Based On "the Liver Responds To The Spring, It Is Mainly Used For Dredging And Regulating Emotions" To Study The Mechanism Of The Pineal Gland Regulating Hippocampal Function In Four Seasons

The theory of "Five Zang conforming to the time rhythm" originated from the thought of"nature and human echo" in Inner Canon of Huangdi,which is an important part of the theory of viscera state in traditional Chinese medicine.Therefore,the function of "liver’s governance of conducting and dispersing Qi and regulating emotion" has the physiological characteristics of changing in response to the changes of Yin and Yang in the four seasons.The research on the modern scientific connotation of "liver’s governance of conducting and dispersing Qi and regulating emotion,and changes with time" has found its correlation with the regulation of hippocampal function by pineal gland melatonin(MT).However,the signal transduction mechanism of pineal gland MT regulating hippocampal function is still unclear.This study intends to combine the theory of "liver’s governance of conducting and dispersing Qi and regulating emotion,and changes with time" with nerve cell signal transduction to further reveal the scientific connotation of "liver ’s governance of conducting and dispersing Qi and regulating emotion,and changes with time".From the perspective of "Five Zang conforming to the time rhythm",it enriches the theoretical connotation of TCM liver viscera and time medicine,and provides theoretical and experimental basis for the clinical prevention and treatment of mental emotional diseases.1 PurposeCombined with the theory of "Five Zang conforming to the time rhythm" and the mechanism of nerve cell signal transduction,the effects of seasonal rhythm on pineal gland-MT,melatonin receptor(MTR)in hippocampus and Gs/Gi-cyclic adenosine monophosphate(cAMP)-protein kinase A(PKA)-cAMP-response element binding protein(CREB)signaling pathway were studied to reveal the micro mechanism of "liver’s governance of conducting and dispersing Qi and regulating emotion,and changes with time".2 Methods2.1 Theoretical researchBy using the methods of literature review and theoretical discussion,we collected and summarized the theoretical and experimental studies on "liver’s governance of conducting and dispersing Qi and regulating emotion,and changes with time",pineal gland MT,hippocampal MTR and Gs/Gi-cAMP-PKA-CREB signaling pathways.Objective to explore the cellular signal transduction mechanism and scientific connotation of "liver’s governance of conducting and dispersing Qi and regulating emotion,and changes with time".2.2 Experimental research2.2.1 Experimental designBased on the time node of the Vernal Equinox(3.21),the Summer Solstice(6.21),the Autumnal Equinox(9.23)and the Winter Solstice(12.22),the 5-week-old male SD rats were selected as the experimental objects,including serum MT,hippocampal MTR,Gs protein,Gi protein,adenylate cyclase(AC);cAMP,PKA and CREB were used as detection indexes to study the expression level of signal transduction in pineal gland hippocampal cells of four seasons physiological group,sham operation group and operation group.By comparing the expression differences between four seasons and groups,the effects of seasonal rhythm and pineal gland MT on MTR Gs/Gi-cAMP-PKA-CREB signaling pathway in hippocampus were investigated.2.2.2 Experimental animalsThirty four SPF grade male SD rats were purchased 42 days before the Vernal Equinox(3.21),the Summer Solstice(6.21),the Autumnal Equinox(9.23)and the Winter Solstice(12.22).The age and weight of rats were 5 weeks and 150-170g.After 7 days of adaptive feeding,rats were randomly divided into physiological group(n=9),sham operation group(n=12)and operation group(n=13).The number of rats in each group was based on the previous experimental study,and the survival rate of rats was about 75%.All operations in sham operation group and operation group were completed within 5 days.Three groups of rats were fed in the same feeding environment.The rats were collected on the evening of the Vernal