RESUMEN

OBJECTIVE To study the protective effect of saikosaponin b2(SSb2)on corticosterone(CORT)induced PC12 cell injury and its mechanism.METHODS ① PC12 cells were divided into the cell control group(24 h of culture with RPMI-1640 medium),CORT group(24 h of culture with CORT 100-800 μmol·L-1)and SSb2 group(24 h of culture with SSb2 1.5625,3.125,6.25,12.5,25,50 and 100 μmol·L-1).MTT assay was used to detect the cell survival rate.②PC12 cells were divided into the cell control group(24 h of culture with RPMI 1640 medium),model group(24 h of culture with CORT 400 μmol·L-1),and model+SSb2 group(3 h pretreatment with SSb2 1.5625,3.125,6.25,12.5 and 25 μmol·L-1,removal of the supernatant before cells were co-incubated with CORT 400 μmol·L-1 and corresponding concentrations of SSb2 for 24 h).MTT assay was used to detect the cell survival rate while micro-plate assay was used to detect the lactate dehydrogenase(LDH)leakage rate of PC12 cells.③PC12 cells were divided into the cell control group,model group and model+SSb2 12.5 μmol·L-1 group.AnnexinV-FITC/PI flow cytometry assay was used to detect PC12 cell apoptosis,ultra-perfor-mance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS)cell metabonomics was used to detect metabolic profile changes and colorimetric assay was employed to detect the glutamic acid content and glutaminase activity in PC12 cells.RESULTS Compared with the cell control group,the cell viability decreased to(55±6)%(P<0.01)when the concentration of CORT was 400 μmol·L-1.When the concentration of SSb2 was higher than 50 μmol·L-1,there was significant toxicity to PC12 cells(P<0.01).②Compared with the cell control group,the cell survival rate was signif-icantly decreased(P<0.01),while the release rate of LDH was significantly increased(P<0.01)in the model group.Compared with the model group,the cell survival rate significantly increased(P<0.05,P<0.01),while the LDH release rate significantly decreased(P<0.01)in the model+SSb2 group.③ Com-pared with the cell control group,cell apoptosis was significantly increased in the model group(P<0.05).Compared with the model group,cell apoptosis was significantly decreased(P<0.05)in the model+ SSb2 group.Metabolomics results show that SSb2 significantly back-regulated nine differential metabo-lites of glutamate,creatine,N-acetylaspartate,L-tyrosine,citric acid,L-isoleucine,lactic acid,glutamine and choline.Further network analysis of the key metabolites regulated by SSb2 yielded five major metabolic pathways:D-glutamine and D-glutamate metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,alanine,aspartate and glutamate metabolism,tyrosine metabolism and arginine biosynthesis.Compared with the cell control group,the content of glutamate and activity of glutaminase were significantly decreased in the model group(P<0.01).Compared with the model group,the content of glutamate(P<0.01)and activity of glutaminase(P<0.05)were significantly increased in the model+SSb2 group.CONCLUSION SSb2 has a neuroprotective effect on CORT-injured PC12 cells,and the mechanism of which is related to inhibition of apoptosis and regulation of metabolic disorders.