| 1.ObjectiveThis article aims at adopting the research method of "measured by time",that is,under the guidance of the concept of "correspondence between human and universe" view of TCM,according to the characteristics of the five internal organs of TCM and the four-time changeBy observing and comparing the seasonal changes of related clock genes and cell signal transduction factors in the tissues of the hypothalamus-pituitary-adrenal(HPA)axis of golden hamsters in the normal group and the model group(with pineal gland removed),further reveals the biological mechanism of " kidney corresponding with winter ",and explores the essential connotation of TCM viscera-state doctrine of kidney.2.Methods2.1 Animal experimentsThe 8-week-old male Syrian golden hamster(SPF)weighing 120g ± 20g,were purchased from Beijing Weitonglihua Eeperimental Animal Technology Co.,Ltd.5 weeks before the spring equinox,summer solstice,autumn equinox,and winter solstice.Then the golden hamsters were randomly divided into a normal group and a model group,with 8 in each group.The model group underwent pineal gland excision,and the normal group did nothing.Under the same breeding conditions,they were raised until the spring equinox,summer solstice,autumn equinox,and winter solstice.It was moved to the dark room at 20:00 on the same day,and began to collect materials at 21:00.The hypothalamus,pituitary,and adrenal tissues of each group of golden hamsters were taken out,and the protein expression of circadian clock genes and cell signal transduction factors in each tissue was detected by Western-Blot.2.2 Indicator detection2.2.1 Effects of four seasons changes on hypothalamic-pituitary-adrenal axis biological clock genes perl and per2 in golden hamster2.2.2 Effects of four seasons changes on signal transduction factors c-fos and c-jun in hypothalamic-pituitary-adrenal axis of golden hamster2.2.3 Effects of four seasons changes on G proteins Gi and Gs of hypothalamic-pituitary-adrenal axis in golden hamster2.3 Statistical analysis methodsMeasurement results are expressed as mean±standard deviation((?)±s),statistical analysis is performed using SAS 8.0,one-way analysis of variance was used between groups;independent paired t test is used for pairwise comparison,or 2 Nonparametric tests of independent samples.The difference was statistically significant at P<0.05.3.Research results3.1 Four seasons changes of protein expression of circadian clock genes Perl and per2 in HPA axis3.1.1 Four seasons changes of protein expression of circadian clock gene Perl and per2 in hypothalamus①The change trend of perl protein expression in the hypothalamus of golden hamsters in the control group during the four seasons was spring>autumn>winter>summer.②The change trend of per2 protein expression in the hypothalamus of golden hamsters in the control group during the four seasons was winter>spring>autumn>summer,and the change trend of per2 protein expression in the hypothalamus tissue of the experimental group was autumn>winter>summer>spring in four seasons.3.1.2 Four seasons changes of protein expression of circadian clock gene per2 in pituitary tissue① The change trend of per2 protein expression in the pituitary tissue of golden hamsters in the control group was spring>winter>autumn>summer in four seasons.The change trend of per2 protein expression in the pituitary tissue of golden hamsters in the experimental group was spring>autumn>winter>summer in four seasons.3.1.3 Four seasons changes of protein expression of circadian clock gene Perl and per2 in adrenal tissue①The trend of perl protein expression in adrenal gland tissue of golden hamsters in the control group during the four seasons was summer>winter>spring>autumn,and the trend of perl protein expression in adrenal gland tissue of golden hamsters in the experimental group during the four seasons was autumn>summer>winter>spring.②In the control group,the expression trend of per protein in adrenal tissue of golden hamster was summer>autumn>spring>winter in four seasons,and there were significant differences in spring,summer and autumn compared with winter(P<0.05,P<0.01,P<0.001);in the experimental group,the expression trend of per2 protein in adrenal tissue of golden hamster was spring>summer>autumn>winter in four seasons.3.2 Four seasons changes of protein expression of c-fos and c-jun proteins in cell signal transduction factors in HPA axis tissues3.2.1 Four seasons changes of protein expression of c-fos and c-jun proteins in cellular signal transduction factors in hypothalamus①The change trend of c-fos protein expression in the hypothalamus of golden hamster in the control group was winter>spring>autumn>summer in four seasons,and that in the hypothalamus of golden hamster in the experimental group was winter>spring>autumn>summer in four seasons.