Antidepressant-like Effects Of Ginseng Total Saponins And Fuzi Total Alkaloids Compatibility And The Mechanisms

BackgroundDepression is a common disease all over the world,which is characterized by a significant and persistent low mood and decreased interest.In severe cases,sleep disorders and even suicide may occur.The pathogenesis of depression is often closely related to heredity,psychology,social environment and brain biochemical disorders,causing serious harm to patients’ families and society.At present,the clinical treatment of depression still mainly relies on traditional antidepressants,but only one-third of the patients’ symptoms have been significantly improved,and but such drugs have a long incubation period,high disease recurrence rate and serious side effects.Therefore,the development of effective antidepressants with low side effects has become a research hotspot.Traditional Chinese medicine has become a promising alternative therapy for depression due to its less adverse reactions and good tolerance,which has broad prospects for development.Studies have shown that the classical prescription Shenfu Decoction has a significant protective effect on the nervous system.And our and other research groups have found that the main active components,ginseng total saponins and Fuzi total alkaloids,can significantly improve depressive-like behavior,and the compatibility of ginseng and Fuzi has the effect of increasing efficiency and reducing side effects.However,as the main components of Shenfu Decoction,the regulatory effects of ginseng total saponins and Fuzi total alkaloids compatibility(GTS-FTA)on depressive-like behavior is still unclear and needs further study.ObjectiveBased on the neurotrophic factor hypothesis and the synaptic plasticity hypothesis of depression,behavioral pharmacology,biochemistry,metabolomics and histomorphology methods were used to explore the improvement effects and possible mechanisms of GTS-FTA on depressive-like behavior in ovariectomized mice from the overall level to the cellular and molecular level.This study provides a theoretical basis for the study of natural Chinese herbal medicine for depression and also provides a new possibility for the clinical drug treatment of depression.MethodsIn this study,we first explored the effects and possible mechanisms of GTS-FTA on depressive-like behaviors induced by ovariectomy.Female ICR mice were used in this part of experiment,and they were randomly divided into 6 groups: sham operation group,ovariectomy group,ovariectomy+GTS-FTA low-,medium-and high-dose groups,and ovariectomy+fluoxetine positive group.Except for the sham operation group,the mice in other groups underwent ovariectomy to remove bilateral ovaries,and the drug was given after a week of recovery.The ovariectomy+low-,medium-and highdose groups of GTS-FTA were given corresponding doses intragastrically for 7consecutive days,respectively;the ovariectomy+fluoxetine group was intraperitoneally injected with fluoxetine for 7 days,and the mice in other groups were given the same dose of blank vehicle intragastrically.24 h after the last administration,behavioral pharmacological experiments,blood collection,prefrontal cortex and hippocampus collection were performed.Open field test and forced swimming test were used to investigate the effects of GTS-FTA on depressive-like behavior.Enzyme-linked immunosorbent assay was used to detect the changes in serum estradiol levels to further verify the establishment of the simulated perimenopausal depression model.Western blot was used to detect the effects of GTS-FTA on the expression levels of BDNF,p CREB,CREB,Akt,mTORC1,LC3,Beclin 1 and p62 in the prefrontal cortex and hippocampus of mice.UHPLC-Q-Exactive quadrupole-Orbitrap MS metabolomics was used to analyze and identify the effects of GTS-FTA on serum differential metabolites and metabolic pathways in mice.Based on the regulatory role of the mTORC1 pathway in depression,our study further explored whether blocking the mTORC1 pathway with rapamycin had any effect on the antidepressant-like effects of GTS-FTA.Female ICR mice were used in this part of experiment,and they were randomly divided into 5 groups: sham operation group,ovariectomy group,ovariectomy+GTS-FTA group,ovariectomy+GTSFTA+rapamycin group and ovariectomy+rapamycin group.Except for the sham operation group,the mice in other groups underwent ovariectomy to remove bilateral ovaries,and the drug was given after a week of recovery.The ovariectomy+GTS-FTA group and the ovariectomy+GTS-FTA+rapamycin