The Antidepressant Mechanism Research Of Saikosaponin A Based On Proteomics

BackgroudDepression is a commonly-occurring neuropsychiatric disease characterized with long-time low mood,thinking retardation and movement inhibition as well as with high incidence,recurrence rate and mortality rate.In recent years,the incidence of depression has been dramatically rising with the increased life and social pressures.A large number of antidepressant drugs have also been applied to the clinical.These antidepressant drugs can alleviate the patient's depressive symptoms to a certain extent,but they cannot completely cure the depression.What's more,these drugs possess the disadvantages of expensive price,slow onset,high recurrence rate and a series of serious side effects.Above factors severely limit the treatment effect of existing antidepressants.Therefore,it is urgent for us to develop safer and more effective antidepressant drugs.Saikosaponin A(SA)is one of the active ingredients extracted from Radix Bupleuri,which has the functions of anti-inflammation,anti-oxidation,anti-allergy,inhibiting tumor cell proliferation and protecting the liver.Current studies have confirmed that SA has a good antidepressant effect,but its specific mechanism of action is still not clear.Therefore,in-depth exploration of the antidepressant mechanism of SA may provide the necessary theoretical and experimental evidences for the development of new antidepressant drugs.ObjectiveTo seek target proteins regulated by SA and clarify its antidepressant mechanism,providing the theory and the experimental evidence for the development of new antidepressant drug.Methods1.The method of chronic unpredictable mild stress(CUMS)combined with separation was adopted to build the depression model and SA was given by gavage once a day.45 rats were randomly divided into control group(C,15),CUMS group(M,15)and SA administration group(SA,15).The whole process of depression model preparation lasted for 10 weeks:one week for adaptation,eight weeks for CUMS procedure and one week for behavior tests.2.After the last behavior test,all rats were decollated and the tissue of hippocampus was obtained.3.The contents of monoamine neurotransmitters(DA,NE,5-OH)in hippocampus were determined by HPLC-MS.4.The proteomic technique isotope-labeled relative and absolute quantitative(iTRAQ)was adopted to screen for the differential expression proteins in hippocampus of rats with or without CUMS exposure and with or without SA administration.5.Western Blotting was used to verify the expression level changes of the screened proteins at the tissue level.6.The cell model of depression was prepared by stimulating PC 12 cell with cortisone.Then,CCK8 was used to detect the effect of SA on the cell activity of PC 12 cells with stress injury and Western Blotting technique was used to detect the influence of SA on the expression of protein PRRT2 in the PC 12 cells with stress injury.Results1.The method of 8-week CUMS exposure combined with separation successfully established rat depression model,resulting in a series of depression-like behaviors:loss of appetite,loss of weight,reduction of activity and decreased preference for sugar water.2.The content of DA in hippocampus of M group was significantly lower than that of C group while that of SA group was obviously increased compared with M group,but there was no significant difference between C group and SA group.3.The result of iTRAQ showed that there were 391 kinds of proteins were significantly differentially expressed before and after CUMS exposure,and 82 kinds of differentially expressed proteins were screened out between M group and SA group.15 differential expression proteins including proline-rich transmembrane protein 2(PRRT2)were identified not only between C group and M group but also between M group and SA group.4.The result of Western Blotting showed that the expression level of PRRT2 in hippocampus of M group was significantly lower than that in C group,while that of SA group was significantly increased compared with M group,which is consistent with iTRAQ result.5.400?M corticosterone could significantly inhibit the proliferation of PC 12 cells,while 5?M SA could significantly reduce the role of corticosterone in inhibiting the proliferation of PC12 cells,relieving the damage of corticosterone to PC12 cell.6.Corticosterone was able to reduce the expression of PRRT2 in PC 12 cells,and 5?M SA had a tendency to up-regulate PRRT2.Conclusions1.Long-term chronic unpredictable mild stress can down-regulated the content of DA in hippocampus,leading to a series of depressive symptoms.2.SA can relieve depression-like symptoms by increasing DA content.3.PRRT2 participated in the regulation of the fusion and release process of synaptic vesicles,which could promote the rapid release of neurotransmitters such as DA,and the decreased expression level of PRRT2 could reduce the released amount of neurotransmitter.Therefore,we conclude that the SA may promote the release of DA by up-regulating the expression of PRRT2,thus playing an anti-depressant role.4.Saikosaponin A may play an anti-depressant role by alleviating the stress damage to nerve cells.