②The change trend of c-jun protein expression in the hypothalamus of golden hamster in the control group in four seasons is spring>autumn>winter>summer,and there is significant difference between summer and winter and spring(P<0.05);the change trend of c-jun protein expression in the hypothalamus of golden hamster in the experimental group in four seasons is autumn>spring>winter>summer,and there is significant difference between the experimental group in spring and the control group in spring There was statistical significance(P<0.05).3.2.2 Four seasons changes of protein expression of c-fos and c-jun in pituitary tissue①The expression trend of c-fos protein in the pituitary of golden hamster in the control group was winter>autumn>spring>summer in four seasons,and there was significant difference between spring and summer and winter(P<0.05,P<0.01);the expression trend of c-fos protein in the pituitary of golden hamster in the experimental group was summer>spring>autumn>winter in four seasons,and there was significant difference between the experimental group and the control group in winter,There was statistical significance(P<0.01).②The change trend of c-jun protein expression in the pituitary of golden hamster in the control group was winter>autumn>spring>summer in four seasons,and there was significant difference between spring and summer and winter(P<0.05);the change trend of c-jun protein expression in the pituitary of golden hamster in the experimental group was autumn>winter>spring>summer in four seasons.3.2.3 Four seasons changes of protein expression of c-fos and c-jun in adrenal tissue① The expression trend of c-fos protein in adrenals of golden hamster in the control group was winter>autumn>spring>summer in four seasons,and that in adrenals of golden hamster in the experimental group was winter>autumn>spring>summer in four seasons.②The expression trend of c-jun protein in adrenals of golden hamster in the control group was winter>spring>autumn>summer in four seasons,and there was significant difference between summer and winter(P<0.05);the change trend of c-jun protein in adrenals of golden hamster in the experimental group was winter>autumn>spring>summer in four seasons.3.3 Four seasons changes in the expression of cell signal transduction factors Gi and Gs proteins in HPA axis tissues3.3.1 Four seasons changes of protein expression of Gi and Gs proteins in cellular signal transduction factors in hypothalamus①The change trend of Gi protein expression in the hypothalamus of golden hamster in the control group was winter>spring>autumn>summer in four seasons,and that in the hypothalamus of golden hamster in the experimental group was spring>summer>winter>autumn in four seasons②The change trend of Gs protein expression in the hypothalamus of golden hamster in the control group in four seasons was winter>autumn>spring>summer,and there was significant difference between spring and summer and winter(P<0.05);the change trend of Gs protein expression in the hypothalamus of golden hamster in the experimental group in four seasons was summer>spring>autumn>winter,and there was significant difference between the experimental group and the control group in summer(P<0.05).3.3.2 Four seasons changes of protein expression of Gi and Gs proteins in cellular signal transduction factors in adrenal tissue①The change trend of Gi protein expression in adrenal tissue of golden hamster in the control group was winter>autumn>spring>summer in four seasons,and that in adrenal tissue of golden hamster in the experimental group was winter>spring>autumn>summer in four seasons.②In the control group,the expression trend of Gs protein in adrenal tissue of golden hamster in four seasons was summer>autumn>spring>winter,and there was significant difference between summer and winter(P<0.05);in the experimental group,the expression trend of Gs protein in adrenal tissue of golden hamster in four seasons was spring>summer>autumn>winter,and there was significant difference between the experimental group and the control group in spring(P<0.05)。4.Conclusion(1)This study is the first to confirm that the biological clock genes perl and per2 in hypothalamus,pituitary and adrenal tissues have a certain seasonal rhythm,and the circadian clock genes perl and per2 participate in the regulation of hypothalamus pituitary adrenal axis on the body.The regularity of "kidney corresponding with the winter" is closely related to the seasonal rhythm change of biological clock gene of hypothalamus pituitary adrenal axis(2)The physiological mechanism of TCM "kidney corresponding with the winter" is closely related to the changes of melatonin in the hypothalamus-pituitary-adrenal axis,G protein-mediated and the third messenger c-fos,c-jun involved in cell signal transduction system. |
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