Int J Pharm Pharm Sci, Vol 7, Issue 12, 178-183Original Article


THE EFFECT OF PROPHYLACTIC INHIBITION OF INDUCIBLE NITRIC OXIDE SYNTHASE BY AMINOGUANIDINEON SERUM LEVELS OF SOME ADIPOCYTOKINES IN PRISTANE-INDUCED ARTHRITISIN RATS

SHATHA H. ALI1, ALI A. KASIM1,2

1Department of Clinical Laboratory Sciences, College of Pharmacy, University of Baghdad, Baghdad-Iraq
Email: ali_a_kasim@yahoo.com

 Received: 07 Sep 2015 Revised and Accepted: 27 Oct 2015

ABSTRACT

Objective: The aim of the present study was to evaluate the effect of prophylactic inhibition of inducible nitric oxide synthase (iNOS) by aminoguanidine (AG) on serum levels of some adipocytokines in pristane-induced arthritis in rats.

Methods: Forty white Albino rats were divided randomly into four groups; each group was composed of ten rats (five male and five female). Group I (AP) has received AG (100 mg/kg/day) i. p. for seven days, and then at day 8, has received single 150 µl pristane dose sc. at the base of rat’s tail. Group II (PC) has only received single 150 µl pristane sc. at the base of rat’s tail, at day 8 from the start of the experiment. Group III (AC) has only received AG (100 mg/kg/day)i. p. for seven days. Group VI (VC) has only received normal saline via i. p. injection for seven days. At the end of the experiment time, rats were sacrificed, and serum samples were obtained and used for the measurement of iNOS, rheumatoid factor (RF), c-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α), interleukine-6 (IL-6), leptin and adiponectin levels, using the corresponding rat ELISA kits.

Results: Administration of pristane for the induction of RA has resulted in significant increase in all of the measured parameters in PC group as compared to VC and AC groups, except for iNOS where the increase was significant as compared to AC group only. Serum levels of RF, CRP and IL-6 in AP group have showed to be significantly elevated as compared to VC and AC groups. Administration of AG has resulted in different levels of reduction in all of the measured parameters in AP group as compared to PC group. The reduction was statistically significant with regard to CRP and leptin.

Conclusion: prophylactic administration of the selective iNOS inhibitor AG, has resulted in a reduction in serum levels of the measured adipocytokines which may reflect a reduction in the severity of PIA.

Keywords: Adipocytokines, Aminoguanidine, iNOS, Pristane, Rheumatoid Arthritis.

© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

INTRODUCTION

Rheumatoid arthritis (RA) is a chronic, progressive, polyarthritic autoimmune disease associated with articular, and extra-articular or systemic effects [1]. It is one of the most common arthritides in western populations, with an estimated prevalence of 0.5-1.0% among adults. RA is more prevalent among women than men with a female preponderance of 2-3:1 [2, 3].

The perception of adipose tissue, as an organ that merely passively store excess fat and act as an energy store has changed. It is now considered as an endocrine organ that has an important role in regulating diverse physiological functions. This regulation is mediated by the action of biological mediators called “adipocytokines”.

More than 50 different adipocytokines are currently recognized to be secreted from adipose tissue. These adipocytokines are implicated in the modulation of a range of physiological functions such as inflammation, glucose homeostasis, lipid metabolism, blood coagulation, angiogenesis, and blood pressure [4]. Adipose tissue produces and releases a variety of proinflammatory and anti-inflammatory factors, including the adipokines leptin, adiponectin, resistin, and visfatin, cytokines such as TNF-α, IL-6, and many others [5].

Nitric oxide (NO) is a free-radical messenger molecule that is produced from the amino acid L-arginine by the enzymatic action of NO synthase (NOS) [6]. Nitric oxide synthase (NOS) is available in both constitutive and inducible forms. Three different nitric oxide synthase (NOS) proteins have been identified, eNOS, iNOS and nNOS (endothelial, inducible and neuronal NOS, respectively) [7].

In autoimmune reactions and other forms of chronic inflammation, NO and NO-derived radicals can harm the host tissues [8]. Among its various biological actions, NO inhibits chondrocytes synthesis of collagen and proteoglycan, stimulates matrix metalloproteinases, and promotes vasodilation, which facilitates the influx of fluid and cells in to an inflammatory site. Furthermore, NO combines with reactive oxygen species, forming peroxynitrite, which promotes chondrocyte apoptosis [9].

Elevated NO production has been reported in RA patients [10, 11], and the increased urinary nitrite (metabolite of NO) concentrations have been shown to diminish after prednisolone treatment [12]. Elevated levels of NO were shown to correlate with several RA disease activity parameters [13]. Increased iNOS expression and NO production have also been detected in animal models of arthritis [14, 15], and the use of NOS inhibitors have been shown to have beneficial effects in experimentally induced arthritis [16, 17].

Aminoguanidine (AG) is a selective inhibitor of iNOS. It is over 50-fold more effective at inhibiting the enzymatic activity of iNOS than endothelial or neuronal isoforms of NOS [18].

Pristane‐induced arthritis (PIA) in rats resembles the human disease such as in the development of symmetrical disease, the presence of serum rheumatoid factors, radiographic changes and chronicity [19]. The aim of the present study was to evaluate the effect of prophylactic iNOS inhibition by AG on serum levels of some adipocytokines in PIA in rats.

MATERIALS AND METHODS

Experimental animals

Forty white Albino rats of comparable age and sex, and weighing 150-200 gm were used in this study; they were obtained from and maintained in the Animal House of the Pharmacy College, University of Baghdad, in a climate‐controlled environment. The animals were housed in polystyrene cages containing wood shavings and fed standard rodent chow and water ad libitum. The study was approved by the ethical committee at the Pharmacy College, University of Baghdad (Ethical approval number 5537; date 30th November 2014).

