Based On The HPA Axis And BDNF-ERK-ECS Signaling Pathway, The Anti-anxiety Effect And Mechanism Of Suanzaoren-Schisandra Chinensis Were Discussed

Background:Anxiety is one of the most common mental disorders in the world,contribute to growing global health problems.Anxiety disorders,a common mental illness in the central nervous system,are a type of neurosis characterized by recurrent acute anxiety,including generalized anxiety and seizures.There are various causes of anxiety disorders,such as dizziness,chest tightness,palpitations,difficulty in breathing and sleep disorders.In mental disorders,anxiety disorders have the highest lifetime prevalence,affecting 30 percent of the population and nearly one in five American adults.In our country,anxiety disorder has become a common disease in modern times,which seriously affects people’s quality of life.Western medicine is effective in the treatment of anxiety disorder,but its medication time is long and its side effects are large.Chinese medicine is safe and effective in treating anxiety disorder,with little side effects.Therefore,the effective treatment of anxiety disorder becomes an urgent problem to be solved.Herb pair is a bridge linking Chinese medicine and prescription.The study of herb pair not only helps to guide clinical medicine and improve clinical efficacy,but also can further reveal the scientific connotation of prescription compatibility theory and enrich the theory of traditional Chinese medicine.Suan zao ren,the first choice of sedatives and tranquilizers,is the mature seed of Ziziphus jujube.Wu wei zi,the fruit of Schisandra chinensis,is one of the common tranquilizers,which can be combined with a variety of traditional Chinese medicine.Suan zao ren Wu wei zi Herb Pair(SWHP)are commonly used in the clinical practice of sedatives and tranquilizers.They are often used in many traditional Chinese medicine prescriptions and modern Chinese medicine compound.There are different proportions of SWHP,and the efficacy is different.In recent years,the studies of SWHP are focused on the changes of the chemical component and sedation and insomnia on the basis of 1:1,but there are no researches on other proportion or on the effect of anti-anxiety.Then,whether it has the potential to resist anxiety?What are the main mechanisms of anti-anxiety?These questions are worth investigating.Objective:The purpose of this study was to screening the optimal extraction method,compatibility proportion and dosage of SWHP,identify the chemical constitutions in the decoction of Suan zao ren,Wu wei zi and SWHP by using HPLC-LTQ Orbitrap MS,clear the anti-anxiety effect of SWHP and probe into the mechanism from HPA and BDNF-ERK-ECS signaling pathways.Methods and results:The main research contents and achieved results are as follows:1.Screening the optimal extraction method,compatibility proportion and dosage of SWHP by using orthogonal experimentDesign orthogonal table using extract method(water extract,alcohol extract,and alcohol-water extract),compatibility proportion(1:1,2:1,3:1)and the dosage(1g/kg,2g/kg,3g/kg)as the examining index according to previous literature and work foundation.Study the pharmacodynamics of mice according to orthogonal table.The EPM showed that SWHP has better anti-anxiety effect when extracted by alcohol-water and the compatibility proportion was 2:1.And the dosage can be set to lg/kg,2g/kg,3g/kg for the next research on the basis of the screening results.2.The Pharmacological Research2.1 The anti-anxiety effect of SWHP(2:1)on miceDetermine the anxiolytic-like effects of SWHP(2:1)by EPM test,LDB test and OFT.According to the EPM test in mice,compared with control groups,SWHP at the dose of 2g/kg and 3g/kg could significantly increase OE%and OT%of mice in EPM.According to the LDB test in mice,compared with control group,SWHP at the dose of 2g/kg could significantly increase time spent and transitions between light and dark compartments.SWHP at the dose of 3g/kg could significantly increase the time spent in the light compartment.According to the OFT in mice,compared with control group,SWHP at the dose of 2g/kg could significantly increase the number and the time spent in the center area.SWHP at the dose of 3g/kg could significantly increase the number of center entries.There was no significant effect on the movement distance of the mice.2.2 The anti-anxiety effect of SWHP(2:1)on rats with RSEvaluate the effect of SWHP(2:1)on restraint stress rats by using the OFT and EPM test.Compared with control groups,RS rats showed an excessive anxiety,since they exhibited a reduction in the time and entries in the center area,in the percentage of time spent and entries on open arms.Compared with RS group,SWHP(1.5 g/kg)could significantly increase the number and the time spent in the center area,SWHP(3.0 g/kg)could significantly increase the time spent in the center area and had no side-effect of activity on rats.In the EPM,compared with RS group,SWHP(1.5 g/kg and 3.0 g/kg)could significantly increase OE%and OT%of rats,SWHP at the dose of 0.75 g/kg could significantly increase OE%.2.3 The anti-anxiety effect of SWHP(2:1)on rats with EBAccording to the results,EB rats showed an excessive anxiety,since they exhibited a reduction in the time and entries in the center area,in the percentage of time spent and entries on open arms.In the OFT,compared with EB group,SWHP(1.5 g/kg)could significantly increase the number and the time spent in the center area,SWHP(3.0 g/kg)could significantly increase the time spent in the center area.In the EPM,compared with EB group,SWHP(1.5 g/kg)could significantly increase OE%and OT%of rats,SWHP at the dose of 3.0 g/kg could significantly increase OE%:3.Mechanism Studies3.1 The mechanism of SWHP(2:1)on DA energy system and 5-HT energy system in miceThe level of monoamine neurotransmitter(5-HT and DA)and their metabolins(5-HIAA,DOPAC and HVA)in cerebral cortex and hippocampus of mice were analyzed by the