Study On The Substance Basis And Mechanism Of Anti-liver Fibrosis Of Apium Graveolens L.Seeds Based On Transcriptomics And Proteomics

Objective:Firstly,the active fractions and monomer components of Apium graveolens L.（celery）seeds and roots against liver fibrosis were screened.Secondly,the active components-targets network and protein interactions network were established to explore the anti-liver fibrosis mechanism of celery seeds.Thirdly,to estimate the potential therapeutic action of apigenin（APG）on liver fibrosis rats and to gain insight into its system-level mechanisms.Methods:1)HSC-LX2 cells activation were induced by TGF-β1 to establish a hepatic fibrosis model in vitro.The antifibrotic activity of extracts and fractions of celery seeds and roots were compared by cell proliferation,apoptosis and the contents of fibrotic indicators in vitro;2)To observe the antifibrotic effects of petroleum ether（PP）,ethyl acetate（PE）,n-butanol（PB）and water（PW）fractions of 60%ethanol extract from celery seeds（60-extract）by detecting the proliferation and apoptosis of HSC-LX2 cells and the contents of fibrotic indicators.The compounds of the PP,PE,PB and PW of 60-extract were determined by GC-TOF-MS and UHPLC-MS/MS.To investigate the correlation between the antifibrotic activity and active ingredients of celery seeds;3)Silica gel,Sephadex LH-20,ODS and polyamide column chromatography were used to separate and purify the compounds of celery seeds,and their structures were identified by ¹H-NMR and ¹³C-NMR.Then the anti-liver fibrosis activity of the isolated compounds were screened by CCK-8 assay in vitro;4)The active ingredients of celery seeds were obtained by TCMSP database,literature and our preceding work.The targets of constituents were obtained by Swiss Target Prediction and BATMAN-TCM databases,Gene Cards and OMIM databases were used to screen the disease targets.The Cytoscape software was used to construct the active components-targets network.The protein interactions network was constructed by the String database and Cytoscape software.GO enrichment and KEGG pathways involved in the targets were analyzed by DAVID,and the anti-liver fibrosis mechanism of active ingredients of celery seeds was analyzed in combination with relevant literatures;5)Male Wistar rats were randomized into the normal group,the model group,the positive control group(Compound Biejiaruangan Troche,BJRG,0.60g·kg^-1),the APG-L(0.15g·kg^-1),APG-M(0.30g·kg^-1)and APG-H(0.60g·kg^-1)groups（n=15）after a week of adjustable feeding.The rats were treated1m L·kg^-1CCl₄（2:5 in peanut oil）orally by gavage twice a week until the end of the ninth week excluding the control rats.Four weeks later,APG and BJRG were respectively administrated by gavage every day.The normal and model rats were received an equivalent volume of solvent.Rats were intraperitoneally injected with zoletil-50 and anesthetized at the end of the experiment.Blood samples,liver and spleen tissues of rats were prepared for follow-up studies.The serum ALT,AST,ALP,LDH,TP,ALB,TB and DB levels were determined with a automatic biochemical analyzer.The serum GSH-PX,SOD,MDA and Hyp levels were detected by the measurement kits.The serum HA,LN,IV-C and PCIII levels were measured by corresponding kits.Histopathological changes of liver were observed by HE and Masson’s trichrome staining.The expressions of TGF-β1and COL-I were detected by immunohistochemistry;6)The differentially expressed genes（DEGs）and proteins（DEPs）were detected by transcriptomics and proteomics.The identifed DEGs and DEPs were classified by the KEGG and GO databases.The key genes were verified using q RT-PCR and Western blot analyses.Results:1)The 60%ethanol extract of celery seeds and 95%ethanol extract of celery roots and its petroleum ether fractions could effectively inhibit the activation of HSC-LX2 cells,reduce the contents of LN,HA and PCIII,and promote the apoptosis of cells.Among them,the petroleum ether fraction of 60%ethanol extract from celery seeds had the strongest effect of anti-liver fibrosis.So celery seeds were taken as the follow-up research object;2)It validated that the inhibition