| [Objective]To investigate the biological mechanism of Danjiania Prosperity San to prevent and treat Anxiety behavior by integrating network pharmacology with transcriptomics and metabolomics in Chinese medicine.[Methods]Part Ⅰ.Integrative pharmacology analysis of Chinese medicineFrom the perspective of "disease-symptom-prescription-target",the TCM integrated pharmacology network computing platform 2.0(TCMIP V2.0,http://www.tcmip.cn/)was used to construct a molecular network of anxiety behaviors.Firstly,we constructed the gene set of anxiety clinical symptom phenotypes in TCMIP V 2.0;secondly,we constructed the core network of "anxiety disorders-clinical symptom phenotypes-Danjiangyaozhuan" by "evidence-related molecular mining and functional analysis" in TCMIP V2.0 platform.target network.Part Ⅱ Experimental Study2.1 Experimental groups and drug administrationTwenty-six male SD rats(200-230g)were fed for 3 days and grouped according to the random number method.The rats were divided into 3 groups:9 rats in the DZXYS group(DZXYS),9 rats in the MOD group(MOD)and 9 rats in the normal control group(CON),and the DZXYS group was given DZXYS aqueous extract daily,while the CON and MOD groups were given equal doses of saline(10 ml/kg)for two weeks.Randomized chronic restraint stress was administered to the DZXYS and MOD groups for 2 consecutive weeks(once daily for 2 h)at random starting times each afternoon except for the CON group.Body weight was measured every 3 days.HPM,OFT,and LDB were performed on days 12,13,and 14,respectively,and serum,thymus,adrenal,hippocampal,and prefrontal cortical tissues were collected after anesthetic execution on day 15.2.2 TranscriptomicsUse Trizol reagent to extract total RNA from brain hippocampal tissue.Using RNA seq for library construction and sequencing,identifying differentially expressed genes,and ultimately conducting bioinformatics analysis.2.4 q-PCR validationEighteen RNA samples with GAPDH as the internal reference gene and five target genes were used to detect the expression of target genes in the sent samples using SYBR GREEN I.2.5 MetabolomicsPlasma was collected and analyzed for plasma metabolome by ultra performance liquid chromatography-tandem quadrupole/time of flight mass spectrometry(UHPLC-Q-TOF/MS),and multivariate statistical analysis was used for pattern identification and searching for differential markers.2.6 Combined transcriptomics and metabolomics analysisMetScape,a plug-in of CytoScape software,was used for combined analysis to construct and analyze the relationship network between "genes" and "compounds".[Results]Part Ⅰ.Pharmacological analysis of Chinese medicine integrated networkThe results of the multidimensional network Anxiety behavior of"herbal-component-target-pathway" showed that:DANJIANJIAYUOYAOAN has a significant impact on neurodegenerative pathways-multiple diseases,hepatitis B,dopaminergic synapses,glutamatergic synapses,MAPK signaling pathway,cAMP signaling pathway,circadian rhythm,Alzheimer’s disease,apoptosis,etc.protein binding,cytoplasmic lysis,cytoplasmic,mitochondrial and other pathways for intervention.(2)Anxiety disease targets and related genes:13 genes were obtained from anxiety and DANJASIA Progressive:PIK3CA,ACSL4,ALDH5A1,ND1,LGALS3,NR4A2,ADCY5,CASR,COMT,DAO,GRIN2A,ESR1,NR3C1 respectively.Part Ⅱ Experimental studies1.Behavioral analysisDuring the modeling period,the rats in the DANJUAN PRYTHAIYAN group and the control group had shiny and smooth fur,normal diarrhea and sensitive daily activities.The rats in the model group became more irritable,with fast weight growth and dull fur.On day 4,day 8 and day 12,the body weight of the control group was significantly different from that of the model group and the Danjianjiangyaozan group,which was statistically significant(P<0.05),and the body weight of the control group grew significantly faster.the EPM results showed that in the OE%group,the model group was lower than the control group,and the difference was statistically significant(P<0.05);the Danjianjiangyaozan group was lower than the control group,and the difference was not statistically significant(P=0.583);Danjian Yiyao San group was higher than the model group,the difference was not statistically significant(P=0.069);in the OT%group,the model group was lower than the control group,the difference was statistically significant(P<0.05);Danjian Yiyao San group was lower than the control group,the difference was not statistically significant(P=0.908);Danjian Yiyao San group was higher than