| ObjectiveAccording to the theory of "heart governing mind" in traditional Chinese medicine,the heart was likened to the monarch and could dominate all life activities and spiritual consciousness activities.Under the guidance of the theory and combining the etiology and pathogenesis of syndrome of heart-Yin deficiency of generalized anxiety disorder(GAD),and based on the principle of "the five internal organs corresponds with five tones,the heart belongs to fire and its tone is related to Zhi-tune,and the Zhi-tune music can regulate the heart”,so this study took "Zhi-tune music from heart to treat GAD" as the breakthrough point.The anxiety behavior and heart-Yin deficiency syndrome of rats were observed.The content of hormones and receptors related to HPA axis in rats were detected.On the one hand,the aim was to establish and evaluate the model of GAD rats with heart Yin deficiency syndrome,to provide experimental research carrier for Zhi-tune music intervention in GAD heart-Yin deficiency syndrome.On the other hand,the theory of "heart governing mind" was used to guide the treatment of GAD with five elements music.And the aim was to reveal the possible targets and mechanisms of anti-anxiety effects of Zhi-tune music from the perspective of HPA axis,relying on advanced enzyme-linked immunoassay and molecular biotechnology.MethodsWith body weight as the factor of block,a randomized block design was used to classify 48 sprague-dawley(SD)rats by weight from low to high,and 6 rats with adjacent body weight were 1 block,a total of 8 blocks(block 1-block 8).Six rats in each block were randomly assigned to the normal,model,Tianwang Buxin pill,diazepam tablet,Zhi-tune music,western music group according to the random number table method.So totally 48 SD rats were randomly divided into 6 groups with 8 rats in each group.The experiment consisted of three parts: modeling,intervention and evaluation.Firstly,it was the establishment and evaluation of GAD model of deficiency of heart-Yin syndrome.In addition to the normal group,the other groups of rats by the method of uncertainty empty bottle stimulation(3 weeks)and the water environment with small platform by sleep deprivation method(72 h)to construct the model of heart-Yin deficiency of GAD rats.After the modeling,the GAD model was successfully verified by the internationally recognized elevated plus maze test and the open field experiment.At the end of the experiment,the microscopic detection indexs were used to verify the scientificity and stability of the GAD model.Referring to the method of syndrome differentiation of heart-Yin deficiency in traditional Chinese medicine,some human qualitative indexs needed for syndrome differentiation were transformed into quantitative indexs of animals(such as heart rate,systolic blood pressure,respiratory rate,rectal temperature,etc.),and then combines the spirit,activity,body type,diet,hair,urine and stool of the rats.The analysis of the macroscopic characterization of the rat was carried out.If the macroscopic indexs was consistent with the symptoms and signs of the heart Yin deficiency syndrome,the rat model of heart Yin deficiency syndrome was initially constructed successfully.At the end of the experiment,the microscopic test indexs of rats and the results of "prescription validation model(Tianwang Buxin Pills)" again verified the reliability of the model.The second was the stage of drug and music intervention.After the model was successfully made,the normal group and the model group were not given any treatment.The Tianwang Buxin pill group was given Tianwang Buxin pill by gastric perfusion,the dosage was 0.74g/kg,once a day,and the diazepam tablet group was given diazepam tablet by gastric perfusion,the dosage was lmg/kg,once a day.The Zhi-tune music group and the western music group played Zhi-tune music and western music respectively once a day and 45 minutes a time.The above interventions are one course of treatment a week,three courses of continuous intervention,a total of 21 days.Finally,it was the evaluation stage of experimental results.At first,the general condition of rats,such as spirit,activity,stool and diet of rats were observed daily.And the weight of rats were measured weekly.Secondly,the ethology results of rats were observed before,after modeling and after 1,2,3 weeks of intervention with elevated plus-maze experiment and open field experiment.Thirdly,the systolic blood pressure,heart rate,breathing,anal temperature results of rats were observed before,after modeling and after 3 weeks of intervention.Lastly,the concentration of corticotropin-releasing hormone(CRH)in hypothalamus,adrenocorticotropic hormone(ACTH)and corticosterone(CORT)in serum were detected by Elisa after 3 weeks of intervention.The expression of type ? CRH receptor(CRH R?)mRNA,type ? CRH receptor(CRH R?)mRNA in hypothalamus and glucocorticoid receptor(GR)mRNA