| BackgroundFarnesoid X receptor(FXR)is a member of the nuclear receptor superfamily and is mainly expressed in organs such as the liver,adrenal gland,and small intestine.As a metabolically regulated gene,FXR is closely related to the metabolism of bile acid(BA),cholesterol,fat,glucose and other substances.It plays an important role in intestinal bacterial growth and liver regeneration.The current study found that FXR agonists upregulate the expression of small heterodimer partner(SHP)by activating FXR,which binds to and inactivates liver receptor homolog-1(LRH-1),thereby inhibiting the expression of cholesterol 7?-hydroxylas(CYP7A1),which is the rate-limiting enzyme in cholesterol synthesis of bile acids,and further reducing the synthesis of BA to reduce cholestatic liver disease.The expression of SHP induced by FXR can alleviate steatosis in nonalcoholic fatty liver disease by inhibiting liver X receptor(LXR)to reduce the synthesis of fat.FXR antagonists modulate gluconeogenesis and improve glucose homeostasis in type 2 diabetic mice by inhibiting FXR binding to the peroxisome proliferator-activated receptor gamma coactivator-la promoter.At present,the discovered FXR antagonists are similar to steroid hormones in structure,which is easy to cause off-target effects,resulting in greater side effects.Therefore,it is of great significance to discover new non-steroidal FXR antagonists.There is no specific drug for acute liver failure caused by paracetamol(APAP).However,as a target gene of FXR,SHP can regulate the expression level of CYP2E1,which is a key enzyme in the metabolism of APAP to the toxic substance NAPQI.Thus,FXR antagonists have potential clinical value in the treatment of acute liver failure caused by APAP.ObjectiveIn this paper,we used computer-aided drug design,pharmacophore construction,virtual screening and activity testing to discover FXR antagonists and investigate whether their down-regulation of the expression level of SHP can alleviate APAP-induced liver failureMethod1.With the help of computer-aided drug design,a pharmacophore model and molecular docking model based on common features are constructed to perform virtual screening of compound libraries.2.Compounds with FXR antagonistic activity were screened at the molecular level by homogeneous time-resolved fluorescence(HTRF).3.HepG2 cells were seeded in culture plates and treated with 2 ?M compounds for 24 h.RT-PCR was used to detect the changes in the mRNA expression levels of SHP,the target genes of FXR,and CYP7A1 to verify whether the compounds had FXR antagonistic activity at the cellular level.4.HTRF was used to verify whether compound 6 has concentration-dependent FXR antagonistic activity.5.FXR agonist GW4064 and compound 6 were added to HepG2 cells,and RT-PCR and double fluorescein reporter assay were used to detect whether compound 6 had antagonistic activity of GW4064 on FXR.6.HepG2 cells were seeded in culture plates and divided into dose-effect relationship groups and time-effect relationship according to experimental groups.RT-PCR was used to detect the changes of mRNA expression level of SHP,the target gene of FXR.7.We injected Kunming male mice at 6-8 weeks intraperitoneally with compound 6(30 mg/Kg)and sacrificed the mice at different times.Total mRNA was extracted from the liver tissue of some mice after fragmentation,and RT-PCR was performed to detect changes in the expression levels of SHP mRNA.8.HepG2 cells were treated with 2 ?M of compound 6 for 24 h.RT-PCR was used to detect changes in the mRNA expression levels of FXR-related genes,as well as changes in the mRNA expression levels of drug metabolism-related genes CYP2C19,CYP2E1,and CYP2C9.Western blot was used to detect changes in SHP protein expression levels.9.Wild C57L/6J male mice aged 6-8 weeks were selected and used for the experiment after 1 week of adaptive feeding,and they were randomly divided into three groups:normal control group,APAPgroup,and compound 6 combined with APAP group.Blood samples were collected from the eyeballs at 4? for 20 min and then centrifuged to obtain serum to measure ALT and AST levels.After the mice were sacrificed,some liver tissues were fixed and stored in 4%paraformaldehyde for HE staining.Results1.Construction of pharmacophore model for virtual screening(1)Six compounds were selected from the literature and patents as training set molecules,and thepharmacophore model of FXR ligands was constructed using Discovery Studio 3.0 software.The final calculation resulted in 10 pharmacophore models.Based on the Rank score,Model 01 was selected as the best pharmacophore model.(2)We used the existing compound library of our group for virtual screening using the Ligand Profiler module in DS3.0 software to obtain 12 compounds for manual screening.2.Test of in vitro activity(1)HTRF screening showed that compounds 6 and 7 had FXR antagonistic activity at the molecular level,of which compound 6 showed concentration-dependent FXR antagonistic activity and had activity compared with GS at the same concentration(2)Pretreatment with compound 6 decreased SHP mRNA expression levels and increased CYP7A1 mRNA expression levels in HepG2 cells(3)RT-PCR and double fluorescein reporter gene results showed that compound 6 reduced the agonistic activity of GW4064 on FXR at the cellular level(4)RT-PCR results showed that compound 6 decreased the SHP mRNA expression level in HepG2 cells in a time-dependent and concentration-dependent manner,significantly decreased the mRNA expression levels of drug metabolizing enzymes CYP2C9,CYP2C19,and CYP2E1 in HepG2 cells,and up-regulated the mRNA expression levels of OATPC,BSEP,and FXR(5)Western blot results showed that pretreatment with compound 6 down-regulated the protein expression level of SHP in HepG2 cells3.Test of in vivo activity(1)Compound 6 decreased SHP mRNA expression levels in the liver of Kunming mice in a time-dependent manner.And it down-regulated the mRNA expression levels of hepatic CYP1A2 and CYP2E1.(2)Pretreatment with compound 6 significantly reduced APAP-induced increase in serum AST levels in mice and alleviated APAP-induced hepatocyte necrosisConclusion1.Construct a pharmacophore model and perform virtual screening to obtain 12 compounds that may have FXR antagonistic activity.After homogeneous time-resolved fluorescence experiment,it is screened that compound 6 and compound 7 have FXR specific antagonistic activity at the molecular level,RT-PCR Further verification showed that only compound.compound 6 has FXR antagonistic activity at the cellular level.And compound.compound 6 has concentration-dependent FXR antagonistic activity at the molecular and cellular levels,and time-dependent FXR antagonistic activity at the cellular and animal levels.2.compound 6 down-regulates expression level of its target gene SHP gene and protein by antagonizing FXR activity,thereby reducing the expression level of the key enzyme CYP2E1 that APAP is metabolized into toxic substance NAPQI to improve acute liver failure in wild mice caused by APAP. |
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