الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
Rheumatoid arthritis is the most common chronic inflammatory arthropathy characterized by synovial hyperplasic with massive infiltration of inflammatory cells primarily affecting the small joints causing pain and swelling that leads to chronic progressive joint destruction causing decline in the physical ability through the release of proinflammatory cytokines as TNF-α, IL-1 and IL-6. The disease affects about 1% of the population with higher frequency in women who are more subjected to the risk of developing the RA than men by 2-3 times.
CDK1 is one of the master regulators of cell division and the only non-redundant pleiotropic cell cycle driver, the protein encoded by this gene is one of the Ser/Ther kinase family which plays role in different cell cycle and immunological mechanisms such as regulating self-renewal of stem cells, chondrocyte proliferation and differentiation together with its role in skeletal development. Although the exact mechanistic role of CDK1 is unknown but it is considered as a potential therapeutic target for bone and joint disease as RA.
PI3Ks are group of enzymes which play role in variety of cellular processes such as signal transduction, vesicular trafficking and differentiation, mast cell functioning, B-cell development and Ab production. In RA, PI3K-Akt pathway was found to be one of the regulatory mechanisms involved in the pathogenesis of the disease. Activation of PI3K-Akt pathway by RA-FLS cytokines leads to promotion of cell migration and invasion together with driving anti-apoptotic response for the FLS. Therefore, modulating this pathway is one of the therapeutic approaches for RA.
RO-33006 is a small inhibitor of many cell cycle proteins with selective activity toward CDK1 by binding to it inhibiting proliferation and induces the arrest of cell cycle at G2/M phase. It helps in inducing cell cycle inhibitory and apoptotic genes in addition to enhancing the downstream signaling of p53 to induce apoptosis. Based on these findings RO-3306 is suggested as a regulator for FLS cell cycle and a treatment for RA.
Vitamin D is a prohormone that is synthesized in the human skin after UV exposure and consumed in diet via the intake of animal-based food. It shows an immune modulatory effect including the interaction between its metabolites and inflammatory cytokines involved in bone resorption and bone loss, controlling adaptive immunity by TLRs, T-cell differentiation and Th17 which play a remarkable role in the pathology of RA and also suppress B-cell proliferation and differentiation, in addition to controlling Ab production. RA prevalence found to decrease in individuals with high intake of vitamin D suggesting it as potential treatment for RA.
So, we aimed in this study to evaluate the effect of RO-3306 inhibitor on cyclin-dependent kinase 1 (CDK-1) and Phosphoinositide 3-kinase (PI3K) in rheumatoid arthritis patients and then correlating this with disease activity and vitamin D.
Summary and conclusion
110
The current study was conducted on 55 subjects who were classified into 40 rheumatoid arthritis patients divided into 10 males and 30 females with different stages of the disease and 15 age matched healthy persons as control group divided into 5 males and 10 females. Venous blood samples obtained from all subjects under study, divided as following: 2ml in plain red-top tube for quantification of serum vitamin D using enzyme linked immunosorbent assay (ELISA) technique and 3ml heparin vacutainers for peripheral blood mononuclear cells (PBMCs) isolation, quantification of CDK1 gene expression either in presence or absence of RO-3306 inhibitor using quantitative real time polymerase chain reaction (qRT-PCR). In addition to assessment of the PI3K levels with and without RO-3306 inhibitor using commercially available ELISA kit.
Vitamin D levels in our study were categorized into 3 levels (sever deficiency, insufficiency and optimal) and insufficient group was the predominant in all the study patients followed by sever deficiency and the optimal and there was no statistically significant difference in mean serum vitamin D concentration in RA patients compared to healthy control subjects also significance was missed when comparing RA patients regarding to gender and disease activity.
The present study showed that there was a statistically significant decrease in the cell cycle regulator cyclin-dependent kinase (CDK1) after addition of RO-3306 inhibitor while there was no statistically significant difference in Phosphoinositde-3 kinase (PI3K) under the effect of adding RO-3306 in comparison with healthy control subjects for both.
Male and female RA patients showed a significant decrease in CDK1 gene expression after RO-3306 addition which was missed in case of PI3K. Also, the current study showed a statistical significance in CDK1 gene expression without R0-3306 of RA patients classified on the base of DAS28 compared to that in healthy controls, this significance was missed after RO-3306 addition in all disease severity stages except for the high disease activity stage. Regarding disease activity score PI3K again did not show any significant reduction in RA patients compared to healthy control candidates.
Correlation analysis clarified that DAS28 was positively correlated with all the laboratory investigations as well as the other parameters of the study including CRP, RF, ESR, RF, Anti-CCP and ANA, CDK1 and PI3K, respectively with being significant for CRP, RF and CDK1.
Our results revealed that vitamin D was negatively correlated with RF, ESR, CRP, Anti-CCP, basal CDK1 and PI3K. On the other hand, we found that it is positively correlated with age, DAS28 and ANA. In addition, CDK1 showed positive correlation with CRP, ESR and PI3K without RO-3306 addition. However, there was a negative correlation between CDK1 and RF, Anti-CCP, ANA and PI3K after adding RO-3306.
Finally, there was a positive non-significant correlation between PI3K and CRP, ESR, ANA, Anti-CCP, respectively and significantly with RF.
Summary and conclusion
111
According to our findings, we concluded that:
▪ The statistical analysis of the data presented by the present work was greatly affected by the low sample size in the RA groups. Most of the statistical significance of the studied parameters (either diagnostic or our proper research) was estimated from the correlation analysis rather than differences between study groups that contained a lot of insignificant variations, in spite of the presence of remarkably different absolute values.
▪ Significant positive correlation recorded between DAS28 RA scoring system with both ESR and CRP might indicate the usefulness of employing both inflammatory markers as reliable components of RA scoring systems in general.
▪ Significant positive correlation recorded between RF and anti-CCP in RA study population with high disease activity could support the possibility that a substantial proportion of milder forms of RA initially negative for RF at the time of diagnosis may seroconvert at advanced stages.
▪ Although it does not seem to be affected by patient gender or disease severity, vitamin D showed significant correlations with most of the study parameters indicating that circulating levels of vitamin D is a significant corner stone in affecting RA pathogenesis, severity and progression and might predict much of the clinical indicators of RA.
▪ Inhibition of CDK1 gene expression by RO-3306 that was not manifested in parallel with PI3K protein activity in addition to being more remarkable in RA patients rather than healthy controls indicates that inhibition of PI3K/Akt/CDK1 signaling pathway is selective and might represent a modulating mechanism in certain inflammatory disorders such as RA.
▪ The RO-3306 suppression of mRNA transcripts of CDK1 as well as PI3K synthesis seems to be influenced by vitamin D level and partially by RA disease activity (low rather than moderate and high disease activity), but neither by any of the RA conventional diagnostic parameters including ESR, CRP, RF and anti-CCP.
▪ The unexpected missing of significant positive correlation between current data of CDK1 and PI3K (either spontaneous or after RO-3306 inhibition) seems to be attributed to differences in assay protocol for both, dose and selective inhibitory behavior for RO-3306 against both or to possible existence of intrinsic feedback loop(s) deviating the kinetics and the outcomes although both of them belong to the PI3K-AKT-CDK1 activation pathway.