Inflammopharmacol DOI 10.1007/s10787-014-0200-5
Inflammopharmacology
RESEARCH ARTICLE
Evening primrose oil and celecoxib inhibited pathological angiogenesis, inflammation, and oxidative stress in adjuvantinduced arthritis: novel role of angiopoietin-1 R. M. El-Sayed • Y. M. Moustafa • M. F. El-Azab
Received: 7 December 2013 / Accepted: 19 February 2014 Ó Springer Basel 2014
Abstract Rheumatoid arthritis is a chronic inflammatory disease characterized by overproduction of inflammatory mediators along with undermined oxidative defensive mechanisms. Pathological angiogenesis was found to play a critical role in the progression of this disease. The current study was carried out to evaluate the anti-angiogenic, anti-inflammatory, and anti-oxidant effects of evening primrose oil (EPO), rich in gamma linolenic acid (GLA), either alone or in combination with aspirin or celecoxib, on adjuvantinduced arthritis. Arthritis was induced by subcutaneous injection of complete Freund’s adjuvant (CFA) in the right hind paw of male albino rats. All treatments were administered orally from day 0 (EPO, 5 g/kg b.w.) or day 4 (celecoxib, 5 mg/kg; aspirin, 150 mg/kg) till day 27 after CFA injection. In the arthritic group, the results revealed significant decrease in the body weight and increase in ankle circumference, plasma angiopoietin-1 (ANG-1) and tumor necrosis factor-alpha (TNF-a) levels. Anti-oxidant status was suppressed as manifested by significant decline in reduced glutathione content along with decreased enzymatic activity of superoxide dismutase and increased lipid peroxidation. Oral
Electronic supplementary material The online version of this article (doi:10.1007/s10787-014-0200-5) contains supplementary material, which is available to authorized users. R. M. El-Sayed Ministry of Health, Arish, Egypt Y. M. Moustafa M. F. El-Azab (&) Department of Pharmacology and Toxicology, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt e-mail: [email protected]; [email protected]
administration of EPO exerted normalization of body weight, ANG-1, and TNF-a levels with restoration of activity as shown by reduced malondialdehyde levels. Moreover, histopathological examination demonstrated that EPO significantly reduced the synovial hyperplasia and inflammatory cells invasion in joint tissues, an effect that was enhanced by combination with aspirin or celecoxib. The joint use of GLA-rich natural oils, which possess anti-angiogenic, anti-inflammatory, and antioxidant activities, with traditional analgesics represents a promising strategy to restrain the progression of rheumatoid arthritis. Keywords Adjuvant-induced arthritis Angiogenesis Angiopoietin-1 Evening primrose oil NSAIDs Oxidative stress TNF-a
Introduction Rheumatoid arthritis is a chronic, destructive inflammatory poly articular joint disease. It is characterized by massive synovial proliferation and subintimal infiltration of inflammatory cells, which along with angiogenesis leads to pannus formation (Koch 1998). This proliferating pannus induces bone erosion and cartilage thinning, leading to the loss of joint function. One of the earliest phenomena observed in rheumatoid arthritis is synovial neovascular formation delivering nutrients and oxygen to this proliferating pannus (Folkman 1995). It has been demonstrated that angiogenesis inhibitors can inhibit the growth of pannus in animal arthritis models (Ferrara and Alitalo 1999). A wide array of growth factors, cytokines, and chemokines governs angiogenesis in the rheumatoid arthritis synovium namely fibroblast growth factors
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(FGFs), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), interleukin-1 (IL-1), tumor necrosis factor-a (TNF-a) and angiopoietins. All these factors induce proliferation, migration, and differentiation of macrophages, lining synovial cells, and endothelial cells (Koch 2000; Walsh and Pearson 2001). TNF-a is considered as a potent angiogenic cytokine with efficacy comparable to fibroblast growth factors (FGFs) (Leibovich et al. 1987). The angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2), and their cellular receptor Tie2, which are all widely expressed in rheumatoid arthritis synovitis, are regulated by both hypoxia and TNF-a (Takahara et al. 2004). Formation of reactive oxygen species (ROS) and lipid peroxides as a result of disease activity may play an important role in rheumatoid arthritis. Furthermore, oxidative stress is thought to contribute to angiogenesis through ROS-mediated stimulation of VEGF expression and enhancing VEGFR2 autophosphorylation (Stone and Collins 2002; UshioFukai 2006). Rat adjuvant-induced arthritis, as an experimental model, resembles rheumatoid arthritis in histological pathology and pannus formation (Remmers et al. 2002; Backlund et al. 2002), thus could be beneficial in screening of new drugs for treatment of rheumatoid arthritis disease. Evening primrose oil (EPO), which is rich in gamma linolenic acid, has been shown to improve rheumatoid arthritis patients (Brzeski et al. 1991). It has been previously reported that monocytes cultured from arthritis patients consuming EPO exhibited a lower secretion of the inflammatory cytokines when compared with control subjects (Watson et al. 1993). Moreover, Miyake et al. have reported gamma linolenic acid as an inhibitor of glioma cell proliferation in vivo through its direct effects on cell cycle and angiogenesis (Miyake et al. 2009). On the other hand, non-steroidal anti-inflammatory drugs (NSAIDs) represent the standard treatment in the management of inflammation accompanying rheumatoid arthritis. Although the anti-angiogenic effect of NSAIDs is well documented in cancer (Ruegg and Mariotti 2003; Ruegg et al. 2004; El-Awady et al. 2005; El-Azab et al. 2009), their therapeutic anti-angiogenic potential in rheumatoid arthritis needs further investigation. Similarly, in spite of the previously reported overexpression of ANG-1 in rheumatoid arthritis patients (Gravallese et al. 2003), its specific role in the pathogenesis of the disease is not extensively studied. In the current research, time course evaluation of ANG-1, inflammation and oxidative stress markers were evaluated. The aim was to explore the underlying mechanisms by which the studied drugs could mediate their ameliorative effect on rheumatoid arthritis progression.
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Materials and methods Chemicals and reagents Complete Freund’s adjuvant (CFA) was purchased from Sigma-Aldrich (St. Louis, USA). Evening primrose oil (EPO) was purchased from NOW FOODS (USA). Celecoxib and aspirin were kindly provided by Pfizer Co. for Pharm. & Chemical Ind. and Sigma Pharmaceutical Co., Egypt, respectively. Rat ANG-1 ELISA kit was purchased from Uscn Life Science Inc. (Wuhan, China) and Rat TNF-a ELISA kit was purchased from Ray Biotech. (Ink, Italy). Commercially available kits (Bio-Diagnostic, Egypt) were used for determining superoxide dismutase activity, reduced glutathione, and malondialdehyde levels. All other chemicals were obtained from Sigma (St. Louis, MO, USA). Animals All animal procedures and experimental protocols were carried out in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Research and Ethics Committee at Suez Canal University. Male Wister rats weighing 160–180 g were obtained from the Egyptian Organization for Biological Products and Vaccines (Vacsera, Egypt) and housed under controlled temperature (25 ± 1 °C) on a 12 h light/dark cycle. Food and water were allowed ad libitum during the study period. Animals were monitored for pain and disability by a person experienced in animal welfare. Induction of rat adjuvant-induced arthritis The animals were acclimatized for 1 week before initiation of the experiment. Arthritis was induced by subcutaneous injection of 0.25 mL CFA in the palmar surface of the right hind paw (Piliero et al. 1966). This procedure induced arthritis in 100 % of the animals. The day of adjuvant injection was designated as day 0. Disease severity was assessed by ankle circumference measurement of the injected paw. Pharmacological treatment One hundred and fourteen adult male albino rats were used in this experiment. Six animals served as normal group and one hundred and eight animals were induced by adjuvant arthritis. Arthritic rats were divided into main six groups, eighteen animals each. These groups were further divided into three sub groups, six rats each. The first group was kept without treatment and served as control. The second group was given aspirin (150 mg/kg p.o.) (Khayyal et al. 2005), from day 4 till the end of the experiment (27 days). The third group was given celecoxib (5 mg/kg p.o.)