the model group,the difference was statistically significant(P<The OFT results showed that the rats stayed in the center of the open field longer in the control group compared with the model group,and the difference between the two groups was statistically significant(P<0.05);compared with the model group,the Danjuania Eugenia San group significantly increased the stay time of the rats in the center of the open field,and the difference between the two groups was statistically significant(P<0.05);compared with the model group,the difference between the control group and the model group was not statistically significant(P<0.05).The LDB results showed that the time of rats in the bright box and the number of crossing the bright and dark boxes were significantly reduced in the model group compared with the control group,and the differences between the two groups were statistically significant(P<0.05);compared with the model group,the time of rats in the bright box and the number of crossing the bright and dark boxes were significantly increased in the Danjiania Eugenia San group,and the rats’ bright box There was a certain trend in the dwell time of the two groups,and the difference was not statistically significant(P>0.05);the difference in the number of rats crossing the bright and dark boxes was statistically significant between the two groups(P<0.05).2.Transcriptomic analysisThe results of differentially expressed genes screening showed that there were 2436 up-regulated genes and 1426 down-regulated genes in the model group compared with the blank group;257 up-regulated genes and 166 down-regulated genes in the Danjiania prodigiosa group compared with the model group;2485 up-regulated genes and 1313 down-regulated genes in the Danjiania prodigiosa group compared with the blank group.KEGG enrichment analysis showed that the enriched pathways were mainly involved in aldosterone synthesis and secretion,neuroactive ligand-receptor interaction,oxidative phosphorylation,arginine and proline metabolism,cGMP-PKG signaling pathway,etc.3.q-PCR validationThe validation of five differentially expressed genes,including LGALS3(Gal-3),CBS,ND 1,NR4A2(Nurr1)and BDNF,showed that Gal-3 levels were significantly higher and BDNF levels were significantly lower in the control group compared with the model group,with statistically significant differences(P<0.05).Compared with the administration group and the model group,the Gal-3 level was significantly lower and the BDNF level was significantly higher in the Danjuana Prolotherapy group.4.Metabolomic analysisA total of 23 endogenous differential metabolites were identified.13 differential metabolites were identified in the control group and the model group,mainly involving tryptophan and glutamate metabolic pathways;16 differential metabolites were identified in the model group and the Danjian Yiyao San group,mainly involving tryptophan,fatty acid and bile acid metabolic pathways;6 metabolites overlapped between the two groups.5.Combined analysis of pharmacology and transcriptomics of Chinese medicine integration networkThe anxiety and Tanjaya prodigiosa crossover genes(PIK3CA,ACSL4,ALDH5A1,ND1,LGALS3,NR4A2,ADCY5,CASR,COMT,CBS,GRIN2A,ESR1,NR3C1)obtained from TCMIP v2.0 platform were intersected with the differential genes obtained from transcriptomics to finally obtain LGALS3(Gal-3),CBS,ND 1,NR4A2(Nurr1)and other 4 differentially expressed genes;find the literature to supplement the related genes BDNF,and finally identify 5 differentially expressed genes.6.Combined transcriptomics and metabolomics analysisThe gene-metabolite association network analysis,LGALS3,ND1,NR4A2 and BDNF genes could not be found in MetScape database.CBS genes and metabolites were mainly involved in bile acid biosynthesis,alanine and threonine metabolism,methionine and cysteine metabolism,saturated fatty acid β-oxidation,selenium amino acid metabolism,urea cycle and aspartate and asparagine metabolism etc.[Conclusion]Firstly,using the TCM integrated pharmacology platform and transcriptomics technology,it was concluded that the differential genes of Danjiania Progressive were associated with LGALS3(Gal-3),CBS,ND 1,NR4A2(Nurr1),etc.We further combined metabolomic analysis and q-PCR to identify Gal-3,CBS and BDNF as the key targets;finally,we concluded that DANJIANJIAEPENGYAOAN prevents neuronal damage and promotes neurogenesis by down-regulating the expression level of Gal-3 gene and up-regulating BDNF expression. |
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