Mechanisms On Ameliorating Drug-induced Liver Injury By Extract From Adventitious Roots Of Oplopanax Elatus Nakai

Objective: Oplopanax elatus Nakai is a traditional Chinese medicine with pharmacological activities such as antioxidant,anti-inflammatory,and anti-cancer.However,the utilization of Oplopanax elatus is difficult because its wild resources are lacking and artificial cultivation is not easy.Adventitious roots(ARs)of O.elatus can be largely and rapidly produced in bioreactor culture systems,but their chemical coumpounds are not extremely clear to date,which affects product development and production of O.elatus ARs.Therefore,in this study,the bioactive compounds of O.elatus ARs were analyzed and identified by using liquid chromatography-mass spectrometry(LC-MS).Furthermore,our previous studies found that O.elatus ARs exerted antioxidant,anti-inflammatory,and antibacterial pharmacological effects,especially high antioxidant and an-inflammatory activities,speculating that ARs may alleviate drug-induced liver injury(DILI),but the specific effect has not been reported yet.Consequently,the effect of O.elatus ARs on DILI was investigated using acetaminophen(APAP)-induced acute liver injury model of mice and its underlying mechanisms were also elaborated.Method:(1)Chemical compound analysis: The ethanolic crude extracts from O.elatus ARs were perified and isolated by using macroporous adsorbent resin.The eluates of water(OEA),50% ethanol(OEB),and 95% ethanol(OEC)were collected and their chemical compounds were determined by using high performance liquid chromatography.After identification of the rich bioactive compounds in OEB,compounds in OEB were analyzed by LC-MS and their information was obtained by MS-DIAL software.(2)Experiment of OEB alleviating of drug-induced liver injury: APAP-induced acute mouse liver injury model used,and six treatment groups were designed: Normal control group(NT),liver injury model group(APAP),positive control group(NAC),OEB low dose group(OEB-L),OEB high dose group(OEB-H)and OEB alone group.After the mice were sacrificed,serum and liver tissue samples were collected.Serum samples were used for determination of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)activities,liver samples were used for determination of oxide dismutase(SOD),catalase(CAT),malondialdehyde(MDA)or glutathione(GSH),and H&E staining for histological observation.(3)Eperiments of anti-oxidative stress and regulating immune microenvironment of OEB: Human liver cancer cell line Hep G2 was used in the anti-oxidative stress experiment,and APAP-induced oxidative stress model was used.After OEB pretreatment,ROS levels were measured by DCFH-DA probe,and the gene and protein expressions of Kelch-like epichlorohydrin-associated protein 1(Keap1)and nuclear factor-associated factor 2(Nrf2)were determined by PCR and Western blotting,respectively.The number of B,T,natural killer(NK),natural killer T(NKT)cells,and neutrophils in the liver of mice treated with OEB was determined by flow cytometry in the immune microenvironment experiment,and the changes in the proportion of immune cells were analyzed.(4)Network pharmacological analysis: The mice were sacrificed at 0.5 h,1 h,2h,and 4 h on day 7 after the mice were continuously gavaged 7 days.Mouse blood was collected,components of OEB in the serum were detected by LC-MS to predict the targets.After collecting the data of the targets related to drug-induced liver injury,the common targets of OEB active components and drug-induced liver injury were screened.Protein interaction network was constructed and core genes were screened.Finally,GO and KEGG pathway enrichment analysis was performed.(5)Transcriptome analysis and experiments of OEB affecting drug metabolism and lipid metabolism: RNA sequencing was performed using liver samples from the APAP-induced liver injury mice,followed by GSEA,GO and KEGG analysis.In the experiment of drug metabolism,LC-MS was used to detect the contents of APAP metabolites [APAP,APAP-CYS and APAP-acetyl cysteine complex(APAP-NAC)] in serum samples,and the activities of CYP2E1 and CYP3A11 in liver samples were determined by liver microsomal incubation assay.The expressions of Cyp2e1 and Cyp3a11 were determined by RT-PCR.In the experiment of lipid metabolism.The levels of triglyceride(TG)and total cholesterol(CHO)in mouse serum of the APAP induced liver