In terms of disease appearance and development. It has been shown that inflammatory disorders like rheumatoid arthritis exhibit diurnal fluctuation. The majority of chronic inflammatory disease symptoms occur during particular hours of the day. Due to the diurnal nature of inflammatory indicators like tumor necrosis factor, this is possible (TNF). In Rheumatoid Arthritis, the expression of several clock genes, including ARNTL2, NPAS2, and DEC2, is dispersed, and TNF is the factor that induces these genes. To prove the hypothesis, TNF regulation on ARNTL2 genes and NPAS2 genes were investigated to prove how they elicit a circadian rhythm in Rheumatoid Arthritis and other inflammatory conditions.
Key Words: Rheumatoid Arthritis, TNF, Circadian Variation.
Introduction
Circadian Rhythms, commonly known as biological clock is the ability of an organism to adapt to various changes that occur around them in a bid to enhance survival (Carlos, 2017). Organisms including human beings have evolved to adapt to changes in the environment such as day/night changes, temperate variations, and these adaptations help them optimize their behavior and physiology. For example, animals know that night time is time to sleep and various wild animals prepare for winter with the help of their biological clock. In recent times circadian rhythms have been proven to exist in all life forms down to the cellular levels in all life forms.
The biological clock is composed of two main parts i.e. the Central clock which is located in the suprachiasmatic nucleus and peripheral clock which is virtually in all cells in the body. On an intracellular molecular basis, the core main genes essential for circadian rhythm regulation are ARNTL which is a transcription factor, circadian locomotors output cycles kaput (CLOCK), ARNTL2 and Neuronal Pas Domain protein 2 (NPAS2). For correct oscillation of the various genes in a circadian rhythm, there are various regulatory genes that either help to express or diminish the effects of the core genes outlined above. These genes are called Periods (PER) and are essential in the up-regulation and down-regulation of the core genes ( Takahashi, 2016). Other regulatory genes are DEC1 and DEC 2 which inhibit the activity of ARNTL/CLOCK (Olkkonen, Vesa-Petteri, & Kuusela, 2017)
The above-outlined components of the circadian clock closely interact in a countercurrent motion with the body's inflammatory system by modulating immune cell function. Any disturbance in their function and oscillation leads to disturbance in the inflammatory system and a dysfunction of inflammatory components lead to disruption in the circadian rhythm. The main aim was thus to prove this interaction, and explain the reason behind variations of symptoms in Rheumatoid arthritis i.e. how fatigue and inflammation correlate.
Method
1. Cell culture-Primary human fibroblast cultures were established and characterized.
2. Cell stimulation-For immunofluorescence human primary fibroblasts were seeded
3. RNA isolation-RNA was isolated. The PCR was performed in iQ5 real-time PCR detection system (Bio-Rad). RPLP0 was used as a housekeeping gene.
4. Immunofluorescence-Human primary fibroblasts were seeded at 1 x 105 cells per well on coverslips placed in 12-well plates containing RPMI-1640 supplemented with antibiotics and 1% FBS. Before stimulations, the cells were synchronized as described above.
5. Plasmids and vectors -DEC1, DEC2, ARNTL, ARNTL2, and NPAS2 cDNA were amplified from human primary fibroblast.
6. Statistical analysis-Transfection experiments were analyzed with one-way ANOVA.
Results
Tumor Necrosis Factor (TNF), a key component in the inflammatory system, regulates the core clock components. It induces the expression of ARNTL2, NPAS2, and DEC2 which are involved in the circadian rhythm. This was demonstrated in the immunofluorescence study whereby, after 24hours of TNF stimulation, the ARNTL2 protein was clearly visible and located in the nuclei.
Discussion
TNF increases the expression of ARNTL2 and NPAS2 in primary human fibroblasts which are the core CLOCK genes expressed in Rheumatoid Arthritis. The study gave evidence that inflammation increases the levels of the core genes. TNF also reduces the expression of the regulatory genes i.e. PER and DBP. The effects of TNF on the core genes thus elicit an overexpression of ARNTL2 and NPAS2 which will not be downregulated due to the inhibition of PER and DBP regulatory genes. A lack of down-regulation is a mismatch of the circadian rhythm and will elicit effects attributed to overexpression of a particular stimulus or underexpression of a particular stimulus. A lack of regulation of the core genes due to TNF may lead to a lack of cortisol release required to rejuvenate the body and replenish energy stores.
Conclusion
Rheumatoid arthritis is characterized by chronic fatigue and joint inflammation. The joint inflammation and pain can be attributed to inflammatory components such as TNF, however, fatigue may correlate with a mismatch of the circadian rhythm due to the expression of TNF. The drugs of choice for rheumatoid arthritis are NSAID, which reduce the inflammation as well as the chronic fatigue and the mechanism of action of this may be attributed to low levels of TNF.
References
Takahashi, J. S. (2016). The transcriptional architecture of the mammalian circadian clock. Nature Reviews Genetics, 164-179.
Carlos, I. (2017). Discoveries of Molecular Mechanisms Controlling the Circadian Rhythm. The Nobel Assembly at Karolinska Institutet , 1-5.
Olkkonen, J., Vesa-Petteri, K., & Kuusela, E. (2017). DEC2 Blocks the Effect of the ARNTL2/NPAS2 Dimer on the Expression of PER3 and DBP. Journal of Circadian Rhythms .15(1), p.6. DOI: http://doi.org/10.5334/jcr.149