rates of PP,PE,PB and PW of 60-extract were 75.14%,73.52%,54.09%and 43.36%,and their percentage of apoptotic cells were 37.5%,4.3%,0.7%and 0.1%at high doses.The contents of fibrotic indexes in the supernatant of PP were closer to that of the normal group.Additionally,122 compounds were identified from the PP,PE,PB and PW of 60-extract.It was manifested that apigenin,aesculetin and 3-n-butylphthalide have major contribution to the overall compouds of celery seeds,and the inhibition effect on the cell proliferation elevated with increased apigenin,aesculetin and 3-n-butylphthalide contents;3)Nine compounds were isolated from celery seeds,and their structures were identified as apigenin,5-methoxypsoralen,apiin,rutin,kaempferol,luteolin,quercetin,mollugin and 3-n-butylphthalide.The proliferation inhibition rates of compounds apigenin,apiin,rutin,kaempferol,quercetin and 3-n-butylphthalide on HSC-LX2 cells were 71.32%,30.87%,15.81%,22.02%,48.28%and 61.93%;4)Seventeen active components of celery seeds were screened out,among which apigenin,aesculetin and 3-n-butylphthalide had many targets,which might play a core role in the pharmacological effects of celery seeds.The 69 targets of active components from celery seeds mainly involved biological processes such as collagen catabolic,inflammatory response and negative regulation of cholesterol storage,by adjusting the TGF-β,NF-κB and PI3K/AKT signaling pathways to play its anti-liver fibrosis effect;5)The serum HA,LN,IV-C and PCIII levels in APG treatment rats notably declined compared with the model rats（P<0.05）.Moreover,the serum ALB,SOD and GSH-PX levels were elevated and MDA level was remarkably decreased after APG treatment（P<0.05）.It was revealed that APG markedly reduced AST,ALT,ALP,LDH,TB,TP,DB and Hyp levels compared with model rats（P<0.05）.Additionally,the results of histopathology and immunohistochemistry revealed that inflammatory and fibrotic livers were mitigated in CCl₄-exposed rats after APG treatment;6)891 genes were down-regulated and 161 genes were up-regulated in the APG-H group.DEGs were enriched in Cytokine-cytokine receptor interaction,PPAR signaling pathway,Focal adhesion and Cell adhesion molecules.In total,332 proteins were down-regulated and 207 proteins were up-regulated in the APG-H group.DEPs were enriched in HIF-1/VEGF/MAPK signaling pathway,Focal adhesion and ECM-receptor interaction.Mainwhile,KEGG analysis indicated that overlapped proteins were enriched in HIF-1/MAPK/e NOS/VEGF/PI3K/AKT signaling pathway,Focal adhesion and Regulation of actin cytoskeleton mostly.The results of q RT-PCR and Western blot revealed that APG markedly reduced the m RNA and protein expression of TGF-β1,α-SMA,HIF-1α,FAK,VEGF,i NOs and p38 MAPK compared with the model group（P<0.05）,suggesting that APG attenuates liver fibrosis by multiple targets and multiple pathways.Conclusion:1)9compounds were isolated from celery seeds,in which 5-methoxypsoralen and mollugin were isolated from this herb for the first time and mollugin was isolated from this genus for the first time.The active constituents of celery seeds were identified by GC-TOF-MS and UHPLC-MS/MS,and apigenin,aesculetin,3-n-butylphthalide might be the potential ingredients of anti-liver fibrosis combined with the pharmacodynamic experiments of monomer compounds,among which apigenin had the strongest biological activity;2)Network pharmacology preliminarily predicted the biological processes and signaling pathways involved in anti-hepatic fibrosis effect of celery seeds.It reflected the characteristics of multi-components,multi-targets and multi-pathways of celery seeds,and provided a new idea and method for pharmacological research on the anti-liver fibrosis effect of active ingredients from celery seeds;3)This work explored potential targets and system-level mechanisms underlying the antifibrotic effect of APG by transcriptomics and proteomics,and established the network of"multi targets-multi pathways".In a word,it suggested that APG could ameliorate CCl₄-induced liver fibrosis via VEGF-mediated FAK phosphorylation through the TGF-β,HIF-1,MAPKs,PI3K/AKT and e NOS pathways.