الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
Rheumatoid arthritis is a progressive, autoimmune disorder that is characterized by articular and extra-articular manifestations affecting more than 1% of world‘s population with higher prevalence among females than males. Incidence of RA is influenced by multiple genetic, hormonal and environmental factors. Multiple cytokines and immune cells are involved in the inflammatory cascade taking place in RA.
Autophagy has multiple functions in the biological system homeostasis, and its dysregulation is implicated in RA pathogenesis. It has a mutual relationship with pro-inflammatory cytokines such as TNF-α and IL-6, where autophagy can increase their production and they are also capable of enhancing autophagic activity. Based on the entangled relationship between autophagy and the immune system, autophagy has the potential to employ a protective therapeutic effect or to provoke and worsen inflammation and immune responses.
The crosstalk between autophagy and apoptosis decides cellular destiny. Autophagy predominates as a survival mechanism to provide cellular protection against nutrients and growth factors deficiency induced apoptosis. The imbalance between both pathways is a major participant in RA pathogenesis.
TNF-α plays a major role in regulating the inflammatory reaction taking place in RA. It induces the production of pro-inflammatory cytokines; IL-1, IL-6 and IL-17 with a subsequent perpetuation of disease progression. TNF-α also interacts with various pro-inflammatory signaling pathways including PI3K/Akt and NF-κB, that are closely interrelated to autophagy/apoptosis balance. PI3K/Akt/mTOR signaling pathway is indispensable for cellular growth, survival, differentiation and angiogenesis. It is an essential regulator of both cellular autophagy and apoptosis and its dysfunction is implicated in synovial hyperplasia.
Therefore, targeting and modulation of the PI3K/Akt/mTOR pathway may provide more understanding of the pathophysiological mechanisms of RA and offering better therapeutic benefits.
3-Methyl adenine is the first discovered pan-PI3K inhibitor and it is the most widely used autophagy inhibitor. However, its poor solubility and low potency required its use at high concentrations to prevent autophagy. Multiple compounds have been investigated for their potential autophagy inhibitory effect but they lacked the selectivity and reproducibility. Thus, the identification of new autophagy inhibitors is an attractive field of investigation.
Inhibition of TNF- α has been considered as an effective therapeutic option for RA. Etanercept is the most commonly prescribed TNF-α inhibitor for RA. Multiple clinical trials have proven its clinical efficacy and safety in different disease stages and in comparison, to conventional DMARDs or other biological agents. Recently an inhibitory
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effect of etanercept on autophagy activation was reported in peripheral blood mononuclear cells (PBMCs) and in fibroblast-like synoviocytes (FLS) isolated from RA patients.
Infliximab is a chimeric mAb that was the first anti-TNF-α antibody to be approved for RA treatment. Its efficacy in RA therapy has been established in many clinical trials revealing its great efficacy in joint damage reduction and alleviation of disease signs and symptoms. Its role as an autophagy modulator has been investigated in Inflammatory bowel diseases, but its contribution to autophagic machinery regulation in RA settings, hasn‘t been fully elucidated yet.
Therefore, the aim of the present study was to examine the possible role of autophagy in regulating the anti-inflammatory effect of the two TNF-α inhibitors, etanercept and infliximab, and their potential inhibitory effects on the autophagy signaling pathway; PI3K/Akt/mTOR. The autophagy inhibitory effects of etanercept and infliximab were compared to the pan-PI3K inhibitor; 3-methyladenine. The prospective beneficial impact of 3-MA on the inflammatory parameters was also assessed in the adjuvant arthritis model in rats.
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: Eight untreated AA rats, receiving the vehicle for all drugs; distilled water intraperitoneally daily.
group 3: Eight AA rats treated subcutaneously with etanercept dissolved in distilled water at a dose of 10 mg/kg every three days for 2 weeks.
group 4: Eight AA rats treated intraperitoneally with infliximab dissolved in distilled water at a dose of 7mg/kg every three days for 2 weeks.
group 5: Eight AA rats treated intraperitoneally with 3-methyladenine dissolved in distilled water at a dose of 10 mg/kg/day for 2 weeks (Sigma, Aldrich, USA).
Arthritis severity was assessed by evaluation of hind paw swelling and calculating the arthrogram score. Tested drugs were administered for 14 days, from the 15th day till the end of the study. On the 29th day, animals were sacrificed by cervical dislocation. Blood samples were collected and used for the determination of the following serum parameters by ELISA:
- Anti-CCP.
- RANKL.
- TNF-α.
- IL-6
The subcutaneous tissue of the hind paw and surrounding the tibiotarsal joints of all rats were removed and used for the following determinations by Western blotting and ELISA:
- Beclin-I
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- LC3-I
- LC3-II
- Caspase-3
- p-Akt.
- PI3K
Results of the current study clearly demonstrated the strong anti-inflammatory effects of etanercept and infliximab as indicated by their significant reduction of serum inflammatory biomarkers as well as by the improvement of severity of arthritis. They caused remarkable inhibition of autophagy parameters; Beclin-1, LC3-I and LC3-II expression, but their effects were inferior to the standard autophagy inhibitor, 3-MA. They also exhibited a significant pro-apoptotic activity, which was negatively correlated to their anti-autophagic effect. Moreover, the TNF-α inhibitors and 3-MA significantly suppressed the PI3K/Akt signaling, with 3-MA having the highest inhibitory action on this pathway. So, etanercept, infliximab and 3-MA are believed to share the ability to induce apoptosis in RA synovial tissues via inhibition of PI3K and Akt phosphorylation and activation. 3-MA showed a significant anti-inflammatory and bone protective effect via reducing IL-6, TNF-α, anti-CCP and RANKL circulating levels in addition to ameliorating the signs of arthritis indicating the potential therapeutic utility of autophagy inhibitors in RA treatment.