الفهرس 
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Abstract
Rheumatoid arthritis (RA) is a chronic, destructive autoimmune inflammatory disease that may affect many tissues and organs, but principally attacks the synovium of joints. Although the main causes of RA are still unknown, the immune system is involved in RA pathogenesis. It starts with joint swelling, stiffness and pain due to synovial hyperplasia and synovitis with neovascularization. The late stage of RA involves joint destruction which affects the quality of human life. Concerning RA management, MTX has long been regarded as the standard “anchor” DMARD. However, tremendous efforts have been directed towards adopting targeted biologics to reduce joint destruction and preserve function in RA patients especially those intolerant and insensitive to DMARDs.
The present study was designed to
Study the comparative effect of both RANI and TCZ against MTX in RA model where RANI was used for the first time in RA treatment.
Study the experimental assessment of TCZ as a monotherapy and as co-therapy with MTX because only few number of studies examined the effect of TCZ and there is also no experimental investigation comparing the anti-rheumatic effect of TCZ and MTX either alone or in combination.
Investigate the underlying mechanisms by which the biological agents (RANI and TCZ) produced their anti-rheumatic effect by examining the crosstalk between angiogenic, inflammatory, apoptotic markers & their relation to bone protection.
Study the toxicity profile of both monoclonal antibodies (RANI and TCZ) especially on soft organs (liver and spleen) as RANI was used for the first time in RA treatment and the toxicity of TCZ on liver & spleen was never investigated experimentally.
For the fulfillment of this goal, this study was conducted as follows:
Animals were divided into 8 groups (6 animals each)
group (1): (Negative control) Normal rats received intra-plantar (3 times/week) and intra-peritoneally (i.p.) (once/week).
group (2): (Positive Control) CFA-induced RA rats were received normal saline (0.9% w/v) as a vehicle intra-plantar (3 times/week) and intra-peritonealy (i.p.) (once/week) normal saline (0.9% w/v) as a vehicle.
group (3): CFA – induced RA rats received MTX (0.75 mg/kg/week i.p.).
group (4): CFA – induced RA rats received TCZ (5 mg/kg/ intra-plantar 3times per week).
group (5): CFA – induced RA rats received RANI (5 mg/kg/ intra-plantar 3times per week).
group (6): CFA – induced RA rats received MTX (0.75 mg/kg/week i.p.) and TCZ (5 mg/kg/ intra-plantar 3times per week).
group (7): Normal rats received TCZ (5 mg/kg/ intra-plantar 3times per week) that acts as TCZ toxic group.
group (8): Normal rats received RANI (5 mg/kg/ intra-plantar 3times per week) that acts as RANI toxic group.
The administration of MTX, TCZ and RANI was used simultaneously for all animal groups and started 24 h post-arthritis induction for 21 day. At the end of the experiment, blood samples were collected and animals were then scarified after anaesthetization. Left hind paw, liver and spleen tissues were dissected out for the required analysis.
The results of our study proved that CFA is a well-established RA-inducing agent. It summarizes many crucial symptoms in RA such as synovitis, neovascularization, cell death (apoptosis), cartilage destruction and bone erosion.
Concerning the comparative effect of both RANI and TCZ against MTX, Our study provided evidence for:
The anti-rheumatic/anti-arthritic activities of MTX, TCZ and RANI alone and MTX-TCZ co-therapy via assessment of arthritis severity (assessing gait score, arthritic index, and ankle diameter and paw thickness).
RANI monotherapy and MTX-TCZ co-therapy preserved histological architecture of the periosteum and articular structure in comparison with MTX or TCZ alone.
group (8): Normal rats received RANI (5 mg/kg/ intra-plantar 3times per week) that acts as RANI toxic group.
The administration of MTX, TCZ and RANI was used simultaneously for all animal groups and started 24 h post-arthritis induction for 21 day. At the end of the experiment, blood samples were collected and animals were then scarified after anaesthetization. Left hind paw, liver and spleen tissues were dissected out for the required analysis.
The results of our study proved that CFA is a well-established RA-inducing agent. It summarizes many crucial symptoms in RA such as synovitis, neovascularization, cell death (apoptosis), cartilage destruction and bone erosion.
Concerning the comparative effect of both RANI and TCZ against MTX, Our study provided evidence for:
The anti-rheumatic/anti-arthritic activities of MTX, TCZ and RANI alone and MTX-TCZ co-therapy via assessment of arthritis severity (assessing gait score, arthritic index, and ankle diameter and paw thickness).
RANI monotherapy and MTX-TCZ co-therapy preserved histological architecture of the periosteum and articular structure in comparison with MTX or TCZ alone.
Regarding the underlying mechanisms by examining the crosstalk between inflammatory, angiogenic, apoptotic markers & their relation to bone protection, our results proved that:
Besides MTX anti-inflammatory effect, RANI and TCZ have also anti-inflammatory effect. They significantly reduced the level of IL-6 cytokine compared with CFA arthritic rats. Furthermore, they showed almost negative expression as compared to CFA group.
MTX, TCZ and RANI alone and MTX-TCZ co-therapy produced their anti-angiogenic effect through reduction of angiogenic markers (VEGF and TGFβ). Interestingly, combined therapy of MTX-TCZ reversed their high levels in the serum into the normal level in comparison with MTX or TCZ alone.
MTX, TCZ and RANI Monotherapy and MTX-TCZ co-therapy reduced apoptosis. Interestingly, RANI alone and MTX-TCZ co-therapy reversed apoptotic Bax/Bcl-2 ratio.
MTX, TCZ and RANI alone and combined therapy of MTX-TCZ provided bone protection. RANI alone and MTX-TCZ co-therapy significantly increased OPG transcription as compared with MTX or TCZ monotherapy.
Indeed, RANI had better effect than MTX or TCZ alone in terms of its effect on bone protection or bone health markers.
MTX-TCZ co-therapy had better effect on bone health than MTX or TCZ monotherapy.
With respect the safety or effect on soft tissues such as liver and spleen, our results revealed that:
RANI had a good safety profile especially on liver and this is a very important issue as the use of the majority of the anti-rheumatic agents is associated with hepatoxicity.
However, RANI needs further investigation regarding its effect on the spleen in RA.
The liver function should be monitored during the course of treatment using MTX or TCZ as monotherapy or co-therapy.
In conclusion. Treatment of RA does not only depend on reduction or inhibition of inflammatory cytokines and immune cells but also targets angiogenesis process which is thought to be an effective future therapeutic strategy such as Ranibizumab (RANI). RANI as a monotherapy anti-angiogenic agent has a better effect on inflammatory, apoptotic and bone erosion markers compared with MTX or TCZ monotherapy. This result matched with its effect on histological structure of bone (RANI reverted CFA-induced histopathological alteration). The combined therapy of MTX-TCZ showed better effect in comparison with MTX or TCZ alone on all inflammatory, angiogenic, apoptotic markers and finally cause bone protection.