Yin Deficiency Of Old Age Blood Loss Insomnia Syndrome Rat Model To Establish And Semen Soup Intervention Effect

Objective:our research is to establish the pathogeny rats model of insomina with blood-deficiency and Yin(血亏阴虚)in the old by combining clinical histories,then we compared this kind of compound model with traditional sleep deprivation model only,and evaluated it following the thought of pathogeny-Surface characteristics of Chinese medicine-Syndrome- therapy:First,we evaluated the scientific model of the factors according to the etiology and pathogenesis of Chinese medicine;Second,we observed the direct,intuitionistic and important surface characteristics of Chinese medicine-syndrome,and confirmed the nature of this model;Third,we probed into syndrome,that was,by Modern scientific and technological means,such as molecule biology,we summarized the insomnia mechanisms with blood-deficiency and Yin in the old reflected by the surface characteristics.Finally,we used prescription SZRT to "rule" for evidence to the contrary,then discussed the effect and possible mechanism of SZRT,and established the efficacy evaluating flatform of Chinese herbal compound using systems biology on the basis of Metabolic network changes.Methods:After experimental studies of SZRT on the role of promoting sleep, healthy Wistar rats(n=100,200g±20g)of Equal numbers of male and female were randomly divided into five groups:environmental control group;sleep deprivation group(single-factor model group);syndrome model(multiple factors model group); SZRT large dose group;SZRT small dose group.Environmental control group were treated with similar water environment of the other sleep deprivation group; Single-factor model group were treated with 48h sleep deprivation by self-made multi-platform water environment methods;Multiple factors model group and SZRT groups were treated with neck back subcutaneously D-galactose for six weeks, intraperitoneal injection of cyclophosphamide and hydrocortisone for five days,48h of sleep deprivation using the self-made multi-platform water environmental methods,modeling were lasted for a total of seven weeks;Since SZRT large and small doses group had producted model for four weeks,we gave intragastric administration for three weeks at the same time,other groups were given isovolumic saline. Detection of specific indicators as follows:1.by the simultaneous detection of weight,food intake,axillary temperature,and claws,and other microscopic magnification camera,Chinese signs of the each group rats wered compared;2.rats were Detected by Histochemistry Detection with the change of cortex, hippocampus site radicals and nitric oxide synthase(NOS);3.rats were examined with changes on blood cell counts by a blood cell analyzer;4.a Morris water maze test were used to examine rats learning and memory abilities change;5.transmission electron microscopy observation of the experiment rat cortical parts ultrastructural changes;6.rats were detected about glutamate(Glu)andγ-ammonia Dimethylolbutyric acid(GABA)content changes by HPLC in cerebral cortex and hypothalamus parts;7.using immunohistochemical staining and semi-quantitative analysis of a retrovirus - polymerase chain reaction(RT-PCR),the experimental group rat were detected on cortical and hippocampal parts ofγ-aminobutyric acid A receptor (GABAAR)a₁ andγ₂ subunit expression changes;8.immunohistochemical staining was used in the cortical in rats star-shaped glial cells expression.Results:1.Compared with the control group,SLAD group and SZRT-large and small dose could significantly extend the threshold dose of pentobarbital sodium inducing sleep time(P＜0.01),significantly increase in the threshold dose of pentobarbital sodium inducing sleep properly rate(P＜0.01).The effect of SZRT high-dose group was more obvious than low-dose group,but no statistical significance(P＞0.05);2.The Chinese surface characteristics showed that,compared to environmental control group,sleep deprivation group appeared insomnia symptoms,severe fatigue performance of the state;Compared to sleep deprivation group,syndrome model group in the event of symptoms of insomnia at the same time,looked gray-faced and sleeping,blood lethargy,appetite decreased,weakness,hair shaft,blood-deficiency and Yin signs,and body weight,food intake,decreased(P＜0.01),but axillary temperature's have not statistically signifcant(P＞0.05);Compared to syndrome model group, SZRT of large and small dose groups apeared improvement of blood blood-deficiency and Yin signs,but not statistically significant(P＞0.05);3.Compared to syndrome model group,the other group rats' malondialdehyde (MDA)content,superoxide dismutase(SOD)and nitric oxide synthase(NOS) activity were significantly decreased in the cerebral cortex,hippocampus(P＜0.01, P＜0.05),and SZRT of large and small dose group exhibited some dose-effect relationship(P＜0.05);4.Compared to syndrome model group,the other rats'whole blood erythrocyte count,white blood cell count,platelet count and hemoglobin level were