PIA induction and evaluation of arthritis

PIA was induced by a single subcutaneous (s. c.) injection of 150μl pristane (2,6,10,14‐tetramethylpentadecane; Sigma-Aldrich, St. Louis, Missouri, USA) at the base of rat’s tail[19] at the age of 8–12 w. Arthritis development and severity were monitored in all four limbs every seven days by the perimeters of ankle and mid-paw, and a macroscopic scoring system. Briefly, the development of arthritis was monitored using a macroscopic scoring system for the four limbs ranging from 0-4 (1 = swelling and redness of one joint, 2 = two joints involved, 3 = more than two joints involved, and 4 = severe arthritis in the entire paw). The scores of the four paws were added, yielding a maximum total score of 16 for each rat [20].

Preparation of AG solution for injection

The daily dose of AG (Aminoguanidine HCl; Sigma-Aldrich, St. Louis, Missouri, USA) was prepared by dissolving the required amount in normal saline, and the dose was administered intraperitoneally (i. p.). AG dosage (100 mg/kg) was selected depending on the levels used effectively in experimental animal models for iNOS blockage [21-23].

Experimental protocol

Forty white Albino rats were enrolled in this experiment. The rats were divided randomly into four groups; each group was composed of ten rats, five of which were male and five were female. The study groups was treated as follows:

Group I (AP): has received AG treatment (100 mg/kg/day) i. p. for seven days, and then at day 8, has received single 150 µl pristane dose sc. at the base of rat’s tail for the induction of RA.

Group II (PC): has received the pristane dose only (single 150 µl pristane sc. at the base of rat’s tail for the induction of RA), at day 8 from the start of the experiment.

Group III (AC): has only received the AG treatment (100 mg/kg/day) i. p. for seven days.

Group VI (VC): has only received the solvent (normal saline) via i. p. injection for seven days.

Preparation of serum samples

After four weeks of last treatment, the animals have been anesthetized by ether, and then the blood was collected by cervical decapitation and transferred into a plain tube and left to clot for ten minutes before being centrifuged at 2,000 rpm for another 10 min. The sera were collected using rubber micropipette and divided into small aliquots in Eppendorf tubes and kept frozen till the time of analysis.

Biochemical analysis

Serum levels of iNOS, rheumatoid factor (RF), c-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), leptin, and adiponectin were measured by quantitative enzyme-linked immunosorbent assay (ELISA), using the corresponding readymade kits purchased from (Elabscience Biotechnology; China), and according to the manufacturer instructions.

Statistical analysis

Analysis of data was performed using GraphPad Prism software version 6.05 for Windows (GraphPad, San Diego, CA, USA). Values are presented as mean±SEM. The significance of difference among groups was tested by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc analysis to compensate for multiple testing. Homogeneity of variance was tested by, Brown-Forsythe’s and Bartlett's Test. Statistical significance was considered whenever the P value was equal or less than 0.05.

RESULTS

Administration of pristane for the induction of RA has resulted in significant increase in all of the measured parameters in PC group as compared to VC and AC groups, except for iNOS where the increase was significant as compared to AC group only [(9.760±1.091 ng/ml vs. 5.100±0.6495 ng/ml for iNOS), (8.828±0.686kIU/lvs. 1.503±0.3833 kIU/land 1.786±0.3642kIU/l for RF), (22.15±1.532 mg/l vs. 7.06±0.760 mg/l and 9.01±1.238 mg/l for CRP), (55.00±4.874 pg/ml vs. 25.98±4.667 pg/ml and 34.79±5.346 pg/ml for TNF-α), (704.3±52.01 pg/ml vs. 175.5±30.71 pg/ml and 170.6±28.43 pg/ml for IL-6), (3.422±0.1923µg/l vs. 1.623±0.1857µg/land 1.998± 0.2108µg/l for leptin) and (47.09±3.464 ng/ml vs. 32.71±4.003 ng/ml and 33.26±3.822 ng/ml for adiponectin) respectively]. In AG treatment group (AP group), serum levels of RF, CRP and IL-6 have showed to be significantly elevated as compared to VC and AC groups [(7.913±0.4628kIU/l vs. 1.503±0.3833kIU/land 1.786±0.3642kIU/l for RF), (15.88 mg/l±1.416 vs. 7.06±0.760 mg/l and 9.01±1.238 mg/l for CRP) and (676.0±58.15 pg/ml vs. 175.5±30.71 pg/ml and 170.6±28.43 pg/ml for IL-6) respectively], as shown in table1.

Administration of AG has resulted in different levels of reduction in all of the measured parameters in AP group as compared to PC group. The reduction was statistically significant with regard to CRP and leptin [(15.88±1.416 mg/l vs. 22.15±1532 mg/l for CRP) and (1.917±0.2208µg/l vs. 3.422±0.1923µg/l for leptin) respectively], as shown in table 1.