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(Khayyal et al. 2005), from day 4 till the end of the experiment. The fourth group was given EPO (5 g/kg p.o.) (Matsuo et al. 1996), from day 0 till the end of the experiment. The fifth and the sixth groups were coadministrated EPO with either aspirin or celecoxib in the same previously mentioned regimen. Animals were scarified by cervical dislocation on days 9, 18 and 27 for the time course evaluation for all parameters. Sample collection Blood samples were collected on EDTA (for plasma) or without EDTA (for serum) through the orbital sinus, under light ether anesthesia, centrifuged at 1,0009g for 15 min. Serum, plasma, and erythrocyte lysate samples were separated and stored at 80 °C. After scarifying, ankle joints (with digits removed) were collected into 10 % neutral buffered formalin for histological analysis. Body weight and ankle circumference To assess the disease progression, body weight and ankle circumference were measured prior to the induction of arthritis, and then every 3 other days till the study was terminated on day 27 post injection of the adjuvant. For ankle circumference determination, two perpendicular diameters of the joint were measured using Vernier caliper. Ankle circumference was determined using the geometric formula as following: rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi a2 þ b2 2p 2 where ‘‘a’’ is the later lateral diameter, and ‘‘b’’ is the anteroposterior diameter and p = 22/7 (Halloran et al. 1996). Determination of plasma ANG-1 Time course evaluation of ANG-1 in plasma was done on days 9, 18 and 27. Standards or samples were added to the appropriate microtiter plate wells pre-coated with a biotinconjugated polyclonal antibody preparation specific for ANG-1. Next, avidin conjugated to horseradish peroxidase (HRP) was added to each well and incubated at room temperature. Then a substrate solution, 3,30 ,5,50 -tetramethyl benzidine (TMB) in buffered solution, was added to each well. Only those wells that contain ANG-1, biotinconjugated antibody and enzyme-conjugated avidin exhibited a change in color. The enzyme-substrate reaction was terminated by the addition of a sulphuric acid solution and the color change was measured spectrophotometrically at 450 nm. The concentration of ANG-1 in the samples was then determined by comparing the O.D. of the samples to the standard curve.
Determination of plasma TNF-a Similar to ANG-1, time course evaluation of TNF-a in plasma was done on days 9, 18 and 27. This assay employed an antibody specific for Rat TNF-a coated on a 96-well plate. Standards and samples were pipette into the wells and TNF-a present in a sample was bound to the wells by the immobilized antibody. The wells were washed and biotinylated anti-rat TNF-a antibody was added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin was pipette to the wells. The wells were again washed, a TMB substrate solution was added to the wells and color developed in proportion to the amount of TNF-a bound. The stop solution changed the color from blue to yellow, and the intensity of the color was measured at 450 nm. Correlation between ankle circumference and plasma TNF-a or body weight Pearson’s equation was used for the determination of correlation coefficient ‘‘r’’ between plasma levels of TNF-a or body weight and ankle circumference in adjuvant-induced arthritic rats. Linear regression was used for curve fitting of plasma TNF-a or body weight versus ankle circumference. Histopathological examination The histopathological examination was done using animals scarified on day 27 after the CFA injection. Ankle joints (with digits removed) were collected into 10 % neutral buffered formalin for at least 24 h. Decalcification was done using 10 % nitric acid. The ankle joint was transected in the longitudinal plane to give approximately equal halves. Joints were processed for paraffin embedding, sectioned and stained with hematoxylin and eosin. Cartilage and bone destruction by pannus formation, mononuclear cell infiltration, and vascularity in synovial tissues in each preparation were evaluated using the following scoring system : mononuclear cell infiltration (0, no infiltration; 1, mild infiltration; 2, moderate infiltration; 3, severe infiltration); cartilage and bone destruction by pannus formation (0, no change; 1, mild change, pannus invasion within cartilage; 2, moderate change pannus, invasion into cartilage/subchondral bone; 3, severe change, pannus invasion into the subchondral bone); and vascularity (0, almost no blood vessels; 1, few blood vessels; 2, some blood vessels; 3, many blood vessels) (Taniguchi et al. 1999). Lipid peroxidation level in plasma Lipid peroxidation is a good way of evaluating oxidative stress-induced damage to tissues. Hence levels of malondialdehyde as thiobarbituric acid-reactive substance
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were measured in plasma by the method of Ohkawa et al. (1979). Briefly, thiobarbituric acid reacts with malondialdehyde in acidic medium at temperature of 95 °C for 30 min to form colored substance. The resultant pink color representative of thiobarbituric acid-reactive substance was measured at 534 nm.
control group. Moreover, the combination of aspirin or celecoxib with EPO showed a further increase in body weight compared to corresponding individual treatment. The body weight of arthritic group treated with EPO was significantly higher than aspirin-treated group (Fig. 1a). Effect on ankle circumference
Superoxide dismutase activity in erythrocyte lysate Erythrocyte lysates were prepared from different samples according to standard protocol (Lippi 2012). The activity of the anti-oxidant enzyme superoxide dismutase was measured in the erythrocyte lysate using standard spectrophotometric assay. Briefly, superoxide dismutase activity was determined by generating superoxide radicals by the photochemical reduction of phenazine methosulphate, which reduced nitroblue tetrazolium into a bluecolored compound, formazone. Superoxide dismutase quenched free oxygen radicals and inhibited reduction of nitroblue tetrazolium, which was measured at 560 nm (Nishikimi et al. 1972).
Subcutaneous injection of CFA in the right hind paw successfully induced adjuvant arthritis as was evident by a significant increase (P B 0.05) in ankle circumference of untreated control rats compared to normal rats. Similarly, a significant increase in ankle circumference, however to less extent, was observed in all treatment groups compared to normal group. Individual treatment with aspirin or celecoxib showed a significant reduction (P B 0.05) in ankle circumference compared to control arthritic group. The treatment with EPO was more effective in reducing ankle circumference compared to aspirin or celecoxib. Although it failed to reach normalization, the combination of EPO with celecoxib revealed the maximum reduction in ankle circumference among all treatment groups (Fig. 1b).
Reduced glutathione Determination of plasma ANG-1 Glutathione is a key anti-oxidant and is used as an indicator of the reduction capacity of the tissue. Glutathione was determined in plasma by the spectrophotometric method, which was based on the use of Ellman’s reagent. Results were expressed in micromoles of glutathione per liter (Beutler et al. 1963). Statistical analysis The quantitative data of continuous variables were expressed as mean ± SEM. Statistical significance was tested by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. A P value B0.05 was considered statistically significant.
Results Effect on body weight Body weights were compared among all groups on day 27 post adjuvant arthritis induction. Current results showed a significant decrease (P B 0.05) in body weight of control arthritic rats compared to normal group. Similarly, arthritic rats treated with aspirin or celecoxib showed a significant reduction in body weight compared to normal group. On the other hand, individual treatment with aspirin, celecoxib or EPO and their combinations revealed significant increase (P B 0.05) in body weight compared to untreated
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Plasma ANG-1 levels were determined as a time course on days 9, 18 and 27 post adjuvant arthritis induction. The progression of adjuvant arthritis showed a significant increase (P B 0.05) in plasma ANG-1 levels with highest level observed on day 9 post induction, where the control arthritic group showed (&2.5 fold) increase in ANG-1 compared to normal level. Interestingly a regression in plasma ANG-1 levels was observed on days 18 and 27 in the arthritic group yet remained higher than normal level. Although individual treatment with aspirin or celecoxib significantly (P B 0.05) reduced ANG-1 level on day 9 compared to arthritic control group, they failed to normalize it. On the other hand, single treatment with EPO significantly (P B 0.05) reduced plasma ANG-1 to reach normal levels. In addition, the combination of EPO with aspirin or celecoxib was superior to corresponding individual treatments in reducing plasma ANG-1 levels. The treatment with aspirin or celecoxib did not show any significant difference compared to arthritic control group on days 18 and 27, while the treatment with EPO either alone or combined with aspirin or celecoxib significantly (P B 0.05) reduced ANG-1 on day 27 to reach normal levels (Fig. 2). Determination of plasma TNF-a Analysis of TNF-a levels in plasma showed persistent increases (&1.5 to 2 fold) in arthritic control group along
Novel role of angiopoietin-1 Fig. 1 Effect of aspirin (150 mg/kg), celecoxib (5 mg/ kg), EPO (5 g/kg) and their combinations on body weight (a) and ankle circumference (b) of adjuvant-induced arthritic rats. Body weight and ankle circumference of rats were measured prior to the induction of arthritis, and then every 3 other days until the study was terminated on day 27 post injection of the adjuvant. Values were compared on day 27 and expressed as mean ± SEM. All data were analyzed using ANOVA followed by Bonferroni post hoc test. *P B 0.05 with respect to normal, #P B 0.05 with respect to control, filled circle P B 0.05 with respect to corresponding individual treatment, open circle P B 0.05 with respect to EPO, (n = 6)
Fig. 2 Effect of aspirin (150 mg/kg), celecoxib (5 mg/kg), EPO (5 g/ kg) and their combinations on time course evaluation of ANG-1 in adjuvant-induced arthritic rats. Plasma samples were collected on days 9, 18 and 27 and analyzed using ELISA kit for rat ANG-1. Values are expressed as mean ± SEM. All data were analyzed using
ANOVA followed by Bonferroni post hoc test. *P B 0.05 with respect to normal, #P B 0.05 with respect to control on corresponding day, filled circle P B 0.05 with respect to corresponding individual treatment (i.e. aspirin or celecoxib), (n = 6)
the study period compared to normal group. Individual treatment with aspirin, celecoxib, or EPO showed a significant (P B 0.05) reduction in TNF-a levels compared to
arthritic control on days 9, 18 and 27. The combination of EPO with aspirin or celecoxib showed a further reduction in plasma TNF-a levels compared to individual treatment
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Fig. 3 Effect of aspirin (150 mg/kg), celecoxib (5 mg/kg), EPO (5 g/ kg) and their combinations on time course evaluation of TNF-a in adjuvant-induced arthritic rats. Plasma samples were collected on days 9, 18 and 27 and analyzed using ELISA kit for rat TNF-a. Values are expressed as mean ± SEM. All data were analyzed using
ANOVA followed by Bonferroni post hoc test. *P B 0.05 with respect to normal, #P B 0.05 with respect to control on corresponding day, filled circle P B 0.05 with respect to corresponding individual treatment (i.e. aspirin or celecoxib), open circle P B 0.05 with respect to EPO, (n = 6)
groups and EPO-treated group as well to reach normal levels (Fig. 3).
or celecoxib (Figs. 4g, 5g) showed further significant (P B 0.05) reduction in the total histopathological scores (Fig. 6) as was evident by normalization of all previously mentioned parameters and resuming normal histological architecture indicating the enhancing effect of EPO on antiinflammatory and anti-angiogenic activities of aspirin and celecoxib.