in hippocampus were detected by RT-PCR after 3 weeks of intervention.ResultsBy the end of the third weekend of intervention,46 rats had completed the experiment,and 2 rats died,all from diazepam tablet group.The experimental results were as follows.1.Comparing results of each index before modelingThere was no obvious abnormality in general condition of rats in the normal group.The results of one-way ANOVA showed that there was no significant difference in body weight among the all groups before modeling(P>0.05).The results of random block variance analysis showed that there were no significant differences in the OE% values,OT% values,the number of horizontal and vertical movements,systolic blood pressure,heart rate,respiratory rate and anal temperature between groups and between block groups before modeling(P>0.05).2.Comparing results of each index after modeling 2.1 General condition and body weight changes 2.1.1 General conditionAfter the establishment of the model,the rats of normal group had no obvious abnormality.But the rats of other groups were depressed,depilated,unresponsive,increased drinking water,decreased food intake,dry stool and weight loss,which were well clustered at one end of the cage,accompanied by behavioral changes such as modification,avoidance and decreased activity.2.1.2 Body weightThe results of one-way ANOVA showed that there was significant difference in body weight between groups after modeling(P<0.05).Further pairwise comparison results showed that there were significant difference in body weight between the other five groups and the normal group(P<0.05).2.2 Ethology resultsThe results of random block variance analysis showed that there were significant differences in OE% values,OT% values,the number of horizontal and vertical movements among groups(P<0.05),but there were no significant difference in those ethology results between block groups after modeling(P>0.05).Further pairwise comparison results in groups showed that there were significant differences in OE% values,OT% values,the number of horizontal and vertical movements between the other five groups and the normal group(P<0.01).2.3 Vital signs resultsThe results of random block variance analysis showed that there were significant differences in the systolic blood pressure,heart rate,respiratory rate and anal temperature among groups(P<0.05),but there were no significant difference in those vital signs results between block groups after modeling(P>0.05).Further pairwise comparison results in groups showed that there were significant differences in the systolic blood pressure,heart rate,respiratory rate and anal temperature between the other five groups and the normal group(P<0.01).3.Comparing results of each index after intervention 3.1 General condition and body weight changes 3.1.1 General conditionThere was no obvious abnormality in the normal group.Rats in the model group were mentally depressed,dim eyes,fewer food,more drinking water,thinner body,bow back,withered and dull coat,slow reaction to activities,like to pile up,still have plenty of stool and urine.In all intervention group,the above-mentioned conditions were improved,with better spirit,glossy coat,increasing diet,weight gain,sensitive action and more decorative behavior.3.1.2 Body weight(1)The results of one-way ANOVA showed that:(1)Comparison among groups:There were significant differences in body weight between groups at the 1st,2nd and 3rd weekend of intervention(P<0.05).(2)Further pairwise comparison results showed that: At the 1st and 2nd weekend of intervention,there were no significant difference in body weight between Tianwang Buxin Pill group,diazepam tablets group,Zhi-tune music group,western music group and model group(P>0.05);At the 3rd weekend of intervention,there was significant difference in body weight between diazepam tablets group and model group(P<0.01),but there were no significant difference in body weight between Tianwang Buxin Pill group,Zhi-tune music group,western music group and model group(P>0.05).At each time point after intervention,there were no significant difference in body weight between Tianwang Buxin pill group and diazepam tablets group,and between Zhi-tune music group and western music group(P>0.05).(2)The results of repeated measurement variance analysis showed that:(1)time,inter-group and interaction effects: There were significant difference in body weight between different time and groups(P<0.01),and there was interaction between time and time effects(P<0.01).