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Original Paper

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Camel Milk Attenuates Rheumatoid Arthritis Via Inhibition of Mitogen Activated Protein Kinase Pathway

Arab H.H.a,b · Salama S.A.a,c · Abdelghany T.M.d · Omar H.A.e,f · Arafa E.-S.A.f,g · Alrobaian M.A.h · Maghrabi I.A.i

Author affiliations

aBiochemistry Division and GTMR Unit, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Taif University, Taif, Saudi Arabia
bDepartment of Biochemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
cDepartment of Biochemistry, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt
dDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt
eSharjah Institute for Medical Research and College of Pharmacy, University of Sharjah, Sharjah, United Arab Emirates
fDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt
gDepartment of Pharmacology and Toxicology, College of Pharmacy and Health Sciences, Ajman University, Ajman, United Arab Emirates
hDepartment of Pharmaceutics, Faculty of Pharmacy, Taif University, Taif, Saudi Arabia
iDepartment of Clinical Pharmacy, Faculty of Pharmacy, Taif University, Taif, Saudi Arabia

Corresponding Author

Hany H. Arab

Biochemistry Division, Department of Pharmacology and Toxicology, Faculty of Pharmacy,

Taif University, Al-Haweiah, Taif, 21974, (KSA)

Tel. +966 582704865, E-Mail hany.arab@pharma.cu.edu.eg, h.arab@tu.edu.sa

Related Articles for ""

Cell Physiol Biochem 2017;43:540–552

Abstract

Background/Aims: Camel milk (CM) has shown beneficial anti-inflammatory actions in several experimental and clinical settings. So far, its effect on rheumatoid arthritis (RA) has not been previously explored. Thus, the current work aimed to evaluate the effects of CM in Adjuvant-induced arthritis and air pouch edema models in rats, which mimic human RA. Methods: CM was administered at 10 ml/kg orally for 3 weeks starting on the day of Freund’s adjuvant paw inoculation. The levels of TNF-α and IL-10 were measured by ELISA while the protein expression of NF-κBp65, COX-2 and iNOS was detected by immunohistochemistry. The expression of MAPK target proteins was assessed by Western blotting. Results: CM attenuated paw edema, arthritic index and gait score along with dorsal pouch inflammatory cell migration. CM lowered the TNF-α and augmented the anti-inflammatory IL-10 levels in sera and exudates of arthritic rats. It also attenuated the expression of activated NF-κBp65, COX-2 and iNOS in the lining of the dorsal pouch. Notably, CM inhibited the MAPK pathway signal transduction via lowering the phosphorylation of p38 MAPK, ERK1/2 and JNK1/2 in rat hind paws. Additionally, CM administration lowered the lipid peroxide and nitric oxide levels and boosted glutathione and total anti-oxidant capacity in sera and exudates of animals. Conclusion: The observed CM downregulation of the arthritic process may support the interest of CM consumption as an adjunct approach for the management of RA.

© 2017 The Author(s). Published by S. Karger AG, Basel


Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune disease associated with robust synovial inflammation that culminates in destructive events in cartilage and bone along with bone outgrowths that restrict the mobility of joints [1]. Inflammation has been regarded as a cardinal player in the pathogenesis of arthritis. Within the inflamed joint compartment, a massive recruitment of immune cells, such as CD4+ T cells, B cells, macrophages and neutrophils has been detected. Mounting evidence revealed the role of proinflammatory cytokines such as TNF-α and IL-6 in creating self-perpetuating joint inflammation and articular destruction [2-4]. In contrast, low levels of the anti-inflammatory IL-10 have been detected in patients with RA [5]. Proinflammatory cytokines have been reported to activate several transcription factors that are intimately linked to the expression of inflammatory and catabolic signals. A prominent member of these transcription factors is the nuclear factor kappa B (NF-κB) which controls the expression of proinflammatory cytokines, including TNF-α itself, cyclo-oxygenase-2 (COX-2) linked with PGE2 synthesis and inducible nitric oxide synthase (iNOS) associated with overshooting of nitric oxide (NO) in RA patients [6, 7]. Additionally, proinflammatory cytokines can activate several inflammatory transduction pathways, including mitogen activated protein kinase (MAPK). The MAPK family includes p38MAPK, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), which have been reported to be activated in the synovium of RA patients [8, 9].

The infiltration of immune cells to joints has been associated with marked production of reactive oxygen species (ROS) such as superoxide, hydrogen peroxide and hydroxyl radicals [10]. These mediators have been reported to mediate the progression of joint destruction in RA [7]. ROS have been implicated in the destructive oxidation of membrane lipids creating lipid peroxides, a hallmark of oxidative stress in RA. Additionally, increased levels of reactive nitrogen species (RNS) such as NO have been widely detected in serum and synovial fluid of RA patients [7, 10] .

The mainstay of therapy for RA includes glucocorticoids, biological therapy and disease modifying anti-rheumatic drugs (DMARDs) that were proven effective in the management of this ailment [11]. Yet, the adverse effects and the high cost of these agents during long therapy are considered major drawbacks. Thus, new therapeutic approaches, in particular with fewer side effects, are under investigation. Camel milk (CM) might represent a potential candidate. CM has displayed distinctive anti-oxidant properties due to its high content of vitamins C and E along with zinc and selenium. The high levels of lactoferrin in CM can explain its marked anti-inflammatory and anti-oxidant properties [12]. Furthermore, increased amounts of IgA, IgM and nano antibodies have been characterized in CM [13]. At the experimental and clinical settings, CM has been reported to exhibit protective and alleviating properties against diabetes [14], diabetic nephropathy [15], alcohol-induced hepatic injury [16], steatohepatitis [17] and inflammatory bowel disease [18]. Furthermore, CM has shown favorable effects on the redox status and immune cells in diabetic mice [19]. Taken together, the marked anti-oxidant and anti-inflammatory features of CM encouraged us to investigate its efficacy in experimentally-induced RA.

Materials and Methods

Animals

Adult Wistar rats (200±20 g; King Fahd medical research center, Jeddah, KSA) were housed 4 per cage at constant temperature (25±2 °C), humidity (60±10%) and a 12/12 h light/dark cycle. Rats were provided with standard rat chow (15% protein, 3.5 % fat, 6.5% crude fibers plus vitamins/minerals mixture) and water ad libitum. Additionally, animals were acclimatized for 7 days before the start of experimental work. All efforts were made to maintain a clean environment for experiments. All procedures pertaining to the animal care and treatments were approved by the Research Ethical Committee of Taif University, Taif, Saudi Arabia and were strictly performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication No. 80-23, revised 1996).