Correlation between ankle circumference and plasma TNF-a or body weight Correlation studies were made to determine the relationship between plasma levels of TNF-a or body weight and ankle circumference in adjuvant-induced arthritic rats with different treatment regimens. A strong positive association (r = 0.9267) existed between TNF-a levels and ankle circumference. On the other hand, a strong negative correlation (r = -0.9684) existed between body weight and ankle circumference (Supplemental Fig. 1). Histopathological examination Histopathological examination of the ankle joint on day 27 revealed prominent infiltration of mono nuclear inflammatory cells with obliteration of joint cleft by these cells as well as cell debris in arthritic control group (score = 3) (Fig. 4b). In addition, the maximum destruction of the cartilage and bone degradation with pannus formation (score = 3) (Fig. 4b) along with extensive increase in vascularity (score = 3) (Fig. 5b) was observed in arthritic control group. Individual treatment with aspirin or celecoxib significantly (P B 0.05) decreased the total score as was evident by inhibition of mononuclear infiltration cells and pannus formation in synovial tissues (Figs. 4c, d, 6) as well as the extent of vascularity (Figs. 5c, d, 6) compared to arthritic control group. The treatment with EPO either alone (Figs. 4e, 5e) or in combination with aspirin (Figs. 4f, 5f)
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Effect on lipid peroxidation To study the differential and the combined effect of the studied NASAIDs and EPO on lipid peroxidation induced by CFA, serum malondialdehyde levels were evaluated in different treatment groups. A significant (P B 0.05) increase in malondialdehyde level was observed in arthritic control group compared to normal group. The individual treatment with aspirin, celecoxib or EPO blunted the mounted levels of malondialdehyde indicating a partial protection against lipid peroxidation in treated groups. The combination of EPO with aspirin or celecoxib produced a significant reduction in malondialdehyde levels compared to aspirin (on day 9) or celecoxib (on days 9, 18 and 27), respectively. Furthermore, daily treatment with aspirin or celecoxib in combination with EPO successfully normalized malondialdehyde levels on day 27 (Fig. 7a). Effect on superoxide dismutase activity On the other hand, the activity of the anti-oxidant enzyme superoxide dismutase significantly decreased in the erythrocytes lysate of untreated arthritic rats (P B 0.05), indicating attenuation of anti-oxidative defence. Surprisingly, the treatment of arthritic rats with aspirin showed
Novel role of angiopoietin-1 Fig. 4 Photomicrographs represent changes in the hind limb of adjuvant-induced arthritic rats. a Normal, b control, c aspirin, d celecoxib, e EPO, f EPO and aspirin, g EPO and celecoxib. Histopathological examination of H&E stained sections from the foot joint revealed infiltration of mononuclear cells and the formation of pannus in synovial tissues (head arrow for pannus and infiltration) (1009) (n = 6)
initial reduction in superoxide dismutase activity on days 9 and 18 that tended to improve, yet to little extent, on day 27 compared to control group. Oppositely, celecoxib treatment showed a significant increase in superoxide dismutase activity on days 18 and 27 compared to control group. Similar results were observed with EPO on days 9, 18 and 27. Furthermore, single treatment with EPO enhanced superoxide dismutase activity to a better extent compared to individual aspirin- or celecoxib-treated groups as well as combined treatment with aspirin (on day 9). However, the combined treatments with EPO and NSAIDs were superior to individual corresponding treatments. Normalization of superoxide dismutase activity was observed on day 27 upon combined treatment with EPO and celecoxib (Fig. 7b).
Effect on reduced glutathione Parallel to superoxide dismutase activity, reduced glutathione level was found to be significantly (P B 0.05) deceased in the plasma of untreated arthritic rats. Individual treatments with aspirin or celecoxib partially upregulated reduced glutathione levels at all time points; however, they failed to normalize it. Similar results were obtained with EPO either alone or in combination with aspirin (on days 9 and 18) as well as in combination with celecoxib (on day 9). On the other hand, restoration of reduced glutathione normal level was observed upon treatment with EPO either alone (on day 27) or in combination with aspirin (on day 27) or celecoxib (on days 18 and 27). Interestingly, combined treatment of EPO with
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R. M. El-Sayed et al. Fig. 5 Photomicrographs represent changes in the hind limb of adjuvant-induced arthritic rats. a Normal, b control, c aspirin, d celecoxib, e EPO, f EPO and aspirin, g EPO and celecoxib. Histopathological examination of H&E stained sections from foot joint showed significant decrease in the vascularity in comparison to control rats (arrows for blood vessels) (1009) (n = 6)
aspirin or celecoxib was superior to corresponding individual treatments at all time point (Fig. 7c).
Discussion Angiogenesis is a critical event in rheumatoid arthritis pathogenesis, and is up-regulated by an intricate balance of pro- and anti-angiogenic regulators, which include members of VEGF and ANG receptor-ligand families (Paleolog 2002). Our study was carried out to evaluate the antiangiogenic effect of EPO and NSAIDs (aspirin and celecoxib, as non-selective and selective cyclooxygenase inhibitor, respectively) on adjuvant-induced arthritic rats. Results of the present study showed that the induction of arthritis was accompanied by a significant weight loss that
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could not be improved by aspirin or celecoxib treatment. Adjuvant-induced arthritis is an immune response to an antigen present on the capsule of the mycobacterium that has been previously reported to produce systemic features of inflammation, such as uveitis, inflammation of the gastrointestinal tract and body weight loss that starts 24–48 h before the clinical onset of arthritis (Prakken et al. 2003). Findings of the present study revealed an anti-inflammatory effect of all individual treatments that has enhanced up on combination as was evident by a significant reduction in rat ankle circumference in treated groups. These results are in accordance with those obtained by Singh et al. (2011) who showed that aspirin produced a significant decrease in the joint diameter as compared to adjuvant-induced arthritic rats on all observation days. Aspirin and celecoxib suppress inflammation by inhibiting the enzyme
Novel role of angiopoietin-1
Fig. 6 Histopathological scores of the hind limb in adjuvant-induced arthritic rats either untreated or treated with different regimens. Monocellular infiltration, pannus invasion into the cartilage and vascularity were measured by microscopic examination on two separate occasions, scores of the sections are expressed as
mean ± SEM. All data were analyzed using ANOVA followed by Bonferroni post hoc test. *P B 0.05 with respect to normal, # P B 0.05 with respect to control, filled circle P B 0.05 with respect to corresponding individual treatment, open circle P B 0.05 with respect to EPO, (n = 6)
cyclooxygenase resulting in decreased prostaglandin synthesis and thromboxanes, the two main classes of lipidderived pro-inflammatory molecules. It worth mentioning that the effect of EPO was superior to either aspirin or celecoxib. The use of EPO as a source of GLA has been investigated, and several clinical applications on inflammatory disorders have been described (Zurier et al. 1996; Rosenstein et al. 2003). The rationale of its therapeutic use is based on the high amount (9 %) of gamma linolenic acid (Kunkel et al. 1981). The antiinflammatory activity of EPO may be attributed to the metabolism of gamma linolenic acid to dihomo gamma linolenic acid (DGLA) and thus competitive inhibition of the 2-series prostaglandins and 4-series leukotrienes (Jantti et al. 1989). DGLA itself cannot be converted to leukotrienes but can form a 15-hydroxyl derivative that blocks the transformation of arachidonic acid to leukotrienes. As a result, increased DGLA intake possibly will suppress inflammation (Voorhees 1983). Previous studies have described the expression of ANG1 and ANG-2 (Scott et al. 2002; Gravallese et al. 2003) and their receptors Tie-1 and Tie-2 (Shahrara et al. 2002; Uchida et al. 2000) in rheumatoid arthritis. Furthermore, overexpression of VEGF and ANG-1 in the arthritic joints was found to be associated with distinct vascular morphology (Szekanecz and Koch 2007). We observed increased ANG-1 concentration in the plasma of early adjuvant-induced arthritis that had decreased dramatically by day 18 post adjuvant administration indicating a vital role of ANG-1 at early stages of disease progression. Our results are in agreement with Shahrara et al. (2002) who demonstrated greater expression of ANG-1, ANG-2, Tie-1 and Tie-2 in rheumatoid arthritis as compared with normal synovium. Moreover, Clavel et al. (2007) reported that