Table 1: Serum levels of the studied parameters

 

VC n=10

AC n=10

PC n=10

AP n=10

ANOVA P-value

iNOS (ng/ml)

6.211±1.239

5.100±0.6495

9.760±1.091b

7.200±0.8868

0.0152

RF (kIU/l)

1.503±0.3833

1.786±0.3642

8.828±0.686a,b

7.913±0.4628a,b

<0.0001

CRP (mg/l)

7.06±0.760

9.01±1.238

22.15±1.532a,b,c

15.88±1.416a,b

<0.0001

TNF-α (pg/ml)

25.98±4.667

34.78±5.346

55.00±4.874a,b

40.11±3.402

<0.0009

IL-6 (pg/ml)

175.5±30.71

170.6±28.43

704.3±52.01a,b

676.0±58.15a,b

<0.0001

Leptin (µg/l)

1.623±0.1857

1.998±0.2108

3.422±0.1923a,b,c

1.917±0.2208

<0.0001

Adiponectin (ng/ml)

32.71±4.003

33.26±3.822

47.09±3.464a,b

44.27±2.356

0.0082

Data are expressed as mean±SEM, VC= Vehicle control group, AC= Aminoguanidine control group, PC= Pristanecontrol group, AP= Treatment group (Aminoguanidine+Pristane), iNOS: Inducible Nitric Oxide Synthase, RF: Rheumatoid Factor, CRP: C - reactive protein, TNF-α: Tumor Necrosis Factor-alpha, IL-6: Interleukon-6. a = Significant difference as compared to VC group, b=Significant difference as compared to AC group, c= Significant difference as compared to AP group. A significant difference between groups is considered using ANOVA test whenever (P<0.05).

DISCUSSION

The detailed mechanisms of RA pathogenesis are not totally understood, and hence, the factors responsible for the initiation and progression of this disease are the core subject of many studies. The articular and synovial adipose tissue represents an important entity of human joints, and local and systemic alteration in the synthesis, release, and receptor action of adipocytokines needs further study to elucidate the potential role of these adipocytokines in RA.

In the present study, the administration of pristane for the induction of AR has resulted in a significant elevation in serum levels of iNOS, RF, CRP, TNF-α, IL-6, leptin and adiponectin, in PC group as compared to VC group (table1).

In addition to macrophages where it is primarily identified, iNOS expression can be induced almost in any cell or tissue, as long as the appropriate agents have induced its synthesis [24]. At the synovium of the inflamed joint macrophages, chondrocytes, osteoclasts and osteoblasts express iNOS [25]. In addition, the inflamed synovium is invaded by T lymphocytes, B lymphocytes, neutrophils, monocytes, and dendritic cells [26] which also participate in iNOS expression.

In the present study, the administration of AG a selective iNOS inhibitor has resulted in small, non-significant decrease in iNOS levels in AC group as compared to VC group. This is very expected since the enzyme is usually not expressed in the normal physiological conditions. Nevertheless, AG treatment has resulted in a profound reduction (still statistically non-significant), in iNOS levels in AP group as compared to PC group. iNOS expression (and action) is controlled by large and different regulatory mechanisms[27, 28]. Some of these regulators are bone derived [29, 30]. On the other hand, adipose tissue has a role in inflammatory pathways, through the secretion of many cytokines that alter the expression of iNOS [31-34]. TNF-α has been found to upregulate iNOS transcription in synovial fibroblasts, articular chondrocytes and osteoblasts cultured from RA patients [10]. IL-6 also has been found to increase iNOS expression [35]. In human and murine chondrocytes, leptin synergizes with IL-1𝛽 and IFN𝛾 for the activation of iNOS [36-38]. In cultured chondrocytes, adiponectin increases the secretion and activity of many proinflammatory mediators, including iNOS [39]. Thus, elevated serum levels of TNF-α, IL-6, leptin and adiponectin, reported in the present study may explain, at least in part, why AG has failed to bring down serum iNOS levels in the treatment group AP to that of VC group.

Rheumatoid factors (RFs) are typically produced during secondary immune responses against infections or immunizations [40]. RFs are detected in 60–80% of RA patients [41]. High titer RF is associated with radiologic erosion, extra-articular manifestations and thus, poorer outcomes [41-44]. RF has proven to be the most useful disease marker of RA, as included in the American College of Rheumatology classification criteria for RA [45].

The animal model that has been used in the present study, PIA rat model, resembles the human disease in the development of symmetrical disease, chronicity, radiographic changes and presence of serum RF [19].

C-reactive protein (CRP) is an acute-phase reactant that is commonly used in the diagnosis and follow-up of disease activity in rheumatology clinics. Even though it is called acute phase reactants, CRP rises in both acute and chronic inflammatory conditions [46].

In the present study, the administration of pristane has resulted in significant increase in CRP levels in both PC and AP groups as compared to VC and AC groups. This is greatly expected since pristane induces both cellular and humoral responses during the course of RA induction [47].

The synthesis of CRP is regulated primarily by interleukin-6 (IL-6), which in turn is controlled by other inflammatory cytokines, for instance, TNF-α and IL-1[48-50]. This occurs in accordance with the results of the present study regarding IL-6 and TNF-α (table1), which has showed a significant increase in IL-6 and TNF-α in pristane-treated groups.

Adipokines have been noted as new mediators of inflammatory processes [51, 52]. Many studies have demonstrated an elevated serum level of many adipokines in patients with RA, and an association between the levels of these adipokines and CRP [53-55]. In the present study serum levels of both leptin and adiponectin (table 1) have been shown to be elevated in pristane-treated groups. Thus, may further contribute to the elevated CRP levels.

Tumor necrosis factor-alpha (TNF-α), exhibits both the proinflammatory and immunoregulatory properties of cytokines. It is produced mainly by monocytes and macrophages, but also by B-cells, T-cells fibroblasts and adipocytes [56].

In the present study, the administration of pristane has resulted in significant increase in serum TNF-α level in PC group, as compared to VC and AC groups (table1). This result agrees with the findings of many clinical and experimental studies which have reported high synovial and serum levels of TNF-α in RA, and that TNF-α plays an important role in inflammation and joint destruction that are hallmarks of the disease [57-61].