Reagents and drugs

Freund Complete Adjuvant (FCA) was obtained from Difco laboratories, USA, whereas Diclofenac was from Sigma-Aldrich, USA. Other chemicals/solvents are of the highest analytical grade. Camel milk (CM) was obtained as a standardized CM product containing 3% fat and 6.3 % milk non-fat solids purchased from Al-Turath Al-Saudia Company, Jeddah, KSA. Notably, the marked anti-inflammatory and antioxidant features of CM are considerably attributed to lactoferrin, an iron binding glycoprotein, which represents about 0.5% of total CM proteins (220 mg/L) [20, 21].

Experimental design and treatment protocol.

To study the anti-arthritic effects of CM, two models of arthritis were investigated in the same rat; adjuvant-induced arthritis and subcutaneous (sc) air pouch edema. Adjuvant arthritis, an immune mediated immunopathy, mimics several features of RA [22] while the air pouch model closely approximates the human synovial cavity and its lining has marked resemblance to the synovium [23]. Moreover, the air pouch has the advantage of providing capacity for quantifying inflammatory mediators as well as exudative and cellular responses [24].

To instigate arthritis in rats, 0.25 mg of heat-killed Mycobacterium butyricum (FCA) was inoculated subcutaneously into the plantar surface of left hind paw (day 0) as previously described [25]. To induce dorsal pouch edema, 10 days later, the back of the animal was shaved, cleansed with 70% ethanol and 20 ml of sterile air was subcutaneously injected under thiopental anesthesia (50 mg/kg; ip). On the 16th day, the same dose of FCA was injected subcutaneously to the optimally responsive 6-day-old pouch [23]. At the end of the experimental period (day 21), animals were anaesthetized and pouch exudates and lining were harvested. Blood samples were obtained via retro-orbital plexus for whole blood and serum separation.

Forty rats were randomly divided into five groups (n=8). Group I (Control gp): normal rats, which received the vehicle only by oral route. Group II (Control + CM gp): normal rats that received Camel milk (CM; 10 ml/kg orally for 21 days). Group I and II also received sterile saline solution into the left hind paw on day zero. Group III (Arthritic gp): rats, which were rendered arthritic and received oral vehicle for 21 days. Group IV (Arthritic + CM): arthritic rats which received CM (10 ml/kg/day orally for 21 days). Group V (Arthritic + DF): arthritic rats which received the reference drug; diclofenac (5 mg/kg/day orally for 21 days). Diclofenac (in 0.5% carboxymethyl cellulose vehicle) and CM were administered by gavage daily starting two hours before injecting FCA on day 0 and were continued up to day 20.

The selected dose of CM is based on our preliminary experiments as well as previous studies reporting the efficacy of 10ml/kg oral dose in amelioration of experimental inflammatory bowel disease [18], ethanol-induced hepatic injury [16] and lipopolysaccharide-evoked lung injury [26]. Diclofenac was chosen as a reference anti-inflammatory agent due to its proven anti-arthritic effects in Adjuvant-induced arthritis [27, 28]. Its selected dose is in accordance with previous literature [27, 28]. The chosen regimen is in accordance with previous reports [23, 29].

Blood and air pouch exudates collection

At the end of the experimental period, rats were anesthetized and whole blood, serum and pouch exudates were collected as previously described [27].

Increase in paw diameter

Using micrometer screw gauge, the dorso-plantar paw diameter was determined on days 0, 4, 8, 14, 19 and 21 following FCA inoculation as previously described [25].

Arthritic index score

A semi-quantitative scoring system was used to express the severity of arthritis as previously reported [30]. A 0-4 score was used as described in Table 1.

Table 1.

Scoring of arthritis

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Gait scoring

The severity of pain resulting from arthritis was expressed using a 0-3 scoring system [31] as follows: 0, normal gait; 1, slight lameness; 2, lameness with weight bearing on toes only; 3, non-weight bearing animals.

Histopathology and immunohistochemical detection of NF-κBp65, COX-2 and iNOS

A routine histopathology protocol was adopted as previously described [32]. In brief, air pouch lining specimens were harvested and fixed in 10% formalin for 24 h. Five micron paraffin embedded sections were stained with hematoxylin and eosin (H&E) and the slides were inspected under the light microscope (Leica Microsystems, Germany) by an observer unaware of sample identity.

For immunohistochemistry, 3-micron sections were placed in xylene and rehydrated in graded ethanol and finally blocked in 5% bovine serum albumin/ Tris buffered saline (TBS) [33]. Primary antibody incubation was performed overnight at 4 °C using rabbit polyclonal antibodies against NF-κBp65, COX-2 or iNOS (Thermo Scientific, IL, USA). Slides were washed with TBS and incubated with secondary antibody. Slides were finally incubated in 0.02% diaminobenzidine (DAB) containing 0.01% H2O2 and counter stained with hematoxylin. Slides were examined under the light microscope (Leica Microsystems, Germany). Densitometric analysis of target protein expression was performed on the digital images using Image J software (Bethesda, USA).

Determination of TNF-α and IL-10

The serum and pouch exudate levels of tumor necrosis factor-α (TNF-α) and interleukin 10 (IL-10) were estimated as described [34] using specific ELISA kits purchased from R&D Systems, USA . All procedures were adopted as instructed by the manufacturer and absorbance was read at 450 nm (ELx800, BioTek, USA).

Western blotting

Soft tissues from rat hind paws were excised and immediately frozen. Then, they were homogenized in ice cold lysis buffer (2% SDS, 100mM Tris HCl) provided with a protease inhibitor and phosphatase inhibitor cocktail. Protein quantitation was performed using Biorad- DC protein assay kit (Biorad, USA). The Western blot protocol was performed as described [35, 36]. Aliquots containing equal protein amounts (30 μg) were separated by SDS-polyacrylamide gel electrophoresis (8% gel). The proteins were transferred to PVDF membranes which were probed overnight at 4°C with specific primary antibodies: rabbit monoclonal anti-phospho-p38MAPK (Thr180/Tyr182), rabbit monoclonal anti- p38MAPK, rabbit monoclonal anti-phospho-ERK1/2 (phospho-p44/42; Thr202/Tyr204)), rabbit monoclonal anti-ERK1/2 (p44/42), rabbit monoclonal anti-phospho-JNK1/2 (Thr 183/185), rabbit polyclonal anti-JNK1/2 and rabbit monoclonal anti- Lamin B (Cell Signaling Technology, USA). Membranes were washed 3 times in TBS/tween20, then, incubated with anti-rabbit IgG HRP-linked secondary antibody (Cell Signaling Technology, USA) for 1h. Following washing, membranes were incubated in Clarity Western ECL substrate (Biorad, CA, USA) and exposed to x-ray film. Equal protein loading was confirmed by probing with anti-Lamin B antibody. Densitometric analysis was performed by normalizing to total corresponding target protein and signals were quantified via Image J software (Bethesda, MD, USA).