(2)The results of time-varying trend chart showed that: the body weight of normal group showed an overall growth trend during the experiment,with the largest growth rate and the fastest growth rate.However,the body weight of normal group decreased at the second weekend of intervention.The body weight of the other five groups decreased after modeling,and was significantly lower than that of the normal group.After intervention,except for the weight of the model group increased slowly,the other groups increased rapidly,especially diazepam tablets tablet group.By the end of intervention,the weight of diazepam tablets tablet,Tianwang Buxin pill,Zhi-tune music,music,western music group were both close to normal group.3.2 Ethology results 3.2.1 Results of elevated plus-maze experiment(1)The results of random block variance analysis showed that:(1)Comparison among groups or block groups: There were significant differences in OE% and OT% values between groups at the 1st,2nd and 3rd weekends of intervention(P<0.05),but there were no significant differences in OE% and OT% values between block groups in any time points after intervention(P>0.05).(2)Further pairwise comparison results among groups showed that: At the 1st,2nd and 3rd weekend after intervention,the OE% and OT% values of model group were significantly different from those of normal group(P<0.05),and the OE% and OT% values of Tianwang Buxin pill,diazepam tablets,Zhi-tune music,western music group significantly different from those of normal group(P<0.05).There were no significant difference in OE% and OT% values between Tianwang Buxin pill group and diazepam tablets at each time point after intervention(P>0.05).At the 1st weekend of intervention,there was no significant difference in OE% values between Zhi-tune music group and western music group(P>0.05),but there was significant difference in OT% values between Zhi-tune music group and western music group(P<0.05);At the 2nd weekend of intervention,there were no significant difference in OE% and OT% values between Zhi-tune music group and western music group(P>0.05);At the 3rd weekend of intervention,there was significant difference in OE% values between Zhi-tune music group and western music group(P<0.05),but there was no significant difference in OT% values between Zhi-tune music group and western music group(P>0.05).(2)The results of repeated measurement variance analysis showed that:(1)time,inter-group and interaction effects: There were significant differences in OE and OT% values between different time and groups(P<0.01),and there was interaction between time and group effects in OE values(P<0.05).(2)Further pairwise comparison results showed that: Firstly,it was OE% values.In the normal,model group,there was no significant difference in OE% value between any time point(P>0.05).In the Tianwang Buxin pill,diazepam tablets and Zhi-tune music group,there was significant difference in OE% value between after modeling and the 1st,2nd and 3rd weekends of intervention(P<0.05).In the western music group,there was significant difference in OE% between after modeling and after the 2nd and 3rd weekends of intervention(P<0.01).Secondly,it was OT% values.In the normal group,it was significantly different from that in the after modeling and at the 2nd weekend of intervention(P<0.05).In the model group,it was significantly different from that in the 1st weekend and the 3rd weekend of intervention(P<0.05).In the Tianwang Buxin pill,diazepam tablets,Zhi-tune music and western music group,there was significant difference in OT% values after modeling and the first,second and third weekends of intervention(P<0.05).(3)The results of time-varying trend chart showed that: The values of OE% and OT% values in the normal group changed steadily with time.The OE% and OT% values of the other five groups showed a downward trend after the establishment of the model,and were significantly lower than those of the normal group.After intervention,except the OE% and OT% values in the model group fluctuated at a relatively low level,the OE% and OT% values in the other four groups showed an overall upward trend.At the end of the intervention,the OE% and OT% values in diazepam tablets group,Tianwang Buxin Pill group,Zhi-tune music group,western music group were similar to those in the normal group,and significantly higher than those in the model group.3.2.2 Results of open field experiment(1)The results of random block variance analysis showed that:(1)Comparison among groups or block groups:There were significant differences in the number of horizontal and vertical movements between groups at the 1st,2nd and 3rd weekends of intervention(P<0.05).Except for the 1st weekend of intervention,there was significant difference in the number of horizontal movements between block groups(P<0.05).There were no significant difference in the number of horizontal and vertical movements between block groups in any time points after intervention(P>0.05).