Explore The Mechanism Of Anti-depression Of Albiflorin By Regulating Intestinal Flora Based On Targeted Metabolomics Research Methods

Depression is a serious emotional disorder mental a significant feature of persistently depressed mood.Currently,the prepared antidepressants used in clinical have certain curative effect,but they have slow onset,low efficacy and strong side effects.Therefore,it is the hotspot to explore the complex pathogenesis of depression,to find a new target on developing the antidepressants with better efficacy and decreased side effects.In our previous work,we systematically explored the antidepressant effects of the total glycosides,peoniflorin and albiflorin,and found that they all have certain antidepressant effects.Moreover,the antidepressant effects depend on the dose of albiflorin,is better than the paeoniflorin and total glycosides.In this study,the classic chronic unpredictable mild stress(CUMS)model is used to investigate the rapid antidepressant effects of albiflorin.The antidepressant molecular pharmacological mechanism of albiflorin is revealed from the metabolites in the hippocampus and gut microbiome of rats using next-generation of targeted metabolomics technology.The antidepressant effects of albiflorin in CUMS model rats has was systematically investigated at different dosage,which were including body weight,1% sugar water preference experiment and open-field test(OFT),compared with antidepressant drug fluoxetine and antidepressant fast-acting drug ketamine.The results showed that in the CUMS models,after 7 days continuous administration albiflorin,the weight loss and reduction of 1% sugar water preference in the CUMS model rats could be significantly reversed,as well as the decrease in the total distance of activity and the number of upright positions in the OFT.However,fluoxetine(10 mg/kg)for 7 days continuously affected the 1% sugar preference of model rats,with no significant difference compared with the CUMS group.A single dose of ketamine(10 mg/kg)and albiflorin(7 mg/kg)can significantly reverse the decrease of 1% sugar water preference in the CUMS model rats,and there is no statistical difference between ketamine and albiflorin,indicating that albiflorin has a rapidly acting antidepressant effect.Carried out a pharmacokinetic study of albiflorin,at the beginning of the experiment,rats were intragastrically administered with albiflorin(7 mg/kg)and the blood was taken from the posterior orbital venous plexus at predetermined time point.Then,the rats were sacrificed and the rat brain hippocampus was quickly separated after collecting blood at each time point.LCMS /MS was used to determine the content of albiflorin in plasma and hippocampus.The results indicated that albiflorin in rats could be detected within 5 min after gavage,and the concentration of albiflorin in the hippocampus reached its peak value at 45 min(Tmax).Pharmacokinetic results further proved the rapid onset of the albiflorin.The antidepressant mechanism of albiflorin on CUMS depression model rats was carried out.The study results of targeted metabolomics in rat hippocampus showed that albiflorin could restore the metabolism disorder of hippocampus,improve the level of metabolic products,and regulation of phenylalanine,tyrosine,glutamate and tryptophan metabolic pathways.Western Blot study results that c AMP-response element binding protein(CREB),Phosphorylated Camp Response Element Binding Protein(p CREB),Tropomyosin-related kinase B receptor(Tr KB)and phosphorylated tropomyosin related kinase B(p Tr KB)in the hippocampus and nucleus accumbens(NAc)of rats were upregulated after continuous administration of albiflorin for 7 days compared with the CUMS model group.In the hippocampus,Brain derived neurotrophic factor(BDNF)expression was significantly upregulated after administration of albiflorin for 7 days,but it was not affected by fluoxetine treatment.In addition,albiflorin significantly reduced the expression level of N-methyl-D-aspartate receptor(NMDA)receptor B subtype NMDA Receptor 2B(GLu N2B),played a similar role to Ketamine.The study results of targeted fecal metabolomics in rats displayed that CUMS induced metabolic disorders in the intestinal flora of rats,and the treatment of albiflorin made the overall intestinal microbial metabolism in rats tend to normal metabolism,regulating chronic stress-induced intestinal metabolic disorders,regulates metabolic pathways such as arachidonic acid and tyrosine.In order to further verify the role of intestinal microbes in the antidepressant effect of albiflorin,we conducted a broad-spectrum antibiotic intervention on the intestinal flora of rats through gavage and drinking water.A model of Antibiotic-induced microbiome depletion(AIMD)was constructed.The fecal bacterial DNA load and the level of intestinal microbial metabolites were significantly lower than those of normal control rats,and the benzoic acid content in AIMD group was significantly reduced,indicating the success of intestinal microbiome clearance.Then,the CUMS depression model was used to evaluate the effect of AIMD on the antidepressant activity of albiflorin.The results demonstrated that AIMD group treated with albiflorin only partially corrected reduction in the number of upright and total distance of activity caused by chronic stress in the OFT tests.The results of targeted metabolomics in the hippocampus revealed that AIMD partially eliminated the repair of the metabolic disorders of hippocampus in CUMS model rats,which affected the metabolism pathway of tryptophan,tyrosine and arachidonic acid in hippocampus.Compared with the common CUMS model rats treated with albiflorin,the response of AIMD-induced CUMS depression model rats to albiflorin treatment was significantly different.In conclusion,albiflorin can be used as an antidepressant candidate with promising application and rapid curative effects.The main antidepressant mechanism of albiflorin is: albiflorin exerts an antidepressant effect by regulating the metabolic pathways of tyrosine,tryptophan,glutamic acid,phenylalanine and arachidonic acid;albiflorin can restore the metabolic disorder induced by CUMS,because the intestinal microorganisms and their metabolic enzymes decomposed albiflorin into the metabolite benzoic acid,which can correct the imbalance of intestinal flora,reshape the composition and function of gut microorganisms,and improve the bioavailability of the albiflorin in the body,leading to exerting the antidepressant effects.