Changes And Effects Of Glial Histone Acetylation In Rat Brain After REM Sleep Deprivation

Numerous studies have confirmed that acute or chronic sleep deprivation can lead to significant increases in peripheral blood leukocyte counts and C-reactive protein and inflammatory factor levels(IL-1?,IL-6,TNF-?)in humans without obvious infection or injury.In addition,animal experiments showed that lack of sleep can cause activation of microglia in the hippocampus and cerebral cortex of mice,and cause local nerve inflammatory response,which aggravates neurological impairment.The hypothalamus is the center of sleep regulation,but the effect of sleep deprivation on the microglia and local neuroinflammation in the hypothalamus and its mechanism are not yet clear.Histone modification is the process of methylation,acetylation,phosphorylation,and ubiquitination under the enzymes.The common histone acetylation is modified at H3K9(Ninth-bit lysine at N-end of H3)K14,K23 and other loci.The level of histone acetylation is mainly regulated by histone deacetylase(HDAC)and histone acetyltransferase(HAT).The study found that histone acetylation plays an important role in the inflammatory reaction,improve the histone acetylation level can reduce the inflammatory reaction of the nervous system;inhibition of microglial activation and promote the transformation of microglia to the M2 phenotype that has the function of tissue repair.However,the role of histone acetylation in the regulation of microglia after sleep deprivation has not been reported.This experiment is mainly to explore the effect of sleep deprivation on the microglia and histone acetylation modification in hypothalamus of rats,and to observe the effect of histone acetylation modification on microglia activation and related inflammatory factors.To find the theoretical basis and the new direction of etiological treatment for patients with clinical sleep disorders.Part I The expression of microglia in different brain regions of rats after REM sleep deprivationObjective:To study the expression of microglia in different brain regions after sleep deprivation.Methods:SD model was made by modified multiple platform method(MMPM).Thirty adult rats were randomly divided into:control group(control,CON),sleep deprivation group for three days(SD3),sleep deprivation group for six days(SD6).The activation of microglia(Iba-1)in the frontal cortex,hippocampus and hypothalamus of different treatment groups were observed by immunofluorescence.Results:Immunofluorescence showed:(1)Frontal cortex:Compared with the control group,the activation of Iba-1 increased(P<0.05),the cell body was bigger and branches became thicker in the SD3 group and SD6 group;(2)Hippocampus:Compared with the control group,the activation of Iba-1 increased(P<0.05),the cell body was bigger and branches became thicker in the SD3 group and SD6 group;(3)Hypothalamus:Compared with the control group,the activation of Iba-1 increased(P<0.05),the cell body was bigger and branches bacame thicker in the SD3 group and SD6 group.Conclusion:After sleep deprivation,the microglia of the frontal cortex,hippocampus and hypothalamus were activated.Part II Effects of histone acetylation modification on microglial activation in rat hypothalamus after REM sleep deprivationObjective:To investigate the effects of REM sleep deprivation on the modification of histone acetylation,and to observe the effects of increasing the level of histone acetylation on the activation of microglia and the expression of related inflammatory factors.Methods:The rats were divided into 5 groups:control group,SD3 group,SD6 group,SD3+SAHA(SAHA is a histone deacetylase inhibitor)group and SD6+SAHA group;Immunofluorescence double staining was used to observe the acetylation of histone H3K9 of hypothalamus microglia in normal rats;The activation of Iba-1 in the hypothalamus region of rats in different groups were observed by immunofluorescence;Western Blot was used to detect the changes in the level of H3K9AC modification;The expression of inflammatory factors(TNF-a,IL-6,NOS2 and IL-10)in the hypothalamus region were detected by RT-PCR.Results:(1)Immunofluorescence double staining showed that the acetylated histone H3K9 site expressed in the control group rats of microglia of the hypothalamus.(2)Immunofluorescence showed:Compared with the control group the activation of Iba-1 increased(P<0.05),the cell body was bigger and branches became thicker of hypothalamus region in the SD3 group and SD6 group;The activation of Iba-1 cells in the hypothalamus area decreased(P<0.05)in the SD3+SAHA group compared with the SD3 group;Compared with group SD6 group,the activation of Iba-1 cells in the hypothalamus area decreased(P<0.05)in the SD6+SAHA group.(3)Western Blot showed that compared with the control group,the H3K9AC level of hypothalamus region in the SD3 group and SD6 group decreased significantly(P<0.01);And its level increased significantly after histone acetylation(P<0.05).(4)RT-PCR results showed:Compared with control group,hypothalamus inflammatory factors TNF-a,IL-6,NOS2,IL-10 levels increased significantly(P<0.05)in the SD3 group and SD6 group;Compared with SD3 group,the expression of IL-6 and NOS2 decreased significantly(P<0.01)in the SD3+SAHA group;Compared with SD6 group,the expression of TNF-a,IL-6 and NOS2 decreased significantly(P<0.05),but the expression of IL-10 increased(P<0.05)in the SD6+SAHA group.Conclusion:REM sleep deprivation can enhance the activity of microglia in the hypothalamus area and also reduces the level of histone acetylation in rats;The improvement of histone acetylation can significantly reduce the activation of microglia and inhibit the expression of proinflammatory factors after sleep deprivation.It is suggested that histone acetylation may be one of the most important mechanisms for regulating the activation of microglia.Part ? Histone acetylation modification could inhibit inflammation effects on congnitive function in rats after REM sleep deprivationObjective:To research whether protein acetylation by inhibiting the inflammatory response to the influence of cognitive function in rats after sleep deprivation.Methods:The rats were randomly divided into 3 groups:CON group,SD6 group and SD6+SAHA group.The rats were tested by Morris water maze for 6 days at fixed time.Sleep deprivation was continued after daily test,and the number of rats crossing the original platform was observed on sixth day.Results:The latent period of the SD6 group was significantly longer than that in the normal group(P<0.05)in the fourth day;and the latency of the SD6+SAHA group was shorter than that in the SD6 group(P<0.05)for sixth day of positioning navigation.In the space exploration experiment carried out at 6 hours after the end of positioning navigation,compared with the control group,the duration of the rats in the target quadrant were significantly reduced significantly reduced(P<0.05)in the SD6 group;Time was no significant change in the target quadrant between the SD6+SAHA group and the SD6 group(P>0.05).Conclusion:Improve the level of histone acetylation modification inhibit inflammation,could improve cognitive function in rats after REM sleep deprivation by Morris water maze experiment.