serum concentrations of VEGF, sFlt-1 and ANG-1 were correlated to parameters of inflammation and to bone destruction in early arthritis. Malik et al. (2010) demonstrated that the ANG–Tie ligand–receptor system is dysregulated in collagen-induced arthritis. Tie1-751, a novel splice variant of the Tie1 receptor, inhibits ANG-1/ VEGF signaling, suggesting that ANG inhibition may be of therapeutic benefit in inflammatory arthritis (Malik et al. 2010). Regarding the anti-angiogenic effect of our studied drugs, both aspirin and celecoxib significantly decreased plasma ANG-1 level at early stage of adjuvant-induced arthritis development (i.e. on day 9). Interestingly, oral administration of EPO either alone or in combination with aspirin or celecoxib successfully maintained normal levels of ANG-1 at all time points (i.e. on days 9, 18 and 27). The anti-angiogenic effect of gamma linolenic acid or cyclooxygenase-2 inhibitors has been previously reported in different settings. Miyake et al. (2009) reported in their study that gamma linolenic acid inhibited glioma cell proliferation in vivo, an effect that involved changes in protein expression of VEGF, VEGF receptors (Flt1 and Flk1), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP9), ERK1 and ERK2. Furthermore, they have shown that gamma linolenic acid altered the expression of several proteins involved in cell cycle control (Miyake et al. 2009). Besides, Wang et al. (2008) reported that celecoxib enhanced tumor cell apoptosis, thereby inhibited the growth and angiogenesis of implanted tumors in a mouse model of human colorectal cancer by inhibiting COX-2, PGE2 synthesis, and VEGF and matrix MMP-2 mRNA expression in tumor tissue (Wang et al. 2008). Also, Refaat et al. (2013) have recently reported that treatment with celecoxib caused a remarkable decrease in VEGF serum levels in adjuvant arthritic rats. That could be
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R. M. El-Sayed et al. Fig. 7 Effect of aspirin (150 mg/kg), celecoxib (5 mg/ kg), EPO (5 g/kg) and their combinations on time course evaluation of lipid peroxidation (a), superoxide dismutase (b), and reduced glutathione (c) in adjuvant-induced arthritic rats. Analyses were done on days 9, 18 and 27. Serum levels of malondialdehyde as thiobarbituric acid-reactive substances were measured spectrophotometrically and expressed as nmol/mL. Superoxide dismutase and reduced glutathione were evaluated spectrophotometrically and expressed as U/mL and mg/dL, respectively. Values are expressed as mean ± SEM. All data were analyzed using ANOVA followed by Bonferroni post hoc test. *P B 0.05 with respect to normal, #P B 0.05 with respect to control on corresponding day, filled circle P B 0.05 with respect to corresponding individual treatment (i.e. aspirin or celecoxib), open circle P B 0.05 with respect to EPO, (n = 6)
explained based on previous observation that the reduction of PGE2 induced by cyclooxygenase inhibitors resulted in less production of VEGF in human synovial fibroblasts (Inoue et al. 2002; Ma et al. 2002). To the best of our knowledge, the current study is the first to report the antiangiogenic effect of EPO rich in gamma linolenic acid in adjuvant-induced arthritis model. This anti-angiogenic effect was found to be mediated through early inhibition of ANG-1. In addition, cyclooxygenase inhibitors used in the current study showed also an anti-angiogenic potential also through ANG-1 inhibition. Previous reports showed a rise in the inflammatory mediator TNF-a during disease progression of adjuvantinduced arthritic rats (Cai et al. 2006; Kushner et al. 2010; Refaat et al. 2013). The time course evaluation of plasma TNF-a levels in arthritic group came in parallel with ankle inflammation that was reported in the current study.
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Surprisingly, the observed increment in TNF-a levels had gradually decreased by day 18 then 27 post complete Freund’s adjuvant administration. The effect of individual and combined regimens used in the current study on TNF-a levels showed a similar pattern to ankle measurements with highest anti-inflammatory effect observed in combination groups. The anti-inflammatory activity of individual treatments used currently is in agreement with previous reports. Purasiri et al. (1997) have shown that dietary gamma linolenic acid suppressed inflammatory cytokines level in human blood lymphocytes. In addition, Khayyal et al. (2005) reported that aspirin as well as celecoxib showed a similar effect in reducing TNF-a in adjuvant-induced arthritic rats. Moreover, El-Ghazaly et al. (2010) reported that treatment with celecoxib was effective in decreasing the elevated levels of TNF-a in irradiated adjuvant-induced arthritic rats. These observed anti-inflammatory and anti-
Novel role of angiopoietin-1
angiogenic activities were confirmed in the current study by histopathological findings which revealed that EPO significantly reduced the synovial hyperplasia and inflammatory cells invasion in joint tissues, an effect that was enhanced by combination with aspirin or celecoxib. Essential fatty acids such as gamma linolenic acid and linolenic acid suppressed ROS generation to a significant degree in a dose-dependent fashion and raised superoxide dismutase activity significantly in hyperglycaemia-induced excess proliferation of retinal vascular endothelial cells (Shen et al. 2012). Also, Arimura et al. (2004) demonstrated that evening primrose extract exposure elicited a rapid increase in the activity of superoxide dismutase and intracellular peroxides levels in Ehrlich ascites tumor cells. In the same way, EPO treatment used in the current study showed indispensable role in reducing ROS in adjuvant arthritic model. The induction of adjuvant arthritis resulted in cartilage destruction that involves numerous and interconnected mechanisms. This include excessive generation of ROS, several cytokines, reduction in the level of reduced glutathione associated with a corresponding rise in malondialdehyde and a rise in the level of nitric oxide indicating a status of oxidative stress (Arend 2001; Henrotin et al. 2003; Jawed et al. 2010; El-Ghazaly et al. 2010). This was confirmed in the present study, where a depressed anti-oxidant status was manifested by a gradual but significant increase in lipid peroxidation that was accompanied by a great reduction in reduced glutathione levels and superoxide dismutase activities along the course of disease progression in arthritic rats. These results are in agreement with a recent study by Rasool and Varalakshmi (2007) who referred such results to the weak free radical defence system against oxidative stress that might explain the pathogenic state associated with arthritis. Regarding lipid peroxidation, all treatments significantly decreased malondialdehyde level on days 18 and 27 while the normalization was observed on day 27 only on combined treatments. In contrast, El-Ghazaly et al. (2010) reported that treatment with celecoxib showed no effect on superoxide dismutase activity and levels of malondialdehyde and reduced glutathione in adjuvant-induced arthritis model in irradiated rats. This could be due to different experimental design. On the other hand, Cha´vez et al. (2010) reported that celecoxib supported the recovery of livers with necrotic and cholestatic damage through antioxidant activities that were manifested by restoration of redox equilibrium and inhibition of lipid peroxidation. We observed that all treatments significantly increased reduced glutathione levels at all time points but the effect of EPO alone or combined with aspirin or celecoxib was superior to individual treatments with cyclooxygenase inhibitors. In the same way, superoxide dismutase activities were gradually resumed in all treatment groups while normalization
was observed only on day 27 up on combination of EPO with celecoxib. These results in agreement with the study of De La-Cruz et al. (1999) who showed that dietary supplementation with EPO decreased tissue oxidative stress, through reduction of lipid peroxidation and increased glutathione in all tissues. They reported that EPO was able to enhance the activities of glutathione reductase and consequently resulted in decreased percentage of oxidized glutathione.
Conclusions The current study showed for the first time that ANG-1 plays a vital role in the early rather than late phase of adjuvant-induced arthritis pathogenesis making it an attractive non-invasive tool for early diagnosis and prediction of disease prognosis. This study also suggest that ANG-1 inhibition may be of therapeutic value in inflammatory arthritis. It can be concluded that early intervention with natural oil rich in gamma linolenic acid, which possess anti-angiogenic, anti-inflammatory, and anti-oxidant activities, with traditional analgesics can improve therapeutic benefits and represent a promising strategy to restrain the progression of rheumatoid arthritis. Current data propose EPO as a potential inhibitor of pathological angiogenesis. Acknowledgments The authors express gratitude to Dr. Hend M. Tag, Assistant Professor of Physiology, Faculty of Science, Suez Canal University, Ismailia, Egypt, for her valuable assistance with photomicrography and histopathology scoring. They also would like to thank Mr. Mohamed I. El-Assaal for his great efforts in keeping the animals under uniform experimental conditions. Celecoxib and aspirin were kindly provided by Pfizer Co. for Pharm. & Chemical Ind. and Sigma Pharmaceutical Co., Egypt, respectively. Conflict of interest
None.
