The Effect Of REM Sleep Deprivation On Th17/Treg Balance In Spleen And IL-17 Related Cytokines In CNS

Objectives:1, To establish the rapid eye movement(REM) sleep deprivation model of rat.2, To investigate the effects of REM sleep deprivation on the proportions of Th17 cells, Tregs and Th17/Treg cells balance in peripheral spleen lymphocytes.3, To observe the morphologic changes of neurocytes, the protein level of IL-17 and the cellular infiltration of Th17 cells in rat brain.4, To test the relative mRNA expression level of IL-17, TGF-?, IL-6, ROR?t,Foxp3, IL-6, IL-1?, TNF-?in rat brain.Thus, we access the effect of REM sleep deprivation on Th17/Treg balance in spleen and the expression of IL-17 related cytokines in CNS in order to explore the brain injury mechanism after sleep deprivation.Method:Male Sprague-Dawley rats were randomly divided into cage control(CC) group and REM sleep deprivation(SD) group. SD group was subdivided into SDld,SD3 d and SD5 d group with twelve rats in each group.1, The REM sleep deprivation model was established by applying modified multiple platforms method(MMPM). General behavior changes were observed and the body weight and the spleen weight of rats were weighed during sleep deprivation.2, In peripheral spleen lymphocytes, the proportion of Th17 cells and Treg cells were analyzed by flow cytometry and then the Th17/Treg ratio was calculated.3, The morphologic changes of brain tissue were observed by hematoxylin and eosin(HE) staining and Nisal staining.4,The protein level of IL-17 and the cellular infiltration of Th17 cells in rat brain were assessed by immunofluorescent staining and the relative mRNA expression level of IL-17, TGF-?, IL-6, ROR?t, Foxp3 and so on were determined by real-time quantitative PCR in rat brain.Results:1, Because of the repeating dipping into the water of head when falling into sleep, the rats on the platform were sleep deprived and the sleep deprivation model was established. The sleep deprivation induced obvious mental behavior changes and the body weight was reduced and the spleen weight was increased. 2, The cytometry analysis of spleen lymphocytes demonstrated that the percentage of Th17 cells in lymphocytes was significantly higher in SD3 and SD5 group than CC and SD1 group(P<0.05).The percentage of CD4~+ cells of lymphocytes in SD5 group was significantly higher than CC(P=0.023)and SD1 group(P=0.017). In CD4~+ cells population, the proportion of Th17 cells in SD3(P=0.005)and SD5 group(P=0.032)was obviously higher than that in CC group,and the percentage of Treg cells in SD3 group and SD5 group was significantly higher than that in CC and SD1 group(P<0.05). Meanwhile, the percentage of Treg cells in SD5 group was significantly higher than that in SD3 group(P=0.046).Comparing to CC group, the Th17/Treg ratio was increased in SD1 and SD3 group, but decreased in SD5 group.3, HE staining and Nisal staining showed that the neurocytes of rat hypothalamus and hippocampal had mild changes after sleep deprivation. The perineural space was enlarged and the cell body and nisal body were reduced. Immunofluorescent staining showed that the protein level of IL-17, the IL-17 expressing cells and the cellular infiltration of Th17 were increased in sleep deprived rat brain.4, The relative mRNA expression levels of proinflammatory cytokines IL-17, IL-6,TNF-?, IL-1 and Th17 specific transcriptional factor ROR?t in SD group were higher than those in CC group(P<0.05). IL-23 and anti-inflammatory cytokines TGF-?showed no significant difference. The mRNA expression level of Treg specific transcriptional factor Foxp3 was decreased after sleep deprivation and significantly elevated until the fifth day(P<0.001).Conclusions:1, The REM sleep deprivation model of rat was successfully established. The REM sleep deprivation induced obvious mental behavior changes, caused the energy imbalance and activated the inmmunol system.2, After the REM sleep deprivation, the proportion of Th17 and CD4~+cells in spleen lymphocytes were elevated. In the CD4~+ cells population,the percentage of Th17 was elevated as the SD time prolonged. The proportion of Tregs was decreased after sleep deprivation and then markedly elevated as the SD time prolonged. So the Th17/Treg cells balance was disrupted by sleep deprivation.3, The neurocytes of rat hypothalamus were damaged after sleep deprivation. Sleep deprivation promoted the expression of proinflammatory cytokine IL-17 and the infiltration of Th17 in brain maybe the potential mechanism of the damage.4,Sleep deprivation may promoted the inflammatory brain injury by elevating the relative mRNA expression level of proinflammtory cytokines(such as IL-17, IL-6,TNF-?, IL-1) and inhibiting the level of anti-inflammatory cytokines(such as TGF-?). In addition, sleep deprivation may upregulated the mRNA expression level of Th17 specific transcriptional factor ROR?t while inhibited the level of Treg specific transcriptional factor Foxp3. So the down-regulation of Foxp3 induced by TGF-? and up-regulation of ROR?t induced by IL-6 plus TGF-? may also be the regulatory mechanism of the elevated IL-17 and its brain damage.