(2)Further pairwise comparison results among groups showed that: At the 1st,2nd and 3rd weekend of intervention,there were significant differences in the number of horizontal and vertical movements between model group and normal group(P<0.05);there were significant differences in the number of horizontal and vertical movements between Tianwang Buxin pill,diazepam tablets,Zhi-tune music group and model group(P<0.05).There was no significant difference in the number of vertical movements between the western music group and the model group at the 1st weekend of intervention(P>0.05),but in the other time points after intervention,there were significant difference in the number of horizontal and vertical movements between western music group and the model group(P<0.05).At each time point after intervention,there were no significant difference in the number of horizontal and vertical movements between Tianwang Buxin pill group and diazepam tablets group,and between Zhi-tune music group and western music group(P>0.05).(2)The results of repeated measurement variance analysis showed that:(1)time,inter-group and interaction effects: There were significant differences in the number of horizontal and vertical movements between different time and groups(P< 0.01),and there was interaction between time and group effects in the number of horizontal movements(P<0.05).(2)Further pairwise comparison results showed that: Firstly,it was the number of horizontal movements.In the normal group,it was significantly different from that in the after modeling and the 1st weekend of intervention in the number of horizontal movements(P<0.05).In the model group,there was no significant difference in the number of horizontal movements between any time point(P>0.05).In the Tianwang Buxin pill,diazepam tablets group,there was significant difference in the number of horizontal movements between after modeling and the 1st,2nd and 3rd weekends of intervention(P<0.05).In the Zhi-tune music group,there was significant difference in the number of horizontal movements between after modeling and the 1st weekends of intervention(P<0.01),and there was significant difference in the number of horizontal movements between the 1st and 2nd weekends of intervention(P<0.05).In the western music group,there was significant difference in the number of horizontal movements between after modeling and the 1st,2nd,3rd weekends of intervention(P<0.05),and between 2nd and 3rd weekends of intervention(P<0.05).Secondly,it was the number of vertical movements.In the normal group,it was significantly different from that in the after modeling and at the 1st weekend of intervention in the number of vertical movements(P<0.05).In the model group,there was no significant difference in the number of vertical movements between any time point(P>0.05).In the Tianwang Buxin pill group,there was significant difference in the number of vertical movements between after modeling and the 1st,2nd and 3rd weekends of intervention(P<0.05).In the diazepam tablets group,there was significant difference in the number of vertical movements between after modeling and the 1st,2nd weekends of intervention(P<0.05).In the Zhi-tune music group,there was significant difference in the number of vertical movements between after modeling and the 1st,3rd weekends of intervention(P<0.05).In the western music group,there was significant difference in the number of vertical movements between after modeling and the 1st,2nd weekends of intervention(P<0.05).(3)The results of time-varying trend chart showed that: The number of horizontal and vertical movements in the normal group varied steadily with time.The number of horizontal and vertical movements in the other five groups decreased after the establishment of the model,and was significantly lower than that in the normal group.After the intervention,except for the relatively low fluctuation of the horizontal and vertical movement times in the model group,the horizontal and vertical movement times in the other four groups increased as a whole,but decreased at the 2nd weekend of intervention.At the end of the intervention,the number of horizontal and vertical movements in the Tianwang Buxin pill,diazepam tablets,Zhi-tune music,and western music group were similar to these in the normal group,and were significantly higher than that in the model group.3.3 Vital signs results 3.3.1 Results of systolic blood pressure and heart rateThe results of random block variance analysis showed that:(1)Comparison among groups or block groups: At the 3rd weekend of intervention,there were significant differences in systolic blood pressure and heart rate between groups(P<0.05),but there were no significant difference in systolic blood pressure and heart rate between block groups(P>0.05).