| Objective: Through clinical research,to observe the effectiveness of Danzhi Xiaoyao Powder in the treatment of generalized anxiety disorder with liver depression and fire,and whether it can promote the regeneration of central nervous system neurons in patients with generalized anxiety disorder,improve the level of 5-HT and improve anxiety symptoms.Through animal experimental studies,to observe whether Danzhi Xiaoyao Powder can regulate Notch signal pathway and promote neurovascular regeneration in hippocampus of patients with generalized anxiety disorder,so as to improve the function of5-HT system in hippocampus and improve anxiety,so as to open up new ideas and methods for the treatment of generalized anxiety disorder with traditional Chinese medicine.Methods: In the clinical study,106 patients with generalized anxiety disorder who met the inclusion criteria were randomly divided into treatment group and control group.53 cases in the treatment group were treated with Danzhi Xiaoyao Powder;53 cases in the western medicine group were treated with buspirone tablets.Both groups were treated for 14 days.The anxiety symptom scores of the two groups before and after treatment were observed by scale,and the changes of 5-HT,NSE and IGF-1 levels in peripheral blood of the two groups were detected by ELISA and intravenous test.Animal experiment: 120 Wistar rats were divided into five groups: control group,model group,DZXYS group,DZXYS + DAPT group and buspirone group.Except the control group,the other groups were given restraint method and uncertain empty bottle stimulation method for 21 days to prepare the generalized anxiety disorder model,and then each group was given corresponding drug intervention treatment for 14 days.The changes of body weight and food intake of rats were continuously monitored throughout the process;The behavioral status of rats was monitored by oft and EPM;After 7 and 14 days of gavage,12 rats in each group were randomly selected.The concentration of 5-HT in hippocampus of rats in each group was observed by ELISA,the expression of 5-HT1 A m RNA in hippocampus was observed by PCR,the regeneration of neurons in hippocampus was observed by immunofluorescence,and the expression of VEGF,VEGFR2 and CD34 in hippocampus was observed by immunohistochemistry,The protein expression of Notch signaling pathway Notch1,Hes1,Hes5 and Dll4 in rat hippocampus was monitored by realtime fluorescence quantitative PCR and Western blot.Results: Clinical study: the HAMA score and TCM syndrome score of GAD patients in the treatment group and the control group were significantly lower than those before treatment(p<0.0001,p<0.0001;p<0.0001,p<0.0001),The curative effects of the two groups were compared according to the score reduction rate of the TCM syndrome integral table of GAD of liver depression and fire transformation type.It was found that the curative effect of the treatment group was better than that of the control group in the improvement of TCM symptoms of liver depression and fire transformation type(p<0.05);In terms of the improvement of serological indexes,the levels of serum 5-HT and IGF-1 in the treatment group and the control group were higher than those before treatment(p<0.01,p<0.05;p<0.05,p<0.05),and the levels of NSE were lower than those before treatment(p<0.05;p<0.05).There was no significant difference between the two groups.Animal experimental findings:2.1 General observation: after the 28 th day of GAD modeling,the food intake of rats in buspirone group,DZXYS group,DZXYS + DAPT group and model group was significantly lower than that in the control group(p<0.0001,p <0.0001,p<0.0001,p<0.0001),and the weight growth rate was significantly lower than that in the control group(p<0.0001,p<0.0001,p<0.0001,p<0.0001).After 7 days of drug intervention,There was no significant difference in food intake between each drug intervention group and the model group.After the 14 th day of intervention,the decrease of appetite in DZXYS and DZXYS + DAPT groups was significantly reversed(p<0.0001,p<0.0001).The body weight growth rate was increased after intervention with buspirone,DZXYS and DZXYS + DAPT for 7 and 14 days respectively(p=0.0103,p<0.0001,p<0.0001;p<0.0001,p<0.0001,p<0.0001),which had a significant time effect.2.2 Behavioral observation: on the 28 th day after modeling,the movement distance of central area in model group,buspirone group,DZXYS group and DZXYS + DAPT group was significantly lower than that in the control group(p<0.0001,p<0.0001,p<0.0001,p<0.0001).Buspirone,DZXYS Compared with the model group,DZXYS + DAPT Group continued to increase significantly(p=0.0010,p=0.0009,p<0.0001;p<0.0001,p<0.0001,p<0.0001),which was highly consistent with the change of residence time in the central area of the open field.The results of elevated cross maze test showed that on the 28 th day after modeling,the OT% and OE% of rats in each group were significantly lower than those in the control group(p<0.0001,p<0.0001,p<0.0001,p<0.0001;p<0.0001,p<0.0001,p<0.0001,p<0.0001).After 7 and 14 days of drug intervention,the OT% value of buspirone,DZXYS,DZXYS + DAPT group was significantly higher than that of the model group(p<0.0001,p=0.0046,p<0.0001;p<0.0001,p<0.0001,p<0.0001),and the OE% value was significantly higher than that of the model group(p=0.0035,p=0.0176,p=0.0048;p<0.0001,p<0.0001,p<0.0001).2.3 Changes in the level of 5-HT system: after 7 and 14 days of drug intervention,the level of 5-HT and the expression