| Learning and memory decline belongs to "forgettery" "amnesia" in Traditional Chinese Medicine(TCM), and are the evidence of brain dysfunction. With the increase of stress in modern life, learning and memory decline has become one of the significant factor which affect people's life. Memory decline can be caused by many factors, in young and middle-aged people, it is mainly caused by unhealthy lifestyle such as lack of exercises, staying up late, drink abuse, in which the focus is the memory decline caused by sleep-disorder and the sleep disorder could be imitated by sleep-deprivation. This study reconstructed animal model by sleep-deprivation to specify the neurobiology mechanism of Sini San's effect on sleep-deprivation rats model learning and memory ability.Part I Theoretical ResearchBy searching on the relationship between sleep and learning and memory of doctors through the ages, find that the formation and consolidation of sleep and learning memory has a strong relation with liver, heart and kidney. In the process of learning and memory, liver, heart, kidney, heart can interrelated each other by qi and blood, meridians and five-zang shen, and they have a division in information processing. People's cognition of the outside world starts from the heart, then form a preliminary impression by thinking and analysing, and judge that by recalling and storing the memory, and finally consolidate the memory in brain. The liver controlling conveyance and dispersion, the regulation of liver soul penetrate in the process, and intercourse between heart and kidney is also important for waxing and waning of yin-yang. As for the learning disorder caused by lack of sleeping, theoretically, the main differenciation types are not just limited to the regulation of heart and kidney. Liver loss dispersion on conveyance and dispersion, lack of nourisment of heart spirit and deficency of kidney essence can be involved.Part II Experimental studyExperiment 1 Sleep-deprivation rats and the evaluation of its learning and memory abilityObjective:Reconstruct learning and memory disorder model, evaluate the model by animal behavior and observe Jiajian Sini San's effect on rats'learning and memory ability. Method: Reconduct learning and memory disorder rats model by sleep-deprivation, record the changes of general condition, weight, Y-maze score and SOD in serum of rats. Results:①changes of general conditions:the control group is normal from the third day of molding to the end, the model group and treatment group show mania, aggressivity, high alert, bite, unhairing, loose stool in different level, the condition of Sini San group and composition treatment group alleviate than model group and Shenghui Decoction group.②changes of weight:recording the weight changes of the 1,3,5,8 day from molding to the end, found that the weight of control group increased, the model group and other 3 treatment group decended in different level, compared to control group, the remaining four groups difference significant, but no difference among the four groups.③Y-maze scores:test the wrong reaction times and active avoidance response rate of the rats on the 0 and 7 day. On the 0 day, there is no difference among groups, on the 7 day, the wrong reaction times of model group and other three treatment groups are more than the control group, there are significant differences among model group, Sini San treatment group and Shenghui decoction treatment group, composition treatment group have significant difference; comparing to the model group, the active avoidence response rate of composition treatment group increases and has significant difference while the Sini San group has significant difference, too.④The expression of SOD in serum:comparing to the control group, the content of SOD in model group decrease and has significant difference; the content of SOD in Shenghui decoction treatment group reduced; whlie in Sini San group and composition group, there is no significant difference. Comparing to the model group, the content of SOD in Sini San group and composition group increase significantly, between both groups, the former group has a lower content of SOD than the composition group. Conclusion:①The rats'memory decline with general conditions and weight changes can be caused by sleep-deprivation.②The soothing liver, tonifying kidney and nourishing heart method can improve the memory decline caused by sleep-deprivation, the soothing liver method can improve part of the memory ability and tonifying kidney and nourishing heart method has no obvious effect.ExperimentⅡBehavioral changes of sleep-deprivation rats and the regulation of Jiajian Sini SanObjective:Observe the effect of Jiajian Sini San on the cognition of sleep-deprivated rats to a new enviroment. Method:use self-made desert field box to do open-field test. Results:on the 0 day of molding, the indexes of open-field test has no significant difference among the groups. On the 7 day, comparing to the control group, the upright number of model group decrease significantly, thus has significant difference; comparing to the model group, the unright number of the three treatment group increase and have significant difference. As for the grid-across number, comparing to the control group, the model group decrease significantly; comparing to the model group, Sini San group increase dramatically, and has significant difference, Shenghui decoction group increases significantly, thus has significant difference. On the 7 day of molding, comparing to the control group, the residence time in central region of the control group induces and has significant difference; and when comparing to the model group, the residence time of Shenghui decoction group and composition group are shorter and have significant difference, the residence time of Sini San group is shorter and has notable difference. As the decoration time, the compositon group is more than the model group, and there are no significant difference among other groups. Conclusion:①Sleep-deprivation can reduce the rats'space exploration ability, excitability and cognition ability, but no evident effect on adaption.②The soothing liver, tonifying kidney and nourishing heart method can improve the emotion and cognition disorder caused by sleep-deprivation, and they can regulate them from different respects.ExperimentⅢThe effect of sleep-deprivation on the gene and immune positive things expression of BDNF in rats'hippocampus and the receptor TrkB, and the regulation of Jiajian Sini San.Objective:Observe the effect of Jiajian Sini San on the expression of BDNF and TrkB related to learning and memory in sleep-deprivated rats. Method:Use immuohistochemistry and real-time PCR to detect the positive material and gene expression of BDNF and TrkB in hippocampal CA3 area and DG area. Conclusion:①Immune positive matter of BDNF can be seen in hippocampal CA3 and DG area, which is claybank granulate, oval or cone, and they distribute in cytoplasm and nucleus of CA3 area pyramidal cell and DG area granulocyte.