(2)Further pairwise comparison results among groups showed that: At the 3rd weekend of intervention,the systolic blood pressure and heart rate of model group and normal group were significantly different(P<0.01).And the systolic blood pressure and heart rate between Tianwang Buxin pill,diazepam tablets tablet,Zhi-tune music,western music group and model group were significantly different(P<0.01).There were no significant difference in systolic blood pressure and heart rate between Tianwang Buxin pill group and diazepam tablets group,and between Zhi-tune music group and western music group(P>0.05).3.3.2 Results of respiratory frequency and anal temperatureThe results of random block variance analysis showed that:(1)Comparison among groups or block groups: At the 3rd weekend of intervention,there were significant differences in respiratory rate and anal temperature between groups(P<0.01),but there were no significant difference in respiratory rate and anal temperature between block groups(P>0.05).(2)Further pairwise comparison results in groups showed that: At the 3rd weekend of intervention,there were significant differences in respiratory rate and anal temperature between model group and normal group(P<0.05).There were significant differences in respiratory rate and anal temperature between Tianwang Buxin pill,diazepam tablets,Zhi-tune music,western music group and model group(P<0.01).And there were no significant difference in respiratory rate and anal temperature between Tianwang Buxin pill group and diazepam tablets group,and between Zhi-tune music group and western music group(P>0.05).3.4 Laboratory Indicators Results 3.4.1 Comparisons of HPA axis-related hormones concentrationsThe results of random block variance analysis showed that:(1)Comparison among groups or block groups: At the 3rd weekend of intervention,there were significant differences in CRH,ACTH and CORT between groups(P<0.01),but there were no significant difference in CRH,ACTH and CORT between block groups(P>0.05).(2)Further pairwise comparison results in groups showed that: There were significant differences in CRH,ACTH and CORT between model group and normal group(P<0.01).There were significant differences in CRH,ACTH and CORT between Tianwang Buxin pill group,diazepam tablets tablets group,Zhi-tune music group,western music group and model group(P<0.05).There were no significant difference in CRH,ACTH and CORT between Tianwang Buxin pill group and diazepam tablets group,and between Zhi-tune music group and western music group(P>0.05).3.4.2 Comparison of gene expression levels of HPA axis-related receptorsThe results of random block variance analysis showed that:(1)Comparison among groups or block groups: At the 3rd weekend of intervention,there were significant differences in gene expression levels of CRH RI mRNA in hypothalamus and GR mRNA in hippocampus between groups(P<0.01),but there were no significant difference in gene expression levels of CRH RII mRNA in hypothalamus between groups(P>0.05).And there were no significant difference in gene expression of CRH RI mRNA,CRH RII mRNA in hypothalamus and GR mRNA in hippocampus between block groups(P>0.05).(2)Further pairwise comparison results in groups showed that:There were significant difference between model group and normal group in CRH RI mRNA in hypothalamus and GR mRNA in hippocampus(P<0.01).There were significant differences in CRH RI mRNA in hypothalamus and GR mRNA in hippocampus between Tianwang Buxin pill,diazepam tablets,Zhi-tune music group and model group(P<0.05).Compared with the model group,the CRH RI mRNA gene expression in hypothalamus of the western music group had significant difference(P<0.05),and the GR mRNA gene expression in hippocampus of the model group had no significant difference(P>0.05).There were no significant difference in CRH RI mRNA in hypothalamus and GR mRNA in hippocampus between Tianwang Buxin pill group and diazepam tablets group,and between Zhi-tune music group and western music group(P>0.05).And there were no significant difference in CRH RII mRNA gene expression in hypothalamus between any groups(P>0.05).ConclusionsIn this study,we constructed the model of GAD with heart-Yin deficiency syndrome by the method of uncertainty empty bottle stress combined with the method of sleep deprivation on small platform in water environment.And the comprehensive evaluation of the model of GAD with heart-Yin deficiency syndrome was carried out from three perspectives of macroscopic representation,micro index and prescription validation.The results showed that the method has successfully constructed the GAD model with heart-Yin deficiency syndrome.Based on the model,Zhi-tune music can effectively improve anxiety behavior and the symptoms of GAD rats with heart-Yin deficiency syndrome,significantly reduced the concentration of CRH in hypothalamus,the concentration of ACTH and CORT in serum,down-regulated the expression level of CRHRI mRNA in hypothalamus,increased the expression level of GR mRNA in hippocampus,indicating that Zhi-tune music exerted anti-anxiety effect by inhibiting the over-activation of HPA axis initiation and enhancing the negative feedback regulation function of hippocampal-HPA axis. |
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