Studies On The Anti-anxiety Effect And Mechanism Of 5,7,3’,4’,5’-pentamethoxy Flavonoid

With the rapidly economic and social development,anxiety disorders have gradually become major burden affecting 300 million people around the world.At present,the treatment of anxiety mainly includes psychological therapy and drug therapy,as well as the combination of the two treatment.5-hydroxytryptamine reuptake inhibitors and benzodiazepines,which can play a role through continuous use of 6-12 months,are used commonly,but they have large side effects,easy to addiction,and cannot achieve a radical cure effect.There are many Chinese herbal medicines recorded to relieve anxiety,which can make up for the deficiency of chemical drugs to a certain extent.Therefore,it is urgent to develop anti-anxiety drugs with low side effects and good effects.Murraya Paniculata,also known as Qili,Qianli,etc.,is an evergreen shrub belonging to Rutaceae and Murraya J.Koenig ex L.Total flavonoids from leaves of murraya paniculata（TFMP）are flavonoids extracted and separated from leaves of murraya paniculata,which are composed of a series of methoxyl flavonoids.Among them,the most abundant flavonoid monomer components are 5,7,3’,4’,5’pentamethoxyflavone（JB）.Studies have shown that JB has good sedative and hypnotic effect,but whether it has anti-anxiety effect is not clear yet.Object:To observe the anti-anxiety effect of 5,7,3’,4’,5’-pentamethoxyflavone and discuss its mechanism.Methods:Kunming mice were randomly divided into Control group,CUMS group,5,7,3’,4’,5’-pentamethoxyflavone（50 and 100 mg/kg）group and Diazepam group（3 mg/kg）.Control group and CUMS group were given 0.5%CMC-Na 20 mL/kg intragastric administration（ig）,50 and 100 mg/kg JB groups and Diazepam group were given corresponding drug 20 mL/kg intragastric administration,once a day,continuous administration for 30 days.In this experiment,Chronic unpredictable mild stimulation was used to establish anxiety models.Control group was fed normally without any treatment,and the other groups were exposure to CUMS for 30 days.Drug intervention was performed half an hour before modeling,during which the body weight and food intake of mice were recorded every day.On day 28,behavioral experiments were conducted.Entering times,staying time and total movement distance of mice in open arms were measured in EPM（Elevated Plus-maze test）.In the open field test（OFT）,the times of crossing,times of standing,the total distance of movement and the time of staying in the center were recorded.The content of Corticosterone（CORT）were determined in serum of mice.The contents of 5hydroxytryptamine（5-HT）,adenosine cyclic phosphate（cAMP）and γ-aminobutyric acid（GABA）in the hippocampus of mice were determined.16S rDNA highthroughput sequencing technology was used to detect the composition,diversity and gene function of gut microflora;Western blot was used to detect protein expression including brain-derived influence factor（BDNF）,tyrosine kinase B receptor（TrkB）,phosphorylated tyrosine kinase B receptor（p-TrkB）,adenosine A2A receptor（A2AR）,Gephyrin and GABA α 2 receptor（GABRA2）in mouse hippocampus.Results:1.The results of body weight and food,water intake showed that compared with the Control group,the body weight of mice in the CUMS group was significantly decreased（P<0.01）,but their food intake was significantly increased（P<0.05 or P<0.01）;Compared with the CUMS group,administration of 50,100 mg/kg JB or 3 mg/kg diazepam could increase the body weight of mice,but there was no statistical significance（P>0.05）.Meanwhile,administration of 50,100 mg/kg JB or 3 mg/kg Diazepam did not affect the amount of food or water consumed by mice.2.The results of EPM showed that compared with the Control group,the percentage of mice entering the open arm and dwelling time in the open arms in the CUMS group decreased（P<0.01）,the dwelling time in the closed arm increased（P<0.01）,and the total movement distance of mice increased（P<0.01）;While compared with the CUMS group,administration of 50 mg/kg JB could increase the percentage of mice entering the open arm and dwelling time in the open arm,and reduce the percentage of mice dwelling time in the closed arm and the total movement distance,but there was no statistical significance（P>0.05）.Administration of 100 mg/kg JB or 3 mg/kg diazepam could significantly increase the percentage of mice entering the open arm and staying time in the open arm（P<0.05 or P<0.01）.At the same time,the percentage of dwelling time and total movement distance of mice in closed arm were decreased（P<0.05 or P<0.01）.3.The results of OFT showed that compared with the Control group,the activity index and total movement distance of the CUMS mice increased（P<0.05 or P<0.01）,and the center residence time decreased significantly（P<0.01）;Compared with the CUMS group,administration of 50 mg/kg JB can reduce the activity index and total movement distance of mice,and increase the time of mice staying in the center,but there is no statistical significance（P>0.05）.Administration of 100 mg/kg JB or 3 mg/kg Diazepam can significantly reduce the activity index and total movement distance,and increase the time of staying in the center（P<0.05 or P<0.01）.4.The results of HPA axis function showed that the content of CORT in the serum of CUMS group mice was significantly higher than that of the Control group（P<0.01）;compared with the CUMS group,administration of 50 mg/kg JB could reduce the content of CORT in the serum of mice,but there was no statistical significance（P>0.05）.Administration of 100 mg/kg JB or 3 mg/kg Diazepam could significantly