TNF-α is one of the key cytokine molecules that causes inflammation in RA, by both direct [58], and indirect action through stimulating the release of other proinflammatory cytokines such as IL-6 [62-64]. Furthermore, TNF-α and leptin control the expression of each other. In one hand, TNF-α enhances the expression of leptin and its receptors [65]. On the other hand, leptin enhances TNF-α expression [66-68]. Thus, TNF-α may be an important contributor to the elevated serum levels of IL-6 and leptin, reported in the present study.

Interlekin-6 (IL-6) plays a role in adaptive immune responses involved in the pathogenesis of RA. In addition, this cytokine is responsible for mediating many of the systemic manifestations of RA [50].

In the present study, the administration of pristane has resulted in significant increase in serum IL-6 levels in PC and AP groups as compared to VC and AC groups (table1). This result agrees with the clinical findings that have reported an excess production of IL-6 in the synovial fluid and blood of RA patients and correlates with the disease activity and joint destruction [69, 70].

IL-6 induces the acute-phase response, particularly the development of CRP [71], which suggests that IL-6 contributes to the elevated serum levels of CRP reported in the present study.

Leptin is mainly produced by white adipose tissue and the circulating levels of leptin correlate positively with the amount of adipose tissue and body mass index (BMI) [72]. However, leptin synthesis is also regulated by the action of inflammatory mediators [73].

In the present study, the administration of pristane has resulted in significant increase in leptin levels in PC group as compared to VC and AC group (table 1). This result is in accordance with many studies that have reported elevated leptin levels in patients with RA and that serum leptin correlates with synovial fluid: serum leptin ratio and disease duration and parameters of RA activity [74-76]. Moreover, leptin has been found to correlate with CRP level in RA patients, which indicates that it may act as a proinflammatory cytokine in RA [53]. Generally, leptin behaves as proinflammatory cytokine due to its synergistic role with IFN-γ and IL-1β on iNOS [36-38].

The actions of leptin in RA are not only targeted to articular tissues, this adipokine also exerts direct modulatory effects on activation, proliferation, maturation and production of inflammatory mediators in a variety of immune cells, it activates monocyte/macrophage cells and increases production of the pro-inflammatory cytokines (TNF-α and IL-6) and reactive oxygen species (ROS) [77].

Some studies have reported conflicting data on serum levels of leptin in RA patients. Leptin levels were reported in some studies to be similar [78, 79], or even lower [80] in RA patients as compared with healthy controls. The discrepancies in the measured leptin levels may be attributed to the fact that, leptin levels are influenced by many factors such as body fat content, BMI, treatment, and disease activity and progression [81].

Adiponectin is produced mainly by white adipose tissue. It has structural homology with collagens VIII and X and complements factor C1q and the proinflammatory cytokine TNF-α, and it circulates in the blood in relatively large amounts in different molecular forms [82, 83].

Adiponectin concentration is decreased in diseases associated with low-grade inflammation [84, 85]. Moreover, its production is suppressed by inflammatory cytokines such as TNF-α and IL-6 [34]. On the other hand, adiponectin exerts a variety of anti-inflammatory effects on metabolic pathways and vasculature [86]. However, adipokines are elevated in RA and other classical inflammatory diseases [87-89].

Despite the discrepancy, adiponectin may have proinflammatory rather than anti-inflammatory properties in RA. Adiponectin shares extensive sequence homologies with the complement factor C1q and the pro-inflammatory cytokine TNF-α [82, 83]. Furthermore, adiponectin has demonstrated to induce the production of IL-6 and pro-MMP1 [89-91], key mediators of destructive arthritis.

In the present study, the administration of pristane has resulted in significant increase in serum adiponectin levels in PC group as compared to VC and AC groups (table1). This result is in total agreement with the findings of many studies that have reported that the amount of adiponectin is increased in the synovial fluid [92], and serum[93]of patients with RA, with higher levels in erosive versus mild RA [93-95], and in chronic versus early RA [96].

Articular adipose tissues may be the main source of adiponectin in RA, yet, serum adiponectin level has been reported to be higher than in synovial fluid in RA[93], suggesting that other systemic sources are more prominent than articular adipose tissues. Furthermore, the negative association of glomerular filtration rate and serum adiponectin level has been reported [97], indicating that impaired renal clearance of adiponectin may lead to increased adiponectin level in inflammatory diseases as well.

Finally, in the present study the administration of the selective iNOS inhibitor AG, has resulted in a reduction in serum levels of RF, CRP, TNF-α, IL-6, leptin and adiponectin, in AP group as compared to PC group. The reduction was statistically significant with regard of CRP and leptin (table 1). This reduction may be attributed to the decrease in the severity of PIA by the administration of AG.

This study is rather limited in terms of statistical power because of the small sample size and further large-scale animal and clinical studies are needed to establish our findings. Also, further work is required to confirm whether the reported effects are confined to AG or the whole members of iNOS inhibitors family of agents.

ACKNOWLEDGEMENT

The data were abstracted from Ph.D. Thesis submitted by Ali A. Kasim to the Department of Clinical Lab. Sciences, College of Pharmacy, University of Baghdad. The authors thank the University of Baghdad for supporting the project.

