Study On The Pharmacodynamic Material Basis And Mechanism Of Shengyu Decoction In Relieving Chemotherapy Drug-induced Myelosuppression

Objective:With the increasing incidence of tumors year by year,the efficacy and safety of drug therapy have become the focus of attention,and chemotherapy-induced myelosuppression（CIM）is a common clinical adverse reaction.In recent years,traditional Chinese medicines for nourishing qi and blood have been used to prevent and treat myelosuppression.Shengyu decoction（SYD）,a traditional blood-tonifying formula,is composed of Radix Rehmanniae Praeparata,Ginseng,Radix Paeoniae Alba,Rhizoma Chuanxiong,Radix Angelicae Sinensis and Radix Astragali.Studies have shown that SYD has the effect of treating CIM.However,there are few reports on its pharmacodynamic material basis and mechanism in the treatment of myelosuppression.Therefore,in this study,the chemical composition of SYD was analyzed in vitro and in vivo;On the basis of component analysis,combined with network pharmacology and molecular docking technology,the possible mechanism of action and pharmacodynamic components of its treated effect for myelosuppression were explored;Verify the activity of the predicted ingredients and initially explore the mechanism of action;The pharmacokinetic behavior of the main pharmacodynamic components was investigated.Through the above research aiming to clarify the pharmacodynamic material basis of SYD in treating myelosuppression,and provide a theoretical basis for the clinical application of SYD.Methods:1.Identification of SYD in vitro and in vivo:Establish a qualitative analysis method for SYD based on LC-QTOF/MS:ZORBAX Eclipse Plus C18 column（150 mm×4.6 mm,3.5μm）,with 0.1%formic acid-water and 0.1%formic acid-acetonitrile were used for gradient elution;Mass spectrometry was collected using ESI source,TOF MS-IDA-MS/MS scanning mode.The mass spectrometry information of the components in the water extract and drug-containing serum of SYD were collected in positive and negative ion modes,respectively.The chemical database of SYD was constructed,and the Peak View^?V.2.2 workstation was used to conduct qualitative research on the chemical components in SYD.2.Network pharmacology and molecular docking study of SYD:Using the Swiss Target Prediction database and TCMSP to search for the targets of absorbed components;Using Gene Cards and OMIM databases to search for target genes related to myelosuppression;Using Venny 2.1 software to get the intersection targets of both components targets and disease targets.The DAVID database was used to conduct GO analysis and KEGG pathway enrichment analysis on intersection targets to predict the biological process and mechanism of SYD.The intersection targets were imported into the STRING platform to construct a PPI network,and the Cytoscape 3.8.2 software was used for visual mapping.The Cytohubba plug-in was used to screen Hub genes.Using i GEMDOCKv 2.1 and Autodock Tools 1.5.6 to conduct molecular docking between the absorbed components and the Hub targets to predict the possible active components of SYD.3.Validation of active components of SYD and its mechanism of regulating the PI3K-Akt signaling pathway of bone marrow stromal cells and affecting cell proliferation and apoptosis:The pharmacodynamics of the active components predicted by molecular docking-paeoniflorin,albiflorin,ferulic acid and calycosin-7-glucoside were validated.The effects of the four components on the proliferation of OP9 cells after treated with 5-FU were investigated,and the component with the strongest pharmacological effect was screened for follow-up mechanism research.OP9 cells were treated with 5-FU to establish a chemotherapy drug-induced bone marrow stromal cell injury model.The control group,5-FU group,5-FU+albiflorin group,5-FU+albiflorin+LY294002 group and 5-FU+LY294002 group were established.Flow cytometry was used to detect apoptosis and cell cycle of OP9 cells;Western Blot was used to detect the expressions of PI3K,p-PI3K,Akt,p-Akt,Bcl-2,Bax,p21 and Cyclin D1 proteins in OP9 cells.To verify whether albiflorin could affect the proliferation and apoptosis of bone marrow stromal cells by activating the PI3K-Akt signaling pathway.4.To study the pharmacokinetic behavior of the effective components of SYD:Establish the UPLC-MS/MS method for the simultaneous determination of four effective components in rat plasma.Liquiritin was used as the internal standard,the plasma samples were precipitated with methanol,and analyzed on a Poroshell 120 SB C18（4.6×250 mm,5μm）column,with acetonitrile-water as the mobile phase for gradient elution,and mass spectrometry using ESI negative ions.The scanning method was MRM,and a systematic methodological verification was carried out.The established method was used to investigate the pharmacokinetic behaviors of four components,paeoniflorin,albiflorin,ferulic acid and calycosin-7-glucoside in rat plasma.Results:1.A LC-QTOF/MS-based qualitative analysis method for the chemical components of SYD in vitro and in vivo was established,and the source of the medicinal materials was identified and attributed.A total of 148 chromatographic peaks were identified in SYD:42 from Rehmanniae Radix Praeparata,including phenethanol glycosides,monoterpene glycosides and furan aldehyde derivatives;37 from Radix Paeoniae Alba,including monoterpene glycosides and flavonoids;22 from Radix Astragali,including flavonoids;32 from Ginseng,including ginsenosides;15 from Chuanxiong Rhizoma and Angelicae Sinensis Radix,including organic acids and lactones.On this basis,the components absorbed of