of 5-HT1 A m RNA in the hippocampus of the model group were significantly lower than those in the control group(p<0.0001,p<0.0001),and the levels of buspirone,DZXYS,DZXYS + DAPT were significantly higher than those in the model group(p=0.0501,p=0.0386,p=0.0212;p=0.0010,p=0.0002,p<0.0001),The expression of5-HT1 A m RNA was significantly higher than that in the model group(p=0.0120,p=0.0137,p=0.0027;p=0.0001,p=0.0002,p<0.0001).Both groups had significant time effect.2.4 New proliferating cells in hippocampal DG area: through Brd U + / nestin +immunofluorescence double staining in hippocampal DG area of rats,it was found that after7 and 14 days of drug intervention,the number of Brd U + / nestin + in model group was significantly higher than that in control group(p<0.0001),and the number of Brd U + / nestin+ in buspirone,DZXYS,DZXYS + DAPT group was significantly lower than that in model group(p=0.0004,p=0.0004,p<0.0001;p<0.0001,p<0.0001,p<0.0001),Two-way ANOVA showed that the number of newborn neural stem cells in this area had significant treatment effect(p<0.0001)and time effect(p<0.05);The double immunofluorescence staining of Brd U + / Neu N + in DG area showed that after 7 and 14 days of drug intervention,the number of Brd U + / Neu N + in the model group was significantly lower than that in the control group(p<0.0001),and the number of Brd U + / Neu N + in buspirone,DZXYS and DZXYS + DAPT groups was significantly higher than that in the model group(p<0.0001,p<0.0001,p<0.0001;p<0.0001,p<0.0001,p<0.0001),There were significant treatment effect(p<0.0001)and significant time effect(p=0.0028);The double immunofluorescence staining of Brd U + / GFAP + in DG area showed that the number of Brd U + / GFAP + in the model group was significantly higher than that in the control group after 7 and 14 days of drug intervention(p<0.0001),and the number of Brd U + / GFAP + in buspirone,DZXYS,DZXYS + DAPT group was significantly lower than that in the model group(p<0.0001,p<0.0001,p<0.0001;p<0.0001,p<0.0001,p<0.0001),There were significant treatment effect(p<0.0001)and significant time effect(p<0.0001).2.5 Angiogenesis in hippocampal DG area: through immunohistochemical staining of CD34,VEGF and VEGFR2 in hippocampal DG area of rats,it was found that the value of VEGF IOD in DG area of model group was significantly lower than that of control group(p<0.0001),and the value of VEGF IOD was significantly higher than that of model group after 7 and 14 days of intervention with buspirone,DZXYS,DZXYS + DAPT(p<0.0001,p<0.0001,p<0.0001;p=0.0045,p=0.0067,p=0.0030),The expression result of AOD value is consistent with that of IOD value;The value of VEGFR2 IOD in the model group was significantly lower than that in the control group(p=0.0016).After 7 days of intervention,it was significantly higher than that in the model group in buspirone group,DZXYS group and DZXYS+ DAPT group(p<0.0001,p<0.0001,p<0.0001),and decreased after 14 days(p=0.0584,p=0.100,p=0.1343);The microvessel density in DG area(CD34 staining)in t he model group was lower than that in the control group,but there was no significant difference.After 7 and 14 days of drug intervention,the microvessel density in buspirone group,DZXYS group and DZXYS + DAPT group was significantly higher than that in the model group(p<0.0001,p<0.0001,p<0.0001;p<0.0001,p<0.0001,p<0.0001).There was a significant treatment effect(p<0.0001),but there was no time effect.2.6 Changes of key targets of Notch signaling pathway in hippocampus: real-time fluorescence quantitative PCR showed that the expression of notch1 mrna,hes1mrna,hes5 mrna and dll4 mrna in the model group was significantly higher than that in the control group(p<0.0001;p<0.0001;p<0.0001;p<0.001).After 7 and 14 days of drug intervention,buspirone,DZXYS The expression of notch1 mrna in DZXYS + DAPT group was significantly lower than that in model group(p=0.0019,p=0.0025,p=0.0002;p<0.0001,p<0.0001,p<0.0001),the expression of Hes1 m RNA in each group was significantly lower than that in model group(p=0.0144,p=0.0083,p=0.0061;p<0.0001,p<0.0001,p<0.0001),and the expression of hes5 mrna in each group was significantly lower than that in model group(p=0.0023,p=0.0494,p=0.0073;p<0.0001,p<0.0001,p<0.0001),After 7 days of drug intervention,the expression of Dll4 m RNA in each group was significantly lower than that in the model group(p<0.0001;p<0.0001;p<0.0001).After 14 days of drug intervention,the expression of Dll4 m RNA in DZXYS and DZXYS + DAPT groups was significantly higher than that in the model group(p=0.0187;p=0.0132).The expression of WB protein was basically consistent with the above PCR results.It was confirmed that the key targets of Notch signaling pathway were significantly up-regulated in the hippocampus of GAD rats,and the regeneration of neurons in DG region was impaired,while DZXYS could inhibit the excessive up-regulation of Notch signaling pathway key targets Notch1,Hes1 and Hes5(down-regulated first and then up-regulated for Dll4),promote the differentiation of neural stem cells into neurons in the hippocampus,promote neuronal regeneration and promote angiogenesis.Conclusion: Danzhi Xiaoyao Powder can regulate Notch signaling pathway,promote neurovascular regeneration in hippocampus of patients with generalized anxiety disorder,improve the function of 5-HT system in hippocampus,reduce the sensitivity of the body to aversive stimuli and improve anxiety symptoms. |
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