Zebrafish Model to Study the Effects of Thymoquinone on Circadian Rhythms via Clock

Background: All living organisms depend on circadian rhythms which are managed by clock1a, bmal1, per2, cry1 and nr1d1 genes. The disruption of this causes various illnesses including chronic diseases over time. Thymoquinone which exists in Nigella sativa produces antioxidant effects together with anti-inflammatory and anti-cancer activities. Its effects on regulating biological clocks has not yet been explored. The research investigates Thymoquinone effects on zebrafish circadian gene expression and evaluates its neuroblastoma cell toxicity. This study investigates how Thymoquinone alters zebrafish circadian gene expression while measuring human neuroblastoma cell toxicity through MTT assay. Materials and Methods: The study administered Thymoquinone to zebrafish using various concentration levels. After the RNA extraction expression levels of clock1a, bmal1, per2, cry1 and nr1d1 were measured using RT-qPCR. Expression changes were determined through 2^−ΔΔCt calculations. The data was adjusted based on the expression of housekeeping genes. The MTT assay tested neuroblastoma cells treated with different Thymoquinone concentrations at 1,10,25,50,100 μg/mL. Spectrophotometry at 570 nm was used to measure cell viability. Results: In zebrafish, Thymoquinone affected the expression of circadian genes by upregulation of bmal1 through nr1d1 and downregulation of per2 and cry1. The thymoquinone demonstrated dose related killing action on neuroblastoma cells and kept the cell viability above 85% at 10 μg/mL concentration, while higher doses (25 and 50μg/mL) showed significant degree of cytotoxicity suggesting its anticancer effects. Conclusion: Our data suggest that thymoquinone alters zebrafish circadian gene expression and exerts selective killing in neuroblastoma cells. A concentration of 27.67 μg/mL was found to be most effective. Thymoquinone serves as both a chronomodulatory agent and an anticancer agent which establishes its potential for future development of circadian-based cancer therapies.