Determination of lipid peroxides, NO, GSH and TAC

Serum and exudate lipid peroxides, expressed as malondialdehyde (MDA), were determined using thiobarbituric acid reaction according to Buege and Aust [37]. Nitric oxide (NO) levels were estimated based on the method of Miranda et al. [38] with the modification of replacing zinc sulfate instead of ethanol for protein precipitation [32]. The anti-oxidant defenses; reduced glutathione (GSH) and total anti-oxidant capacity (TAC) were determined according to the colorimetric method of Beutler et al. [39] and the specific TAC kit (Cayman, USA), respectively.

Statistical analysis

Statistical analysis of parametric data (expressed as mean ± SEM) was performed using one-way analysis of variance (ANOVA), followed by Tukey-Kramer test for multiple comparisons between groups. The statistical significance for non-parametric data (expressed as median) was carried out using Kruskal-Wallis analysis of variance followed by the rank-based Mann–Whitney U-test for group comparisons. For both cases, SPSS programme, version 17, was utilized.

Results

Camel milk mitigates Adjuvant-induced arthritis

The potential anti-arthritic actions of CM were studied with the aid of Adjuvant-induced arthritis model, a well recognized immune-mediated model of arthritis [22]. Injection of FCA into rats exhibited significant arthritis features as evidenced by a sharp increase in hind paw diameter on day 4 that continued till the end of the study (21st day; Fig. 1A). The animals also showed increased arthritic index scores revealing significant paw swelling and hyperemia involving the entire paw and ankle (Fig. 1B). Additionally, the rats suffered severe arthritis pain that caused gait anomalies expressed as increased scores of animal gait (Fig. 1C). Interestingly, CM mitigated the development of paw edema by 41%, 39%, 47% and 51% on the 8th, 14th, 19th and 21st day, respectively, as compared to the arthritic group. CM also attenuated the severity of arthritis as demonstrated by lowering of the arthritic index scores by 53.2% together with ameliorating the arthritic pain as seen by lowering of the gait scores by 60% inhibition, as compared to the arthritic group. These actions were similar to those afforded by the reference drug; diclofenac.

Fig. 1.

Camel milk ameliorates Adjuvant-induced arthritis in rats. (A) Effect of Camel milk administration for 21 days on paw edema in Adjuvant arthritic rats. (A) Effect of Camel milk administration for 21 days on paw edema in adjuvant arthritic rats. (B) Effect of Camel milk on Arthritic index score on day 21. (C) The impact on animal’s gait score on day 21. Parametric values are expressed as mean ± SEM (n= 6-8) for (A) while non-parametric data in (B) and (C) are expressed as medians (n=6-8). *Significant difference vs control gp at p<0.05, # Significant difference vs arthritic gp at p<0.05. AA; Adjuvant arthritis, CM; Camel milk, DF; diclofenac.

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Camel milk attenuate pouch leukocyte influx and histopathologic alterations

Histopathology of the pouch lining from arthritic group revealed diffuse inflammatory cell infiltration through the entire membrane thickness with some focal areas of suppuration (Fig. 2A). The migration of immune cells and the histopathologic alterations were mitigated by CM and DF (Fig. 2B,C). In this regard, lining from CM and DF groups displayed resolving areas of fibroblasts, whereas, that of DF was characterized with a dense granulation tissue. Additionally, injection of FCA into the sc pouch of pre-sensitized animals provoked a spike in infiltration of leukocytes as evidenced by detection of 21 ± 2.95 x 103 cells/mm3 in dorsal exudates (Fig. 2D). Administration of CM afforded 38.7% inhibition of leukocyte influx; an event that was similar to diclofenac that showed 46.7% inhibition.

Fig. 2.

Camel milk mitigates pouch lining histopathologic changes and exudate leukocyte infiltration in arthritic rats. (A-C) Representative photomicrographs of pouch lining harvested on day 21 and stained with H&E (magnification × 200). (a) Pouch lining of arthritic gp revealed massive recruitment of inflammatory cells (m). (B,C) Lining of Camel milk- and diclofenac-treated arthritic rats, respectively, displaying attenuated influx of leukocytes (m) and newly formed blood capillaries (hollow arrow) along with resolving regions of fibroblasts (f). (D) Leukocyte count in pouch exudate of arthritic rats. Values are expressed as mean ± SEM (n= 6-8). # Significant difference vs arthritic gp at p<0.05. AA; Adjuvant arthritis, CM; Camel milk, DF; diclofenac.

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Camel milk suppresses arthritis-associated inflammatory response

Injection of FCA resulted in a marked inflammatory response in arthritic rats as evidenced by a 5-fold increase in serum TNF-α together with lowering of the anti-inflammatory IL-10 levels (28.8%), as compared to control rats (Fig. 3). The levels of the aforementioned cytokines in the exudate of arthritic animals were 257.8 ± 28.9 pg/ml and 71.6 ± 8.8 pg/ml, respectively. Notably, CM resulted in a 29.6% and 39.7% inhibition of TNF-α in sera and exudates, respectively as compared to arthritic rats. Additionally, it normalized the serum levels of IL-10 and boosted the exudate levels by 197.4%.

Fig. 3.

Camel milk attenuates systemic and local inflammation in arthritic rats. (A) Levels of TNF-α in sera and exudates of arthritic rats. (B) Levels of IL-10 in sera and exudates of arthritic rats. Values are expressed as mean ± SEM (n= 6-8). *Significant difference vs control gp at p<0.05, # Significant difference vs arthritic gp at p<0.05. AA; Adjuvant arthritis, CM; Camel milk, DF; diclofenac.

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Camel milk downregulates the protein expression of NF-κBp65, COX-2 and iNOS in pouch lining

To further characterize the inflammatory milieu of arthritic rats, the protein expression of activated NF-κBp65, COX-2 and iNOS, proinflammatory signals were detected in the dorsal pouch lining. Arthritic rats exhibited marked expression of activated NF-κB p65, COX2 and iNOS as manifested by intense brown staining of the relevant target proteins (Fig. 4-4-6). Administration of CM attenuated the expression of the three inflammatory proteins as demonstrated by 69.7%, 41.1%, and 36.7% inhibition of the optical density for NF-κBp65, COX-2 and iNOS expression, respectively. These mitigation patterns were similar to DF.

Fig. 4.

Camel milk down-regulates NF-κBp65 protein expression in pouch lining of arthritic rats. (A-C) Representative images of pouch lining of arthritic rats (magnification × 100) showing immunohistochemical staining of NF-κBp65 expression. (D) Optical density of NF-κBp65 expression in pouch lining. Values are expressed as mean ± SEM (n= 4). # Significant difference vs arthritic gp at p<0.05. AA; Adjuvant arthritis, CM; Camel milk, DF; diclofenac.

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Fig. 5.