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Inflammopharmacology
RESEARCH ARTICLE
Evening primrose oil and celecoxib inhibited pathological angiogenesis, inflammation, and oxidative stress in adjuvantinduced arthritis: novel role of angiopoietin-1 R. M. El-Sayed • Y. M. Moustafa • M. F. El-Azab
Received: 7 December 2013 / Accepted: 19 February 2014 Ó Springer Basel 2014
Abstract Rheumatoid arthritis is a chronic inflammatory disease characterized by overproduction of inflammatory mediators along with undermined oxidative defensive mechanisms. Pathological angiogenesis was found to play a critical role in the progression of this disease. The current study was carried out to evaluate the anti-angiogenic, anti-inflammatory, and anti-oxidant effects of evening primrose oil (EPO), rich in gamma linolenic acid (GLA), either alone or in combination with aspirin or celecoxib, on adjuvantinduced arthritis. Arthritis was induced by subcutaneous injection of complete Freund’s adjuvant (CFA) in the right hind paw of male albino rats. All treatments were administered orally from day 0 (EPO, 5 g/kg b.w.) or day 4 (celecoxib, 5 mg/kg; aspirin, 150 mg/kg) till day 27 after CFA injection. In the arthritic group, the results revealed significant decrease in the body weight and increase in ankle circumference, plasma angiopoietin-1 (ANG-1) and tumor necrosis factor-alpha (TNF-a) levels. Anti-oxidant status was suppressed as manifested by significant decline in reduced glutathione content along with decreased enzymatic activity of superoxide dismutase and increased lipid peroxidation. Oral
Electronic supplementary material The online version of this article (doi:10.1007/s10787-014-0200-5) contains supplementary material, which is available to authorized users. R. M. El-Sayed Ministry of Health, Arish, Egypt Y. M. Moustafa M. F. El-Azab (&) Department of Pharmacology and Toxicology, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt e-mail: [email protected]; [email protected]
administration of EPO exerted normalization of body weight, ANG-1, and TNF-a levels with restoration of activity as shown by reduced malondialdehyde levels. Moreover, histopathological examination demonstrated that EPO significantly reduced the synovial hyperplasia and inflammatory cells invasion in joint tissues, an effect that was enhanced by combination with aspirin or celecoxib. The joint use of GLA-rich natural oils, which possess anti-angiogenic, anti-inflammatory, and antioxidant activities, with traditional analgesics represents a promising strategy to restrain the progression of rheumatoid arthritis. Keywords Adjuvant-induced arthritis Angiogenesis Angiopoietin-1 Evening primrose oil NSAIDs Oxidative stress TNF-a
Introduction Rheumatoid arthritis is a chronic, destructive inflammatory poly articular joint disease. It is characterized by massive synovial proliferation and subintimal infiltration of inflammatory cells, which along with angiogenesis leads to pannus formation (Koch 1998). This proliferating pannus induces bone erosion and cartilage thinning, leading to the loss of joint function. One of the earliest phenomena observed in rheumatoid arthritis is synovial neovascular formation delivering nutrients and oxygen to this proliferating pannus (Folkman 1995). It has been demonstrated that angiogenesis inhibitors can inhibit the growth of pannus in animal arthritis models (Ferrara and Alitalo 1999). A wide array of growth factors, cytokines, and chemokines governs angiogenesis in the rheumatoid arthritis synovium namely fibroblast growth factors
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(FGFs), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), interleukin-1 (IL-1), tumor necrosis factor-a (TNF-a) and angiopoietins. All these factors induce proliferation, migration, and differentiation of macrophages, lining synovial cells, and endothelial cells (Koch 2000; Walsh and Pearson 2001). TNF-a is considered as a potent angiogenic cytokine with efficacy comparable to fibroblast growth factors (FGFs) (Leibovich et al. 1987). The angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2), and their cellular receptor Tie2, which are all widely expressed in rheumatoid arthritis synovitis, are regulated by both hypoxia and TNF-a (Takahara et al. 2004). Formation of reactive oxygen species (ROS) and lipid peroxides as a result of disease activity may play an important role in rheumatoid arthritis. Furthermore, oxidative stress is thought to contribute to angiogenesis through ROS-mediated stimulation of VEGF expression and enhancing VEGFR2 autophosphorylation (Stone and Collins 2002; UshioFukai 2006). Rat adjuvant-induced arthritis, as an experimental model, resembles rheumatoid arthritis in histological pathology and pannus formation (Remmers et al. 2002; Backlund et al. 2002), thus could be beneficial in screening of new drugs for treatment of rheumatoid arthritis disease. Evening primrose oil (EPO), which is rich in gamma linolenic acid, has been shown to improve rheumatoid arthritis patients (Brzeski et al. 1991). It has been previously reported that monocytes cultured from arthritis patients consuming EPO exhibited a lower secretion of the inflammatory cytokines when compared with control subjects (Watson et al. 1993). Moreover, Miyake et al. have reported gamma linolenic acid as an inhibitor of glioma cell proliferation in vivo through its direct effects on cell cycle and angiogenesis (Miyake et al. 2009). On the other hand, non-steroidal anti-inflammatory drugs (NSAIDs) represent the standard treatment in the management of inflammation accompanying rheumatoid arthritis. Although the anti-angiogenic effect of NSAIDs is well documented in cancer (Ruegg and Mariotti 2003; Ruegg et al. 2004; El-Awady et al. 2005; El-Azab et al. 2009), their therapeutic anti-angiogenic potential in rheumatoid arthritis needs further investigation. Similarly, in spite of the previously reported overexpression of ANG-1 in rheumatoid arthritis patients (Gravallese et al. 2003), its specific role in the pathogenesis of the disease is not extensively studied. In the current research, time course evaluation of ANG-1, inflammation and oxidative stress markers were evaluated. The aim was to explore the underlying mechanisms by which the studied drugs could mediate their ameliorative effect on rheumatoid arthritis progression.
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Materials and methods Chemicals and reagents Complete Freund’s adjuvant (CFA) was purchased from Sigma-Aldrich (St. Louis, USA). Evening primrose oil (EPO) was purchased from NOW FOODS (USA). Celecoxib and aspirin were kindly provided by Pfizer Co. for Pharm. & Chemical Ind. and Sigma Pharmaceutical Co., Egypt, respectively. Rat ANG-1 ELISA kit was purchased from Uscn Life Science Inc. (Wuhan, China) and Rat TNF-a ELISA kit was purchased from Ray Biotech. (Ink, Italy). Commercially available kits (Bio-Diagnostic, Egypt) were used for determining superoxide dismutase activity, reduced glutathione, and malondialdehyde levels. All other chemicals were obtained from Sigma (St. Louis, MO, USA). Animals All animal procedures and experimental protocols were carried out in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Research and Ethics Committee at Suez Canal University. Male Wister rats weighing 160–180 g were obtained from the Egyptian Organization for Biological Products and Vaccines (Vacsera, Egypt) and housed under controlled temperature (25 ± 1 °C) on a 12 h light/dark cycle. Food and water were allowed ad libitum during the study period. Animals were monitored for pain and disability by a person experienced in animal welfare. Induction of rat adjuvant-induced arthritis The animals were acclimatized for 1 week before initiation of the experiment. Arthritis was induced by subcutaneous injection of 0.25 mL CFA in the palmar surface of the right hind paw (Piliero et al. 1966). This procedure induced arthritis in 100 % of the animals. The day of adjuvant injection was designated as day 0. Disease severity was assessed by ankle circumference measurement of the injected paw. Pharmacological treatment One hundred and fourteen adult male albino rats were used in this experiment. Six animals served as normal group and one hundred and eight animals were induced by adjuvant arthritis. Arthritic rats were divided into main six groups, eighteen animals each. These groups were further divided into three sub groups, six rats each. The first group was kept without treatment and served as control. The second group was given aspirin (150 mg/kg p.o.) (Khayyal et al. 2005), from day 4 till the end of the experiment (27 days). The third group was given celecoxib (5 mg/kg p.o.)