significantly increased(P＜0.01),and SZRT of large and small dose group exhibited some dose-effect relationship(P＜0.05);5.In the positioning navigation experiments,compared to syndrome model group,environmental group and SZRT of large and small dose group rats' incubation period of four days and two days were significantly reduced(P＜0.01).In the search space experiments,the platform quadrant swimming distance percentage of the total distance,the wall 20%and 40%from the percentage of the regional swimming results showed the same trend to the positioning navigation experiments;Compared to syndrome model group,the environment control group and SZRT of large and small dose groups' number of crossing the location of the original platforms increased significantly(P＜0.01).SZRT of large and small dose group exhibited dose-effect relationship.Two model group showed no significant difference(P＞0.05);6.After 48h of sleep deprivation,under the electron microscope showed that: sleep deprivation rat cerebral cortex neurons of minor differences with gathered side were becoming nuclear-week gap widened slightly,the majority of mitochondrial highly edemaed,and the mitochondrial matrix mitochondrial ridge disappeared;Syndrome model group rat cerebral cortex neurons in cell cytoplasm edemaed,connecting widened,we could see that single neurons with different quality cohesion,more edge,showed apoptotic morphology,cell disappeared;SZRT of large and small dose group could improve the rat neurons pathological changes;7.Compared to environmental control group,sleep deprivation group rats' amino acid neurotransmitter Glu,GABA contents were significantly increased in cerebral cortex and hypothalamus site(P＜0.01),but GABA increased more significantly, Glu/GABA proportional reducted;Compared to environmental control group, syndrome model group rats' contents of Glu and GABA were significantly increased (P＜0.01),but Glu increased more markedly,Glu/GABA ratio increased;Compared to syndrome model group,SZRT of large and small dose group rats'amino acid neurotransmitter in cerebral cortex and hypothalamus site,Glu,GABA were significantly reduced,but Glu reduced more significantly,and GIu/GABA ratio decreased,with a certain dose-effect relationship(P＜0.05);8.Compared to syndrome model group,environmental control group and SZRT of large and small dose group rats' GABAARa₁ andγ₂ subunit immunochemistry absorbance in cerebral cortex and hippocampus accumulated significantly lower (P＜0.01),and GABAARa₁,γ₂ mRNA and protein expression were significantly reduced(P＜0.01),SZRT of large and small dose group exhibited dose-effect relationship;Compared to the sleep-deprived group,syndrome model group showed weaker expression(P＜0.05);9.Compared to syndrome model group,environmental control group,SZRT of large and small dose group rats' GFAP expression in the cortical showed weaker after 48h of sleep deprivation;Compared to the sleep-deprived group syndrome model group showed stronger expression.Conclusion:1.SZRT have significantly sedative and hypnotic effects,and the role of high-dose is Obvious than small doses; 2.The rats surface characteristics,D-galactose + cyclophosphamide, hydrocortisone + sleep deprivation,this composite model is closer to clinical blood loss elderly patients with insomnia Yin-performance,is a viable,reliable animal model of the compound;3.The ability of learning and memory in old rats and pathological damage are relevant to sleep deprivation and blood-deficiency and Yin.Increasing of the level of free radicals and reducing of blood is one of the pathological mechanisms of insomina with blood-deficiency and Yin in the old.Disproving prescription SZRT can be effective to improving learning and memory abilities and repairing brain injury of syndrome model rats,and its pathological mechanism is relevant to Anti-free radicals and the role of the blood;4.In cortex and hypothalamus,the improvement of the content of Glu,GABA, and the ratio of Glu/GABA may be one of the neurobiology pathological mechanisms of insomina with blood-deficiency and Yin in the old.SZRT can protection organizations by downward adjusting the content of Glu,GABA,and the ratio of Glu/GABA in cerebral cortex and hypothalamus,This may be one of the mechanisms of treatment on insomina with blood-deficiency and Yin in the old;5.Sleep deprivation may increase obviously the rats' GABAAR a₁ andγ₂ subunit expression in cerebral cortex and hippocampus.SZRT downward adjusting GABAAR a₁ andγ₂ subunit expression in the rat brain may be one of the mechanisms of treatment on insomina with blood-deficiency and Yin in the old;6.Sleep deprivation may increase GFAP expression of the rats in the cortical, indicating that astrocytes can be excited,and may be involved in sleep regulation.Tts expression increasing obviously may be one of the mechanisms of insomina with blood-deficiency and Yin in the old.SZRT downward adjusting the expression of GFAP may be one of the mechanisms of treatment on insomina with blood-deficiency and Yin in the old.