CONFLICT OF INTERESTS

Declared None

REFERENCES

  1. Choy E. Understanding the dynamics: pathways involved in the pathogenesis of rheumatoid arthritis. Rheumatology 2012;51(Suppl 5):v3-v11.
  2. Gabriel SE, Crowson CS, O'Fallon WM. The epidemiology of rheumatoid arthritis in Rochester, Minnesota, 1955-1985. Arthritis Rheum 1999;42:415-20.
  3. Neovius M, Simard JF, Askling J. Nationwide prevalence of rheumatoid arthritis and penetration of disease-modifying drugs in Sweden. Ann Rheum Dis 2011;70:624-9.
  4. Mac Dougald OA, Burant CF. The rapidly expanding family of adipokines. Cell Metab 2007;6:159-61.
  5. Fantuzzi G. Adipose tissue, adipokines, and inflammation. J Allergy Clin Immunol 2005;115:911-9.
  6. Bredt DS. Endogenous nitric oxide synthesis: biological functions and pathophysiology. Free Radic Res 1999;31:577-96.
  7. Bryan NS, Bian K, Murad F. Discovery of the nitric oxide signaling pathway and targets for drug development. Front Biosci 2009;14:1-18.
  8. Nagy G, Clark JM, Buzas EI, Gorman CL, Cope AP. Nitric oxide, chronic inflammation and autoimmunity. Immunol Lett 2007;111:1-5.
  9. Lotz M, Hashimoto S, Kuhn K. Mechanisms of chondrocyte apoptosis. Osteoarthritis Cartilage 1999;7:389-91.
  10. Grabowski PS, England AJ, Dykhuizen R, Copland M, Benjamin N, Reid DM, et al. Elevated nitric oxide production in rheumatoid arthritis. Detection using the fasting urinary nitrate: creatinine ratio. Arthritis Rheum 1996;39:643-7.
  11. Ali AM, Habeeb RA, El-Azizi NO, Khattab DA, Abo-Shady RA, Elkabarity RH. Higher nitric oxide levels are associated with disease activity in Egyptian rheumatoid arthritis patients. Rev Bras Reumatol 2014;54:446-51.
  12. Stichtenoth DO, Fauler J, Zeidler H, Frolich JC. Urinary nitrate excretion is increased in patients with rheumatoid arthritis and reduced by prednisolone. Ann Rheum Dis 1995;54:820-4.
  13. Onur O, Akinci AS, Akbiyik F, Unsal I. Elevated levels of nitrate in rheumatoid arthritis. Rheumatol Int 2001;20:154-8.
  14. Cuzzocrea S. Role of nitric oxide and reactive oxygen species in arthritis. Curr Pharm Des 2006;12:3551-70.
  15. Vuolteenaho K, Moilanen T, Knowles RG, Moilanen E. The role of nitric oxide in osteoarthritis. Scand J Rheumatol 2007;36:247-58.
  16. Weinberg JB, Granger DL, Pisetsky DS, Seldin MF, Misukonis MA, Mason SN, et al. The role of nitric oxide in the pathogenesis of spontaneous murine autoimmune disease: increased nitric oxide production and nitric oxide synthase expression in MRL-lpr/lpr mice, and reduction of spontaneous glomerulonephritis and arthritis by orally administered NG-monomethyl-L-arginine. J Exp Med 1994;179:651-60.
  17. Connor JR, Manning PT, Settle SL, Moore WM, Jerome GM, Webber RK, et al. Suppression of adjuvant-induced arthritis by selective inhibition of inducible nitric oxide synthase. Eur J Pharmacol 1995;273:15-24.
  18. Corbett JA, McDaniel ML. The use of aminoguanidine, a selective iNOS inhibitor, to evaluate the role of nitric oxide in the development of autoimmune diabetes. Methods 1996;10:21-30.
  19. Vingsbo C, Sahlstrand P, Brun JG, Jonsson R, Saxne T, Holmdahl R. Pristane-induced arthritis in rats: a new model for rheumatoid arthritis with a chronic disease course influenced by both major histocompatibility complex and non-major histocompatibility complex genes. Am J Pathol 1996;149:1675-83.
  20. Nordquist N, Olofsson P, Vingsbo-Lundberg C, Petterson U, Holmdahl R. Complex genetic control in a rat model for rheumatoid arthritis. J Autoimmun 2000;15:425-32.
  21. Parlakpinar H, Ozer MK, Acet A. Effect of aminoguanidine on ischemia-reperfusion-induced myocardial injury in rats. Mol Cell Biochem 2005;277:137-42.
  22. Xu J, Li N, Dai DZ, Yu F, Dai Y. The endothelin receptor antagonist CPU0213 is more effective than aminoguanidine to attenuate isoproterenol-induced vascular abnormality by suppressing overexpression of NADPH oxidase [correction of oxidas], ETA, ETB, and MMP9 in the vasculature. J Cardiovasc Pharmacol 2008;52:42-8.
  23. Ozturk A, Firat C, Parlakpinar H, Bay-Karabulut A, Kirimlioglu H, Gurlek A. Beneficial effects of aminoguanidine on skin flap survival in diabetic rats. Exp Diabetes Res 2012;8. doi.org/10.1155/2012/721256. [Article in Press]
  24. Pautz A, Art J, Hahn S, Nowag S, Voss C, Kleinert H. Regulation of the expression of inducible nitric oxide synthase. Nitric Oxide 2010;23:75-93.
  25. Wimalawansa SJ. Nitric oxide and bone. Ann N Y Acad Sci 2010;1192:391-403.
  26. Taylor PC, Mehta P, Tull T. Aetiopathology of rheumatoid arthritis. Medicine 2010;38:163-6.
