الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
Rheumatoid arthritis is a chronic, progressive, inflammatory autoimmune disease associated with articular, extra-articular and systemic effects affecting about 1% of the population with a female: male ratio of 3:1. It is characterized by polyarthritis with often progressive joint damage, disability, immunologic abnormalities, systemic inflammation, increased co-morbidity and premature mortality. Dysfunctional intracellular signaling involving deregulated activation of the Janus Kinase/Signal Transducers and Activators of Transcription (JAK/STAT) and “cross-talk” between JAK/STAT and the stress-activated protein kinase/mitogen-activated protein kinase (SAPK/MAPK) and Phosphatidylinositide-3-Kinase/AKT/mammalian target of rapamycin (PI-3K/AKT/mTOR) pathways play a critical role in rheumatoid arthritis. This is exemplified by immune-mediated chronic inflammation, up-regulated matrix metalloproteinase gene expression, induction of cartilage apoptosis and “apoptosis-resistance” in rheumatoid synovial tissue. Toll like receptors (TLRs) are at least 12 mammalian family members of transmembrane receptors, which play a key role in both innate and adaptive immune responses. When exposed to an immunogenic stimulus, such as a microbial pathogen, the initiation of the inflammatory and immune response is mediated by TLRs which result in the activation of cells of the innate immune system including monocytes, macrophages and dendritic cells. Activation of TLRs results in the rapid expression of pro-inflammatory cytokines such as TNF-α, and chemokines mediated via activation of NF-κB. These mediators orchestrate an immune response that recruits neutrophils, monocytes and lymphocytes. Macrophages and dendritic cells take up and process the pathogen and migrate to peripheral lymphoid tissue where antigen presenting cells initiate the generation of adaptive immunity resulting in the generation of cellular immunity and antibodies. Tofacitinib is a potent, orally active and approved JAK inhibitor for the treatment of RA. it is a reversible, competitive inhibitor that binds to the adenosine triphosphate (ATP) binding site in the catalytic cleft of the kinase domain of JAK. As a result of binding to the ATP site, tofacitinib inhibits the phosphorylation and activation of JAK, thereby preventing the phosphorylation and activation of STATs, and thus the activation of gene transcription leads to decrease cytokine production and modulation of the immune response. Dipeptidyl peptidase-4 (DPP-4) inhibitors, as sitagliptin, are a class of antidiabetic drugs that inhibit the degradation of insulinotropic incretins, mainly glucagon-like peptide-1(GLP-1). Soluble DPP-4 has been shown to enhance TLR pathway and to augment inflammatory reactions that were ameliorated by DPP-4 inhibitors. Also, sitagliptin has been reported to promote cardioprotection in type 2 diabetic rats via modulation of JAK/STAT pathway. However, its effect on signaling pathways in autoimmune diseases has not been investigated.
The aim of the present study is to investigate the effects of sitagliptin on JAK/STAT pathway and the TLR/NF-κB signaling pathways and to compare its efficacy with tofacitinib. The possible beneficial outcome of the combination of both drugs on
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inflammatory parameters as well as on biochemical alterations and drug related effects in the adjuvant arthritis model will also be evaluated.
To develop a rat model of adjuvant arthritis,Sprague Dawley rats were injected with 0.1 ml suspension of heat-killed Mycobacterium butyricum (Difco Laboratories Co, USA), 12 mg/ml in Freund’s incomplete adjuvant (FIA) (Sigma Aldrich Co-USA), intradermally at the base of the tail. chronic inflammation was allowed to progress in all animals for 14 days. Animals were divided into 5 groups as follows:
group 1: Eight non-arthritic healthy control rats.
group 2: Sixteen untreated AA rats, 8 of them received the vehicle for tofacitinib (methyl cellulose) and 8 received distilled water (vehicle for sitagliptin) orally daily.
group 3: Eight AA rats treated orally with tofacitinib dissolved in methyl cellulose at a dose of 6.2 mg/kg/day (Sigma, Aldrich, USA) .
group 4: Eight AA rats treated orally with sitagliptin dissolved in distilled water at a dose of 100 mg/kg/day (MSD, USA).
group 5: Eight AA rats treated orally with the combination of both sitagliptin and tofacitinib in the aforementioned doses.
Severity and progression of atrithis was assessed by evaluation of hind paw swelling. Tested drugs were administered daily for 10 days, from the 15th day till the end of the study (24th day from adjuvant injection). At te end of the experimental period, rats were sacrificed and blood samples were collected for measurement of the following serum parameters:
1- Anti-CCP.
2- RANKL.
3- TNF-α.
4- IL-6.
5- Lipid profile (Total cholesterol, Triglycerides, LDL and HDL).
6- Fasting blood glucose level (FBGL).
7- Liver function tests (ALT and AST).
8- Renal function tests (Ceatinine and Urea).
Tissues of the tibiotarsal joints were isolated, weighed and pepared for determination of :
1- JAK-1, JAK-2 and JAK-3.
2- STAT-3.
3- TLR-4.
4- NF-κB.
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The results of the study clearly suggested that tofacitinib and sitgliptin given individually and in combination,were effective in suppressing clinical severity of arthritis, decreasing the studied parameters of inflammation in the adjuvant model of arthritis. Tofacitinib caused more inhibition of JAK1 and JAK3 than sitagliptin, while, JAK2 was equally inhibited by both drugs, indicating a limited risk of possible hematopoietic side effects, with sitagliptin treatment. Although the effect of sitagliptin treatment, on some of the biochemical alterations, in the adjuvant arthritis model, was variable, its favorable impact was quite evident, upon combination with tofacitinib, by normalization of blood glucose and serum lipids, which could offer protection against CVD, in RA patients. Moreover, by causing partial reversal of the elevated serum transaminases and SCr levels, in the combined therapy group, sitagliptin may also have a protective effect against tofacitinib’s related hepato and nephrotoxicity. Tofacitinib was superior to sitagliptin, in most of the studied inflammatory parameters, except for NF-kB/TLR4 pathway, which was more significantly inhibited by sitagliptin. Inhibition of TLR4 protein expression by tofacitinib, in the present study, is a novel finding that needs further investigations. Such inhibition may represent another point in the huge complex interrelationships between JAK/STAT and NF-kB/TLR4 signaling pathways.
Thus the combination of both drugs provided higher therapeutic efficacy compared with either agents alone, this means that the combination therapy has more anti-inflammatory action and lesser side effects as compared to each drug alone. Also in this study new anti-inflammatory mechanism of action for both drugs has been suggested.