| RATIONALES: Depression is a common mental illness.It is characterized by persistent depression and cognitive dysfunction.It is also a complex disorder involving multiple mechanisms.At present,antidepressant western medicines used clinically can improve the symptoms of depression,but it is difficult to achieve comprehensive results.Research and development of new antidepressants based on traditional Chinese medicine and its prescriptions has become a research hotspot.In recent years,our research group has taken the classic prescription Xiaoyaosan(XYS)as the research object,and carried out in vivo and in vitro pharmacological activities and serum pharmacological studies,and found that Bupleuri Radix is the major drug in the XYS prescription.Bupleuri Radix has a greater contribution to its antidepressant effect.Bupleuri Radix,as the most frequently used antidepressant,previous studies have shown that petroleum ether fraction of Radix Bupleuri has good antidepressant effect,but the best medicinal effect site has not been screened through pharmacological experiments.After the drug is absorbed into the blood through oral administration,it can be transported to various target organs through the blood circulation to exert its effect.The brain is an important target organ for depression,if the drug can pass through the blood-brain barrier,it may directly enter the brain to exert its effect.In addition,different pathological conditions of the body may also affect the in vivo process of the drug.Therefore,the differences in metabolic characteristics and pharmacokinetic behavior of the blood components of the best active site of Radix Bupleuri after administration under different body conditions,the differences in chemical components and contents in different brain regions,and the change of blood-brain barrier permeability have become essential to investigate.The research content provides a reference for the development of new antidepressants.Objective: To clarify the antidepressant active site of Radix Bupleuri,in vivo metabolic characteristics and differences in pharmacokinetic behavior of the blood components under different body states.To explore the differences in chemical components reaching the brain tissue,and study the effect of active components on blood-brain barrier permeability.Through the above studies,comprehensivelyevaluates the in vivo process of the active site of Radix Bupleuri,and provides a scientific basis for the drug to exert its effect and the metabolism of the drug in the body.Methods: 1.SD rats were randomly divided into control group,CUMS model group,positive drug group(PC,venlafaxine hydrochloride,35 mg/kg),petroleum ether fraction of Radix Bupleuri low-polarity site(B,15 g/kg)group,ethyl acetate fractions of Radix Bupleuri Mid-polarity site(BE,15 g/kg)and the big polar site of Radix Bupleuri(BW,15 g/kg)group.To screen the best active site of Radix Bupleuri antidepressant,behavioral indicators such as body weight,sucrose preference,and open field,and the content of neurotransmitters in serum were compared.2.Rats in group K and group M were administered orally with petroleum ether fraction of Radix Bupleuri(50 g/kg),and serum samples were collected at 0 h,1 h,1.5 h,2.5 h,and 12 h.UHPLC-Q Exactive Orbitrap-MS high resolution mass spectrometry combined with Compound discover 3.0 analysis software was used to deeply characterize the prototype compounds in the serum and metabolites of polyacetylenes and saikosaponins,and compare the differences of blood component types and concentrations under different body conditions.3.Rats in group K and group M were administered orally with petroleum ether fraction of Radix Bupleuri(50 g/kg),and serum samples were collected at 0、0.08、0.16、0.33、1、2.5、4、6、9、12、24、36、48 h.Metabolomics technology was used to analyze the differences in endogenous metabolic profiles of rat serum at different administration time points in the model group,model group,and control group.The relative distance value was used to find the time point of the best drug effect,and then the differential metabolites related to disease and drug effect were identified according to VIP>1,?pcorr?>0.58 and P<0.05,and ROC curve was used to evaluate the diagnostic performance of differential metabolites.UHPLC-Q Exactive Orbitrap-MS analysis technology was used to compare the pharmacokinetic behavior of chemical components in different organism states through heatmap analysis,and to classify exogenous compounds in the model group by cluster analysis,while the relative distance value is used as an index of pharmacodynamics to perform correlation analysis with the pharmacokinetic data of the model group to findpotential pharmacodynamic component groups and pharmacokinetic markers related to pharmacodynamics.Finally,PK-PD tudy analysis was performed to reveal the relationship between pharmacokinetics,time,and efficacy.4.Rats were randomly divided into control group(K group),the CUMS model group(CUMS group),control drug group(PK group,50 g/kg),model drug group(PM group,50 g/kg).The chemical constituents in hippocampus,cortex,striatum,hypothalamus,and pituitary tissue were characterized 12 h after administration in the PK and PM groups,and the types and contents of chemical constituents in different body states were compared.5.The rats were randomly divided into the control group(K),the CUMS model group(M),the