| Part One The effect of REM sleep deprivation on autophagy of hippocampus in ratsObjective:To investigate whether autophagy is involved and its level change in hippocampus of rats after REM sleep deprivation.Methods:We selected the SD rats as the research object.The experimental animals were divided into REM sleep deprivation group,water environment control group and normal blank control group.The modified multi-platform sleep deprivation method was used to establish the sleep deprivation model needed for this experiment.Using fluorescence to observe the formation of autophagy in the hippocampus of three groups.The expression of autophagy-related protein in hippocampus was detected by western blot.Results:Autophagy was found in the hippocampus of the three groups under immunofluorescence.And we have a quantitative examination of the autophagy related proteins----LC3B and P62.Compared with the experimental group,the expression of LC3B in the hippocampus of the experimental group was significantly increased?p<0.05?,and the expression of P62 protein was decreased?p<0.05?.There was no significant difference in the expression levels of LC3B and P62 protein between the two control groups?p<0.05?.Conclusion:The autophagy activity of hippocampus in rats after REM sleep deprivation was activated.Part Two To selece time and concentration of orexin in hippocampal neurons and detect the expression of autophagyObjective:To observe the effect of orexin on cell viability and autophagy level in vitro hippocampal neurons.Methods:we go into the cellular level to have a deeply analysis,using the original generation of rats hippocampus cells,first step is to give different concentration gradient(0mol/L?10-11 mol/L?10-9 mol/L?10-7 mol/L)of orexin for 4 hour and 10-7 mol/L orexin for different time gradient?0h?2h?4h?6h?8h?to stimulate the cells,according to the cell activity and the expression of autophagy-related protein P62 to select the appropriate stimulation conditions.Results:With the increase of orexin concentration,the activity of hippocampal neurons increased gradually.The concentration of 10-7 mol/L orexin in the selected group was different from that in the blank group?P<0.05?.At the same time,the activity of hippocampal neurons increased with the prolongation of intervention time.The cell viability at 4 h was statistically significant compared with the blank control group?P<0.05?.The expression of P62 protein,which is negatively correlated with autophagy activity,increased with the prolongation of stimulation time and intervention concentration of orexin.Same as cell viability,we discover a significant differences in orexin 10-7 mol/L group and intervention 4h group compared with the blank group?P<0.05?.Conclusion:Orexin can increase the activity rate of cells and inhibit the autophagy activity in hippocampal neurons.The function is time and concentration dependent.Part Three:Research on possible mechanism of orexin effect on autophagy levels in hippocampal neuronsObjective:To explore the possible mechanism of orexin affecting the autophagy activity in hippocampal neurons.Methods:At this part,we still choose hippocampal neurons as the study subjects.Acording to the results of part 2,we regard the specific stimulus(10-7 mol/L?4h)as a condition.The experiment was divided into the control group,Orexin group,Orexin receptor block group?almorexant?group,autophagic activator?rapamycin?group,orexin+almorexant group and orexin+rapamycin group.By detecting the expression levels of autophagy-related proteins Beclin1 and P62 in the hippocampal neurons,and the specimens were observed by electron microscopy to identificate of changes in autophagy levels.Results:The expression of beclin1 protein in the orexin group was lower than that in the blank control group?P<0.05?.The expression of beclin1 protein was significantly increased in the orexin receptor block group and the autophagic activator group?P<0.05?.In the addition of orexin+almorexant or the addition of orexin+rapamycin,we can observed that the expression level of beclin1 protein in these two groups was not different from that of the control group?P>0.05?,while have a clear increase compared with the orexin group?P<0.05?.In contrast,the expression level of protein P62,which was negatively correlated with autophagic activity during the experiment,was increased after stimulation with orexin,which was statistically significant compared with the control group?P<0.05?.In the two groups?orexin+almorexant?orexin+rapamycin?,the levels of P62 protein is significantly lower than those in the control group?P<0.05?,but there was no significant difference compared with the control group?P>0.05?.Conclusion:Orexin inhibition of hippocampal neuronal autophagy levels can be blocked by orexin receptor blockers and mTOR inhibitors. |
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