injury model,and the expression of genes related to peroxisome proliferator-activated receptor(PPAR)of the lipid metabolism pathway(Pparα,Fabp1,Acaa1 b,Apoc3)in mouse liver samples.Results:(1)The 74 compounds were identified in OEB by LC-MS,among which the most compounds were quinic acid coumpounds.These include quinic acid,3-caffeoylquinic acid,3-p-coumaroylquinic acid,trans-5-caffeoylquinic acid,cis-5-caffeoylquinic acid,5-p-coumaroylquinic acid,5-feruloylquinic acid,1-feruloylquinic acid,1,4-dicaffeoylquinic acid,3,5-dicaffeoylquinic acid and 3,4-dicaffeoylquinic acid.In addition,amino acid compounds,organic acid compounds,and hydroxycinnamic acid compounds were also detected.(2)The results of experiment of allivating APAP-induced liver injury indicated that OEB obviously reduced the activities of ALT and AST in serum.Pathological analysis showed that the inflammatory infiltration in the liver of mice pretreated with OEB was reduced,indicating that OEB had a good hepatoprotective effect.(3)The results of anti-oxidative stress experiment showed that OEB reduced the ROS level in APAP-induced oxidative stress of Hep G2 cells,inhibited the expression of Keep1 and promoted the expression of Nrf2,indicating that OEB had good antioxidative stress ability.In the experiment of regulating immune microenvironment,OEB treatment reduced the proportion and number of immune cells such as NK,NKT,and neutralparticles in the liver,indicating that OEB had a significant effect on the composition of the immune microenvironment in the liver,speculating that OEB could further inhibit the occurrence of aseptic inflammation in the liver.(4)The results of network pharmacological analysis showed that 17 of the 74 OEB components were blood entry components,and the 7 components were confirmed as the main active components of OEB after comparison with the prototype components of blood entry.The 7 active compounds of the blood entry components were 3-caffeoylquinic acid,5-caffeoylquinic acid,quinic acid,3-feruloylquinic acid,trans-4-coumaric acid,3,5-dicaffeoylquinic acid,and 3,4-dicaffeoylquinic acid.The results of network pharmacological analysis showed that the 7 active compounds had93 targets,among which 17 targets(MMP2,PYGL,PRKCD,ABCB1,TTR,PTPN22,PTGER2,NR1H4,FABP4,PPARA,FFAR1,UGT2B7,SERPINA6,CA3,MMP9,F3,ESR1)shared common targets with drug-induced liver injury.GO analysis was performed on common targets,and 46 enrichment results were obtained,namely,the negative regulation of inflammation response cell response to reactive oxygen species,I kappa B signaling transduction,interleukins,protein,and tumor necrosis factor,cholesterol homeostasis,glucose balance,and bindings of bile acid,organic acid,steroid,and fatty acid.(5)In the experiment of OEB affecting metabolic process,RNA sequencing analysis showed that OEB pretreatment changed liver drug metabolism and lipid metabolism in APAP-induced liver injury model.Specifically,the concentrations of APAP and its metabolites,such as APAP-CYS and APAP-NAC,in serum of OEB pretreated mice were decreased,and activity of CYP2E1 and CYP3A11 and expression of Cyp2e1 and Cyp3a11 in liver were also reduced.Serum TG and CHO levels of OEB pretreated mice decreased and lipid accumulation was alleviated.OEB inhibited the expression of genes(Pparα,Fabp1,Acaa1 b,and Apoc3)relected to PPAR signaling pathway.Conclusion:(1)74 compounds were identified from OEB by LC-MS,among which quinic acid compounds(11)were the most abundant.(2)OEB has a good effect on the improvement of APAP-induced liver injury.OEB could inhibit the oxidative stress induced by APAP and could change the immune microenvironment,thereby reducing the occurrence of liver inflammation.(3)7 types of prototype components in OEB were iditified,and network pharmacological analysis showed that these 7 components had 93 targets,which that had 17 co-targets with drug-induced liver injury and were mainly enriched in inflammatory reaction,oxidation,and lipid metabolism.(4)OEB reduced the content of APAP and its metabolites in serum and inhibited the activity of CYP2E1 and CYP3A11 and expression of CYP2E1 and CYP3A11 in the liver.OEB decresed the levels of TG and CHO in liver,inhibited the expression of Pparα,Fabp1,Aca1 b,and Apoc3 in PPAR lipid metabolism signaling pathway,thereby regulating lipid metabolism disorder caused by APAP and alleviating liver injury.