control administration group of petroleum ether fraction of Radix Bupleuri(CK,50 g/kg),and the model administration group of petroleum ether fraction of Radix Bupleuri(CM,50 g/kg)group.Rats were administered with oral administration for 28 days at the same time as modeling.Immunohistochemical method was used to determine the expression of blood-brain barrier structure related proteins ZO-1,AQP4,CX43 in hippocampus,prefrontal cortex,striatum,hypothalamus and pituitary.RT-q PCR was used to measure the expression levels of functional barrier-related genes MDR1,BCRP,and MRP1.The effects of petroleum ether fraction of Radix Bupleuri on the structure and function of blood-brain barrier in different brain regions were compared under different body conditions.Results: 1.Compared with the control group,the model group’s body weight,sucrose preference,and the crossing number were significantly reduced(P <0.01,P<0.001),and the levels of Trp,5-HT,GABA,GABA/Glu,and Tyr in the serum were significantly reduced(P < 0.05,P <0.01),indicating that CUMS modeling was successful.Comparing the behavioral indicators of different polar sites of Radix Bupleuri,the results showed that different polar sites of Radix Bupleuri can improve the depression-like behavior of CUMS rats and the low polar site of Radix Bupleuri has the strongest antidepressant effect.Comparing the levels of neurotransmitters in serum of different polar sites of Radix Bupleuri,the results showed that the neurotransmitter regulation of different sites of Radix Bupleuri has its own characteristics and the levels of neurotransmitters in the low-polarity part of RadixBupleuri are closer to that in the control group,and the effect is stronger.Based on the above results of behavior and neurotransmitter,this study selected the low-polarity part of Radix Bupleuri with the strongest antidepressant activity for further research.2.Through further separation and purification of petroleum ether fraction of Radix Bupleuri,two polyacetylenic compounds RB-9 and RB-10 were separated and identified,of which RB-9 was a new compound.UHPLC-Q Exactive Orbitrap-MS technology was used to analyze serum samples from control rats and model rats.A total of 70 prototype compounds(C1-C70)and 201 metabolites(M1-M50,MA1-MA14,MB1-MB14,MC1-MC15,MD1-MD15,MS1,MS2)were found,among which the prototype compounds included 26 known compounds reported in the literature,26 polyacetylene prototype compounds,1 Prosaikegenin analog,17 unknown prototype compounds,and metabolites included 148 polyacetylene metabolites,3 saikosaponin metabolites,and 50 other metabolites with no predicted structure.In addition,comparing the differences in the types and concentrations of drug absorption in different body states,it is found that there were certain differences in the absorption and metabolism of drugs in vivo.Under physiological conditions,248 compounds were detected in serum,including 69 prototype compounds and 179metabolites;in CUMS pathological conditions,239 compounds were detected in serum,including 51 prototype compounds and 188 metabolites,of which 1 prototype compound and 22 metabolites were detected only in serum samples of the model administration group.3.After a single administration of petroleum ether fraction of Radix Bupleuri,for endogenous compounds,compare the endogenous metabolic profiles of rat serum between control group and model group and at different time points after model administration,and the results showed that the metabolic profiles of serum at 4h,6h,and 9h after model administration were close to the control group,and the efficacy was better,and the efficacy of the drug: M6h> M9h> M4 h.By establishing a mathematical model,the relative distance value was used as an effect indicator to reflect the overall change of endogenous compounds at different time points in the body.In addition,multivariate statistical analysis was performed on the endogenous compounds in the blank serum,model serum and model serum of 4h,6h,9h aftermodel administration.Twenty endogenous differential metabolites related to drug efficacy,such as sphingomyelin,sphingosine,and L-palmitoyl carnitine were screened and identified,and they were related to the sphingolipid metabolism pathway.ROC curve analysis showed that the AUC of the 20 different metabolites were all greater than 0.7,which was expected to become a marker for the diagnosis of depression.For exogenous compounds,it is found that there were obvious differences in the pharmacokinetic curves of chemical components under different organism states through heatmap analysis;compounds in the model group were classified into 6 types of compounds through cluster analysis;the correlation analysis between these 6 types of compounds and the efficacy index(relative distance value)showed that the sixth type of compounds had a positive correlation with the efficacy and are the group of potential medicinal ingredients.The first class of compounds C38 and C68,and the sixth class of compounds C12,C61,MA3-3,MB1-4,MC13-4,MD15-2,M4,M35,and M43 with correlation coefficients greater than 0.4 were exogenous pharmacokinetic markers.The integrated PK-PD binding model of CUMS depressed rats shows that there was a significant lag in the drug effects and blood concentrations of the first,second,third,and fourth compounds.Relevantly,the fifth and sixth compounds had a longer peaking