Study On The Neurobiological Mechanism Of Generalized Anxiety Disorder Treated With Shugan Tiaoshen Acupuncture

Objective:To observe the effects of the Shugan Tiaoshen acupuncture(SGTS)on the anxiety state and the autonomous functional activity of brain regions in patients with Generalized anxiety disorder(GAD).At the same time,an animal model of anxiety was established,with neurotransmitter disorders as the entry point,both in terms of behavioural and molecular biology.The aim is to observe the effects of SGTS on the dorsal medical prefrontal cortex-amygdala,an important target area in the anxiety model rats,initially.And to investigate the central mechanism of improving GAD by sparing the liver and regulating the spirit of acupuncture.In order to provide a scientific basis for the treatment of this disease by SGTS.Methods:1.Clinical studies:A before-and-after controlled design using GAD patients themselves was used,with 18 cases included.The Hamilton Anxiety Inventory(HAMA)and functional magnetic resonance imaging(fMRI)scans were used before and after 4 weeks of acupuncture treatment,respectively.Relevant software is also applied to pre-process and analyse the scale assessment and test data and scan data.2.Experimental studies:Forty-eight SD male rats were selected and randomly divided into blank control group,model group,electro-acupuncture group and shamacupuncture group according to their body weight and behavioral results,12 rats in each group were housed in separate cages.The blank control group was not given any stimulation,and the rest of the groups were subjected to 21 days of unpredictable chronic emotional stress(CES)to establish a rat anxiety model.After successful modeling,the electro-acupuncture group was given electro-acupuncture to "Baihui","Neiguan","Shenmen" and "Taichong".And the sham-acupuncture group was given shallow acupuncture at 1 mm next to the corresponding acupuncture points.All interventions were administered once daily for 25min/dose,and rats were executed after 3 weeks of treatment.The rats were examined for weekly weight changes throughout the process.The rats were observed for anxiety before,after and after the intervention using the open-field test(total distances,average speeds and central zone dwell time)and the elevated cross-maze test(percentage of times entering the open-arm area and percentage of central time entering the open-arm area).The expression levels of IGF-1 in the dorsal medial prefrontal lobe of rats and amygdala NR2 B,Glu R1,Glu R2,IGF-1,GABAAR1 m RNA were detected by Real-time PCR.The expression levels of IGF-1 in the dorsal medial prefrontal lobe of rats and amygdala NR2 B,Glu R1,Glu R2,IGF-1,GABAAR1 proteins were detected by Western Blot.The average fluorescence intensity of NR2 B in rat amygdala was detected by immunofluorescence method.Results:1.Clinical studies:(1)The HAMA index scores of GAD patients were significantly lower after treatment,and there was a statistically significant difference compared to before treatment(P<0.01).(2)fMRI findings:The immediate as well as cumulative effects of acupuncture can alter ALFF values in mood-related brain regions in GAD patients.Single acupuncture stimulation significantly reduced ALFF values in the amygdala,angular gyrus,lingual gyrus,prefrontal and thalamus of GAD patients(GRF corrected,P-voxel<0.05,P-cluster<0.05),with no brain regions with significantly elevated ALFF values.Compared with the pre-treatment period,the brain areas with increased ALFF values in GAD patients after 4 sessions of acupuncture treatment mainly included the middle cingulate gyrus,postcentral gyrus,supplementary motor area,and middle frontal gyrus(GRF corrected,P-voxel<0.05,P-cluster<0.05).And there were no brain areas with significantly decreased ALFF values.(3)Results of correlation analysis: Pre-treatment prefrontal ALFF values were positively correlated with pre-treatment HAMA scale scores(rs=0.550,p=0.018).Post-acupuncture amygdala ALFF values were negatively correlated with pre-treatment HAMA scale scores(rs=-0.497,p=0.036).2.Experimental studies:(1)The rats in the anxiety model group gradually lost weight,and there was a statistical difference(P<0.05)between them and the blank control group at the end of modeling.A significant increase in the total distance of movement,average speeds in the model group(P<0.05)and a significant decrease in the central zone dwell time,percentage of dwell time into the open arm zone and percentage of times into the open arm zone(P<0.05).Compared with the model group,the rats in the electroacupuncture group showed significant reversal of all the above indices(P<0.05).(2)Dorsal medical prefrontal-amygdala related neurotransmitter m RNA and protein expression levels:IGF-1 m RNA and protein expression were significantly lower in the dorsomedial prefrontal lobe of rats in the model group compared with the blank control group(P<0.01).The expression of GABAAR1 m RNA and protein in the amygdala of rats in the model group was significantly lower(P<0.05),NR2 B and Glu R2 m RNA and protein expression were significantly higher(P<0.05),and Glu R1 and IGF-1 m RNA and protein expression were not significantly different(P>0.05).Compared with the model group,m RNA and protein expression of IGF-1(P<0.01)and GABAAR1(P<0.01)and IGF-1(P<0.05)in the dorsal medial prefrontal lobe and amygdala of rats were significantly higher in the electroacupuncture group.NR2B(P<0.05),Glu R2(P<0.01),Glu R1(P<0.05)m RNA and protein expression were significantly reduced in the amygdala.(3)Changes in mean fluorescence intensity of NR2 B in the amygdala: Compared with the blank control group,the mean fluorescence intensity of NR2 B was significantly higher in the model group rats(P<0.01).Compared with the model group,the mean fluorescence intensity of NR2 B in rats in the electroacupuncture group was significantly lower(P<0.01).Conclusions:1.Clinical studies:(1)SGTS can improve HAMA scores in GAD patients,indicating that acupuncture can effectively improve mood disorders in GAD patients.(2)Analysis of fMRI scan results showed that immediate acupuncture reduced ALFF values in the amygdala,angular gyrus,lingual gyrus,prefrontal and thalamus.The cumulative effect of needling increases ALFF values in the middle cingulate gyrus,postcentral gyrus,supplementary motor area,and middle frontal gyrus.The prefrontal-amygdala loop is an important target area for the regulation of anxiety disorders by acupuncture.The central mechanism of acupuncture for GAD may be associated with reduced or enhanced autonomic function activity in the above-mentioned brain regions and is closely related to HAMA scale scores.2.Experimental studies:(1)Electroacupuncture can significantly improve weight changes and anxiety behavior in rats in anxiety model.(2)Electroacupuncture can reduce neurotoxicity in the dorsal medical prefrontal-amygdala region of rats in the anxiety model by regulating changes in the neurotransmitters NR2 B,Glu R2,IGF-1and GABAAR1.In contrast,the differences in IGF-1 and Glu R1 between the blank control and model groups on the amygdala were not significant,and their mechanism of action still needs further study.