Camel milk down-regulates COX-2 protein expression in pouch lining of arthritic rats. (A-C) Representative images of pouch lining of arthritic rats (magnification × 100) showing immunohistochemical staining of COX-2 expression. (D) Optical density of COX-2 expression in pouch lining. Values are expressed as mean ± SEM (n=4). # Significant difference vs arthritic gp at p<0.05. AA; Adjuvant arthritis, CM; Camel milk, DF; diclofenac.

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Fig. 6.

Camel milk downregulates iNOS protein expression in pouch lining of arthritic rats. (A-C) Representative images of pouch lining of arthritic rats (magnification × 100) showing immunohistochemical staining of iNOS expression. (D) Optical density of iNOS expression in pouch lining. Values are expressed as mean ± SEM (n= 4). # Significant difference vs arthritic gp at p<0.05. AA; Adjuvant arthritis, CM; Camel milk, DF; diclofenac.

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Camel milk suppresses activation of MAPK transduction in hind paws of arthritic rats

MAPK pathway plays a pivotal role in the development of inflammation and tissue destruction in RA [8, 9]. Western blot analysis revealed activation of MAPK signaling in hind paws of arthritic rats as seen by increased phosphorylation of p38MAPK, ERK1/2 and JNK1/2 proteins, compared to control rats (Fig. 7A). CM inhibited the phosphorylation of the above MAPK proteins by 51.9%, 36.6% and 37.4%, respectively (Fig. 7B-D). These actions were similar to DF.

Fig. 7.

Camel milk suppresses the activation of mitogen activated protein kinase (MAPK) pathway in hind paw homogenates of arthritic rats. (A) Western blot analysis depicting the expression of phosphorylated and total protein forms of p38 MAPK, ERK1/2 and JNK1/2. Lamin B was utilized to prove equal loading of total protein lysate. (B) Relative expression of phospho-p38MAPK. (C) Relative expression of phospho-ERK1/2. (D) Relative expression of phospho-JNK1/2. The control value was set as 1.0. Data were extracted from 3 independent experiments and values were expressed as mean ± SEM.*Significant difference vs control gp at p<0.05, # Significant difference vs arthritic gp at p<0.05. AA; Adjuvant arthritis, CM; Camel milk, DF; diclofenac.

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Camel milk inhibits oxidative stress markers and augments the anti-oxidant defenses

To delineate the redox status of arthritic animals, the levels of lipid peroxides expressed as MDA and NO together with GSH and TAC were determined in the sera (or blood for GSH) and exudates of arthritic rats. Sera of arthritic rats showed a 3.9 and 4.7 fold rises in lipid peroxides and NO levels, respectively, with a concomitant decrease of blood GSH (60.3%) and TAC (71.9%), as compared to control group (Fig. 8). Likewise, the exudates of arthritic animals revealed marked oxidative stress where significant levels of MDA (7.48 ± 0.88 μmol/L ), NO (157.7 ± 17.38 μmol/L), GSH (483.45 ± 72.4 μmol/L) and TAC (1.28 ± 0.15 mmol/L) were detected. In both sera and exudate of animals, CM alleviated the levels of MDA and NO and boosted the anti-oxidant GSH and TAC levels; events that were similar to DF actions.

Fig. 8.

Camel milk alleviates systemic and local oxidative stress markers in arthritic rats. The Levels of lipid peroxides expressed as MDA (A), NO (B), GSH (C) and TAC (D) were determined in sera (blood for GSH) and exudates of arthritic rats. Values are expressed as mean ± SEM (n= 6-8). *Significant difference vs control gp at p<0.05, # Significant difference vs arthritic gp at p<0.05. AA; Adjuvant arthritis, CM; Camel milk, DF; diclofenac.

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Discussion

The current study reports, for the first time, the promising anti-arthritic actions of CM in immune-mediated Adjuvant arthritis and air pouch edema models that share several features of human RA. Adjuvant arthritis was used to delineate the systemic inflammatory responses triggered by FCA in animals whereas the air pouch edema model recapitulates the local events in RA synovial compartment [22, 23]. Our data revealed a robust inflammatory response in arthritic rats marked with elevated TNF-α levels and protein expression of activated NF-κB p65, COX-2 and iNOS proinflammatory signals. Additionally, the anti-inflammatory IL-10 levels were diminished. These findings are in concert with previous studies [5, 27]. Cytokines have been described for mediating the progression of arthritis [4, 40]. TNF-α triggers the release of ROS and drives osteoclastogenesis and the expression of matrix metalloproteinases culminating in articular degradation [2, 3]. The observed decline in IL-10 may indicate severe inflammatory response in arthritic rats since IL-10 serves as an anti-inflammatory signal which attenuates the release of proinflammatory cytokines from activated macrophages and neutrophils [5]. The pathogenesis of RA also involves activation of NF-κB pathway which plays a central role in inflammation, hyperalgesia and tissue injury in RA [6]. Driven by ROS and diverse cytokines, activated NF-κB is involved in the transcription of several proinflammatory genes including COX-2, iNOS and TNF-α under a pro-oxidant environment [41]. Following degradation of the inhibitory IκB subunit, active NF-κB heterodimer (p50 and p65 subunits) translocates to the nucleus to regulate target genes transcription. Thus, activated NF-κBp65 has been classically regarded as an activation index for NF-κB [40]. In RA patients, intense expression of COX-2, induced by proinflammatory cytokines, has been described in synovium lining cells, fibroblasts and macrophage with consequent overshooting of PGE2. The synovium-resident cells also display an enhanced iNOS expression which is driven by NF-κB and several cytokines including TNF-α [7]. Virtually, iNOS plays a crucial role in mediating irreversible bone erosion in the joints of RA patients probably via stimulation of bone osteoclast activity [7, 40].

The present findings revealed that CM exerted marked anti-inflammatory effects as evidenced by downregulation of TNF-α, COX-2, iNOS and their upstream effector NF-κB together with augmenting the IL-10 anti-inflammatory signal. These multipronged anti-inflammatory mechanisms are in accord with previous reports that described CM marked anti-inflammatory actions in several experimental models [16, 26, 42]. A prominent anti-inflammatory component of CM is the lactoferrin protein which has been characterized to abrogate TNF-α, IL-1 and IL-6 in mononuclear cells in vitro and in vivo in response to LPS stimulation. Lactoferrin has been also reported to enhance the levels of IL-10 in a rat model of colitis [12]. The observed downregulation of activated NF-κB p65 in pouch lining confirms the effective anti-inflammatory actions of CM [43]. Meanwhile, the observed downregulation of TNF-α, COX-2 and iNOS in arthritic rats is likely due to inhibition of their upstream NF-κB transcription factor [7, 40].