②The protein expression of BDNF:the results of CA3 area shows the MOD of BDNF of model group and three treatment groups decrease comparing to the control group, the difference is significant; comparing to the model group, the MOD of composition group increases and has significant difference, the Sini San group and Shenghui decoction group has no significant difference. The DG area results show the MOD of BDNF in model group and other three treatment group decrease compared to the control group, the difference is significant; comparing to the model group, there is no significant difference among Sini San group, Shenghui decoction group and composition group.③Gene expression of BDNF:comparing to the control group, hippocampal BDNF mRNA of model group decrease, the difference is notable; and there is no significant difference between model group and Sini San group or Shenghui decoction group; but in composition group, the hippocampal BDNF mRNA expression increase dramatically, and has significant difference.④The protein expression of TrkB:The results from CA3 area show that TrkB MOD of model group and the other three treatment groups decrease compared to the control group, the difference is significant; comparing to the model group, the MOD of Sini San group and compostion group increase and has notable difference, and Sini San group has a lower expression than the composition group. The results from DG area show TrkB positive materials MOD of model group and the three treatment groups decrease compared to the control group, and the difference is notable; comparing to the model group, three treatment groups MOD have no significant difference.⑤Gene expression og TrkB:comparing to the control group, mRNA of TrkB in model group decreases and has significant difference; there is no significant difference among Sini San, Shenghui decoction and composition groups; comparing to the model group, the mRNA expression of TrkB in Sini San group and composition group increase remarkably, the difference is notable; the mRNA expression of TrkB in Shenghui decoction has no evident increase. Conclusion:①Sleep-deprivation can decrease protein and gene expression of hippocampal BDNF and TrkB.②The composition can increase the gene expression of hippocampal BDNF and TrkB.③The soothing liver regulatory point of is narrow relatively and the method can improve expression of TrkB.Experiment IV Gene expression of nonreceptor tyrosine kinase Fyn in sleep-deprived rats'hippocampus and the intervention of Jiajian Sini SanObjective:To observe the effect of Jiajian Sini San on Fyn gene expression in sleep-deprived rats'hippocampus, identify the relationship between memory improvement and Fyn, BDNF, TrkB. Method:The gene expression of Fyn will be detected by realtime fluorescence quantitative PCR. Results:compared to the control group, Fyn mRNA expression in model group hippocampus decreases remarkably with very significant difference; compared to the model group, Fyn mRNA expression in Shenghui decoction group hippocampus increases, and has significant difference, the compostion Fyn mRNA increases significantly with notable difference, when there is no significant difference in Sini San group. Conclusion:①Sleep-deprivation can decrease the gene expression of Fyn in hippocampus.②Tonifying kidney and nourishing heart method and composition can improve the sleep-deprivation induced learning and memory decline by regulating the gene expression of Fyn, especially the composition group.③The gene expression of Fyn, BDNF and its receptor in sleep-deprivation rats' hipocampus has consistency, and the regulatory function on learning and memory decline of the three protein may share the same signal path.Experiment Y The protein expression of ERK and pERK in. sleep-deprivated rats' hippocampus and the regulatory function of Sini San.Objective:To observe the effect of Jiajian Sini San on ERK and pERK expression in sleep-deprivated rats'hippocampus, and identify the relationship between the effect and nonreceptor tyrosine kinase and brain-derived neurotrophic factors. Method:Observe the expression of ERK and its homocystein thiolactone by using western blot. Results:①The protein expression of ERK2:comparing to the control group, the expression of ERK2 with Shenghui and composition group increase; in model group and Sinisan group,there is no difference. comparing to the model group, the expression of ERK2 with Shenghui and composition group increase with siginificant difference.②The protein expression of pERK2: comparing to the control group, the pERK2 in composition groups improve while expression of the model group induces; comparing to the model group Shenghui and composition group increase.Conclusion:①Sleep-deprivation can significantly decrease the protein expression of ERK2 and pERK2 which are related to memory-formation.②The composition and tonifying kidney and nourishing heart method can improve ERK2 and pERK2 decline in sleep-deprivated rats'hippocampus, more obvious in pERK2 expression.③ERK2 and its Homocystein thiolactone, together with BDNF and Fyn, may participate in learning and memory decline caused by sleep-deprivation.SummaryThis study is based on sleep theory, five-zang spirit theory, to explore the mechanism of sleep formation and learning memory from liver, heart and kidney. People's cognition of the outside world starts from the heart, then make a preliminary impression by thinking and analysing, then to judge that by recalling and storing the memory, and finally solid the memory in brain. The liver controlling conveyance and dispersion, the regulation of liver soul penetrate in memory formation and consolidation. Five-zang spirit, especially the liver, heart and kidney are of key importance to sleep-induced learning and memory disorder. By analysing the experimental results, we make conclusions as followings:①Deprive sleep from rats for 7 days continuously can cause learning and memory impairment.②The composition of Sini San and Shenghui decoction can improve the sleep-deprivation-induced learning and memory impairment by regulating the signalling transduction pathways ERK-pERK/BDNF /TrkB/Fyn in hippocampus.③The effect target of Sini San and Shenghui decoction is narrow, they improve memory ability from behavior, some proteins and genes.④The joint action of soothing liver and tonifying kidney, nourishing heart on memory improvement is compre-hensive and stable. |
PDF Full Text Request