The Role Of Melatonin In Apoptosis Of Rat Thymocytes And Regulation Of Mitochondrial Pathway-related Proteins

ObjectiveTo investigate the anti-apoptosis effect of melatonin on rat thymocytes in vitro. So as to explore its anti-apoptosis mechanism and the role in differentiation and development of thymocytes, as well as the regulation of immunological function, therefore to enrich the research of neuroendocrine-immune system.Methodsâ‘ Rat thymocytes were incubated in vitro and divided into six groups. They were M1, M2, M3, M4, M5 and normal control group. The melatonin supplementation concentration of each group was 10^-3, 10^-4, 10^-6, 10^-8, 10^-10 and 0 mol/L.â‘¡Cells were collected at 4 and 8 hours, then detected by Annexin V-FITC/PI flow cytometry to observe the proportion of apoptotic cells.â‘¢Semiquantitative RT-PCR and Western blot were applied to observe the mRNA and protein expression level of Bax, Bim and Bcl-x_L.Resultsâ‘ Double-immunofluorescent labeling for Annexin V-FITC/PI and flow cytometric analysis showed increased FITC⁺/ PI^- thymocytes in 8-hour groups compared to 4-hour's. The proportion of FITC⁺/ PI^- thymocytes decreased in M1, M2, M3 groups.â‘¡The results of Semiquantitative RT-PCR and Western blot in rat thymocytes both showed the levels of Bax were higher in 8-hour-groups than that of 4-hour-groups. But in groups incubated for 4 hours, melatonin showed no effect on the levels of Bax. The same results came with Bim. â‘¢Contrast to Bax or Bim, the results of semiquantitative RT-PCR and Western blot, which were correspondent with that of flow cytometric analysis, showed the levels of Bcl-x_L didn't change when incubated longer. But in the presence of melatonin, we noticed an increase in its level as the concentrations of melatonin were 10^-3, 10^-4, 10^-6 mol/L.ConclusionWe observed an increase in thymocytes apoptosis when incubated in vitro and could be reduced by certain concentrations of melatonin. The mRNA and protein levels of Bax and Bim increased as the increase of culture time, but showed no difference in the presence of melatonin. Further more, certain concentrations of melatonin upregulated the mRNA and protein levels of Bcl-x_L when different incubation time made no change on them. In summary, the present results suggest that the antiapoptotic effect of melatonin on thymocytes could involve the consequence of an inhibition of the mitochondrial pathway, presumably through the regulation of Bcl-xL protein levels.