  27. Chiou WF, Chen CF, Lin JJ. Mechanisms of suppression of inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells by andrographolide. Br J Pharmacol 2000;129:1553-60.
  28. Casper I, Nowag S, Koch K, Hubrich T, Bollmann F, Henke J, et al. Post-transcriptional regulation of the human inducible nitric oxide synthase (iNOS) expression by the cytosolic poly(A)-binding protein (PABP). Nitric Oxide 2013;33:6-17.
  29. Zheng H, Yu X, Collin-Osdoby P, Osdoby P. RANKL stimulates inducible nitric-oxide synthase expression and nitric oxide production in developing osteoclasts: An autocrine negative feedback mechanism triggered by RANKL-induced interferon-β via NF-κB that restrains osteoclastogenesis and bone resorption. J Biol Chem 2006;281:15809-20.
  30. Kahles F, Findeisen HM, Bruemmer D. Osteopontin: a novel regulator at the crossroads of inflammation, obesity and diabetes. Mol Metab 2014;3:384-93.
  31. Mazurek T, Zhang L, Zalewski A, Mannion JD, Diehl JT, Arafat H, et al. Human epicardial adipose tissue is a source of inflammatory mediators. Circulation 2003;108:2460-6.
  32. Vuolteenaho K, Koskinen A, Kukkonen M, Nieminen R, Paivarinta U, Moilanen T, et al. Leptin enhances synthesis of proinflammatory mediators in human osteoarthritic cartilage--mediator role of NO in leptin-induced PGE2, IL-6, and IL-8 production. Mediators Inflammation 2009. doi: 10.1155/2009/345838. [Epub  13 Aug 2009]
  33. Tsay TB, Yang MC, Chen PH, Lin CT, Hsu CM, Chen LW. TNF-alpha decreases infection-induced lung injury in burn through negative regulation of TLR4/iNOS. J Surg Res 2013;179:106-14.
  34. Cai X, Li X, Li L, Huang XZ, Liu YS, Chen L, et al. Adiponectin reduces carotid atherosclerotic plaque formation in ApoE-/-mice: roles of oxidative and nitrosative stress and inducible nitric oxide synthase. Mol Med Rep 2015;11:1715-21.
  35. Fischer P, Hilfiker-Kleiner D. Survival pathways in hypertrophy and heart failure: the gp130-STAT3 axis. Basic Res Cardiol 2007;102:279-97.
  36. Otero M, Lago R, Lago F, Reino JJ, Gualillo O. Signalling pathway involved in nitric oxide synthase type II activation in chondrocytes: synergistic effect of leptin with interleukin-1. Arthritis Res Ther 2005;7: R581-91.
  37. Otero M, Lago R, Gomez R, Lago F, Gomez-Reino JJ, Gualillo O. Phosphatidylinositol 3-kinase, MEK-1 and p38 mediate leptin/interferon-gamma synergistic NOS type II induction in chondrocytes. Life Sci 2007;81:1452-60.
  38. Bao JP, Chen WP, Feng J, Hu PF, Shi ZL, Wu LD. Leptin plays a catabolic role on articular cartilage. Mol Biol Rep 2010;37:3265-72.
  39. Lago R, Gomez R, Otero M, Lago F, Gallego R, Dieguez C, et al. A new player in cartilage homeostasis: adiponectin induces nitric oxide synthase type II and pro-inflammatory cytokines in chondrocytes. Articular Cartilage Osteoarthritis 2008;16:1101-9.
  40. Song YW, Kang EH. Autoantibodies in rheumatoid arthritis: rheumatoid factors and anticitrullinated protein antibodies. Q J Med 2010;103:139-46.
  41. Nell VP, Machold KP, Stamm TA, Eberl G, Heinzl H, Uffmann M, et al. Autoantibody profiling as early diagnostic and prognostic tool for rheumatoid arthritis. Ann Rheum Dis 2005;64:1731-6.
  42. Jonsson T, Arinbjarnarson S, Thorsteinsson J, Steinsson K, Geirsson AJ, Jonsson H, et al. Raised IgA rheumatoid factor (RF) but not IgM RF or IgG RF is associated with extra-articular manifestations in rheumatoid arthritis. Scand J Rheumatol 1995;24:372-5.
  43. Jonsson T, Steinsson K, Jonsson H, Geirsson AJ, Thorsteinsson J, Valdimarsson H. Combined elevation of IgM and IgA rheumatoid factor has high diagnostic specificity for rheumatoid arthritis. Rheumatol Int 1998;18:119-22.
  44. Pai S, Pai L, Birkenfeldt R. Correlation of serum IgA rheumatoid factor levels with disease severity in rheumatoid arthritis. Scand J Rheumatol 1998;27:252-6.
  45. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315-24.
  46. Saxena A, Cronstein BN. 57-Acute phase reactants and the concept of inflammation. In: O'Dell GS, Firestein RC, Budd SE, Gabriel IB, McInnes JR. editor. Kelley's Textbook of Rheumatology (Ninth Edition). Philadelphia, USA: W. B. Saunders; 2013. p. 818-29.
  47. Hoffmann MH, Tuncel J, Skriner K, Tohidast-Akrad M, Turk B, Pinol-Roma S, et al. The rheumatoid arthritis-associated autoantigen hnRNP-A2 (RA33) is a major stimulator of autoimmunity in rats with pristane-induced arthritis. J Immunol 2007;179:7568-76.
  48. Calabro P, Chang DW, Willerson JT, Yeh ET. Release of C-reactive protein in response to inflammatory cytokines by human adipocytes: linking obesity to vascular inflammation. J Am Coll Cardiol 2005;46:1112-3.
