| Sleep is an important physiological procedure,which take over 1/3 of our life. The sleep disorder now was under serious situation, many people in society have poor sleep quality and less sleep time, and now there were a lot of shift workers while the circadian rhythm disorder will also impact sleep regulation. Patients with sleep disorders are often associated with psychological disorders, but there are few patients who have been diagnosed and treated. This social situation deserves our attention, there were a number of clinical trials which determined that the lack of sleep will affect the cognitive function,including reduced concentration, working memory impairment, perceptual retardation and so on; lack of sleep would inhibit the immune function, had pro- inflammatory effect;epidemiological investigation showed that lack of sleep or rhythm disorders increased the incidence of heart disease; because that sleep restriction and circadian rhythm disorder would affect the metabolism of sugar and hormone secretion, the incidence of diabetes increased significantly; sleep disorder often happened associated with neurodegenerative disease occurrence and development, and now as a breakthrough point for improving the treatment for neurodegenerative diseases.The present study mostly choose rodents for experiment as their sleep cycles andstages was similar to humans'. Now there were acute and chronic sleep deprivation simulating social status, and those varied methods for sleep deprivation have their own characteristics and shortcomings.It is known that, there were a lot of controversial studies about the fundamental mechanisms of the harmness sleep deprivation caused, the physiological mechanism of sleep still has not been determined by now, which maked it harder. In considering, we believe that we can find some new views through epigenetics which is research on gene transcription regulation. We started our study from histone modifications after sleep deprivation, and contact the actual results, grab the proinflammatory effects as a breakthrough point for further study. Our experimental design is as follows:Objective1. Through REM sleep depriving rats for different length of time, changes of H3K9 and H3K4 tri-methyl were observed in different parts of rat brain including hippocampus,hypothalamus, ventromedial prefrontal cortex and the raphe nuclei; 2. Verify the inflammatory effect promoted by sleep deprivation of defferent time; 3. The mechanism of proinflammatory effect produced by sleep deprivation was studied preliminarily associated with H3K4 trime and H3K9 trime changes.Method Using Western Blot to test the H3K9 trime and H3K4 trime level; Observe the morphological changes of microglial cells by immunofluorescence staining, combined with RT-PCR technique to detect the m RNA level of proinflammatory factors including IL-1?, IL-6, TNF-? and i NOS and n NOS; Applicating Western Blot to detect the expression of PPAR-? and ESET/SETdb1 protein combined with immuno- fluorescence double staining results for analysis.Results1. After REM sleep deprivation, rats in SD groups were easy irritable and anxious, their aggressive behavior increased; the growth of sleep deprived rats' body weight was lower than normal rats while their feeding were similar, these rats in SD groups also appeared slow growth trend compared with control group.2. After REM sleep deprivation, H3K9 trime level of SD groups were lower than control group in hippocampus and ventral medial prefrontal cortex, while the level of H3K4 trime were higher than control group, and the results between SD 1d, SD 3d, SD 6d groups had no statistically significant difference. In hypothalamus, H3K9 trime levels were lower in SD groups than that in the Control group, the H3K4 trime level in the SD groups showed a tendency to increase first and then decrease compared to the Control group. In raphe nuclei fractions, H3K9 trime level in SD groups were significantly higher than that in control group which appeared to be gradually rising, the SD groups' H3K4 trime levels were higher than control group and its trend seemed to be gradually reducing.3. After REM sleep deprivation, in hippocampus the activation of microglia in SD groups was significantly higher than that in normal control group, and there were no difference in the activation degree between SD1 d, SD3 d and SD6 d groups. In the hypothalamus, the activation of SD1 d group was significantly less than that of Control group, and the activation of SD3 d and SD6 d groups was significantly higher than that in the control group. After REM sleep deprivation, in hippocampus the proinflammatory cytokine gene expression were increased in varying degrees, the m RNA expression level of IL-1? in SD3 d group increased most significantly, TNF-? m RNA level only of SD6 d group increased significantly than control group, i NOS m RNA level of SD groups showed a rising trend, n NOS and IL-6 m RNA levels increased obviously,and the elevated levels of three SD groups did not differ. At the same time, in hypothalamus m RNA levels of IL-1?in SD groups were lower than that of control group, but not significant, TNF-? m RNA level in the SD1 d and SD6 d group increased compared with control group, while it in SD3 d group decreased, n NOS and IL-6 m RNA levels in SD6 d group increased obviously.4. After REM sleep deprivation, the expression of PPAR-? in SD groups were lower than that in control group, and the protein expression of ESET/SETdb1 was higher. In the hypothalamus, SD 1d and SD3 d group's PPAR-? protein expression were higher than control group, but SD 6d group's PPAR-? protein expression was lower than control group,ESET/SETdb1 protein expression SD groups were higher than that of control group. The results of double immunofluorescence staining showed that the expression of H3K9trimein microglial cells was relatively stable, the double rate can reach more than 90%, when the microglia was activated,the expression of H3K9 trime significantly decreased.Conclusion REM sleep deprivation significantly affected the behavior and body weight of rats, and the related gene and mechnism regulated by histone modification were complicated. Its pro-inflammatory effects were not related to the duration of sleep deprivation in the hippocampus while long time of sleep deprivation could produce pro- inflammatory effect in the hypothalamus. REM sleep deprivation may be through the interaction of ESET and PPAR-? directly or indirectly influencing H3K9 trimethyl, and thus regulate the target genes like pro inflammatory cytokines or microglia activation. |
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