| Objective: To observe the changes of amino acid neurotransmitters in PCPA inomnia rats and influence the expression of Clock gene,to explore the mechanism of the hypnotic effect of Cnidii Fructus hypnotic active constituent(SCZ)on on sleep deprived rats.Method: Build the insomnia model by intraperitoneal injection of PCPA(para-chlorophenylalanine).with SCZ of low(25mg/kg),medium(50mg/kg)and high(100mg/kg),conentrations were also given respectively at the same time,and in diazepam positive group,intragastric administration of PCPA was performed in insomnia rats for 3days,The expressions of GABA and GLU receptor proteins in hippocampal dentate gyrus of rats were detected by immunohistochemistry.The levels of GABA,GLY and GLU,ASP in the hippocampus,brain stem,hypothalamus were detected by high performance liquid chromatography(HPLC).The expression levels of Clock,Bmal1,Cry1,Cry2,Per1,Per2 and Per3 were detected by real-time PCR.To analyze the relationship between the contents of clock gene and neurotransmitter in Cochinchinensis hypnotic active components in insomnia rats.Results:1.Effects of the active components of Cnidii Fructus on Amino Acid neurotransmitters in the Hippocampus,Brain Stem and Hypothalamus of rats with PCPA Insomnia(1)The content of GLU in the hippocampus,the model control group was significantly higher than the blank control group(P<0.01).The GLU content in the diazepam positive control group was significantly lower than the model control group(P<0.01).The GLU content in the SCZ high and middle dose groups was significantly lower than the model control group(P<0.01,P<0.05),there was no significant difference inASP content between the groups,but the diazepam positive control group ? each administration group was decreased than the model control group,the content of GABA,GLY was significantly lower than the blank control group(P<0.01).The content of GABA and GLY in the diazepam positive control group was significantly higher than that the model control group(P<0.01,P<0.001).The content of GABA and GLY in the SCZ high dose group was significant higher than model control group(P<0.05).It was shown that SCZ can significantly increase the content of GABA and GLY inhibitory neurotransmitters in hippocampus of rats with PCPA insomnia,and reduce the GLU content.(2)The content of GLU and ASP in Brain stem,the control group were significantly higher than the blank control group(P<0.05,P<0.01).The content of GLU in the SCZ high and middle dose groups were significantly lower than the model control group(P<0.01).The ASP content in the diazepam positive control was significantly lower than the model control group(P<0.01).The ASP content in the SCZ high and middle dose group was significantly lower than the model control group(P<0.01,P<0.05).The GABA and GLY content model control group was significantly lower than the blank control group(P<0.05).the content of GABA and GLY in the diazepam positive control group was significantly higher than the model control group(P<0.01,P<0.05).The GABA content in the SCZ high and low dose groups was significantly higher than the model control group(P<0.01,P<0.05),The content of GLY in the SCZ high and middle dose groups was significantly higher than the model control group(P<0.05).It was shown that SCZ can significantly increase the content of GABA and GLY in brain stem of rats with PCPA insomnia,and reduce the content of GLU and ASP.(3)The content of GLU and ASP in the hypothalamus,the the control group was significantly higher than the control group(P<0.01).The content of GLU and ASP in the diazepam positive control group was significantly lower than the model control group(P<0.001,P<0.01).The content of GLU in the SCZ high and middle-dose group was significantly lower than the model control group(P<0.01,P<0.05).The ASP content in the SCZ high-and middle-dose group was significantly lower than the model control group(P<0.01,P<0.001),The content of GABA,GLY in the model control group was significantly lower than the blank control group(P<0.05).The content of GABA and GLY in the diazepam positive control group was significantly higher than the model control group(P<0.001,P<0.01).The content of GABA in the SCZ high and middle dose group was significantly higher than the model control group(P<0.01).The content of GLY in SCZ high dose group was significantly higher than the model control group(P<0.001).It was shown that SCZ can significantly increase the content of GABA and GLY inhypothalamus of rats with PCPA insomnia and reduce the content of GLU and ASP.(4)GABA content in hippocampus was significantly higher than the brain stem(P<0.01).GLU content in hippocampus was significantly higher than the brain stem and hypothalamus(P<0.001,P<0.01).The content of ASP and GLY in hypothalamus was significantly higher than the control group in Hippocampus and brain stem(P<0.01).It was shown that GABA and GLU were widely distributed in the hippocampus.2.Effect of the active components of Cnidii Fructus on the Expression of GABA and GLU Receptor Proteins in dentate gyrus of the hippocampus on Insomnia rats induced by PCPAThe GLU integral optical density of the hippocampal dentate gyrus in the model control group was significantly higher than the blank control group(P<0.01),and the administration group was significantly lower than the model control group(P<0.01),The GABA integral optical density in the hippocampal dentate gyrus in the control group was significantly lower than the blank control group(P<0.05),The diazepam positive control of GABA content in the group was significantly higher than that the model control group(P<0.01),and the administration group was significantly higher than the model control group(P<0.01).3.Effects of the Active Components of Cnidii Fructus on hippocampus Clock Gene Expression in Insomnia ratsThe expression of Clock and Bmal1 gene in the model control group was significantly higher than the blank control group(P<0.05),The expression of Clock gene in the diazepam positive control group were significantly lower than the model control group(P<0.01,P<0.05),The expression of Clock and Bmal1 gene in SCZ middle dose group was significantly lower the model control group(P<0.01),and the expression levels of Cry1,Per1,Per2,Per3 gene in control group were significantly lower than in blank control group(P<0.05,P<0.01,P<0.05,P<0.05).The expression levels of Cry1,Per1,Per2,and Per3 in the diazepam positive control group were significantly higher than in the model control group(P<0.01,P<0.01,P<0.05,P<0.05).The expression levels of Cry1,Per1,Per2,and Per3 genes in the high dose group were significantly higher than in the model control group(P<0.05,P<0.01,P<0.05,P<0.01).The Cry2 gene expression level in the SCZ middle dose group was significantly higher than the model control group(P<0.05),the Perl gene expression level in the SCZ low dose group was significantly higher than the model control group(P<0.01).It was showed that SCZ significantly reduced the expression of Clock and Bmal1 gene in insomnia rats with PCPA,and increased the expression levels of Cry1,Cry2,Per1,Per2,Per3 genes.Conclusion:1.SCZ can reduce the contents of GLU and ASP excitatory amino acid neurotransmitters in hippocampus,brain stem and hypothalamus of PCPA insomnia rats.and increased the content of GLY and GABA inhibitory amino acid neurotransmitters of PCPA insomnia rats.2.SCZ significantly increased GABA receptor expression of inhibitory amino acid neurotransmitters in the dentate gyrus of PCPA insomnia rats,and decreased the expression of excitatory amino acid neurotransmitter GLU receptor.3.SCZ regulates the sleep-wake cycle of the hippocampal clock gene,by down-regulating expression of the positive regulators Clock and Bmal1,and up-regulating the expression of Cry1,Cry2,Per1,Per2,and Per3 genes. |
PDF Full Text Request