Novel role of angiopoietin-1
(Khayyal et al. 2005), from day 4 till the end of the experiment. The fourth group was given EPO (5 g/kg p.o.) (Matsuo et al. 1996), from day 0 till the end of the experiment. The fifth and the sixth groups were coadministrated EPO with either aspirin or celecoxib in the same previously mentioned regimen. Animals were scarified by cervical dislocation on days 9, 18 and 27 for the time course evaluation for all parameters. Sample collection Blood samples were collected on EDTA (for plasma) or without EDTA (for serum) through the orbital sinus, under light ether anesthesia, centrifuged at 1,0009g for 15 min. Serum, plasma, and erythrocyte lysate samples were separated and stored at 80 °C. After scarifying, ankle joints (with digits removed) were collected into 10 % neutral buffered formalin for histological analysis. Body weight and ankle circumference To assess the disease progression, body weight and ankle circumference were measured prior to the induction of arthritis, and then every 3 other days till the study was terminated on day 27 post injection of the adjuvant. For ankle circumference determination, two perpendicular diameters of the joint were measured using Vernier caliper. Ankle circumference was determined using the geometric formula as following: rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi a2 þ b2 2p 2 where ‘‘a’’ is the later lateral diameter, and ‘‘b’’ is the anteroposterior diameter and p = 22/7 (Halloran et al. 1996). Determination of plasma ANG-1 Time course evaluation of ANG-1 in plasma was done on days 9, 18 and 27. Standards or samples were added to the appropriate microtiter plate wells pre-coated with a biotinconjugated polyclonal antibody preparation specific for ANG-1. Next, avidin conjugated to horseradish peroxidase (HRP) was added to each well and incubated at room temperature. Then a substrate solution, 3,30 ,5,50 -tetramethyl benzidine (TMB) in buffered solution, was added to each well. Only those wells that contain ANG-1, biotinconjugated antibody and enzyme-conjugated avidin exhibited a change in color. The enzyme-substrate reaction was terminated by the addition of a sulphuric acid solution and the color change was measured spectrophotometrically at 450 nm. The concentration of ANG-1 in the samples was then determined by comparing the O.D. of the samples to the standard curve.
Determination of plasma TNF-a Similar to ANG-1, time course evaluation of TNF-a in plasma was done on days 9, 18 and 27. This assay employed an antibody specific for Rat TNF-a coated on a 96-well plate. Standards and samples were pipette into the wells and TNF-a present in a sample was bound to the wells by the immobilized antibody. The wells were washed and biotinylated anti-rat TNF-a antibody was added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin was pipette to the wells. The wells were again washed, a TMB substrate solution was added to the wells and color developed in proportion to the amount of TNF-a bound. The stop solution changed the color from blue to yellow, and the intensity of the color was measured at 450 nm. Correlation between ankle circumference and plasma TNF-a or body weight Pearson’s equation was used for the determination of correlation coefficient ‘‘r’’ between plasma levels of TNF-a or body weight and ankle circumference in adjuvant-induced arthritic rats. Linear regression was used for curve fitting of plasma TNF-a or body weight versus ankle circumference. Histopathological examination The histopathological examination was done using animals scarified on day 27 after the CFA injection. Ankle joints (with digits removed) were collected into 10 % neutral buffered formalin for at least 24 h. Decalcification was done using 10 % nitric acid. The ankle joint was transected in the longitudinal plane to give approximately equal halves. Joints were processed for paraffin embedding, sectioned and stained with hematoxylin and eosin. Cartilage and bone destruction by pannus formation, mononuclear cell infiltration, and vascularity in synovial tissues in each preparation were evaluated using the following scoring system : mononuclear cell infiltration (0, no infiltration; 1, mild infiltration; 2, moderate infiltration; 3, severe infiltration); cartilage and bone destruction by pannus formation (0, no change; 1, mild change, pannus invasion within cartilage; 2, moderate change pannus, invasion into cartilage/subchondral bone; 3, severe change, pannus invasion into the subchondral bone); and vascularity (0, almost no blood vessels; 1, few blood vessels; 2, some blood vessels; 3, many blood vessels) (Taniguchi et al. 1999). Lipid peroxidation level in plasma Lipid peroxidation is a good way of evaluating oxidative stress-induced damage to tissues. Hence levels of malondialdehyde as thiobarbituric acid-reactive substance
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were measured in plasma by the method of Ohkawa et al. (1979). Briefly, thiobarbituric acid reacts with malondialdehyde in acidic medium at temperature of 95 °C for 30 min to form colored substance. The resultant pink color representative of thiobarbituric acid-reactive substance was measured at 534 nm.
control group. Moreover, the combination of aspirin or celecoxib with EPO showed a further increase in body weight compared to corresponding individual treatment. The body weight of arthritic group treated with EPO was significantly higher than aspirin-treated group (Fig. 1a). Effect on ankle circumference
Superoxide dismutase activity in erythrocyte lysate Erythrocyte lysates were prepared from different samples according to standard protocol (Lippi 2012). The activity of the anti-oxidant enzyme superoxide dismutase was measured in the erythrocyte lysate using standard spectrophotometric assay. Briefly, superoxide dismutase activity was determined by generating superoxide radicals by the photochemical reduction of phenazine methosulphate, which reduced nitroblue tetrazolium into a bluecolored compound, formazone. Superoxide dismutase quenched free oxygen radicals and inhibited reduction of nitroblue tetrazolium, which was measured at 560 nm (Nishikimi et al. 1972).
Subcutaneous injection of CFA in the right hind paw successfully induced adjuvant arthritis as was evident by a significant increase (P B 0.05) in ankle circumference of untreated control rats compared to normal rats. Similarly, a significant increase in ankle circumference, however to less extent, was observed in all treatment groups compared to normal group. Individual treatment with aspirin or celecoxib showed a significant reduction (P B 0.05) in ankle circumference compared to control arthritic group. The treatment with EPO was more effective in reducing ankle circumference compared to aspirin or celecoxib. Although it failed to reach normalization, the combination of EPO with celecoxib revealed the maximum reduction in ankle circumference among all treatment groups (Fig. 1b).
Reduced glutathione Determination of plasma ANG-1 Glutathione is a key anti-oxidant and is used as an indicator of the reduction capacity of the tissue. Glutathione was determined in plasma by the spectrophotometric method, which was based on the use of Ellman’s reagent. Results were expressed in micromoles of glutathione per liter (Beutler et al. 1963). Statistical analysis The quantitative data of continuous variables were expressed as mean ± SEM. Statistical significance was tested by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. A P value B0.05 was considered statistically significant.
Results Effect on body weight Body weights were compared among all groups on day 27 post adjuvant arthritis induction. Current results showed a significant decrease (P B 0.05) in body weight of control arthritic rats compared to normal group. Similarly, arthritic rats treated with aspirin or celecoxib showed a significant reduction in body weight compared to normal group. On the other hand, individual treatment with aspirin, celecoxib or EPO and their combinations revealed significant increase (P B 0.05) in body weight compared to untreated
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Plasma ANG-1 levels were determined as a time course on days 9, 18 and 27 post adjuvant arthritis induction. The progression of adjuvant arthritis showed a significant increase (P B 0.05) in plasma ANG-1 levels with highest level observed on day 9 post induction, where the control arthritic group showed (&2.5 fold) increase in ANG-1 compared to normal level. Interestingly a regression in plasma ANG-1 levels was observed on days 18 and 27 in the arthritic group yet remained higher than normal level. Although individual treatment with aspirin or celecoxib significantly (P B 0.05) reduced ANG-1 level on day 9 compared to arthritic control group, they failed to normalize it. On the other hand, single treatment with EPO significantly (P B 0.05) reduced plasma ANG-1 to reach normal levels. In addition, the combination of EPO with aspirin or celecoxib was superior to corresponding individual treatments in reducing plasma ANG-1 levels. The treatment with aspirin or celecoxib did not show any significant difference compared to arthritic control group on days 18 and 27, while the treatment with EPO either alone or combined with aspirin or celecoxib significantly (P B 0.05) reduced ANG-1 on day 27 to reach normal levels (Fig. 2). Determination of plasma TNF-a Analysis of TNF-a levels in plasma showed persistent increases (&1.5 to 2 fold) in arthritic control group along
Novel role of angiopoietin-1 Fig. 1 Effect of aspirin (150 mg/kg), celecoxib (5 mg/ kg), EPO (5 g/kg) and their combinations on body weight (a) and ankle circumference (b) of adjuvant-induced arthritic rats. Body weight and ankle circumference of rats were measured prior to the induction of arthritis, and then every 3 other days until the study was terminated on day 27 post injection of the adjuvant. Values were compared on day 27 and expressed as mean ± SEM. All data were analyzed using ANOVA followed by Bonferroni post hoc test. *P B 0.05 with respect to normal, #P B 0.05 with respect to control, filled circle P B 0.05 with respect to corresponding individual treatment, open circle P B 0.05 with respect to EPO, (n = 6)
Fig. 2 Effect of aspirin (150 mg/kg), celecoxib (5 mg/kg), EPO (5 g/ kg) and their combinations on time course evaluation of ANG-1 in adjuvant-induced arthritic rats. Plasma samples were collected on days 9, 18 and 27 and analyzed using ELISA kit for rat ANG-1. Values are expressed as mean ± SEM. All data were analyzed using
ANOVA followed by Bonferroni post hoc test. *P B 0.05 with respect to normal, #P B 0.05 with respect to control on corresponding day, filled circle P B 0.05 with respect to corresponding individual treatment (i.e. aspirin or celecoxib), (n = 6)
the study period compared to normal group. Individual treatment with aspirin, celecoxib, or EPO showed a significant (P B 0.05) reduction in TNF-a levels compared to
arthritic control on days 9, 18 and 27. The combination of EPO with aspirin or celecoxib showed a further reduction in plasma TNF-a levels compared to individual treatment
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Fig. 3 Effect of aspirin (150 mg/kg), celecoxib (5 mg/kg), EPO (5 g/ kg) and their combinations on time course evaluation of TNF-a in adjuvant-induced arthritic rats. Plasma samples were collected on days 9, 18 and 27 and analyzed using ELISA kit for rat TNF-a. Values are expressed as mean ± SEM. All data were analyzed using
ANOVA followed by Bonferroni post hoc test. *P B 0.05 with respect to normal, #P B 0.05 with respect to control on corresponding day, filled circle P B 0.05 with respect to corresponding individual treatment (i.e. aspirin or celecoxib), open circle P B 0.05 with respect to EPO, (n = 6)
groups and EPO-treated group as well to reach normal levels (Fig. 3).
or celecoxib (Figs. 4g, 5g) showed further significant (P B 0.05) reduction in the total histopathological scores (Fig. 6) as was evident by normalization of all previously mentioned parameters and resuming normal histological architecture indicating the enhancing effect of EPO on antiinflammatory and anti-angiogenic activities of aspirin and celecoxib.
