Effect Of Pineal Gland And Melatonin On CD4～+CD25～+Treg Cells Development And Foxp3 Expression Of Rat Thymus

Objective To investigate the influence of pinealectomy(Px)and melatonin (MLT) supplementation giving rise to CD4+CD25+Treg cells development and Foxp3 expression in rat thymus; To analyse the effects of pineal gland and melatonin on the differentiation and development of thymocytes, as well as regulation of immunological function and tumors progression, thereafter enrich the research of neuroendocrine-immune system.Methods①The pinealectomized rat models were established: 120 clean male rats of 2 months old were divided into five groups: normal control group, sham pinealectomized group, pinealectomized group, pinealectomy +7.5 mg·kg-1·day-1 MLT intraperitoneal injection group, pinealectomy +15 mg·kg-1·day-1 MLT intraperitoneal injection group, and the thymus was taken out at 4th week and 8th week, rats of pinealectomized group were injected with normal sodium in the same volume as other groups;②Double-immunofluorescent labeling for CD4 and CD25 and flow cytometric analysis were used to analyse the number of double positive cells in thymus and peripheral blood after Px and MLT supplementation;③Semiquantitative reverse transcription- polymerase chain reaction (RT-PCR) and the gel imaging analysis system were applied to analyse the level of thymic Foxp3 mRNA after Px and MLT supplementation;④Immunohistochemistry (IHC) and the quantitative automatic image analysis were used to observe and analyse the changes of Foxp3 positive cells in thymus after Px and MLT supplementation.Results①Double-immunofluorescent labeling for CD4 and CD25 and flow cytometric analysis showed no difference between the number of double positive cells in thymus of Px rat models and normal/sham group. After MLT supplementation the number of double positive cells increased dependently on time and dose, except that of Px rats with high dose melatonin at 8th week;②F low cytometric analysis showed: the number of double positive cells in rat peripheral blood significantly increased in models of Px at 4th week compared to normal and sham group, and then recovered to normal level after MLT supplementation. But the results showed no significant changes in all groups at 8th week;③The results of Semiquantitative RT-PCR and IHC in rat thymus both showed that Foxp3 expression increased in Px rats compared to nomal/sham group at 4th weeks, which returned to normal level after MLT supplementation. On the other hand, Foxp3 expression showed no significant difference in all groups at 8th week.Conclusion The differentiation and development of CD4+CD25+Treg cells in rat thymus changed along with the influence of growth and metabolism of thymocytes by pineal gland and MLT. Px made no difference between the development of CD4+CD25+Treg cells in rat thymus, but resulted in significant increase of thymic output and Foxp3 expression. But all the effects disapeared at 8th week. MLT supplementation could reverse the abnormity. The influence of thymic CD4+CD25+Treg cells development might be one of the mechanisms by which the pineal gland and melatonin promote rat immunological function and inhibit the tumors progression.