| Section One Heart hiding god plays an important role in rgulating vigilance to sleep.The essence of insomnia about heart not hiding god lies in distraction of the mind,and the main symptom is insomnia,which is difficult or even sleepless,and the position is mainly related to the heart.The etiology of insomnia about heart not hiding god including two aspects of deficiency and excess,heart deficiency of qi,blood,yin,and yang,heart fire,phlegm fire,food stagnation and blood stasis.The main pathogenesis is Xie Rao Xin Shen or Xin Shen Shi Yang.The remedies for insomnia about heart not hiding god include dispelling evil for tranquilization(clearing heart fire,eliminating phlegm,harmonizing stomach,dispersing blood stasis),nourishing for tranquilization(nourishing heart qi,nourishing heart blood,nourishing heart yin,warming heart yang),and tranquilization with heavy prescription.The insomnia of heart not hiding god can be differentiated into heart fire hyperactivity,phlegm fire disturb heart,heart stomach discord,blood stasis block heart,insufficiency of heart qi,blood,yin and yang.There are many single herbs for treating insomnia about heart not hiding god.However,each herb has its own characterstics,we should respectively select the calming herb of clearing heart fire,dispelling phlegm,dispersing blood stasis,nourishing qi,blood and yin of heart according to the clinical pathogenesis of insomnia about heart not hiding god.Only in this way can the exact curative effect be received.Coptis chinensis is the best remedy for insomnia,which has been reported in many ancient and modern literatures.Berberine is the main component of its which plays the role of clearing heart fire for tranquilization,while other herbs do not contain this component.Moreover,the fiery evil disturbing the god is a common pathogenic factor for the heart not hiding god,and the work of Huang Lian’s clearing heart fire for tranquilization is just aimed at the consistent disease factor of fiery evil disturbing the god.Therefore,in this study,Huang Lian was the first choice for the experimental study.Section Two Objective: To explore the modeling methods and evaluation criteria of insomnia rats with heart not hiding god.Methods: P-chlorophenylalanine(PCPA)combined with caffeine was used to replicate the insomnia rat model,and the success of the model was evaluated from general conditions,water maze experiment,pentobarbital sodium induced sleep experiment,cardiovascular related factors and electroencephalogram(EEG)monitoring.Results: After the modeling,compared with the normal group,the body weight obviously decreased,and heart rate,respiratory rate and systolic blood pressure of the rats increased significantly in the combined group.Compared with the normal group,the sleep latency time of the combined group was significantly prolonged(P<0.01),which was higher than that of the PCPA group and the caffeine group(P<0.01).The duration of sleep was significantly shorter in the combined group(P < 0.01)than in the PCPA group and caffeine group(P < 0.01).Compared with the normal group,the escape incubation period of rats in the heart without storing spirit group and the PCPA group was significantly prolonged(P < 0.01).The frequency of crossing the target quadrant of rats in the combined group and PCPA group decreased(P< 0.01).Compared with the normal group,the content of ANP,BNP,ET-1,IL-6,TNF-αand IL-17 A in plasma,myocardium and brain tissues of the rats in the combined group increased to different degrees,and were higher than that of the PCPA group and the caffeine group.Conclusion: PCPA combined with caffeine can impair learning and memory ability of rats,disrupt cardiovascular function,and cause abnormal changes in EEG.EEG showed significant reduction in TST,significantly prolonged sleep latency,significantly decreased SWS,and decreased REMS,which were similar to PSG characteristics of patients with insomnia of heart not hiding god sleep.Therefore,PCPA combined with caffeine could be used to construct a rat model of insomnia of heart not hiding god.Section Three Objective: To explore the mechanism of berberine for tranquilization based on transcriptome sequencing technology.Methods: 60 SD rats were randomly divided into normal group,model group,diazepam group and berberine low,medium and high dose group,with 10 rats in each group.The high,medium and low dose groups were given 200,100 and 50mg/kg berberine by gavage,respectively.The diazepam group was given 0.92mg/kg of diazepam solution by gavage.The model group and the normal group were given the same volume of normal saline by gavage for 7 consecutive days.After the treatment,pentobarbital sodium sleep experiment and water maze experiment were conducted.Plasma levels of ANP,BNP,ET-1,IL-6,TNF-αand IL-17 A were determined by Elisa.The hippocampal tissues of the normal group,the model group,the berberine M group and the diazepam group were selected for transcriptome sequencing.Padj < 0.05 and |log2foldchange|≥2 were used as screening criteria for significantly differentially expressed genes(log2foldchange≥2 was a significantly up-regulated gene,and log2foldchange≤-2 was a significantly down-regulated gene.Padj < 0.05 was used as the screening criterion for significant enrichment of GO and KEGG.PCR and Western blot was used to validate the key genes.Results: Compared with the model group,the sleep latency and sleep duration of the rats in the low,medium and high dose of berberine group and the diazepam group were all shortened and prolonged(P<0.01).The escape latency of the rats was significantly shortened and the number of times of penetrating the platform increased(P < 0.01).Compared with the model group,the contents of ANP,BNP,ET-1,IL-6,TNF-α and IL-17 A in plasma of the low,medium and high dose of berberine group and the diazepam group were decreased,and the medium dose of berberine group was superior to the diazepam group(P < 0.01).Compared with the model group,164 DEGs were screened out in the normal group,including 146 up-regulated genes and 18down-regulated genes.In the medium dose of berberine group,164 DEGs were screened out,among which 159 were up-regulated and 5 were down-regulated.In the diazepam group,123 DEGs were screened out,of which 111 were up-regulated and 12 were down-regulated.According to padj < 0.05,and compared with the model group,the normal group had significantly enriched 564 up-regulated(BP 384,CC 89,MF 91)and320 down-regulated(BP 150,CC 107,MF 63)GO terms.In the berberine medium dose group,there were significantly enriched 558 up-regulated(BP 398,CC 87,MF 73)and343 down-regulated(BP 148,CC 130,MF 65)GO terms.In the diazepam group,there were significantly enriched 830 up-regulated(BP 619,CC 109,MF 102)and 243down-regulated(BP 110,CC 82,MF 51)GO terms.According to padj < 0.05,and compared with the model group,the normal group had significantly enriched 84up-regulated and 13 down-regulated KEGG pathways.In the berberine medium dose group,there were significantly enriched 88 up-regulated and 15 down-regulated KEGG pathways.In the diazepam group,there were significantly enriched 89 up-regulated and 14down-regulated KEGG pathways.Compared with the normal group,the expression levels of key genes Ar,Erb B2,Erb B4 and Grin2 a in the model group were significantly decreased,while the expression levels of Ar,Erb B2,Erb B4 and Grin2 a were significantly increased after medium dose of berberine and treatment with diazepam(P < 0.01).Conclusion: The number of down-regulated DEGs were more than that of up-regulated DEGs in the hippocampus of rats in model group.After treatment of medium dose of berberine and diazepam,the number of up-regulated DEGs were higher than that of down-regulated DEGs,and the effective targets of berberine may be more extensive than diazepam.The medium dose of berberine and diazepam play more roles in the treatment of insomnia of heart not hiding god,and the number of up-regulated KEGG pathways were more than that of down-regulated KEGG pathways.The genes expression of Ar,Erb B2,Erb B4 and Grin2 a in hippocampus of rats was upregulated by medium dose of berberine and Erb B signaling pathway was activated,which may be potential effective targets and signaling pathway of berberine’s sedative effect. |
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