SYD were analyzed.Of the 148chemical constituents,33 compounds could be absorbed ias prototypes.The prototype components are mainly phenethyl alcohol glycosides,flavonoids,monoterpene glycosides,lactones and organic acids.2.Based on network pharmacology and molecular docking technology,the signaling pathways,targets and pharmacodynamic components of SYD in the treatment of myelosuppression were predicted.Targets related to 33absorbed components were retrieved from TCSMP and Swiss databases,of which 5components lacked targets data,so a total of 28 effective components were obtained,and subsequent network construction and molecular docking were carried out by these 28components.After summarize and deduplicate,113 compound-related targets were obtained.From the Gene Cards and OMIM databases,a total of 7649 myelosuppression-related targets were retrieved,and a total of 2946 disease targets were screened with"score≥4.0"as the criterion.A total of 62 intersecting targets were obtained by the intersection of them.GO analysis showed that SYD was related to biological processes such as negative regulation of apoptosis and positive regulation of cell proliferation.KEGG pathway enrichment analysis showed a total of 51 significantly enriched pathways in KEGG,and the main pathways related to myelosuppression were enriched in PI3K-Akt signaling pathway,HIF-1 pathway,VEGF pathway and estrogen pathway,etc.The 62 intersection targets were imported into the STRING platform to establish a PPI network,and the Cytoscape 3.8.2 software was used for visualization.The PPI network has a total of 58 nodes（4 target proteins are not involved）,374 interaction lines.6 Hub genes were screened by Cytohubba plug-in:TNF,IL-6,VEGFA,SRC,HRAS and STAT3.The 28 absorbed components of SYD were molecularly docked with IL-6,TNF,SRC,HRAS,STAT3 and VEGFA.Among them,ferulic acid,oxypaeoniflorin,paeoniflorin,albiflorin and calycosin-7-glucoside have good binding with the 6 hub targets.3.Validation of effective components of SYD and its mechanism of regulating cell proliferation and apoptosis of BMSCs via PI3K-Akt signaling pathway.After OP9cells were treated with 5-FU:the cells were blocked in the G1 phase,and the apoptosis rate was significantly increased;The protein expression and phosphorylation levels of PI3K and Akt were significantly decreased;the expressions of Bcl-2 and Cyclin D1 were significantly decreased,and the expressions of p21 and Bax were significantly increased.The revesal effect of paeoniflorin,albiflorin,calycosin-7-glucoside and ferulic acid in5-FU-induced proliferation inhibition of OP9 cell was significant.Among them,albiflorin had the strongest reversal effect and was selected to act the follow-up mechanism study.After 5-FU-injured OP9 cells were treated with albiflorin:the protein expressions and phosphorylation levels of PI3K and Akt were significantly increased;the expressions of Bcl-2 and Cyclin D1 were significantly increased,and the expressions of p21 and Bax were significantly decreased;G1 Phase cell cycle arrest was significantly reduced,and the rate of apoptosis was significantly reduced.However,the reversal effect of albiflorin was significantly attenuated after treated with the PI3K-Akt pathway inhibitor LY294002.It is suggested that albiflorin could reverse 5-FU-induced apoptosis and cell cycle arrest in BMSCs by activating the PI3K-Akt pathway,thereby increasing the phosphorylation level of Akt and affecting the expression of apoptosis-related and cell cycle-related proteins.4.Pharmacokinetic study of the active components of SYD:An UPLC-MS/MS method for the simultaneous determination of four active components in rat plasma was established and applied to pharmacokinetic studies.Under the determined conditions:the peak shapes of ferulic acid,calycosin-7-glucoside,paeoniflorin,albiflorin and internal standard were good;The LLOQ were 1,1,50,and 2ng/m L,respectively;Specificity,precision,accuracy,linearity,exraction recovery,matrix effects,residue and stability were all met the methodological requirements.The C_maxof ferulic acid,calycosin-7-glucoside,albiflorin and paeoniflorin were 1571.07±1329.48 ng/m L,21.16±19.31 ng/m L,1469.91±721.08 ng/m L and 2777.38±1719.53ng/m L,respectively;t_1/2were 0.76±0.25 h,4.41±3.39 h,9.39±1.12 h,4.27±3.15 h,respectively;AUC_（0-t）were 1622.36±1201.40 ng·h/m L,21.04±19.12 ng·h/m L,1752.35±442.22 ng·h/m L,4361.98±2284.52 ng·h/m L,respectively;T_maxwere 0.14±0.04 h,0.16±0.07 h,0.29±0.14 h,0.29±0.14 h,respectively.And all of them could be rapidly absorbed within 5 min.Conclusion:The LC-QTOF/MS method established in this study was used to identify the chemical constituents in the extracts of SYD and the serum of rats after oral administration of SYD,preliminarily elucidated the chemical substance basis of SYD.The potential active ingredients of SYD and their possible targets were predicted.Four pharmacological components,ferulic acid,paeoniflorin,calycosin-7-glucosidee and albiflorin,were screened and verified,of which albiflorin had the strongest effect.Albiflorin could activate the PI3K-Akt pathway,thereby reducing 5-FU-induced apoptosis and promoting cell re-entry into the cell cycle.The PI3K-Akt pathway may be one of the mechanisms by which albiflorin alleviates the inhibition of proliferation and the increase of apoptosis in OP9 cells induced by 5-FU.The established UPLC-MS/MS method was used to investigate the pharmacokinetic behaviors of four pharmacodynamic components,albiflorin,paeoniflorin,calycosin-7-glucosidee and ferulic acid in rat plasma.It will provide a basis for rational clinical application.