The current findings demonstrated that Adjuvant arthritis triggered the activation of p38, ERK 1/2 and JNK 1/2 MAPKs in hind paws of animals, events which are in harmony with previous literature [40]. In fact, the three MAPKs have been identified to play a key role in the regulation of cytokine, chemokine and prostaglandin synthesis. Dysregulated activation of MAPKs has been implicated in RA pathogenesis, so, MAPKs can serve as pivotal molecular targets for therapeutic intervention [8, 9]. Activation of p38 MAPK contributes to RA pathogenesis via driving the expression of adhesion proteins in inflammatory cells and through generation of TNF-α [9]. The ERK family is involved in macrophage generation of TNF-α and COX-2-dependent production of PGE2 in RA human fibroblast like synoviocytes [8]. A crucial role of JNK in RA pathogenesis is through matrix metalloproteinases(MMPs)-mediated cartilage degradation [8, 9]. Interestingly, administration of CM attenuated the activation of the three MAPKs. These data indicate that the suppression of MAPK activation is, at least partly, involved in CM alleviation of AA in rats. In fact, a previous study by Zhu et al. [26] described the efficacy of CM for downregulation of MAPK pathway in LPS-evoked respiratory distress in rats.

The current data affirmed the systemic and local oxidative stress marked with increased lipid peroxides and NO with concomitant decline of GSH and TAC anti-oxidant defenses. These findings are in line with previous reports [27, 29]. Virtually, aberrant generation of ROS and RNS by activated neutrophils and macrophages causes damage to joints likely via upregulation of MMPs and stimulation of osteoclast activity [7]. Lipid peroxides can trigger joint damage via releasing lysosomal enzyme into synovial cavity with resultant exacerbation of arthritis [7]. Within RA synovial compartment, excessive production of NO by iNOS-expressing macrophages and fibroblasts has been characterized [7]. NO reacts with superoxide anion to generate peroxynitrite, a robust oxidizing agent that instigates DNA fragmentation and lipid peroxidation [44]. The observed decline of GSH level is probably due to its consumption during detoxification of peroxides e.g., H2O2 via the action of glutathione peroxidase [27, 45].

The current data revealed that CM suppressed local and systemic oxidative stress and boosted the GSH and TAC anti-oxidant defenses. These findings support the premise that CM anti-oxidant actions are involved in the attenuation of immune-mediated arthritis. The powerful anti-oxidant activity of CM has been partly implicated in the mitigation of tissue damage in several experimental models [14-16, 18, 46]. Agents with marked anti-oxidant actions have been reported to curb the inflammatory events in experimental RA [5, 27, 40]. CM has displayed distinctive anti-oxidant properties owing to its high content of vitamins C and E along with zinc, selenium and other trace elements [47, 48]. The high content of lactoferrin in CM efficiently scavenges free iron in damaged tissues and abrogates the production of hydroxyl radicals [12, 49]. A likely explanation for the mitigation of the redox imbalance is secondary to the observed abrogation of leukocyte influx [27]. In the same context, a major factor for blunting oxidative stress is the observed inhibition of MAPK and NF-κB pathways [40]. The CM lowering of NO overshooting is possibly due to the observed downregulation of iNOS along with its upstream effector NF-κB [27, 40, 50].

Conclusion

In conclusion, the current study has described the anti-inflammatory actions of CM in Adjuvant arthritis and air pouch edema models in rats. These promising effects were mainly linked to the inhibition of MAPK pathway, which controls the synthesis of proinflammatory signals. Thus, the current data may show the interest of CM consumption as an adjunct approach for the management of RA. Since the marked antioxidant/anti-inflammatory actions of CM are essentially ascribed to lactoferrin [12, 49], future studies are warranted to correlate the amount of lactoferrin in CM to the anti-arthritic activity. Further studies are also needed to establish the efficacy of CM in the clinical setting.

Acknowledgements

The current work was supported by the Deanship of Scientific Research, Taif University, KSA (grant number: 1-435-3285 to Hany H. Arab). The authors would like to express their gratitude to Prof. Adel Kholoussy (Department of Pathology, Cairo University, Egypt) for his help in histopathology/ immunohistochemistry.

Disclosure Statement

The authors declare that there are no conflicts of interest.


