| RATIONALES: Depression is a chronic and frequently psychiatric illness and is ranked by the World Health Organization as one of the heaviest diseases in society.Synthetic chemical drugs are effective in the treatment of depression,however,they have been found to produce severe side-effects.Traditional Chinese medicine has mild antidepressant effects and less side-effect.Through the guidance of serum pharmacochemistry in our previous work,it was clear that the most components of the active fraction of Xiaoyaosan(XYS)in serum are from Bupleuri Radix.And this proves that Bupleuri Radix is the major drug in the XYS prescription.In addition,the obtained results in our further studies showed that the petroleum ether fraction of Bupleuri Radix(PBR)has an antidepressant effect in a middle-high dosage and it induces not much influence in the liver or kidney in CUMS rats.However,it can produce liver and kidney damage in healthy rats in high dosage.Through further analysis,it was found that the degree of the antidepressant effect of PBR was different in different dosages,and the toxic response was also different in pathological and physiological conditions.However,it is not clear now that the antidepressant mechanism of PBR in CUMS rats and the toxic mechanism of liver or kidney damage caused by PBR in healthy rats.Therefore,it is need to research the antidepressant and toxic mechanism of PBR in pathological and physiological conditions and the correlation of the efficacy/toxicity and dosages.Objective: To established the metabonomics research methods of serum,hippocampus and liver of rats by UPLC-MS and to clear the antidepressant and toxic mechanism of petroleum ether fraction of Bupleuri Radix in pathological and physiological conditions and the correlation of the efficacy/toxicity and the dosages.Methods: The serum,hippocampus and liver metabolic profiles of CUMS rats and healthy rats given different dosages of PBR were obtained by Xcalibur 3.0 of UHPLC-Q Exactive Orbitrap-MS technique.And the LC-MS raw data were imported into Compound Discoverer 2.0 to obtain the peak data,then the peak data was imported into excel to solve peak area normalization.The differential variables were screened through principal component analysis by SIMCA-P 13.0.The differential metabolites were identified by HMDB database and relavant metabolic pathways were analyzed through KEGG database and related literature.The linear correlation of the differential metabolites in CUMS rats or the relavant toxic metabolites in healthy rats given with PBR and the dosage were discussed.Results: 1.The results of principal component analysis of serum,hippocampus and liver metabolic profiles in CUMS rats showed an obvious separation between the model group and control group,and the separation was more obvious in the groups of CUMS rats given higher dosage of PBR compared with the model group.Studies have shown that 22 differential metabolites were altered in serum of CUMS rats given with PBR,for example Tryptophan,Glutamic aicd,4-Acetamidobutanoic acid,sphinganine and so on,which involved in amino acid metabolism,glycometabolism,sphingolipid metabolism,glycerophospholipid metabolism,and fatty acid metabolism.Seven differential metabolites were altered in hippocampus of CUMS rats given with PBR,for example Leucine,Isoleucine,cis-Aconitic acid and so on,which involved in amino acid metabolism and energy metabolism.Seventeen differential metabolites were altered in liver of CUMS rats given with PBR,which involved in amino acid metabolism,energy metabolism,sphingolipid metabolism and β oxidation of fatty acid.Taken together,the regulated metabolic pathways of PBR in serum,hippocampus and liver metabolic profiles were complementary and mutually corroborative.The PBR produces an antidepressant effect through regulating glycometabolism,amino acid metabolism,energy metabolism,sphingolipid metabolism,glycerophospholipid metabolism,and fatty acid metabolism.The results of the correlation analysis of the efficacy and the dosage showed that the percentage of the degree of the differential metabolites returning to normal is positively correlated with the dosage in CUMS rats following PBR administration,and means that the degree of the metabolites returning to normal was greater with giving higher dosage.Also,the percentage of the degree of the differential metabolites returning to normal is greatest in D6 dosage of PBR,suggesting that the differential metabolites may return to normal better in D6 dosage.2.The PBR can significantly altered the metabolic profiles of serum and liver in healthy rats,but produce not much influence in hippocampus metabolic profiles,suggesting that petroleum ether fraction of Bupleuri Radix does not induce toxic reactions in hippocampus.The amino acid metabolism,sphingolipid metabolism,energy metabolism,fatty acid β oxidation,glycerol phospholipid metabolism and bile acid metabolism were significantly affected in healthy rats given with PBR.The PBR might cause liver damage by altering hepatic cell survival and structural plasma membrane permeability in liver tissue,or cause hepatic cell damage by accumulating lipid peroxidation products and triggering an inflammatory reaction.The results of the correlation analysis of the toxicity and the dosage showed that PBR could induce a greater influence with giving higher dosage of PBR in healthy rats.3.The amino metabolism,energy metabolism,sphingolipid metabolism,glycerophospholipid metabolism and fatty acid metabolism were identical regulated pathways in both CUMS rats and healthy rats following PBR administration,but some regulated target sites of metabolites of PBR were different,suggesting that PBR affected different sites of action in the five pathways under different physiological conditions.In the same regulated differential metabolites in both CUMS rats and healthy rats given with PBR,the altered metabolites were reversed to a level close to normal in CUMS rats given with PBR,except that acyl cartinines were reversed to a level less than normal in high dosage of PBR,suggesting that PBR produced a comparatively mild drug-induced toxic reaction in CUMS rats.Bile acid metabolism were only altered in healthy rats given with PBR,and it was the most significant of the regulated pathways of PBR in the different physiological states,suggesting that bile acid metabolism may be the regulated site of PBR in pathological and physiological states so that it can be seen a differential responses of PBR in the different body conditions.Conclusion: The petroleum ether fraction of Bupleuri Radix produces an antidepressant effect through regulating glycometabolism,amino acid metabolism,energy metabolism,sphingolipid metabolism,glycerophospholipid metabolism,and fatty acid metabolism.The petroleum ether fraction of Bupleuri Radix may affect different sites of action in subjects with depression and healthy subjects,and thus produced differential effective and toxic responses in different physiological conditions.In addition,the effective and toxic responses of PBR were positively correlated with the dosage. |
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