time.It may be speculated that different components in petroleum ether fraction of Radix Bupleuri have different in vivo processes and different targets for their activity.Each component may synergize to make the effect last longer and have a more comprehensive effect.4.After a single administration of petroleum ether fraction of Radix Bupleuri for 12 h,the types and relative contents of compounds that entered the different brain regions were examined,and the differences in the types and amounts of compounds absorbed into the brain in normal rats and CUMS models were compared.The results showed that after the administration of petroleum ether fraction of Radix Bupleuri,it mainly existed in the form of metabolites in the brain,and under different body states,the number and relative content of chemical components in different brain regions were different.The number of drugs in hippocampus and cortex was the most,and the striatum and pituitary tissue was the least.The class D polyacetylene metabolites wasmore likely to enter the brain under physiological and pathological conditions.In addition,under physiological conditions,class B polyacetylene metabolites easily entered the hippocampus,cortex,striatum,and hypothalamus through the blood-brain barrier,and C-type polyacetylene metabolites easily passed through the hippocampus and cortex;under pathological conditions,Class C polyacetylene metabolites easily entered the hippocampus and striatum,and class B polyacetylene metabolites easily entered the cortical tissue.In terms of relative content,under physiological conditions,the compound absorbed more in the striatum and hypothalamus.Compounds with higher content in each brain area may protect the homeostasis of the environment within the blood-brain barrier through interactions.In the pathological state,the compound was absorbed more in the hippocampus and cortical tissue,and the compound with higher content in each brain area may be the material basis for the antidepressant effect;while in different body states,the difference in absorption of the compound in the pituitary tissue was not obvious.The unique compounds in the pituitary tissue may play an important role in the protection of brain tissue and the occurrence of drug effects.5.After 28 days administration of petroleum ether fraction of Radix Bupleuri,the expressions of structural related proteins AQP4,CX43,and ZO-1 in different brain regions were determined by immunohistochemistry.At the same time,the expression levels of MDR1,BCRP,and MRP1 genes related to functional barriers were determined using RT-q PCR technology.The result showed that compared with group K,the expression of AQP4 and CX43 in hippocampus and cortex of group M were significantly reduced(P <0.05,P <0.01),the expression of ZO-1 in striatum was significantly reduced(P <0.05),and the expression of AQP4 and ZO-1 in the hypothalamus tissues were significantly reduced(P <0.05),indicating that CUMS modeling caused damage to the blood-brain barrier and increased blood-brain barrier permeability in the hippocampus,cortex,striatum,and hypothalamus.At the same time,the expression of efflux transport genes in hippocampus,cortex,striatum,and hypothalamus were increased,which increased the functional barrier of the blood-brain barrier.After administering petroleum ether fraction of Radix Bupleuri to CUMS model rats,the expression of structural protein related in hippocampus,cortex,striatum,and hypothalamus of the CM group all changed back to varying degrees,which reduced the blood-brain barrier permeability and improved blood-brain barrier damage.The expression of efflux transporters were significantly reduced,allowing drugs to enter the brain selectively.Compared with the group K,the expression of MDR1 in hippocampal tissue of the CK group was significantly increased,the expression of AQP4 was significantly increased,and the expression of MRP1 was significantly reduced in the cortex,the expression of striatum AQP4,CX43,ZO-1were significantly increased,and the expression of CX43 in hypothalamus was significantly increased to maintain the steady state of the brain environment.In this study,there were no significant differences in the expression of pituitary tissue before or after modeling and the expression of structural related proteins before and after administration.The expression of BCRP in group K was significantly increased after administration to protect the homeostasis.Conclusion: Based on the above studies,this paper had screened the best active site of Radix Bupleuri: petroleum ether fraction of Radix Bupleuri.The similarities and differences in the blood components after the oral administration of petroleum ether fraction of Radix Bupleuri in different body states were systematically characterized,and further conducted pharmacokinetic studies on these compounds.At the same time,pharmacodynamic studies were conducted with clear endogenous differential metabolites,and correlation studies were performed to clarify the relationship between pharmacokinetics,time,and efficacy,and finally revealed brain-entering components and its effect on the blood-brain barrier in brain tissues in different body states.This study provides a basis for revealing the in vivo process of petroleum ether fraction of Radix Bupleuri and antidepressant mechanisms.S... |
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