  49. Pearle AD, Scanzello CR, George S, Mandl LA, DiCarlo EF, Peterson M, et al. Elevated high-sensitivity C-reactive protein levels are associated with local inflammatory findings in patients with osteoarthritis. Osteoarthritis Cartilage 2007;15:516-23.
  50. Hunter CA, Jones SA. IL-6 as a keystone cytokine in health and disease. Nat Immunol 2015;16:448-57.
  51. Koerner A, Kratzsch J, Kiess W. Adipocytokines: leptin--the classical, resistin--the controversial, adiponectin--the promising, and more to come. Best Pract Res Clin Endocrinol Metab 2005;19:525-46.
  52. Tilg H, Moschen AR. Adipocytokines: mediators linking adipose tissue, inflammation and immunity. Nat Rev Immunol 2006;6:772-83.
  53. Yoshino T, Kusunoki N, Tanaka N, Kaneko K, Kusunoki Y, Endo H, et al. Elevated serum levels of resistin, leptin, and adiponectin are associated with C-reactive protein and also other clinical conditions in rheumatoid arthritis. Intern Med 2011;50:269-75.
  54. Allam A, Radwan A. The relationship of serum leptin levels with disease activity in Egyptian patients with rheumatoid arthritis. Egypt Rheumatol 2012;34:185-90.
  55. Del Prete A, Salvi V, Sozzani S. Adipokines as potential biomarkers in rheumatoid arthritis. Mediators Inflammation 2014;2014:11.
  56. Takayanagi H. Osteoimmunology and the effects of the immune system on bone. Nat Rev Rheumatol 2009;5:667-76.
  57. Chu CQ, Field M, Feldmann M, Maini RN. Localization of tumor necrosis factor alpha in synovial tissues and at the cartilage-pannus junction in patients with rheumatoid arthritis. Arthritis Rheum 1991;34:1125-32.
  58. Matsuno H, Yudoh K, Katayama R, Nakazawa F, Uzuki M, Sawai T, et al. The role of TNF-alpha in the pathogenesis of inflammation and joint destruction in rheumatoid arthritis (RA): a study using a human RA/SCID mouse chimera. Rheumatology (Oxford) 2002;41:329-37.
  59. Schulz M, Dotzlaw H, Neeck G. Ankylosing spondylitis and rheumatoid arthritis: serum levels of TNF-alpha and Its soluble receptors during the course of therapy with etanercept and infliximab. BioMed Res Int 2014. doi.org/10.1155/2014/675108. [Article in Press]
  60. Osta B, Roux JP, Lavocat F, Pierre M, Ndongo-Thiam N, Boivin G, et al. Differential effects of IL-17A and TNF-alpha on osteoblastic differentiation of isolated synoviocytes and on bone explants from arthritis patients. Front Immunol 2015;6:151.
  61. Ranganathan P. Rheumatoid arthritis: biomarkers of response to TNF inhibition in RA. Nat Rev Rheumatol 2015;11:446-8.
  62. Nishimoto N, Ito A, Ono M, Tagoh H, Matsumoto T, Tomita T, et al. IL-6 inhibits the proliferation of synovial fibroblastic cells from rheumatoid arthritis patients in the presence of soluble IL-6 receptor. Int Immunol 2000;12:187-93.
  63. Choy EH, Panayi GS. Cytokine pathways and joint inflammation in rheumatoid arthritis. N Engl J Med 2001;344:907-16.
  64. Hashizume M, Hayakawa N, Mihara M. IL-6 trans-signalling directly induces RANKL on fibroblast-like synovial cells and is involved in RANKL induction by TNF-alpha and IL-17. Rheumatol (Oxford) 2008;47:1635-40.
  65. Gan L, Guo K, Cremona ML, McGraw TE, Leibel RL, Zhang Y. TNF-alpha up-regulates protein level and cell surface expression of the leptin receptor by stimulating its export via a PKC-dependent mechanism. Endocrinology 2012;153:5821-33.
  66. Agrawal S, Gollapudi S, Su H, Gupta S. Leptin activates human B cells to secrete TNF-alpha, IL-6, and IL-10 via JAK2/STAT3 and p38MAPK/ERK1/2 signaling pathway. J Clin Immunol 2011;31:472-8.
  67. Kopec-Medrek M, Kotulska A, Widuchowska M, Adamczak M, Wiecek A, Kucharz EJ. Plasma leptin and neuropeptide Y concentrations in patients with rheumatoid arthritis treated with infliximab, a TNF-alpha antagonist. Rheumatol Int 2012;32:3383-9.
  68. Lee SM, Choi HJ, Oh CH, Oh JW, Han JS. Leptin increases TNF-alpha expression and production through phospholipase D1 in Raw 264.7 cells. PloS one 2014;9:e102373. doi: 10.1371/journal.pone.0102373. [Article in Press]
  69. Madhok R, Crilly A, Watson J, Capell HA. Serum interleukin 6 levels in rheumatoid arthritis: correlations with clinical and laboratory indices of disease activity. Ann Rheum Dis 1993;52:232-4.
  70. Sack U, Kinne RW, Marx T, Heppt P, Bender S, Emmrich F. Interleukin-6 in synovial fluid is closely associated with chronic synovitis in rheumatoid arthritis. Rheumatol Int 1993;13:45-51.
  71. Marnell L, Mold C, Du Clos TW. C-reactive protein: ligands, receptors and role in inflammation. Clin Immunol 2005;117:104-11.
  72. Jequier E. Leptin signaling, adiposity, and energy balance. Ann N Y Acad Sci 2002;967:379-88.
  73. Gualillo O, Eiras S, Lago F, Dieguez C, Casanueva FF. Elevated serum leptin concentrations induced by experimental acute inflammation. Life Sci 2000;67:2433-41.
