli+LA Means 635.83x[10.sup.10] SEM 143.7x[10.sup.10] P- value P<0.05 Values are means (M)[+ or -] standard error (SE) for 6 different rats. SEM: Standard error of the mean, E. coli: Escherichia coli, NS: Nigella sativa, LA: Lactobacillus Table 3: Effect of different treatments on S. aureus count in organs (lung and spleen) of Wistar rats after S. aureus challenge Group Control S. aureus S. aureus+NS Means 00 412.7x[10.sup.10] 54.25x[10.sup.10] SEM 00 168.5x[10.sup.10] 22.14x[10.sup.10] P- value P<0.01 Group S. aureus+gum S. aureus+(NS+gum) Means 331.3x[10.sup.10] 115.65x[10.sup.10] SEM 135.25x[10.sup.10] 47.2x[10.sup.10] P- value P<0.05 P<0.01 Group S. aureus+LA Means 29.38x[10.sup.10] SEM 11.99x[10.sup.10] P- value P<0.05 Values are means (M)[+ or -] standard deviation (SD) for 6 different rats. SEM: Standard error of the mean, NS: Nigella sativa, S. aureus: Staphylococcus aureus, LA: Lactobacillus
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|Title Annotation:||RESEARCH ARTICLE|
|Author:||Abd-El-Hafez, Salama Moustafa; Ismael, Alaa Bassuny; Nasir, Omaima; Soliman, Mohamed; Mohamed, Essam|
|Publication:||National Journal of Physiology, Pharmacy and Pharmacology|
|Date:||Sep 1, 2016|
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