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  5. Liu M, Mao W, Guan H, Li L, Wei B, Li P: Effects of taurochenodeoxycholic acid on adjuvant arthritis in rats. Int Immunopharmacol 2011; 11: 2150-2158.
  6. Makarov SS: NF-kappa B in rheumatoid arthritis: a pivotal regulator of inflammation, hyperplasia, and tissue destruction. Arthritis Res 2001; 3: 200-206.
  7. Phillips DC, Dias HK, Kitas GD, Griffiths HR: Aberrant reactive oxygen and nitrogen species generation in rheumatoid arthritis (RA): causes and consequences for immune function, cell survival, and therapeutic intervention. Antioxid Redox Signal 2010; 12: 743-785.
  8. Paunovic V, Harnett MM: Mitogen-activated protein kinases as therapeutic targets for rheumatoid arthritis. Drugs 2013; 73: 101-115.
  9. Ralph JA, Morand EF: MAPK phosphatases as novel targets for rheumatoid arthritis. Expert Opin Ther Targets 2008; 12: 795-808.
  10. Bogdan C: Nitric oxide and the immune response. Nat Immunol 2001; 2: 907-916.
  11. Kahlenberg JM, Fox DA: Advances in the medical treatment of rheumatoid arthritis. Hand Clin 2011; 27: 11-20.
  12. Legrand D, Elass E, Carpentier M, Mazurier J: Lactoferrin: a modulator of immune and inflammatory responses. Cell Mol Life Sci 2005; 62: 2549-2559.
  13. el Agamy EI, Ruppanner R, Ismail A, Champagne CP, Assaf R: Antibacterial and antiviral activity of camel milk protective proteins. J Dairy Res 1992; 59: 169-175.
  14. Agrawal RP, Jain S, Shah S, Chopra A, Agarwal V: Effect of camel milk on glycemic control and insulin requirement in patients with type 1 diabetes: 2-years randomized controlled trial. Eur J Clin Nutr 2011; 65: 1048-1052.
  15. Agrawal RP, Dogra R, Mohta N, Tiwari R, Singhal S, Sultania S: Beneficial effect of camel milk in diabetic nephropathy. Acta Biomed 2009; 80: 131-134.
    External Resources
  16. Darwish HA, Abd Raboh NR, Mahdy A: Camel’s milk alleviates alcohol-induced liver injury in rats. Food Chem Toxicol 2012; 50: 1377-1383.
  17. Korish AA, Arafah MM: Camel milk ameliorates steatohepatitis, insulin resistance and lipid peroxidation in experimental non-alcoholic fatty liver disease. BMC Complement Altern Med 2013; 13: 264.
  18. Arab HH, Salama SA, Eid AH, Omar HA, Arafa el SA, Maghrabi IA: Camel’s milk ameliorates TNBS-induced colitis in rats via downregulation of inflammatory cytokines and oxidative stress. Food Chem Toxicol 2014; 69: 294-302.
  19. Badr G, Sayed LH, Omar HE, Abd El-Rahim AM, Ahmed EA, Mahmoud MH: Camel Whey Protein Protects B and T Cells from Apoptosis by Suppressing Activating Transcription Factor-3 (ATF-3)-Mediated Oxidative Stress and Enhancing Phosphorylation of AKT and IkappaB-alpha in Type I Diabetic Mice. Cell Physiol Biochem 2017; 41: 41-54.
  20. El-Agamy E, Nawar M, Shamsia S, Awada S, Haenlein G: Are camel milk proteins convenient to the nutrition of cow milk allergic children? . Small Rumin Res 2009; 82: 1-6.
    External Resources
  21. Khalesi M, Salami M, Moslehishad M, James Winterburn, Moosavi-Movahedi AA: Biomolecular content of camel milk: A traditional superfood towards future healthcare industry. Trend Food Sci Technol 2017; 62: 49-58.
    External Resources
  22. Taurog JD, Argentieri DC, McReynolds RA: Adjuvant arthritis. Methods Enzymol 1988; 162: 339-355
  23. Tate G, Mandell BF, Laposata M, Ohliger D, Baker DG, Schumacher HR, Zurier RB: Suppression of acute and chronic inflammation by dietary gamma linolenic acid. J Rheumatol 1989; 16: 729-734.
    External Resources
  24. Yoshino S, Cromartie WJ, Schwab JH: Inflammation induced by bacterial cell wall fragments in the rat air pouch. Comparison of rat strains and measurement of arachidonic acid metabolites. Am J Pathol 1985; 121: 327-336.
    External Resources
  25. Newbould BB: Chemotherapy of Arthritis Induced in Rats by Mycobacterial Adjuvant. Br J Pharmacol Chemother 1963; 21: 127-136.
  26. Zhu WW, Kong GQ, Ma MM, Li Y, Huang X, Wang LP, Peng ZY, Zhang XH, Liu XY, Wang XZ: Short communication: Camel milk ameliorates inflammatory responses and oxidative stress and downregulates mitogen-activated protein kinase signaling pathways in lipopolysaccharide-induced acute respiratory distress syndrome in rats. J Dairy Sci 2016; 99: 53-56.
  27. Arab HH, El-Sawalhi MM: Carvedilol alleviates adjuvant-induced arthritis and subcutaneous air pouch edema: modulation of oxidative stress and inflammatory mediators. Toxicol Appl Pharmacol 2013; 268: 241-248.
  28. Suresh P, Kavitha Ch N, Babu SM, Reddy VP, Latha AK: Effect of ethanol extract of Trigonella foenum graecum (Fenugreek) seeds on Freund’s adjuvant-induced arthritis in albino rats. Inflammation 2012; 35: 1314-1321.
  29. Agha AM, Mansour M: Effects of captopril on interleukin-6, leukotriene B(4), and oxidative stress markers in serum and inflammatory exudate of arthritic rats: evidence of antiinflammatory activity. Toxicol Appl Pharmacol 2000; 168: 123-130.
  30. Kimpel D, Dayton T, Fuseler J, Gray L, Kannan K, Wolf RE, Grisham M: Splenectomy attenuates streptococcal cell wall-induced arthritis and alters leukocyte activation. Arthritis Rheum 2003; 48: 3557-3567.
  31. Bush KA, Kirkham BW, Walker JS: The kappa-opioid agonist, asimadoline, alters cytokine gene expression in adjuvant arthritis. Rheumatology (Oxford) 2001; 40: 1013-1021.
  32. Darwish HA, Arab HH, Abdelsalam RM: Chrysin alleviates testicular dysfunction in adjuvant arthritic rats via suppression of inflammation and apoptosis: Comparison with celecoxib. Toxicol Appl Pharmacol 2014; 279: 129-140.
  33. Arab HH, Mohamed WR, Barakat BM, Arafa el SA: Tangeretin attenuates cisplatin-induced renal injury in rats: Impact on the inflammatory cascade and oxidative perturbations. Chem Biol Interact 2016; 258: 205-213.
  34. Omar HA, Mohamed WR, Arafa el SA, Shehata BA, El Sherbiny GA, Arab HH, Elgendy AN: Hesperidin alleviates cisplatin-induced hepatotoxicity in rats without inhibiting its antitumor activity. Pharmacol Rep 2016; 68: 349-356.
  35. Omar HA, Mohamed WR, Arab HH, Arafa el SA: Tangeretin Alleviates Cisplatin-Induced Acute Hepatic Injury in Rats: Targeting MAPKs and Apoptosis. PLoS One 2016; 11:e0151649.
  36. Salama SA, Arab HH, Omar HA, Maghrabi IA, Snapka RM: Nicotine mediates hypochlorous acid-induced nuclear protein damage in mammalian cells. Inflammation 2014; 37: 785-792.
  37. Buege JA, Aust SD: Microsomal lipid peroxidation. Methods Enzymol 1978; 52: 302-310.
  38. Miranda KM, Espey MG, Wink DA: A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide 2001; 5: 62-71.
  39. Beutler E, Duron O, Kelly BM: Improved method for the determination of blood glutathione. J Lab Clin Med 1963; 61: 882-888.
    External Resources
  40. Rosillo MA, Alcaraz MJ, Sanchez-Hidalgo M, Fernandez-Bolanos JG, Alarcon-de-la-Lastra C, Ferrandiz ML: Anti-inflammatory and joint protective effects of extra-virgin olive-oil polyphenol extract in experimental arthritis. J Nutr Biochem 2014; 25: 1275-1281.
  41. Foxwell B, Browne K, Bondeson J, Clarke C, de Martin R, Brennan F, Feldmann M: Efficient adenoviral infection with IkappaB alpha reveals that macrophage tumor necrosis factor alpha production in rheumatoid arthritis is NF-kappaB dependent. Proc Natl Acad Sci U S A 1998; 95: 8211-8215.
    External Resources
  42. Abdel-Salam BK: Modulatory effect of whey proteins in some cytokines involved in wound healing in male diabetic albino rats. Inflammation 2014; 37: 1616-1622.
  43. Ebaid H, Abdel-Salam B, Hassan I, Al-Tamimi J, Metwalli A, Alhazza I: Camel milk peptide improves wound healing in diabetic rats by orchestrating the redox status and immune response. Lipids Health Dis 2015; 14: 132.
  44. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J: Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007; 39: 44-84.
  45. Agha AM, Gad MZ: Lipid peroxidation and lysosomal integrity in different inflammatory models in rats: the effects of indomethacin and naftazone. Pharmacol Res 1995; 32: 279-285.
  46. Agrawal RP, Sharma P, Gafoorunissa SJ, Ibrahim SA, Shah B, Shukla DK, Kaur T: Effect of camel milk on glucose metabolism in adults with normal glucose tolerance and type 2 diabetes in Raica community: a crossover study. Acta Biomed 2011; 82: 181-186.
    External Resources
  47. Ozdemir G, Inanc F: Zinc may protect remote ocular injury caused by intestinal ischemia reperfusion in rats. Tohoku J Exp Med 2005; 206: 247-251.
  48. Ozturk A, Baltaci AK, Mogulkoc R, Oztekin E, Sivrikaya A, Kurtoglu E, Kul A: Effects of zinc deficiency and supplementation on malondialdehyde and glutathione levels in blood and tissues of rats performing swimming exercise. Biol Trace Elem Res 2003; 94: 157-166.
  49. Haversen L, Ohlsson BG, Hahn-Zoric M, Hanson LA, Mattsby-Baltzer I: Lactoferrin down-regulates the LPS-induced cytokine production in monocytic cells via NF-kappa B. Cell Immunol 2002; 220: 83-95.
  50. Wahl SM, McCartney-Francis N, Chan J, Dionne R, Ta L, Orenstein JM: Nitric oxide in experimental joint inflammation. Benefit or detriment? Cells Tissues Organs 2003; 174: 26-33.

