Protective Effect And Mechanism Of Saikosaponin B2 On PC12 Cell Injury Induced By Corticosterone

Rationales::Depression is a complex mental disease,which is a serious threat to human health.Depression has a variety of pathogenesis,but the existing antidepressants and molecular targets are still focused on the hypothesis of monoamine neurotransmitters.At present,antidepressants have caused a variety of side effects,so people pay more attention to traditional Chinese medicine in the drug treatment of depression.Bupleurum is a commonly used traditional Chinese medicine,which has the effect of reconciling exterior and interior,soothing the liver,relieving depression,rising Yang and lifting depression.Saikosaponin（SS）is the main bioactive component of bupleurum,which has many pharmacological effects.In terms of antidepressant effect,there are many reports about total saikosaponin（TSS）,saikosaponin a（SSa）and saikosaponin d（SSd）.At the same time,studies have shown that saikosaponin b2（SSb2）has a variety of pharmacological effects.It was found that SSb2 inhibited the activation of IKKβ/IκBα?/NF-κB signaling pathway and the increase of pro-inflammatory factors,and exerted anti-inflammatory effect on LPS induced macrophages;in addition,SSb2 alleviated H₂O₂induced L02 cell injury by inhibiting mitochondrial apoptosis pathway.In addition,SSb2 has the pharmacological effects of alleviating liver injury,anticancer and antiviral.Li et al.found that SSd was almost completely converted to SSb2during the decoction of Bupleurum.At the same time,the previous research of the research group found that in the treatment of depressed rats,the antidepressant effect of Bupleurum-white peony pair was significantly better than that of Bupleurum or Peony alone.The in vivo pharmacokinetic study showed that Bupleurum-white peony paired Significantly improve the bioavailability of SSb2.In addition,studies have found that SSd has a protective effect on CORT-induced PC12 cell damage.Therefore,it is speculated that SSb2 may have a protective effect on CORT-induced PC12cell damage.However,there is no report on the role of SSb2 in the treatment of depression.The stress response of HPA axis with significantly increased glucocorticoid level is one of the important researches in the pathogenesis of depression.Corticosterone（CORT）,the last effector of HPA axis,is the main glucocorticoid secreted by stress response.When CORT reaches the stress level,it can reduce the release of 5-HT and lead to neurodegeneration.PC12 can be injured by high concentration of CORT,and has been widely used as an in vitro model for the study of injured nerve cells and depression like syndrome.Metabonomics can reflect the metabolism of biological system,analyze potential biomarkers and metabolic network,and provide an effective method for screening disease-related biomarkers and studying drug mechanism.At present,more and more researchers use metabonomics technology to study the mechanism of drug action,including the metabonomics research of CORT on PC12 cell damage.Due to the low level of cell metabolites,ultra performance liquid chromatography quadrupole/time of flight mass spectrometry（UPLC-Q/TOF-MS）has the advantages of high speed,high selectivity,high sensitivity and rich structural information.In recent years,a large number of metabolic markers have been rapidly identified and analyzed by UPLC-Q/TOF-MS technology combined with multivariate statistical methods.Therefore,this project is based on metabolomics and molecular biology techniques to explore the neuroprotection and mechanism of SSb2 on CORT-injured PC12 cells.Objective:To investigate the protective effect of SSb2 on PC12 cells injured by corticosterone（CORT）,and to explore the neuroprotective mechanism of SSb2 based on metabonomics and molecular biology.Methods:1.PC12 cells were injured by CORT was used to evaluate the neuroprotective effect of SSb2 by MTT assay,LDH release rate was determined by microplate method,apoptosis was detected by Hoechst 33342/PI double staining method and Annexin V-FITC method/PI flow cytometry.2.Metabolic changes were detected by LC-MS metabonomics.PCA of multivariate statistical analysis method was used to analyze the metabolic profiles of all groups of samples;PLS-DA was used to analyze the principal component analysis of each group of sample data;OPLS-DA was used to analyze the data of model group and control group to screen out the difference variables.According to VIP value>1 and P<0.05,the differential metabolites and the differential metabolites regulated by ssb2were screened between the control group and the model group;the metabolic pathways involved in the differential metabolites regulated by SSb2 were enriched and analyzed by online software metaboanalyst 4.0 and KEGG The database was used to analyze the association of different metabolites regulated by SSb2,and to study the metabolites and metabolic pathways regulated by SSb2 in PC12 cells injured by CORT.3.Mitochondrial membrane potential detection kit（JC-1）to detect mitochondrial membrane potential（MMP）,enzyme-linked immunosorbent assay to detect TNF-α,IL-1?and IL-6 levels.Western blot technology to study the effect of SSb2 on CORT damage the role of Bax/Bcl-2,cytochrome C and caspase-3 proteins in the mitochondrial apoptosis pathway in PC12 cells and the expression of p-p38,p-IκBα,and p-p65proteins in the p38/NF-κB pathway.Results:1.SSb2 can improve the survival rate of PC12 cells injured by CORT,and its mechanism may be related to inhibiting apoptosis,reducing the release of LDH.2.The results of LC-MS metabonomics showed that compared with the blank group,19 differential metabolites were found in CORT injury group,and SSb2 could callback 9 of them:glutamic acid,creatine,n-acetylaspartic acid,L-tyrosine,citric acid,L-isoleucine,lactic acid,glutamine and choline,involving five major metabolic pathways,namely D-glutamine and D-glutamic acid metabolism,phenylalanine metabolism Biosynthesis of tryptophan,tyrosine,alanine,aspartate and glutamate,tyrosine and arginine.3.SSb2 significantly inhibits the expression of p-p38,p-IκBα,and p-p65 in the p38/NF-κB signaling pathway in PC12 cells induced by CORT,reduces the release of TNF-α,IL-1?and IL-6;inhibits The decreased level of MMP in the mitochondria apoptosis pathway and the expression of Bax/Bcl-2,Cytochrome C and Cleaved caspase-3,thus exerts a neuroprotective effect.Conclusion:SSb2 can protect PC12 cells from CORT induced injury,and its mechanism is closely related to reducing mitochondrial apoptosis pathway,inhibiting p38/NF-κB signaling pathway and regulating metabolism.