Study On Seasonal Changes Of MTR-DAG-IP₃-PKC-c-fos/c-jun-5-HT And NE In Hippocampus Based On The Principle Of “liver Responds To Spring, Regulates Emotions And Moods??

"Five zang organs corresponding to time" refers to the change law of coordination and resonance between the functional activities of the five zang organs and the changes of yin and Yang in the four seasons."Liver corresponding to spring" is an important part of the theory of"five zang organs corresponding to time".Liver zang-xiang is the main system to regulate activities of qi in the body.In the adaptive regulation process of "corresponding with time",it is mainly manifested in the rhythmic changes of four seasons with strong function of "liver controlling dispersion" in spring and summer and weak function in autumn and winter.The previous research of the research group shows that the theory of "liver corresponding to spring,controlling dispersion and regulating emotion" in traditional Chinese medicine has scientific connotation,and its internal basis is related to the four seasons regulation of hippocampal nervous system by pineal gland melatonin,but there is still a lack of research on specific regulation mechanism.Through animal experiments,from the perspective of pineal melatonin regulating hippocampal cell signal transduction and neurotransmitters in four seasons,this paper discusses the biological basis of "liver corresponding to spring,controlling dispersion and regulating emotion".1 PurposeCombining seasonal climate change,modern neuroendocrinology and the theory of"liver corresponding to spring" of traditional Chinese medicine,a cross study was conducted to observe the melatonin receptor(MTR)-diacylglycerol(DAG)-inositol triphosphate(IP3)-protein kinase C(PKC)-c-fos/c-jun and neurotransmitter 5-hydroxytryptamine(5-HT)and noradrenaline(NE)in rat hippocampus To explore the regulation mechanism of pineal melatonin on hippocampal emotion related neuroendocrine network in different seasons,so as to reveal the biological basis of "liver corresponding to spring,controlling dispersion and regulating emotion".2 Methods2.1 Theoretical researchSelect the methods of literature collation and logical analysis,collect the relevant theoretical,experimental and clinical research literature of "five zang organs corresponding to time" and "liver corresponding to spring,controlling dispersion and regulating emotion",and explore the relevant scientific connotation of "liver corresponding to spring,controlling dispersion and regulating emotion",so as to provide a feasible theoretical basis for experimental research.2.2 Experimental research2.2.1 Experimental designThe spring equinox,summer solstice,autumn equinox and winter solstice were set as the experimental time nodes.SD male rats were randomly divided into physiological control group,pseudo operation group(surgical trauma stimulation without pineal gland removal)and operation group(pineal gland removal).The levels of serum melatonin,hippocampal MTR,second messenger DAG and IP3,PKC,third messenger c-fos and c-jun,and neurotransmitters 5-HT and NE were measured in each group in four seasons.Through the intergroup comparisons in the same season and intragroup comparisons in different seasons,this paper discusses the changes of seasons and the regulatory mechanism of pineal melatonin on the hippocampal emotion regulatory chain of MTR-DAG-IP3-PKC-c-fos/c-jun neurotransmitter.2.2.2 Seasonal settingThis experiment used the seasonal climate simulation system box to control the four seasons environment of the experiment.The temperature and humidity in four seasons were set according to the ground climate,monthly average temperature and average relative humidity data in Beijing.The data were from China Meteorological Center.The light exposure was dynamically set according to the flag raising and lowering schedule of Tiananmen Square regional management committee from 2020 to 2021.2.2.3 Experimental animalsMale SD rats aged 5 weeks were purchased 43 days before spring equinox,summer solstice,autumn equinox and winter solstice.Adaptive feeding was carried out for 7 days in each season,and they were completely randomly divided into physiological control group,pseudo operation group and operation group.The rats in the sham operation group and the operation group completed the modeling 31 days before the spring equinox,summer solstice,autumn equinox and winter solstice.The three groups of rats were fed for 31 days under the same conditions of the seasonal climate simulation system box,and the materials were taken on the evening of spring equinox,summer solstice,autumn equinox and winter solstice.2.2.4 Index detectionThe levels of serum melatonin and hippocampal MTR,DAG,IP3,5-HT and NE were detected by enzyme-linked immunosorbent assay;The protein expression levels of p-PKC,c-fos and c-jun in hippocampus were detected by western blot;The levels of c-fos mRNA and c-jun mRNA were detected by quantitative real-time PCR.2.2.5 Statistical methodSPSS 20.0 was used to analyze the experimental data.The measurement data were described by x±s.If the data of intergroups and intragroups were normally distributed and consistent with the homogeneity of variance,one-way ANOVA was used;If it did not conform to normal distribution or variance homogeneity,nonparametric test would be used.3 Results3.1 In the physiological control group,the levels of serum melatonin and hippocampal MTR had seasonal rhythms of lower in spring and summer and higher in autumn and winter.3.2 In the physiological control group,the expression level of DAG-IP3-PKC-c-fos/c-jun signaling pathway in hippocampus had seasonal rhythms of higher in spring and summer and lower in autumn and winter.3.3 In the physiological control group,the expression levels of hippocampal neurotransmitters 5-HT and NE had seasonal rhythms of higher in spring and summer and lower in autumn and winter.3.4 Effects of pinealectomy on serum melatonin and hippocampal