| Background:The mechanism of depression is complicated,and its pathogenesis at molecular level is still unclear.The response rate of depression to first-line drugs is low.The synergistic effect of multi-target traditional Chinese medicine has important guiding and promoting effects on improving the efficiency of anti-depression treatment.Since the Tang and Song Dynasties,the traditional Chinese medicine,Kai-Xin-San(KXS)has become the representative and basic prescription with the benefit of nourishing and calming down the mind.We have carried out in-depth research on the pharmacodynamics and mechanism of action of KXS in classical depressive animals and cell models.However,the effect mechanism and target of KXS has not been identified at clinical level.Objective:Shen-Zhi-Ling(SZL)is a Chinese medicine formulated from a KXS decoction that is commonly used to treat depression caused by dual deficiencies in the heart and spleen.The aim of this study is to systematically screen the molecular markers involved in the pathophysiological process of depression and the target of SZL at the clinical level.We expect to elucidate the molecular mechanism of action and related biological pathways of anti-depression effect of SZL.Methods:Serum samples were collected from depression patients(DPs),DPs who underwent 8 weeks of SZL treatment and healthy controls(HCs).MiRNAs differentially expressed in serum were identified by bioinformatics analysis.The target miRNAs related to depression and antidepressant effects of SZL were selected for RT-PCR validation in order to identify potential molecular therapeutic targets from post-transcriptional regulatory levels.Based on the discovery of a new molecular target miR-1281 from the miRNA expression profile,we established SH-SY5Y human neuroblastoma cell line,a model of corticosterone injury-simulated depression,and treated these cells with different concentrations of KXS to investigate miR-1281 and its predicted target gene expression in neurons.The ability of miR-1281 to degrade wild-type/mutant ADCY1 and DVL1 was detected by double luciferase reporter to validate the regulatory function of miR-1281 on ADCY1 and DVL1.Lentiviral expression vector overexpressing miR-1281 was constructed and treated with different concentrations of KXS.Using CCK8,flow cytometry,PCR,we analyzed the effect of changes in expression levels of miR-1281 on neuronal proliferation and apoptosis and its target gene expression.Western blot was used to detect the effect of miR-1281 overexpression on cAMP/PKA/ERK/CREB and Wnt/(3-catenin signal transduction pathway.Using unlabeled quantitative(label-free)proteomic technique combined with LC-MS/MS,we analyzed serum samples from HCs,DPs and SZL(treated with SZL for 8 weeks).Through bioinformatics analysis,the differentially expressed proteins in the serum were screened and selected,and the key differentially expressed proteins related to the anti-depression effects of SZL were selected for western blot verification.The relevant biological pathways were predicted.Based on the results of proteomics,the anti-platelet aggregation activity(THR,ADP,AA,and COLL as an inducer)of KXS was evaluated by the turbidimetric method.The competitive enzyme-linked immunosorbent assay was used to determine the effect of KXS on thrombin-induced platelet cAMP,5-HT and BDNF.Flow cytometry was used to analyze the effect of KXS on thrombin-induced platelet P-selectin.Fluorescence spectrophotometry was used to determine the effect of KXS on the resting state and thrombin-induced platelet Ca2+ ion.Western blot was used to determine the effect of KXS on expression level of platelet BDNF and prothrombin.The blood coagulation instrument was used to detect the changes of five blood coagulation parameters after ginseng treatment.Results:MiRNAs differentially expressed in clinical peripheral serum samples were screened by Microarray.A total of 112 miRNAs(including up-regulation of 23 and down-regulation of 89)that are potentially associated with depression were identified.Among them,a total of 45 miRNAs(17 were up-regulated and 28 were down-regulated)were significantly differentially expressed.These miRNA could be either biomarkers for the onset of depression or a target for anti-depression of SZL.Subsequently,qRT-PCR was used to verify 10 differentially expressed candidate miRNAs in more serum samples,and the results showed that 6 miRNAs(miR-1281,miR-365a-3p,miR-2861,miR-16-5p,miR-1202 and miR-451a)were consistent with the results of microarray.Among them,miRNA-1281 was highly expressed in depressed patients,and the expression level was about 4 times that of normal control.However,it was significantly down-regulated after treatment with SZL.MicroRNA-gene-pathway-net analysis showed that miR-1281-regulated genes are mostly key nodes in the classical signaling pathway related to depression,suggesting that the abnormal regulation of miR-1281 may be related to the onset of depression and the antidepressant effect of SZL.Further cell functional studies showed that the expression level of miRNA-1281 was increased in SH-SY5Y cells induced by corticosterone,while the expression levels of predicted target genes ADCY1 and DVL1 were decreased.After KXS intervention,ADCY1/DVL1 were reduced/increased in a dose-dependent manner.The