  74. Lee SW, Park MC, Park YB, Lee SK. Measurement of the serum leptin level could assist disease activity monitoring in rheumatoid arthritis. Rheumatol Int 2007;27:537-40.
  75. Targonska-Stepniak B, Majdan M, Dryglewska M. Leptin serum levels in rheumatoid arthritis patients: relation to disease duration and activity. Rheumatol Int 2008;28:585-91.
  76. Olama SM, Senna MK, Elarman M. Synovial/serum leptin ratio in rheumatoid arthritis: the association with activity and erosion. Rheumatol Int 2012;32:683-90.
  77. Lam QL, Lu L. Role of leptin in immunity. Cell Mol Immunol 2007;4:1-13.
  78. Anders HJ, Rihl M, Heufelder A, Loch O, Schattenkirchner M. Leptin serum levels are not correlated with disease activity in patients with rheumatoid arthritis. Metabolism 1999;48:745-8.
  79. Hizmetli S, Kisa M, Gokalp N, Bakici MZ. Are plasma and synovial fluid leptin levels correlated with disease activity in rheumatoid arthritis? Rheumatol Int 2007;27:335-8.
  80. Popa C, Netea MG, Radstake TR, van Riel PL, Barrera P, van der Meer JW. Markers of inflammation are negatively correlated with serum leptin in rheumatoid arthritis. Ann Rheum Dis 2005;64:1195-8.
  81. Neumann E FK, Muller-Ladner U. 58-Acute-phase responses and adipocytokines. In: Watts RA CP, Denton C, Foster H, Isaacs J, Muller-Ladner U. editor. Oxford Textbook of Rheumatology (Fourth Edition). Oxford, UK: Oxford University Press; 2013. p. 424-30.
  82. Shapiro L, Scherer PE. The crystal structure of a complement-1q family protein suggests an evolutionary link to tumor necrosis factor. Curr Biol 1998;8:335-8.
  83. Oh DK, Ciaraldi T, Henry RR. Adiponectin in health and disease. Diabetes Obes Metab 2007;9:282-9.
  84. Weyer C, Funahashi T, Tanaka S, Hotta K, Matsuzawa Y, Pratley RE, et al. Hypoadiponectinemia in obesity and type 2 diabetes: close association with insulin resistance and hyperinsulinemia. J Clin Endocrinol Metab 2001;86:1930-5.
  85. Beauloye V, Zech F, Tran HT, Clapuyt P, Maes M, Brichard SM. Determinants of early atherosclerosis in obese children and adolescents. J Clin Endocrinol Metab 2007;92:3025-32.
  86. Ouchi N, Walsh K. Adiponectin as an anti-inflammatory factor. Clin Chim Acta 2007;380:24-30.
  87. Rovin BH, Song H, Hebert LA, Nadasdy T, Nadasdy G, Birmingham DJ, et al. Plasma, urine, and renal expression of adiponectin in human systemic lupus erythematosus. Kidney Int 2005;68:1825-33.
  88. Rho YH, Solus J, Sokka T, Oeser A, Chung CP, Gebretsadik T, et al. Adipocytokines are associated with radiographic joint damage in rheumatoid arthritis. Arthritis Rheum 2009;60:1906-14.
  89. Choi HM, Lee YA, Lee SH, Hong SJ, Hahm DH, Choi SY, et al. Adiponectin may contribute to synovitis and joint destruction in rheumatoid arthritis by stimulating vascular endothelial growth factor, matrix metalloproteinase-1, and matrix metalloproteinase-13 expression in fibroblast-like synoviocytes more than proinflammatory mediators. Arthritis Res Ther 2009;11: R161.
  90. Ehling A, Schaffler A, Herfarth H, Tarner IH, Anders S, Distler O, et al. The potential of adiponectin in driving arthritis. J Immunol 2006;176:4468-78.
  91. Tan W, Wang F, Zhang M, Guo D, Zhang Q, He S. High adiponectin and adiponectin receptor 1 expression in synovial fluids and synovial tissues of patients with rheumatoid arthritis. Semin Arthritis Rheum 2009;38:420-7.
  92. Schaffler A, Ehling A, Neumann E, Herfarth H, Tarner I, Scholmerich J, et al. Adipocytokines in synovial fluid. JAMA 2003;290:1709-10.
  93. Senolt L, Pavelka K, Housa D, Haluzik M. Increased adiponectin is negatively linked to the local inflammatory process in patients with rheumatoid arthritis. Cytokine 2006;35:247-52.
  94. Giles JT, Allison M, Bingham CO. 3rd. Scott WM Jr, Bathon JM. Adiponectin is a mediator of the inverse association of adiposity with radiographic damage in rheumatoid arthritis. Arthritis Rheum 2009;61:1248-56.
  95. Ebina K, Fukuhara A, Ando W, Hirao M, Koga T, Oshima K, et al. Serum adiponectin concentrations correlate with severity of rheumatoid arthritis evaluated by the extent of joint destruction. Clin Rheumatol 2009;28:445-51.
  96. Laurberg TB, Frystyk J, Ellingsen T, Hansen IT, Jorgensen A, Tarp U, et al. Plasma adiponectin in patients with active, early, and chronic rheumatoid arthritis who are steroid and disease-modifying antirheumatic drug-naive compared with patients with osteoarthritis and controls. J Rheumatol 2009;36:1885-91.
  97. Targonska-Stepniak B, Dryglewska M, Majdan M. Adiponectin and leptin serum concentrations in patients with rheumatoid arthritis. Rheumatol Int 2010;30:731-7.