Correlation between ankle circumference and plasma TNF-a or body weight Correlation studies were made to determine the relationship between plasma levels of TNF-a or body weight and ankle circumference in adjuvant-induced arthritic rats with different treatment regimens. A strong positive association (r = 0.9267) existed between TNF-a levels and ankle circumference. On the other hand, a strong negative correlation (r = -0.9684) existed between body weight and ankle circumference (Supplemental Fig. 1). Histopathological examination Histopathological examination of the ankle joint on day 27 revealed prominent infiltration of mono nuclear inflammatory cells with obliteration of joint cleft by these cells as well as cell debris in arthritic control group (score = 3) (Fig. 4b). In addition, the maximum destruction of the cartilage and bone degradation with pannus formation (score = 3) (Fig. 4b) along with extensive increase in vascularity (score = 3) (Fig. 5b) was observed in arthritic control group. Individual treatment with aspirin or celecoxib significantly (P B 0.05) decreased the total score as was evident by inhibition of mononuclear infiltration cells and pannus formation in synovial tissues (Figs. 4c, d, 6) as well as the extent of vascularity (Figs. 5c, d, 6) compared to arthritic control group. The treatment with EPO either alone (Figs. 4e, 5e) or in combination with aspirin (Figs. 4f, 5f)
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Effect on lipid peroxidation To study the differential and the combined effect of the studied NASAIDs and EPO on lipid peroxidation induced by CFA, serum malondialdehyde levels were evaluated in different treatment groups. A significant (P B 0.05) increase in malondialdehyde level was observed in arthritic control group compared to normal group. The individual treatment with aspirin, celecoxib or EPO blunted the mounted levels of malondialdehyde indicating a partial protection against lipid peroxidation in treated groups. The combination of EPO with aspirin or celecoxib produced a significant reduction in malondialdehyde levels compared to aspirin (on day 9) or celecoxib (on days 9, 18 and 27), respectively. Furthermore, daily treatment with aspirin or celecoxib in combination with EPO successfully normalized malondialdehyde levels on day 27 (Fig. 7a). Effect on superoxide dismutase activity On the other hand, the activity of the anti-oxidant enzyme superoxide dismutase significantly decreased in the erythrocytes lysate of untreated arthritic rats (P B 0.05), indicating attenuation of anti-oxidative defence. Surprisingly, the treatment of arthritic rats with aspirin showed
Novel role of angiopoietin-1 Fig. 4 Photomicrographs represent changes in the hind limb of adjuvant-induced arthritic rats. a Normal, b control, c aspirin, d celecoxib, e EPO, f EPO and aspirin, g EPO and celecoxib. Histopathological examination of H&E stained sections from the foot joint revealed infiltration of mononuclear cells and the formation of pannus in synovial tissues (head arrow for pannus and infiltration) (1009) (n = 6)
initial reduction in superoxide dismutase activity on days 9 and 18 that tended to improve, yet to little extent, on day 27 compared to control group. Oppositely, celecoxib treatment showed a significant increase in superoxide dismutase activity on days 18 and 27 compared to control group. Similar results were observed with EPO on days 9, 18 and 27. Furthermore, single treatment with EPO enhanced superoxide dismutase activity to a better extent compared to individual aspirin- or celecoxib-treated groups as well as combined treatment with aspirin (on day 9). However, the combined treatments with EPO and NSAIDs were superior to individual corresponding treatments. Normalization of superoxide dismutase activity was observed on day 27 upon combined treatment with EPO and celecoxib (Fig. 7b).
Effect on reduced glutathione Parallel to superoxide dismutase activity, reduced glutathione level was found to be significantly (P B 0.05) deceased in the plasma of untreated arthritic rats. Individual treatments with aspirin or celecoxib partially upregulated reduced glutathione levels at all time points; however, they failed to normalize it. Similar results were obtained with EPO either alone or in combination with aspirin (on days 9 and 18) as well as in combination with celecoxib (on day 9). On the other hand, restoration of reduced glutathione normal level was observed upon treatment with EPO either alone (on day 27) or in combination with aspirin (on day 27) or celecoxib (on days 18 and 27). Interestingly, combined treatment of EPO with
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R. M. El-Sayed et al. Fig. 5 Photomicrographs represent changes in the hind limb of adjuvant-induced arthritic rats. a Normal, b control, c aspirin, d celecoxib, e EPO, f EPO and aspirin, g EPO and celecoxib. Histopathological examination of H&E stained sections from foot joint showed significant decrease in the vascularity in comparison to control rats (arrows for blood vessels) (1009) (n = 6)
aspirin or celecoxib was superior to corresponding individual treatments at all time point (Fig. 7c).
Discussion Angiogenesis is a critical event in rheumatoid arthritis pathogenesis, and is up-regulated by an intricate balance of pro- and anti-angiogenic regulators, which include members of VEGF and ANG receptor-ligand families (Paleolog 2002). Our study was carried out to evaluate the antiangiogenic effect of EPO and NSAIDs (aspirin and celecoxib, as non-selective and selective cyclooxygenase inhibitor, respectively) on adjuvant-induced arthritic rats. Results of the present study showed that the induction of arthritis was accompanied by a significant weight loss that
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could not be improved by aspirin or celecoxib treatment. Adjuvant-induced arthritis is an immune response to an antigen present on the capsule of the mycobacterium that has been previously reported to produce systemic features of inflammation, such as uveitis, inflammation of the gastrointestinal tract and body weight loss that starts 24–48 h before the clinical onset of arthritis (Prakken et al. 2003). Findings of the present study revealed an anti-inflammatory effect of all individual treatments that has enhanced up on combination as was evident by a significant reduction in rat ankle circumference in treated groups. These results are in accordance with those obtained by Singh et al. (2011) who showed that aspirin produced a significant decrease in the joint diameter as compared to adjuvant-induced arthritic rats on all observation days. Aspirin and celecoxib suppress inflammation by inhibiting the enzyme
Novel role of angiopoietin-1
Fig. 6 Histopathological scores of the hind limb in adjuvant-induced arthritic rats either untreated or treated with different regimens. Monocellular infiltration, pannus invasion into the cartilage and vascularity were measured by microscopic examination on two separate occasions, scores of the sections are expressed as
mean ± SEM. All data were analyzed using ANOVA followed by Bonferroni post hoc test. *P B 0.05 with respect to normal, # P B 0.05 with respect to control, filled circle P B 0.05 with respect to corresponding individual treatment, open circle P B 0.05 with respect to EPO, (n = 6)
cyclooxygenase resulting in decreased prostaglandin synthesis and thromboxanes, the two main classes of lipidderived pro-inflammatory molecules. It worth mentioning that the effect of EPO was superior to either aspirin or celecoxib. The use of EPO as a source of GLA has been investigated, and several clinical applications on inflammatory disorders have been described (Zurier et al. 1996; Rosenstein et al. 2003). The rationale of its therapeutic use is based on the high amount (9 %) of gamma linolenic acid (Kunkel et al. 1981). The antiinflammatory activity of EPO may be attributed to the metabolism of gamma linolenic acid to dihomo gamma linolenic acid (DGLA) and thus competitive inhibition of the 2-series prostaglandins and 4-series leukotrienes (Jantti et al. 1989). DGLA itself cannot be converted to leukotrienes but can form a 15-hydroxyl derivative that blocks the transformation of arachidonic acid to leukotrienes. As a result, increased DGLA intake possibly will suppress inflammation (Voorhees 1983). Previous studies have described the expression of ANG1 and ANG-2 (Scott et al. 2002; Gravallese et al. 2003) and their receptors Tie-1 and Tie-2 (Shahrara et al. 2002; Uchida et al. 2000) in rheumatoid arthritis. Furthermore, overexpression of VEGF and ANG-1 in the arthritic joints was found to be associated with distinct vascular morphology (Szekanecz and Koch 2007). We observed increased ANG-1 concentration in the plasma of early adjuvant-induced arthritis that had decreased dramatically by day 18 post adjuvant administration indicating a vital role of ANG-1 at early stages of disease progression. Our results are in agreement with Shahrara et al. (2002) who demonstrated greater expression of ANG-1, ANG-2, Tie-1 and Tie-2 in rheumatoid arthritis as compared with normal synovium. Moreover, Clavel et al. (2007) reported that
