Study On The Effect And Underlying Mechanism Of Ganmai Dazao Decoction Combined With Mesenchymal Stem Cells In Treatment Of Depressive Disorder

Depressive disorder(DD)is one of the most common,burdensome,and costly psychiatric disorders worldwide in human.It is characterized by emotional,cognitive,somatic,and behavioral symptoms.Inflammatory immune response and gut microbiota disorder may play significant roles in the pathogenesis of DD.Gan-Mai-Da-Zao decoction(GMZ),which was first presented by Zhang Zhong-jing in the book Synopsis of Golden Chamber,is a classic prescription in Traditional Chinese Medicine(TCM)for the treatment of DD.It is composed of Glycyrrhiza uralensis,Triticum aestivum,and Ziziphus jujube.Research in pharmacology of TCM also remind us that alteration of gut microbiota may be associated with the therapeutic effect of GMZ on DD.Considering that GMZ is often combined-used with classic antidepressants in clinical application,it may play a more prominent clinical effect when combined with other antidepressants treatments.Recently,mesenchymal stem cell(MSC)has drawn great attention because of its therapeutic effects via immunomodulation in neurological diseases.As a result,we select MSC as a kind of immunomodulator to combine with GMZ in the treatment of DD.To investigate the therapeutic effect of GMZ and MSC,this study mainly focuses on the following three aspects:1.Effects of GMZ combined with MSC on CUMS depression modelMSC were injected stereotactically to the left and right hippocampus at the number of 1×105 per side,the survival time and location of MSC were observed by in vivo imaging experiments.Chronic unpredictability mild stress(CUMS)stimulation was used to establish a CUMS depression model.The sucrose preference test,tail suspension test(TST)and open field test(OFT)were used for behavioral evaluation,ELISA were used to detect levels of 5-HT in brain.CUMS-derived mouse depression model were then treated with GMZ+MSC,GMZ,MSC or Fluoxetine(Flx),the treatment efficacy of depressive-like symptoms were further investigated by behavioral evaluation.Furthermore,the antidepressant effect of GMZ combined with Flx were detected at the same time to confirm the effect of GMZ.It was found that:1)MSC can maintain for 21 days after being transplanted into the brain;2)GMZ+MSC combined therapy can effectively alleviate depression-like symptoms in mice,including increasing sucrose preferences rate,decreasing total immobility time in TST,gaining weight,increasing food intake,increasing the numbers of horizontal and vertical locomotor activity in OFT,and increasing levels of 5-HT in brain.Furthermore,the antidepressant effect of GMZ+MSC is better than that of Flx;3)In addition,it further confirmed the antidepressant effect of GMZ.2.The role of GMZ in improving DD based on regulating gut microbiota homeostasisThe composition of gut microbiota were detected by 16S rRNA sequencing,and the data were analyzed by community composition,alpha diversity,and beta diversity.In addition,the possible mechanism of GMZ in improving depressive symptoms was confirmed by the fecal microbiota transplantation(FMT)model.We made a preliminary exploration of the mechanism by which the gut microbiota regulated depressive emotions.Glutamic acid levels in mouse brain homogenates,serum and gut homogenates were detected with commercial kits;Short-chain fatty acid(SCFA)levels in feces were assessed with GC-MS;Pathological changes in colorectal tissue were obsvered with HE staining;The levels of colorectal barrier proteins expression,mucin levels and intestinal barrier integrity were assessed with western blot(WB),qRT-PCR and FITC-dextran assay,respectively;Intestinal antimicrobial peptides and inflammation levels were detected by qRT-PCR.These data suggested that:1)The gut microbiota homeostasis of mice in CUMS model group was significantly disturbed.Compared with the control group,the relative abundances of Firmicutes and Bacteroidetes at the phylum level,Barnesiella,Bacteroides and Anaerobacterium at the genus level were significantly changed in CUMS group.PCoA analysis found that the gut microbiota of the two groups of mice was significantly different,and ADONIS analysis showed that the difference was statistically significant.KEGG analysis suggested that the above flora changes may be related to a variety of metabolic pathways including glutamate synapses signaling pathway.Correlation analysis revealed a significant correlation between gut microbiota disturbance and depressive-like mood in CUMS model group;2)FMT results suggested that microbiota disturbance can induce depressive emotions;3)GMZ alleviate depressive symptoms may through regulating gut microbiota homeostasis;4)The underlying mechanism of the bacteria-gut-brain axis regulation involves many aspects including SCFA levels,intestinal barrier integrity,immune inflammation status,and glutamate levels.3.The role of MSC in improving DD based on regulating immune inflammationEffects of MSC on the central and peripheral inflammatory factors IL-1? and IL-6 in CUMS depression model were detected by ELISA.To evaluate the immune regulation effect of MSC on LPS-induced microglial inflammatory activation model in vitro,MSC and BV2 cells were co-cultured with transwell,pro-inflammatory factors such as IL-1?,IL-6,TNF-? and anti-inflammatory factors such as Argl were detected in BV2 cells by ELISA,qRT-PCR,immunofluorescence(IF)and WB.In addition,a potential mechanism of MSC on regulating immune inflammation in central nervous system was preliminary explored.Results showed that:1)MSC effectively inhibited the levels of inflammatory cytokines IL-1? and IL-6 in CUMS depression model in vivo;2)MSC can play an immunoregulatory role on LPS-activated microglia,it can decrease the levels of IL-1?,IL-6,TNF-?,and increase Argl expression;3)MSC secreted a large amount of TGF-?1 and TGF-?2 with or without LPS stimulation.The level of anti-inflammatory factor Argl can also be increased in BV2 cells in the present of TGF-?1 recombinant proteins.These results reminded us that the mechanism of MSC immune regulation may be related to the effect of TGF-?1 secreted by MSC;In addition,with or without LPS stimulation,blocking TGF-? signaling under MSC conditioned medium co-incubation can significantly reduce the Argl gene level in BV2 cells,further suggesting that MSC exerted immunomodulatory effects through secreting TGF-?1.Conclusion:1.GMZ combined with MSC treatment exhibit a promising effect on CUMS-induced depression model in mice,and its antidepressant effect is even better than Flx.2.There was a significant correlation between gut microbiota disturbance and depressive-like behavior in CUMS model.GMZ can alleviate depression by regulating the homeostasis of gut microbiota.3.MSC can alleviate depression by inhibiting central nervous inflammation and promoting microglial cells convert into Arg1high anti-inflammatory state.The underlying mechanism is related to the effect of TGF-?1 secreted by MSC.