MTR:3.4.1 Intergroup comparisons in the same seasonIn spring,compared with the physiological control group,the levels of melatonin and MTR in the operation group decreased(P<0.01);In summer,compared with the physiological control group,the levels of melatonin and MTR in the operation group decreased(melatonin:P<0.01;MTR:P<0.05);In autumn,compared with the physiological control group,the levels of melatonin and MTR in the operation group decreased(P<0.01);In winter,compared with the physiological control group,the levels of melatonin and MTR in the operation group decreased(P<0.01).3.4.2 Intragroup comparisons in different seasonsIn the pseudo operation group,the levels of melatonin and MTR had seasonal rhythms of lower in spring and summer and higher in autumn and winter,which were consistent with the seasonal rhythms of the physiological control group.In the operation group,the levels of melatonin and MTR were higher in autumn,lower in spring,summer and winter and higher in spring,summer,autumn and lower in winter,respectively,which were different from those in the physiological control group.3.5 Effects of pinealectomy on hippocampal DAG-IP3-PKC-c-fos/c-jun signaling pathway3.5.1 Intergroup comparisons in the same seasonIn spring,compared with the physiological control group,the levels of DAG,IP3,c-jun protein and c-jun mRNA in the operation group decreased(P<0.01),and the levels of p-PKC,c-fos protein and c-fos mRNA increased(P<0.01);In summer,compared with the physiological control group,the levels of DAG,IP3,c-jun protein and c-jun mRNA in the operation group decreased(DAG,IP3,c-jun mRNA:P<0.01;c-jun protein:P<0.05),and the levels of p-PKC,c-fos protein and c-fos mRNA increased(P<0.01);In autumn,compared with the physiological control group,the level of DAG in the operation group decreased(P<0.01),and the levels of IP3,p-PKC,c-jun protein,c-jun mRNA,c-fos protein and c-fos mRNA increased(P<0.01);In winter,compared with the physiological control group,the level of DAG,IP3,p-PKC,c-jun protein,c-jun mRNA,c-fos protein and c-fos mRNA increased(P<0.01).3.5.2 Intragroup comparisons in different seasonsIn the sham operation group,the level of DAG-IP3-PKC-c-fos/c-jun signal pathway had seasonal rhythms of higher in spring and summer and lower in autumn and winter,which were consistent with the seasonal rhythms of the physiological control group.In the operation group,the expression rhythm of DAG,IP3 and c-fos mRNA were higher in winter and lower in spring,summer and autumn;There was no seasonal expression rhythm in the levels of p-PKC and c-fos protein;The expression rhythm of c-jun protein and c-jun mRNA were lower in spring and higher in summer,autumn and winter,which were different from that of physiological control group.3.6 Effects of pinealectomy on hippocampal neurotransmitters 5-HT and NE:3.6.1 Intergroup comparisons in the same seasonIn spring,compared with the physiological control group,the level of 5-HT in the operation group decreased and the level of NE increased(P<0.01);In summer,compared with the physiological control group,the levels of 5-HT and NE in the operation group decreased(P<0.01);In autumn,compared with the physiological control group,the level of 5-HT in the operation group decreased and the level of NE increased(P<0.01);In winter,compared with the physiological control group,the levels of 5-HT and NE in the operation group increased(P<0.01).3.6.2 Intragroup comparisons in different seasonsIn the pseudo operation group,the levels of 5-HT and NE had seasonal rhythms of higher in spring and summer and lower in autumn and winter,which were consistent with the seasonal rhythms of the physiological control group.In the operation group,there was no seasonal rhythm in the expression level of 5-HT;The seasonal secretion rhythm of hippocampal NE was lower in summer and autumn and higher in spring and winter,which was different from that of physiological control group.4 Conclusion4.1 Theoretical research4.1.1 The theory of "liver responding to spring" was constructed in Huangdi Neijing,developed in later generations and perfected in modern times.4.1.2 The essence of liver zang-xiang is the time regulation system of human body that changes according to four seasons.4.1.3 "Liver corresponding to spring,controlling dispersion and regulating emotion" is the embodiment of "the liver should spring" in emotion regulation4.1.4 Put forward the research hypothesis that the biological mechanism of "liver corresponding to spring,controlling dispersion and regulating emotion" may be related to the direct effect of pineal melatonin on the hippocampal nervous system in the four seasons.Seasonal changes can be mediated by pineal melatonin to regulate the seasonal changes of emotion through the related emotion regulation chain of hippocampal signal transduction pathway-neurotransmitters.4.2 Experimental research4.2.1 The expression of serum melatonin and hippocampal MTR has seasonal rhythms of lower in spring and summer and higher in autumn and winter;The expression of DAG-IP3-PKC-c-fos/c-jun-neurotransmitters 5-HT and NE in hippocampus has seasonal rhythms of higher in spring and summer and lower in autumn and winter.Melatonin hippocampal MTR has a negative regulatory effect on DAG-IP3-PKC-c-fos/c-jun signal pathway.4.2.2 Pineal melatonin may be one of the mediators of seasonal changes on the regulation of hippocampal MTR-DAG-IP3-PKC-c-fos/c-jun-neurotransmitter.Pineal melatonin has different levels,degrees and directions of regulation on this hippocampal emotion regulation chain,showing the complexity of regulation and seasonal selectivity.4.2.3 The theory of "liver corresponding to spring,controlling dispersion and regulating emotion" in traditional Chinese medicine has scientific connotation,and its internal basis may be related to the four seasons regulation of pineal gland melatonin on hippocampal MTR-DAG-IP3-PKC-c-fos/c-jun-neurotransmitter 5-HT and NE emotional regulation chain.