Study On Antagonistic Effect Of BaP On AhR And Nrf2 Signaling Pathways By Effective Constituents Of Sonchus Arvensis

Objective Based on the methods of network pharmacology and molecular docking,the effects of effective constituents of Sonchus arvensis（SA）and Benzo（a）pyrene（Ba P）on AhR and Nrf2 signaling pathways were clarified.The main constituents of SA alcohol extract were Apigenin（Api）and Quercetin（Que）by liquid chromatography-mass spectrometry（LC-MS）.Further in vitro experiments were conducted to explore the effect of SA effective constituents antagonistic Ba P on AhR and Nrf2 signaling pathways.Methods（1）Searched the literature,screened the effective constituents of SA（OB≥30%,DL≥0.18,Caco-2>0 and longer HL）and predicted the target,constructed the"constituents-target"network.CTD database predicted Ba P target and intersected with the SA target,PPI and biological function enrichment analysis of the targets（PValue<0.01,FDR<0.01）.Molecular docking analysis of docking sites.（2）Api and Que were identified in the main constituents of SA alcohol extract by LC-MS.（3）MTT method was used to determine the optimal concentration range of Ba P,Api and Que on Hep G2 cells.（4）The effects of different concentrations of Api and Que antagonistic Ba P on BPDE-DNA and 8-hydroxy-2-deoxyguanosine（8-OHd G）were detected by kits.The fluorescence intensity of reactive oxygen species（ROS）was detected by molecular devices to determine whether Api and Que could protect against Ba P-induced cytotoxicity.（5）The expressions of AhR,Nrf2 and downstream genes and proteins at different concentrations of Ba P(0.1,1,10 and 100μmol·L^-1)were detected by q PCR and Western Blot to clarify the effect of Ba P on AhR and Nrf2signaling pathways.（6）q PCR and Western blot were used to detect the effects of different concentrations of Api(25,50,100,150 and 200μmol·L^-1),Api(25,50,100μmol·L^-1)+Ba P(1μmol·L^-1)on the expression of AhR,Nrf2 and downstream genes and proteins.Immunofluorescence was used to detect the fluorescence intensity of CYP1A1 and HO-1 proteins in Hep G2 cells treated with Api+Ba P,so as to determine whether Api can antagonize the effects of Ba P on AhR,Nrf2 and downstream genes and proteins.（7）q PCR and Western blot were used to detect the effects of different concentrations of Que(25,50,100,150 and 200μmol·L^-1),Que(25,50,100μmol·L^-1)+Ba P(1μmol·L^-1)on the expression of AhR,Nrf2 and downstream genes and proteins.Immunofluorescence was used to detect the fluorescence intensity of CYP1A1 and HO-1 proteins in Hep G2 cells treated with Que+Ba P,so as to determine whether Que can antagonize the effects of Ba P on AhR,Nrf2 and downstream genes and proteins.（8）Hep G2 cells were treated with AhR agonist（CAY10465）,Ba P,Ba P+AhR inhibitor（CH-223191）and Ba P+ROS inhibitor（NAC）,q PCR and Western blot was used to detect the effect of Ba P activated AhR on Nrf2.Hep G2 cells were treated with Nrf2 agonist（t BHQ）,Ba P and Ba P+Nrf2 inhibitor（ML-385）,q PCR and Western blot was used to detect the effect of Ba P activated Nrf2 on AhR.Finally,immunocoprecipitation was used to determine whether there was interaction between AhR and Nrf2.Results（1）A total of 10 effective constituents and 336 targets were screened from SA,Quercetin（307）and Apigenin（158）were the constituents with higher degrees.Through biological function enrichment analysis,the effective constituents of SA and Ba P by affecting DNA transcription,RNase transcription regulation,cell proliferation,apoptosis and signaling pathways regulation.According to the molecular docking results,the effective constituents and Ba P can increase the ability of ligands to target proteins by forming hydrogen bonds,aromatic ring stacking forces,van der Waals forces,σbonds,sulfur bonds,and hydrophobic interactions with AhR and Keap1proteins.（2）The main constituents of the SA alcohol extract contain Api and Que.（3）According to the MTT results,the concentration of Ba P was 0.1,1,10 and100μmol·L^-1,and the concentration of Api and Que was 25,50,100,150 and200μmol·L^-1for subsequent experiments.（4）Ba P increased the contents of BPDE-DNA adduct and 8-OHd G（P<0.05）.Api and Que could inhibit the production of BPDE-DNA adduct and 8-OHd G caused by Ba P（P<0.05）.Ba P could enhance the fluorescence intensity of ROS in Hep G2 cells（P<0.05）.Medium and high concentrations of Api and Que antagonize Ba P and reduce the fluorescence of ROS（P<0.05）.（5）Different concentrations of Ba P could activate AhR signaling pathway（concentration dependent）to metabolize Ba P.ROS was produced in the metabolic process,which leads to the activation of antioxidant Nrf2 signaling pathway and protects cells from oxidative damage caused by Ba P.（6）Api was a partial agonist of AhR,and Api metabolizes Ba P by activating CYP1A1,1A2 and 1B1.With the increase of Api concentration,Nrf2 nuclear transcription was promoted,and different concentrations of Api could activate the expression of Nrf2 signaling pathway genes and proteins（P<0.05）;medium and high concentrations of Api could antagonize Ba P and inhibit the expression of Nrf2 signaling pathway genes and proteins（P<0.05）.Immunofluorescence was used to detect the effect of different concentrations of Api on the expression of CYP1A1 and HO-1 protein.The fluorescence intensity of proteins were consistent with the expression results of genes and proteins.（7）Que was a low-affinity ligand for AhR,and Que eliminates Ba P-induced cytotoxicity by regulating the CYP1A families.Medium and high concentrations of Que could antagonize Ba P and inhibit the expression of antioxidant pathway genes and proteins（P<0.05）.Immunofluorescence was used to detect the effect of different concentrations of Que on the expression of CYP1A1 and HO-1 protein.The fluorescence intensity of proteins were consistent with the expression results of genes and proteins.（8）AhR and ROS produced during Ba P metabolism co-regulated the expression of Nrf2 protein,and Nrf2 could affect the expression of AhR protein.Immunoprecipitation indicated that AhR interacted with Nrf2.Conclusions Network pharmacology research shows that SA and Ba P are closely related to AhR and Nrf2 signaling pathways.At the same time,SA and Ba P bind to AhR and Nrf2 proteins through various forces.In vitro experiments show that SA effective constituents Api and Que metabolize Ba P through CYP1A family,CYP1B1and CYP1A family respectively,enhanced the body’s anti-genotoxicity to exert the detoxification and anti-oxidant effect,inhibited the cytotoxicity caused by Ba P,and reduced the contents of BPDE-DNA adduct and 8-OHd G.In addition,AhR interacted with Nrf2.