Author Contacts

Hany H. Arab

Biochemistry Division, Department of Pharmacology and Toxicology, Faculty of Pharmacy,

Taif University, Al-Haweiah, Taif, 21974, (KSA)

Tel. +966 582704865, E-Mail hany.arab@pharma.cu.edu.eg, h.arab@tu.edu.sa


Article / Publication Details

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Abstract of Original Paper

Received: March 27, 2017
Accepted: June 29, 2017
Published online: September 20, 2017
Issue release date: October 2017

ISSN: 1015-8987 (Print)
eISSN: 1421-9778 (Online)

For additional information: http://www.karger.com/CPB


Open Access License / Drug Dosage / Disclaimer

This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND). Usage and distribution for commercial purposes as well as any distribution of modified material requires written permission. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

References

  1. Hitchon CA, El-Gabalawy HS: Oxidation in rheumatoid arthritis. Arthritis Res Ther 2004; 6: 265-278.
  2. Brennan FM, McInnes IB: Evidence that cytokines play a role in rheumatoid arthritis. J Clin Invest 2008; 118: 3537-3545.
  3. Wang Y, Wang H, Jiang J, Zhao D, Liu Y: Comparative Efficacy and Acceptability of Anti-TNF-Alpha Therapy in Ankylosing Spondylitis: A Mixed-Treatments Comparison. Cell Physiol Biochem 2016; 39: 1679-1694.
  4. Xu D, Yan S, Wang H, Gu B, Sun K, Yang X, Sun B, Wang X: IL-29 Enhances LPS/TLR4-Mediated Inflammation in Rheumatoid Arthritis. Cell Physiol Biochem 2015; 37: 27-34.
  5. Liu M, Mao W, Guan H, Li L, Wei B, Li P: Effects of taurochenodeoxycholic acid on adjuvant arthritis in rats. Int Immunopharmacol 2011; 11: 2150-2158.
  6. Makarov SS: NF-kappa B in rheumatoid arthritis: a pivotal regulator of inflammation, hyperplasia, and tissue destruction. Arthritis Res 2001; 3: 200-206.
  7. Phillips DC, Dias HK, Kitas GD, Griffiths HR: Aberrant reactive oxygen and nitrogen species generation in rheumatoid arthritis (RA): causes and consequences for immune function, cell survival, and therapeutic intervention. Antioxid Redox Signal 2010; 12: 743-785.
  8. Paunovic V, Harnett MM: Mitogen-activated protein kinases as therapeutic targets for rheumatoid arthritis. Drugs 2013; 73: 101-115.
  9. Ralph JA, Morand EF: MAPK phosphatases as novel targets for rheumatoid arthritis. Expert Opin Ther Targets 2008; 12: 795-808.
  10. Bogdan C: Nitric oxide and the immune response. Nat Immunol 2001; 2: 907-916.
  11. Kahlenberg JM, Fox DA: Advances in the medical treatment of rheumatoid arthritis. Hand Clin 2011; 27: 11-20.
  12. Legrand D, Elass E, Carpentier M, Mazurier J: Lactoferrin: a modulator of immune and inflammatory responses. Cell Mol Life Sci 2005; 62: 2549-2559.
  13. el Agamy EI, Ruppanner R, Ismail A, Champagne CP, Assaf R: Antibacterial and antiviral activity of camel milk protective proteins. J Dairy Res 1992; 59: 169-175.
  14. Agrawal RP, Jain S, Shah S, Chopra A, Agarwal V: Effect of camel milk on glycemic control and insulin requirement in patients with type 1 diabetes: 2-years randomized controlled trial. Eur J Clin Nutr 2011; 65: 1048-1052.
  15. Agrawal RP, Dogra R, Mohta N, Tiwari R, Singhal S, Sultania S: Beneficial effect of camel milk in diabetic nephropathy. Acta Biomed 2009; 80: 131-134.
    External Resources
  16. Darwish HA, Abd Raboh NR, Mahdy A: Camel’s milk alleviates alcohol-induced liver injury in rats. Food Chem Toxicol 2012; 50: 1377-1383.
  17. Korish AA, Arafah MM: Camel milk ameliorates steatohepatitis, insulin resistance and lipid peroxidation in experimental non-alcoholic fatty liver disease. BMC Complement Altern Med 2013; 13: 264.
  18. Arab HH, Salama SA, Eid AH, Omar HA, Arafa el SA, Maghrabi IA: Camel’s milk ameliorates TNBS-induced colitis in rats via downregulation of inflammatory cytokines and oxidative stress. Food Chem Toxicol 2014; 69: 294-302.
  19. Badr G, Sayed LH, Omar HE, Abd El-Rahim AM, Ahmed EA, Mahmoud MH: Camel Whey Protein Protects B and T Cells from Apoptosis by Suppressing Activating Transcription Factor-3 (ATF-3)-Mediated Oxidative Stress and Enhancing Phosphorylation of AKT and IkappaB-alpha in Type I Diabetic Mice. Cell Physiol Biochem 2017; 41: 41-54.
  20. El-Agamy E, Nawar M, Shamsia S, Awada S, Haenlein G: Are camel milk proteins convenient to the nutrition of cow milk allergic children? . Small Rumin Res 2009; 82: 1-6.
    External Resources
  21. Khalesi M, Salami M, Moslehishad M, James Winterburn, Moosavi-Movahedi AA: Biomolecular content of camel milk: A traditional superfood towards future healthcare industry. Trend Food Sci Technol 2017; 62: 49-58.
    External Resources
  22. Taurog JD, Argentieri DC, McReynolds RA: Adjuvant arthritis. Methods Enzymol 1988; 162: 339-355
  23. Tate G, Mandell BF, Laposata M, Ohliger D, Baker DG, Schumacher HR, Zurier RB: Suppression of acute and chronic inflammation by dietary gamma linolenic acid. J Rheumatol 1989; 16: 729-734.
    External Resources
  24. Yoshino S, Cromartie WJ, Schwab JH: Inflammation induced by bacterial cell wall fragments in the rat air pouch. Comparison of rat strains and measurement of arachidonic acid metabolites. Am J Pathol 1985; 121: 327-336.
    External Resources
  25. Newbould BB: Chemotherapy of Arthritis Induced in Rats by Mycobacterial Adjuvant. Br J Pharmacol Chemother 1963; 21: 127-136.