HPLC-ECD.Compared with control group,SWHP(2g/kg)could significantly decrease the concentration of DA,DOPAC,5-HT and 5-HIAA.SWHP(3g/kg)could significantly decrease the concentration of DA,5-HT and 5-HIAA.3.2 The mechanism of SWHP(2:1)on rats with RSThe level of serum hormones in hypothalamic-pituitary-adrenal axis(for short,HPA axis)of the rats were measured by ELISA methods.According to the results,the level of CRF,ACTH and CORT were increased in RS rats compared to the control group.Compare with the RS group,SWHP(1.5 g/kg and 3.0 g/kg)could significantly decrease the level of CRF,ACTH and CORT.The protein levels of ERK1/2,CREB,phosphorylated ERK1/2(p-ERK1/2),phosphorylated CREB(p-CREB)and BDNF in the hippocampus(HP)and prefrontal cortex(PFC)were examined by Western blot analysis.The results showed that the RS rats resulted in the increase of p-ERK1/2 and p-CREB in the HP and PFC compared with the control group.SWHP(1.5 g/kg and 3.0 g/kg)could significantly decrease the levels of p-ERK1/2 and p-CREB compared with the RS group in the HP and PFC.RS rats resulted in the decrease of BDNF in the HP and PFC compared with the control group.SWHP(1.5 g/kg and 3.0 g/kg)could significantly increase the level of BDNF compared with the RS group in the HP and PFC,In the endocannabinoids system(ECS),the CB1 and CB2 positive levels on hippocampal CA1 were analyzed by immunohistochemical method.The results showed that the rats exposure to stress resulted in the decrease of the CB1 and CB2 positive levels compared with the control group.SWHP(1.5 g/kg and 3.0 g/kg)could significantly increase the CB1 and CB2 positive levels compared with the RS group.3.3 The mechanism of SWHP(2:1)on rats with EBThe level of serum hormones in hypothalamic-pituitary-adrenal axis(for short,HPA axis)of the rats were measured by ELISA methods.According to the results,the level of CRF,ACTH and CORT were increased in EB rats compared to the control group.Compare with the EB group,SWHP(1.5 g/kg)could significantly decrease the level of CRF,ACTH and CORT,SWHP(3.0 g/kg)could significantly decrease the level of CRF and CORT.The protein levels of ERK1/2,CREB,phosphorylated ERK1/2(p-ERKl/2),phosphorylated CREB(p-CREB)and BDNF in the hippocampus(HP)and prefrontal cortex(PFC)were examined by Western blot analysis.The results showed that the EB rats resulted in the increase of p-ERK1/2 and p-CREB in the HP and PFC compared with the control group.SWHP(1.5 g/kg and 3.0 g/kg)could significantly decrease the levels of p-ERK1/2 and p-CREB compared with the EB group in the HP and PFC.EB rats resulted in the decrease of BDNF in the HP and PFC compared with the control group.SWHP(1.5 g/kg)could significantly increase the level of BDNF compared with the EB group in the HP and PFC.In the endocannabinoids system(ECS),the CB1 and CB2 positive levels on hippocampal CA1 were analyzed by immunohistochemical method.The results showed that the rats exposure to EB resulted in the decrease of the CB1 and CB2 positive levels compared with the control group.SWHP(1.5 g/kg and 3.0 g/kg)could significantly increase the CB1 and CB2 positive levels compared with the EB group.4.Identify the chemical constitutions in the decoction of Suan zan ren,Wu wei zi and SWHP by using HPLC-LTQ Orbitrap MSEstablished the stable condition of HPLC-LTQ Orbitrap MS.Analyzed the component of Suan zao ren,Wu wei zi and SWHP under the positive/negative ion mode,which had a good separation degree and comprehensive cracking information.The components were identified according to the secondary quality spectrum fragments and the related studies reported by Suan zao ren and Wu wei zi.A total of 77 compounds were identified(44 compounds belonged to Suanzaoren,most of them were alkaloids,flavonoids and saponins;32 compounds belonged to Wuweizi,most of them were lignans;1 characteristic compound belonged to SWHP,and 29 compounds were the same with Suanzaoren and Wuweizi).It was found that the species of Suanzaoren and Wuweizi were greatly reduced with the characteristic ingredients produced(Gomisin L2 and the unknown components)after the application of 2:1,and the reason for it was to be further explored.At the same time,the unknown new ingredients needed further analysis and identification.This part of the experiments laid a good foundation for the further exploration of the indicative components and the reasons for the change of composition.Conclusion:This subject is aimed at the problem of anxiety,a common neurological disease.Guided by the compatibility theory of compound Chinese medicine,the study has first screened out the optimal extraction method,compatibility proportion and dosage of SWHP by using the orthogonal experiment on the basis of the analysis of the law of Suanzaoren and Wuweizi on the clinical use of drugs in ancient and modern times.Three classical mice behavior tests(EPM,LDB,OFT)were used to preliminarily evaluate the anti-anxiety effects of SWHP,and the effects on the DA energy system and 5-HT energy system.The anti-anxiety effects of the SWHP were completely verified and discussed from the two points of "body binding" and "psychological stimulation" by the restraint stress and the empty bottle stress induced anxiety animal models.The mechanism of HPA axis and BDNF-ERK-ECS signal pathway is studied.The preliminary analysis and identification of the chemical constituents in the single decoction and the combined decoction were made by using the modern separation technique,and the experimental basis was provided for further study on the material basisThrough the above study,combining the effect,mechanism and chemical together,the anti-anxiety effect of SWHP was explored in an all-round way,and the theory of SWHP was enriched.From the multiple angles of the neurotransmitter-HPA axis-ERK-ECS pathway,the new direction of the study of anxiety disorder mechanism was opened up,and the theoretical basis for developing new and unique anti-anxiety drugs from the theory was provided.