reduce the content of CORT in the serum of mice（P<0.05）.5.The results of neurotransmitter expression in the hippocampus showed that compared with the Control group,the contents of 5-HT and GABA in the hippocampus of the CUMS group mice were significantly decreased（P<0.05 or P<0.01）;Compared with the CUMS group,administration of 50 mg/kg JB could increase the contents of 5-HT and GABA in the hippocampus of mice,but there was no statistical significance（P>0.05）.Administration of 100 mg/kg JB or 3 mg/kg Diazepam could significantly increase the contents of 5-HT and GABA in the hippocampus of mice（P<0.05 or P<0.01）6.The 16S rDNA high-throughput sequencing results showed that:（1）The number of feature number（OUT）in the Control group was 1372,the number of CUMS group was 1159,and the JB group was 1241;The number of endemic species in the Control group is 623,the number of CUMS group is 425,the JB group is 477.The number of species in the three groups is 539.（2）At the phylum level,Proteobacteria,Actinobacteriota,Deferribacterota and Cyanobacteria are the bacteria with significant differences.Compared with the Control group,the relative abundance of Proteobacteria and Cyanobacteria in CUMS group decreased,while the relative abundance of Actinobacteriota and Deferribacterota increased;Compared with the CUMS group,the relative abundance of Proteobacteria and Cyanobacteria in JB group increased,while the relative abundance of Actinobacteriota and Deferribacterota decreased.At the genus level,Lachnospiraceae_NK4A136_Group,Ligilactobacillus,Alistipes,and Lachnospiraceae_UCG₀01 are different.Further analysis of the relative abundance of the them revealed that compared with the Control group,the relative abundance of Lachnospiraceae_UCG₀01 and Ligilactobacillus decreased in CUMS mice,while the relative abundance of Alistipes and Lachnospiraceae decreased_NK4A136_group increased;Compared with CUMS group,the relative abundance of Lachnospiraceae_UCG₀01 and Ligilactobacillus increased in JB mice,while the relative abundance Alistipes and Lachnospiraceae increased_NK4A136_Group decreases.（3）Compared with Control group,the Alpha diversity index（ACE,Chaol,Shannon and Sampson）of CUMS group were significantly lower（P<0.05 or P<0.01）;Compared with CUMS group,JB could significantly increase Sampson index（P<0.05）,and ACE,Chao1,Shannon index,but there was no statistical difference（P>0.05）;Beta diversity resulted showed that the distance between the Control group and the CUMS group was large,and the bacterial community structure changed significantly;the distance between CUMS group and JB group is large,which indicated that JB has significantly changed the intestinal flora environment,and the composition of the flora in JB group and Control group has a large overlap.（4）Significant difference between groups:the specific flora of CUMS group is Marvinbryantia,Alistipes_sp_cvl and Eubacterium_brachy_group;The specific strains of JB group are Proteobacteria,Escherichia Shigella and GCA₉00066575.（5）The results of functional gene prediction showed that compared with the Control group,the pathways with significant differences in the model group were carbon fixation of photosynthetic organisms,chemical carcinogenesis and GABAergic synapses.Compared with the CUMS group,the pathways with significant differences in the JB group were pentose,glucuronic acid conversion,caffeine metabolism and ascorbic acid metabolism.Similarly,JB affected GABAergic synapses.7.The results of cAMP content in the hippocampus showed that compared with the Control group,the cAMP content in the hippocampus of the CUMS group mice decreased significantly（P<0.01）;Compared with the CUMS group,administration of 50 mg/kg JB could increase the content of cAMP in the hippocampus of mice,but there was no statistical significance（P>0.05）.Administration of 100 mg/kg JB could significantly increase the content of cAMP in the hippocampus of mice（P<0.01）,but 3 mg/kg Diazepam had no effect on cAMP content in the hippocampus（P>0.05）.8.Western blot detection of GABAergic synaptic associated protein showed that compared with the Control group,the expression of A2AR、GABRA2 and Gephyrin protein significantly decreased（P<0.05 or P<0.01）.Compared with the CUMS group,administration of 50 mg/kg JB increased the contents of GABRA2 and Gephyrin,but there were not statistically significant（P>0.05）.Administration of 100 mg/kg JB or 3 mg/kg Diazepam could significantly increase relative expression of GABRA2 and Gephyrin（P<0.05 or P<0.01）,while there is no effect on relative expression of A2aR in Diazepam and 50 mg/kg JB.9.Western blot showed that compared with the Control group,the expression of BDNF in the hippocampus of CUMS group was significantly decreased（P<0.01）,but relative expression of p-TrkB was not changed（P>0.05）;Compared with the CUMS group,administration of 50 mg/kg JB could increase relative expression of BDNF in the hippocampus of mice,but there was no statistical significance（P>0.05）.Administration of 100 mg/kg JB or 3 mg/kg Diazepam could significantly increase the relative expression of BDNF in the hippocampus of mice（P<0.05）.Conclusion:1.JB could activate A2AR/Gephyrin/GABRA2 pathway,maintain GABAergic synaptic homeostasis and neural plasticity,improve the expression of neurotransmitters,alleviate the hyperfunction of HPA axis,and thus improve the anxiety caused by stress in mice.2.JB may also increase the composition of gut microbiota species,improve the relative abundance of intestinal flora species,and improve intestinal microecology,which may be one of the mechanisms of its anti-anxiety effect.