  26. Zhu WW, Kong GQ, Ma MM, Li Y, Huang X, Wang LP, Peng ZY, Zhang XH, Liu XY, Wang XZ: Short communication: Camel milk ameliorates inflammatory responses and oxidative stress and downregulates mitogen-activated protein kinase signaling pathways in lipopolysaccharide-induced acute respiratory distress syndrome in rats. J Dairy Sci 2016; 99: 53-56.
  27. Arab HH, El-Sawalhi MM: Carvedilol alleviates adjuvant-induced arthritis and subcutaneous air pouch edema: modulation of oxidative stress and inflammatory mediators. Toxicol Appl Pharmacol 2013; 268: 241-248.
  28. Suresh P, Kavitha Ch N, Babu SM, Reddy VP, Latha AK: Effect of ethanol extract of Trigonella foenum graecum (Fenugreek) seeds on Freund’s adjuvant-induced arthritis in albino rats. Inflammation 2012; 35: 1314-1321.
  29. Agha AM, Mansour M: Effects of captopril on interleukin-6, leukotriene B(4), and oxidative stress markers in serum and inflammatory exudate of arthritic rats: evidence of antiinflammatory activity. Toxicol Appl Pharmacol 2000; 168: 123-130.
  30. Kimpel D, Dayton T, Fuseler J, Gray L, Kannan K, Wolf RE, Grisham M: Splenectomy attenuates streptococcal cell wall-induced arthritis and alters leukocyte activation. Arthritis Rheum 2003; 48: 3557-3567.
  31. Bush KA, Kirkham BW, Walker JS: The kappa-opioid agonist, asimadoline, alters cytokine gene expression in adjuvant arthritis. Rheumatology (Oxford) 2001; 40: 1013-1021.
  32. Darwish HA, Arab HH, Abdelsalam RM: Chrysin alleviates testicular dysfunction in adjuvant arthritic rats via suppression of inflammation and apoptosis: Comparison with celecoxib. Toxicol Appl Pharmacol 2014; 279: 129-140.
  33. Arab HH, Mohamed WR, Barakat BM, Arafa el SA: Tangeretin attenuates cisplatin-induced renal injury in rats: Impact on the inflammatory cascade and oxidative perturbations. Chem Biol Interact 2016; 258: 205-213.
  34. Omar HA, Mohamed WR, Arafa el SA, Shehata BA, El Sherbiny GA, Arab HH, Elgendy AN: Hesperidin alleviates cisplatin-induced hepatotoxicity in rats without inhibiting its antitumor activity. Pharmacol Rep 2016; 68: 349-356.
  35. Omar HA, Mohamed WR, Arab HH, Arafa el SA: Tangeretin Alleviates Cisplatin-Induced Acute Hepatic Injury in Rats: Targeting MAPKs and Apoptosis. PLoS One 2016; 11:e0151649.
  36. Salama SA, Arab HH, Omar HA, Maghrabi IA, Snapka RM: Nicotine mediates hypochlorous acid-induced nuclear protein damage in mammalian cells. Inflammation 2014; 37: 785-792.
  37. Buege JA, Aust SD: Microsomal lipid peroxidation. Methods Enzymol 1978; 52: 302-310.
  38. Miranda KM, Espey MG, Wink DA: A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide 2001; 5: 62-71.
  39. Beutler E, Duron O, Kelly BM: Improved method for the determination of blood glutathione. J Lab Clin Med 1963; 61: 882-888.
    External Resources
  40. Rosillo MA, Alcaraz MJ, Sanchez-Hidalgo M, Fernandez-Bolanos JG, Alarcon-de-la-Lastra C, Ferrandiz ML: Anti-inflammatory and joint protective effects of extra-virgin olive-oil polyphenol extract in experimental arthritis. J Nutr Biochem 2014; 25: 1275-1281.
  41. Foxwell B, Browne K, Bondeson J, Clarke C, de Martin R, Brennan F, Feldmann M: Efficient adenoviral infection with IkappaB alpha reveals that macrophage tumor necrosis factor alpha production in rheumatoid arthritis is NF-kappaB dependent. Proc Natl Acad Sci U S A 1998; 95: 8211-8215.
    External Resources
  42. Abdel-Salam BK: Modulatory effect of whey proteins in some cytokines involved in wound healing in male diabetic albino rats. Inflammation 2014; 37: 1616-1622.
  43. Ebaid H, Abdel-Salam B, Hassan I, Al-Tamimi J, Metwalli A, Alhazza I: Camel milk peptide improves wound healing in diabetic rats by orchestrating the redox status and immune response. Lipids Health Dis 2015; 14: 132.
  44. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J: Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007; 39: 44-84.
  45. Agha AM, Gad MZ: Lipid peroxidation and lysosomal integrity in different inflammatory models in rats: the effects of indomethacin and naftazone. Pharmacol Res 1995; 32: 279-285.
  46. Agrawal RP, Sharma P, Gafoorunissa SJ, Ibrahim SA, Shah B, Shukla DK, Kaur T: Effect of camel milk on glucose metabolism in adults with normal glucose tolerance and type 2 diabetes in Raica community: a crossover study. Acta Biomed 2011; 82: 181-186.
    External Resources
  47. Ozdemir G, Inanc F: Zinc may protect remote ocular injury caused by intestinal ischemia reperfusion in rats. Tohoku J Exp Med 2005; 206: 247-251.
  48. Ozturk A, Baltaci AK, Mogulkoc R, Oztekin E, Sivrikaya A, Kurtoglu E, Kul A: Effects of zinc deficiency and supplementation on malondialdehyde and glutathione levels in blood and tissues of rats performing swimming exercise. Biol Trace Elem Res 2003; 94: 157-166.
  49. Haversen L, Ohlsson BG, Hahn-Zoric M, Hanson LA, Mattsby-Baltzer I: Lactoferrin down-regulates the LPS-induced cytokine production in monocytic cells via NF-kappa B. Cell Immunol 2002; 220: 83-95.
  50. Wahl SM, McCartney-Francis N, Chan J, Dionne R, Ta L, Orenstein JM: Nitric oxide in experimental joint inflammation. Benefit or detriment? Cells Tissues Organs 2003; 174: 26-33.
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