serum concentrations of VEGF, sFlt-1 and ANG-1 were correlated to parameters of inflammation and to bone destruction in early arthritis. Malik et al. (2010) demonstrated that the ANG–Tie ligand–receptor system is dysregulated in collagen-induced arthritis. Tie1-751, a novel splice variant of the Tie1 receptor, inhibits ANG-1/ VEGF signaling, suggesting that ANG inhibition may be of therapeutic benefit in inflammatory arthritis (Malik et al. 2010). Regarding the anti-angiogenic effect of our studied drugs, both aspirin and celecoxib significantly decreased plasma ANG-1 level at early stage of adjuvant-induced arthritis development (i.e. on day 9). Interestingly, oral administration of EPO either alone or in combination with aspirin or celecoxib successfully maintained normal levels of ANG-1 at all time points (i.e. on days 9, 18 and 27). The anti-angiogenic effect of gamma linolenic acid or cyclooxygenase-2 inhibitors has been previously reported in different settings. Miyake et al. (2009) reported in their study that gamma linolenic acid inhibited glioma cell proliferation in vivo, an effect that involved changes in protein expression of VEGF, VEGF receptors (Flt1 and Flk1), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP9), ERK1 and ERK2. Furthermore, they have shown that gamma linolenic acid altered the expression of several proteins involved in cell cycle control (Miyake et al. 2009). Besides, Wang et al. (2008) reported that celecoxib enhanced tumor cell apoptosis, thereby inhibited the growth and angiogenesis of implanted tumors in a mouse model of human colorectal cancer by inhibiting COX-2, PGE2 synthesis, and VEGF and matrix MMP-2 mRNA expression in tumor tissue (Wang et al. 2008). Also, Refaat et al. (2013) have recently reported that treatment with celecoxib caused a remarkable decrease in VEGF serum levels in adjuvant arthritic rats. That could be
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R. M. El-Sayed et al. Fig. 7 Effect of aspirin (150 mg/kg), celecoxib (5 mg/ kg), EPO (5 g/kg) and their combinations on time course evaluation of lipid peroxidation (a), superoxide dismutase (b), and reduced glutathione (c) in adjuvant-induced arthritic rats. Analyses were done on days 9, 18 and 27. Serum levels of malondialdehyde as thiobarbituric acid-reactive substances were measured spectrophotometrically and expressed as nmol/mL. Superoxide dismutase and reduced glutathione were evaluated spectrophotometrically and expressed as U/mL and mg/dL, respectively. Values are expressed as mean ± SEM. All data were analyzed using ANOVA followed by Bonferroni post hoc test. *P B 0.05 with respect to normal, #P B 0.05 with respect to control on corresponding day, filled circle P B 0.05 with respect to corresponding individual treatment (i.e. aspirin or celecoxib), open circle P B 0.05 with respect to EPO, (n = 6)
explained based on previous observation that the reduction of PGE2 induced by cyclooxygenase inhibitors resulted in less production of VEGF in human synovial fibroblasts (Inoue et al. 2002; Ma et al. 2002). To the best of our knowledge, the current study is the first to report the antiangiogenic effect of EPO rich in gamma linolenic acid in adjuvant-induced arthritis model. This anti-angiogenic effect was found to be mediated through early inhibition of ANG-1. In addition, cyclooxygenase inhibitors used in the current study showed also an anti-angiogenic potential also through ANG-1 inhibition. Previous reports showed a rise in the inflammatory mediator TNF-a during disease progression of adjuvantinduced arthritic rats (Cai et al. 2006; Kushner et al. 2010; Refaat et al. 2013). The time course evaluation of plasma TNF-a levels in arthritic group came in parallel with ankle inflammation that was reported in the current study.
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Surprisingly, the observed increment in TNF-a levels had gradually decreased by day 18 then 27 post complete Freund’s adjuvant administration. The effect of individual and combined regimens used in the current study on TNF-a levels showed a similar pattern to ankle measurements with highest anti-inflammatory effect observed in combination groups. The anti-inflammatory activity of individual treatments used currently is in agreement with previous reports. Purasiri et al. (1997) have shown that dietary gamma linolenic acid suppressed inflammatory cytokines level in human blood lymphocytes. In addition, Khayyal et al. (2005) reported that aspirin as well as celecoxib showed a similar effect in reducing TNF-a in adjuvant-induced arthritic rats. Moreover, El-Ghazaly et al. (2010) reported that treatment with celecoxib was effective in decreasing the elevated levels of TNF-a in irradiated adjuvant-induced arthritic rats. These observed anti-inflammatory and anti-
Novel role of angiopoietin-1
angiogenic activities were confirmed in the current study by histopathological findings which revealed that EPO significantly reduced the synovial hyperplasia and inflammatory cells invasion in joint tissues, an effect that was enhanced by combination with aspirin or celecoxib. Essential fatty acids such as gamma linolenic acid and linolenic acid suppressed ROS generation to a significant degree in a dose-dependent fashion and raised superoxide dismutase activity significantly in hyperglycaemia-induced excess proliferation of retinal vascular endothelial cells (Shen et al. 2012). Also, Arimura et al. (2004) demonstrated that evening primrose extract exposure elicited a rapid increase in the activity of superoxide dismutase and intracellular peroxides levels in Ehrlich ascites tumor cells. In the same way, EPO treatment used in the current study showed indispensable role in reducing ROS in adjuvant arthritic model. The induction of adjuvant arthritis resulted in cartilage destruction that involves numerous and interconnected mechanisms. This include excessive generation of ROS, several cytokines, reduction in the level of reduced glutathione associated with a corresponding rise in malondialdehyde and a rise in the level of nitric oxide indicating a status of oxidative stress (Arend 2001; Henrotin et al. 2003; Jawed et al. 2010; El-Ghazaly et al. 2010). This was confirmed in the present study, where a depressed anti-oxidant status was manifested by a gradual but significant increase in lipid peroxidation that was accompanied by a great reduction in reduced glutathione levels and superoxide dismutase activities along the course of disease progression in arthritic rats. These results are in agreement with a recent study by Rasool and Varalakshmi (2007) who referred such results to the weak free radical defence system against oxidative stress that might explain the pathogenic state associated with arthritis. Regarding lipid peroxidation, all treatments significantly decreased malondialdehyde level on days 18 and 27 while the normalization was observed on day 27 only on combined treatments. In contrast, El-Ghazaly et al. (2010) reported that treatment with celecoxib showed no effect on superoxide dismutase activity and levels of malondialdehyde and reduced glutathione in adjuvant-induced arthritis model in irradiated rats. This could be due to different experimental design. On the other hand, Cha´vez et al. (2010) reported that celecoxib supported the recovery of livers with necrotic and cholestatic damage through antioxidant activities that were manifested by restoration of redox equilibrium and inhibition of lipid peroxidation. We observed that all treatments significantly increased reduced glutathione levels at all time points but the effect of EPO alone or combined with aspirin or celecoxib was superior to individual treatments with cyclooxygenase inhibitors. In the same way, superoxide dismutase activities were gradually resumed in all treatment groups while normalization
was observed only on day 27 up on combination of EPO with celecoxib. These results in agreement with the study of De La-Cruz et al. (1999) who showed that dietary supplementation with EPO decreased tissue oxidative stress, through reduction of lipid peroxidation and increased glutathione in all tissues. They reported that EPO was able to enhance the activities of glutathione reductase and consequently resulted in decreased percentage of oxidized glutathione.
Conclusions The current study showed for the first time that ANG-1 plays a vital role in the early rather than late phase of adjuvant-induced arthritis pathogenesis making it an attractive non-invasive tool for early diagnosis and prediction of disease prognosis. This study also suggest that ANG-1 inhibition may be of therapeutic value in inflammatory arthritis. It can be concluded that early intervention with natural oil rich in gamma linolenic acid, which possess anti-angiogenic, anti-inflammatory, and anti-oxidant activities, with traditional analgesics can improve therapeutic benefits and represent a promising strategy to restrain the progression of rheumatoid arthritis. Current data propose EPO as a potential inhibitor of pathological angiogenesis. Acknowledgments The authors express gratitude to Dr. Hend M. Tag, Assistant Professor of Physiology, Faculty of Science, Suez Canal University, Ismailia, Egypt, for her valuable assistance with photomicrography and histopathology scoring. They also would like to thank Mr. Mohamed I. El-Assaal for his great efforts in keeping the animals under uniform experimental conditions. Celecoxib and aspirin were kindly provided by Pfizer Co. for Pharm. & Chemical Ind. and Sigma Pharmaceutical Co., Egypt, respectively. Conflict of interest
None.
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