Discussion On The Anti-anxiety Effect Of Regulating Spirit Acupuncture Technique Based On The HPA Axis Neuromodulation:A Clinical And Mechanism Research

Objective1.To evaluate the effect and safety of regulating spirit acupuncture technique in the treatment of women with mild to moderate generalized anxiety disorder(GAD)through Hamilton Anxiety Scale(HAMA),Anxiety Self-Rating Scale(SAS)and Pittsburgh Sleep Quality Index(PSQI)scores,and assess the function of HPA axis by testing serum adrenocorticotropic hormone(ACTH)and Cortisol(CORT)in the prospective case analysis research.2.With the chronic unpredictable mild stress(CUMS)mouse anxiety model,we aim to research the anti-anxiety mechanism of regulating spirit acupuncture technique based on the neuromodulation of HPA axis.Methods1.Clinical research:Prospective case analysis research methods were adopted.24 qualified participants with GAD were recruited and treated with regulating spirit acupuncture technique.The main acupoints were four spirit points,DU24,GB13,HT7,SP6,LI4 and LR3.Lifting-thrusting and twirling reinforcement-reduce methods were carried out at every 15 minutes interval.Retaining the needles for 30 minutes per time.Treatments last for four weeks,three times a week.All participants were scored before,during and after the treatment with HAMA,SAS and PSQI to evaluate the effect of acupuncture in the treatment of GAD.The reduction rate of HAMA score served as the main efficacy index.SAS scores were observed in the two-month follow-up to evaluate the recurrence of GAD.Adverse reactions were recorded for safety assessment.The serum ACTH and CORT levels of patients before and after treatment were compared to investigate the effect of regulating spirit acupuncture technique on HPA axis function in GAD patients.2.Animal experiment:Adult 8-week-old female C57BL/6J mice were randomly divided into 5 groups including control group,model group,manipulation acupuncture(MA)group,electroacupuncture(EA)group and sham acupuncture group.Except the control group,the remaining 4 groups were prepared by CUMS intervention for 28 days to anxiety model mice.The success of model was evaluated by the changes in the behavior of the mice with open field test(OFT)and elevated plus maze test(EPMT).At the same time,forced swimming test and sugar water test were used to rule out depression-like behaviors of the model mice.After modeling,mice in the MA group were received acupuncture for 15 minutes once a day.The EA group was adopted electroacupuncture with density wave pattern and 2 Hz frequency for 15 minutes.Press needles were applied to the acupoint skin without pricking the mice of sham acupuncture group for 15 minutes.The model group received the same grasping.The intervention last for 21 days with a total 15 acupuncture(acupuncture for 5 days and rest for 2 days).After the intervention,the following experiments were carried out:(1)The OFT and EPMT were used to evaluate the anxiety-like behavior of mice.(2)The levels of serum corticotropin releasing hormone(CRH),ACTH and CORT were detected in each group to evaluate the effect of regulating spirit acupuncture technique on the HPA axis of CUMS anxiety mice through Enzyme-linked immunosorbent assay(ELISA).(3)Western blotting was used to detect the neuronal activity,neural excitability and inhibitory receptor proteins,and neural activity related indicators in the prefrontal cortex(PFC),hippocampus(HIP)and basolateral amygdala(BLA)to evaluate the anti-anxiety curative effect and the neuromodulation of HPA axis.The detection proteins included:phosphorylated and total ?-gamma-aminobutyric acid receptors(p-GABRB1,GABRB1),phosphorylated and total N-methyl-D-aspartate type 1 receptors(P-NR1,GluN1),glutamate ionotropic receptor AMPA1(GluAl),phosphorylated calcineurin-dependent kinase(p-CAMKII),phosphorylated and total cyclic phosphate gland glycoside response element binding proteins(p-CREB,CREB),brain-derived neurotrophic factor(BDNF),phosphorylated and total glucocorticoid receptors(p-GR,GR)and mineralocorticoid receptor(MR).All data were processed by software SPSS 23.1.0 and two-sided testing were used for all hypothesis testing.P<0.05 served as a standard with statistical difference while P<0.01 served as a statistically significant difference standard.Results1.Clinical research:(1)Regulating spirit acupuncture technique improved the HAMA,SAS and PSQI scores of GAD patients for 2 and 4 weeks of treatment both mild and moderate anxiety patients(P<0.01).Regulating spirit acupuncture technique improved mental and physical symptoms(P<0.01).(2)After the treatment,the level of CORT,an important serum indicator of HPA axis in GAD patients,was significantly reduced(P<0.01).(3)The total clinical effective rate was 87.5%without adverse reactions.(4)There was no increase in the SAS scores of GAD patients during the follow-up for 1 month and 2 months(P>0.05)and no anxiety recurrence.2.Animal experiment:(1)Intervention of the core acupoints of regulating spirit acupuncture technique improved the anxiety-like behaviors in CUMS anxiety model mice.In the EMPT,time of rearing in open arms were significantly reduced in MA and EA groups(P<0.01).And the time spent in open arms were reduced in MA and EA groups(P<0.05).In the OFT,the distance and time in central zone were significantly increased(P<0.01,P<0.05).(2)The CUMS anxiety model mice in this experiment did not have associated depression-like behaviors.In the sugar water test,there was no statistical difference in sugar water consumption within 1 hour between model group and control group(P>0.05).In the forces swimming test,there was no statistical difference in floating time(P>0.05)and struggle time(P>0.05)between model group and control group.(3)After the CUMS model establishment,the serum ACTH content and CORT content of the model group mice increased significantly(P<0.05).Regulating spirit acupuncture technique improved the abnormal hyperactivity of the HPA axis of CUMS mice with the significantly reduction of CRH level(P<0.01),ACTH level(P<0.05)and CORT level(P<0.01).The MA group was better than the EA group in effect.(4)Regulating spirit acupuncture technique activated PFC,BLA,and HIP brain regions,and improved the imbalance of excitatory and inhibitory nerve function.The acupuncture group reduced the expression of FosB(P<0.05),p-GABRB1(P<0.05)and GluN1(P<0.05)in the HIP,while increased the expression of p-GABRB 1(P<0.05)in PFC and GluN1(P<0.05)in BLA.(5)The MA group increased the expression of p-CREB,BDNF and p-GR proteins(P<0.05)in these three brain regions.The curative effect of the MA group was better than that of the EA group.Conclusion1.Regulating spirit acupuncture technique improved the anxiety symptoms of female mild to moderate GAD patients and the abnormal hyperactivity of the HPA axis.The curative effect was stable and last for 3 months with good safety,but not recurrence or adverse reactions.2.Regulating spirit acupuncture technique exerted anti-anxiety effect in CUMS anxiety model mice by balancing the excitatory and inhibitory nerve functions of PFC,HIP and BLA brain regions,increasing the expression of p-GR,reducing the level of serum CORT,playing a central nervous system protection role and regulating the hyperactive state of HPA axis.