Hücre kültürü modellerinde metilfenidatın gonadotropin salgılatıcı hormon ve kisspeptin üzerine nöroendokrin etkilerinin araştırılması

Methylphenidate (MPH) is widely used among children for attention deficit hyperactivity disorder. Though there are discussions about the growth-related effects of its use, the neuroendocrine mechanisms of MPH around puberty have not been studied. This study aims to investigate the role of MPH on gonadotropin-releasing hormone (GnRH) and Kisspeptin (Kiss1) expression and secretion in vitro using cell culture techniques. GnRH and Kiss1 secreting cells, GT1-7 and rHypoE-8 cell lines respectively, were maintained in DMEM containing 10% FBS and 1% PSN at 37°C under 5% CO2 humidified atmosphere. The effect of MPH on proliferation and migration at different doses (1 nM-200 μM) for 24 hours was assessed by trypan blue dye exclusion and wound healing (scratch) assays, respectively. To investigate GnRH expression and secretion, cells were cultured in maintenance medium without FBS with or without MPH (200 μM) and samples at different were obtained to investigate GnRH expression by real-time qRT-PCR and GnRH release to the medium by ELISA. Normal distribution of the data was analyzed by Shapiro-Wilk normality test. Data from dose-response experiments were either analyzed one-way ANOVA or Kruskal-Wallis followed by Dunnet's multiple comparison or Dunn's multiple comparison tests, respectively. Real-time qPCR and ELISA data were analyzed by Student's t-test and 2-ΔΔCT method. There were no significant differences between untreated cells and MPH-treated cells in terms of proliferation and migration. As the proliferation and migration of the cells were not affected by the different concentrations of MPH, the highest concentration of 200 μM was selected to assess gene expression and hormone secretion. GnRH expression has remained comparable to the control arm with no statistical difference at any time point. GnRH release was significantly higher under MPH compared to the control group at 2 hours after treatment (p<0.05), while no differences were found between groups at other time points. Kiss1 expression was assessed for time points of 30 minutes, 2 hours, and 24 hours. Kiss1 expression was significantly increased in the first 30 minutes of MPH treatment. As, MPH affected GnRH and Kiss1 expression at multiple time points, to further investigate the response of the cells, the recovery in the expression after the cessation of MPH treatment was analyzed. It was observed that after the cessation of MPH treatment there is no statistical significance between MPH treatment and control arms in GnRH expression. Though the cellular evidence suggests that MPH affects the expression and release of GnRH and Kiss1 to some extent, it is far from reflecting the accumulating clinical data. Lack of statistical difference should not be interpreted as no differences between the MPH use over neuronal cell lines. When results are modeled for larger data sets (n= 30-60) statistically the majority of the timepoints become significant in comparison to the control arm. Our findings suggest puberty-delaying effects of MPH may be partly through GnRH and Kiss1 expression and release. Further investigations are needed to understand a medication that possibly have direct effects on 40 million people worldwide.