levels of ADCY1 and DVL1 in HEK293 cells transfected with the mutant ADCY1 and DVL1 plasmid were significantly higher than those transfected with wild-type plasmid,indicating that wild-type ADCY1 and DVL1 can specifically bind to and be degraded by miR-1281.ADCY1 and DVL1 may be the target genes of miR-1281.After transfection with miR-1281 lentivirus,the survival rate of SH-SY5Y cells decreased and apoptosis was observed.After intervention with KXS,the survival rate was significantly increased,and the early apoptosis was significantly inhibited.At the same time,in the SH-SY5Y cell line overexpressing miRNA-1281,the expression of ADCY1 and DVL1 was down-regulated,and the levels of PKA,pERK1/2,CREB,p-CREB and BDNF proteins in the cAMP signaling pathway mediated by ADCY1 were significantly decreased.After the intervention with KXS,the protein level was significantly increased in dose-related manner,while the expression of GSK-3P protein in the wnt signaling pathway mediated by DVL1 was increased,and the expression levels of pGSK-3? and ?-catenin were decreased.KXS intervention could reverse such changes.A total of 878 proteins were identified by label-free proteomics combined with LC-MS/MS.Compared with healthy controls,31 proteins were significantly differentially expressed in the depressed group(>1.5 or<0.67 times,p<0.05).Among them,12 proteins showed a reverse expression trend after 8 weeks of administration of SZL,and the difference between groups was significant.These 12 proteins are mainly localized in platelet-rich alpha particles,which are enriched in platelet degranulation,platelet cytoplasmic Ca2+ elevation,complement and coagulation cascade,platelet activation.We further verified by Western blot that the dynamic changes in the expression levels of VWF,SERPINA1,APOC3,and A2M were consistent with the proteomic results.Based on the results of proteomics studies,we further investigated the inhibitory effects of different concentrations of KXS on THR,ADP,AA,and collagen-induced platelet aggregation,and found that KXS could significantly inhibit THR and ADP-mediated platelet aggregation in a dose-dependent manner compared with the blank control group.Inhibition of collagen-induced platelet aggregation only occurred when high concentration of KXS was used.KXS had no inhibitory effect on AA-induced platelet aggregation.Using Fura-2 fluorescent labeling to observe the effect of KXS on thrombin-activated platelet cytosolic free calcium mobilization,we found that 150?g/mL of KXS can significantly reduce thrombin-mediated increase of platelet cytosolic Ca2+ levels in the presence or absence of external calcium.The study of the effects of KXS on the nucleotide system and release during platelet aggregation also confirmed that in vitro incubation with different concentrations of KXS significantly increased platelet cAMP levels,decreased platelet 5-HT content and inhibited the release of BDNF due to thrombin-induced platelet activation.At the same time,its pharmaceutical preparation SZL can increase serum BDNF levels and decrease serum prothrombin and 5-HT levels in patients with mild depression.However,it does not have effect on prothrombin time,activated partial thromboplastin time,thrombin time,and international standardization ratio.Conclusion:We screened and validated miRNAs and target proteins associated with depression and antidepressant effects of SZL based on serum samples of RCT clinical study.MiR-1281 was identified in peripheral clinical samples as a new microRNA that may be associated with antidepressant effects of SZL.Proteomic studies suggest that the biological pathways of anti-depression of SZL are related to platelet activation,lipid metabolism,coagulation cascade,and immune response.Furthermore,SZL has a significant effect on the expression of cardiovascular proteins,e.g.,VWF,SERPINAl,APOC3,suggesting that it is involved in the regulation of cardiovascular function in depressed patients.Based on the discovery of clinical-level miRNA expression profiles,it was demonstrated in vitro that KXS may activate cAMP/PKA/ERK/CREB and Wnt/?-catenin signal transduction pathways by down-regulating miR-1281 that targets ADCY1 and DVL1 to achieve its role in neuronal cell protection.Based on the discovery of clinical level proteomics,we investigated another possible mechanism of KXS's antidepressant effect,which is the inhibition of thrombin-induced platelet activation.In vitro studies demonstrated that KXS inhibits platelet activation,thereby decreasing platelet 5-HT content,reducing the release of platelet BDNF and related contents through inhibition of platelet intracellular calcium release,extracellular calcium influx,reduction of cAMP levels.These results are consistent with previous proteomics studies and may explain its biological effect of antidepressant function.It is suggested that while SZL can regulate depression-related molecular networks,it also shows regulatory effect on platelet activation,which is one of the mechanisms of depression-mediated cardiovascular disease.There is also no relevant bleeding risk at the clinical level,suggesting that SZL may become a potential preferred drug for treating psycho-cardiological abnormality of patients with depression disorder. |
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