Equinox(3.21),the Summer Solstice(6.21),the Autumnal Equinox(9.23)and the Winter Solstice(12.22).The rats were exposed to natural photoperiod in doors,10±1h in spring,14.5±0.5h in summer,13±1h in autumn and 9±0.5h in winter.The relative humidity was 40%-50%.2.2.3 Detection methodThe expression of MT and MTR,AC,cAMP,PKA and CREB in the hippocampus was detected by enzyme linked immunoassay(ELISA);the expression of GS and GI in hippocampus was detected by Western blot(WB);the real-time quantitative poly chain reaction was used,the expression of CREB mRNA was detected by RT-qPCR..2.2.4 Statistical methodsAll data were presented in the form of mean ± standard deviation(x±s).SPSS 24.0 statistical software was used for statistical analysis of each index data.If the data conform to the normal distribution,the one-way ANOVA method is used for statistical analysis;if the data do not conform to the normal distribution,the nonparametric test method is used for statistical analysis.P<0.05 was considered to have significant statistical significance.3 Results3.1 The expression levels of serum MT and hippocampal MTR in physiological group in four seasons:the expression of serum MT and hippocampal MTR in four seasons had the rhythm of winter>autumn>spring>summer(serum MT:P<0.01;hippocampal MTR:P<0.05).3.2 The expression levels of Gs/Gi-cAMP-PKA-CREB signaling pathway in the four seasons of the physiological group:Gs/Gi in the four seasons had a seasonal rhythm of winter>autumn>summer>spring(P<0.01);the expression levels of AC,camp,PKA,CREB and CREB mRNA in the four seasons had a seasonal rhythm of winter>autumn>spring/Summer(P<0.01).3.3 Effect of pinealectomy on serum MT and hippocampal MTR:compared with the physiological group,the expression rhythm of serum MT and hippocampal MTR in the operation group changed to winter>autumn>spring/summer spring(P<0.01);the expression levels of serum MT and hippocampal MTR in the four seasons operation group decreased(P<0.01).3.4 Effects of pinealectomy on Gs/Gi-cAMP-PKA-CREB signaling pathway in hippocampus:compared with physiological group,the expression rhythm of Gs/Gi in operation group changed to autumn>winter>summer>spring(P<0.01);the expression rhythm of AC,camp,PKA,CREB and CREB mRNA in operation group changed to autumn>winter>spring/Summer(P<0.01);Gs/Gi,AC,camp,PKA,CREB and CREB mRNA in spring operation group changed to autumn>winter>spring/summer(P<0.01)The expression levels of AC,camp,PKA,CREB and CREB mRNA in summer operation group were decreased(P<0.01;P<0.05);the expression levels of AC,camp,PKA,CREB and CREB mRNA in summer operation group were decreased(P<0.01;P<0.05);the expression levels of Gs/Gi,AC,camp,PKA,CREB and CREB mRNA in autumn operation group were decreased(P<0.05)The expression levels of Gs/Gi,AC,camp,PKA,CREB and CREB mRNA in winter operation group were lower than those in control group(P<0.01).4 Conclusions4.1 Theoretical research conclusionIt is hypothesized that the microscopic mechanism of "liver’s governance of conducting and dispersing Qi and regulating emotion" may be related to the direct regulation of pineal gland MT on MTR in hippocampus,and the signal transduction mechanism of pineal gland MT may be related to the Gs/Gi-cAMP-PKA-CREB signal pathway in hippocampus.4.2 Experimental research conclusion4.2.1 The expression of MT,MTR and Gs/Gi-cAMP-PKA-CREB signal pathway in the hippocampus were lower in spring and summer,but higher in autumn and winter in seasonal rhythm.4.2.2 Pineal MT may be one of the mediators of seasonal rhythm regulating hippocampal function.4.2.3 Pineal gland MT has direct regulation on hippocampal function,and its regulation mechanism is related to hippocampal MTR and Gs/Gi-cAMP-PKA-CREB signaling pathway,and has the physiological characteristics of seasonal selectivity,degree difference and regulation complexity.4.2.4 The micromechanism of "liver’s governance of conducting and dispersing Qi and regulating emotion,and changes with time" is related to pineal gland MT hippocampal MTR and Gs/Gi-cAMP-PKA-CREB signaling pathway.