group were given GTS-FTA intragastrically for 7 consecutive days,respectively;while the mice in other groups were given the same dose of blank vehicle intragastrically.24 h after the last administration,mTORC1 antagonist rapamycin was intraperitoneally injected into the ovariectomy+GTS-FTA+rapamycin group and ovariectomy+rapamycin group,and mice in other groups were intraperitoneally injected with the same dose of blank vehicle.After 30 minutes,behavioral pharmacological experiments,prefrontal cortex and hippocampus collection and the whole brain tissues collection after saline perfusion were performed.We investigate the effects of rapamycin on the antidepressant-like effects of GTS-FTA by open field test,forced swimming test,and tail suspension test.Western blot was used to detect the changes of BDNF,p CREB,CREB,Akt,mTORC1,LC3,Beclin 1 and p62 expression levels in the prefrontal cortex and hippocampus of mice after rapamycin administration.Golgi-Cox staining was used to investigate the effects of rapamycin on the density changes of dendritic spines in hippocampal CA1,CA3 and DG subregions of ovariectomized mice induced by GTS-FTA.ResultsThe results of the study on the antidepressant-like effect of GTS-FTA showed that the spontaneous activity of ovariectomized mice was not affected,but the immobility time in the forced swimming test was significantly shortened by the administration of GTS-FTA and fluoxetine.Except for the sham operation group,the estradiol levels of mice in each group decreased significantly,and no significant changes occurred after administration.Besides,the decrease of BDNF,CREB phosphorylation,Akt,mTORC1,p62 levels and the increase of LC3-Ⅱ/LC3-Ⅰ in the prefrontal cortex and hippocampus of mice induced by ovariectomy were most significantly reversed by the medium-dose of GTS-FTA,but there was no significant difference in the expression of Beclin 1 in each group.The results of the metabonomics experiment showed that 15 serum potential differential metabolites(9 of which belong to lipids and lipid molecules)were identified,which may be involved in 6 metabolic pathways.The results of the study on the effects of rapamycin on the antidepressant-like effects of GTS-FTA showed that the improvement effects of GTS-FTA on the immobility time in the forced swimming test and tail suspension test were significantly antagonized by the mTORC1 antagonist rapamycin.Rapamycin alone also significantly reduced the increase of immobility time induced by ovariectomy.At the same time,compared with the GTS-FTA group,the improvement of mTORC1,LC3-Ⅱ/LC3-Ⅰ and p62 levels in the prefrontal cortex and BDNF,Akt,mTORC1 and p62 levels in the hippocampus were significantly antagonized by rapamycin.There was also a tendency to antagonize the changes in the expression levels of other proteins,but there was no statistical difference.Finally,Golgi-Cox staining results showed that the dendritic spines density in the CA1,CA3 and DG subregions of the hippocampus was significantly downregulated in the ovariectomy group compared with the sham operation group,which was restored by GTS-FTA.However,the ameliorating effect induced by GTS-FTA was antagonized by rapamycin.Rapamycin alone did not cause significant changes in the protein levels of the CREB-BDNF,mTORC1 and autophagy pathways in the prefrontal cortex and hippocampus,as well as dendritic spines density in the CA1,CA3 and DG subregions in the hippocampus of ovariectomized mice.Conclusion1.GTS-FTA low-,medium-and high-dose significantly improved the depressivelike behavior of mice induced by ovariectomy without affecting the spontaneous activity.2.The improvement of depressive-like behavior in mice with ovariectomy by GTS-FTA could be closely related to the CREB-BDNF signaling pathway,mTORC1 signaling pathway and autophagy regulation in the prefrontal cortex and hippocampus.Among them,the effect of the medium-dose was the most significant.At the same time,15 serum potential biomarkers and 6 metabolic pathways were involved.3.The antidepressant-like effects of GTS-FTA could be antagonized by rapamycin,which was closely related to the mTORC1 signaling pathway and autophagy in the prefrontal cortex.Moreover,this antagonism is also closely related to the hippocampal synaptic plasticity mediated by the CREB-BDNF pathway and mTORC1 pathway.In conclusion,this study provided a theoretical basis for the study of the antidepressant-like effects of the GTS-FTA,and provided a new direction for the development of new indications for traditional Chinese medicine compounds.