Study On FXR-mediated Anti-cholestasis Effect And Mechanism Of The Seeds Of Herpetospermum Pedunculosum

Bile acids(BAs)homeostasis plays an important role in maintaining human normal physiological functions.Cholestasis is a pathological condition in which the formation,secretion and excretion of bile in the liver were obstructed,resulting in humpered bile flow from liver to duodenum and bile accumulation in blood circulation.Cholestasis may lead to jaundice,skin itching,and hypercholesterolemia,and was aggravated to liver fibrosis,cirrhosis and eventually liver failure without prompt and effective therapy.Farnesoid X Receptor(FXR),an endogenous ligand of BAs,is involved in the regulation of BAs biosynthesis,transport and metabolism.It is of great significance to discovery new medicines targeting FXR for the maintaining of BAs homeostasis and the treatment of cholestatic liver diseases.Herpetospermum pedunculosum(Ser.)C.B.Clarke(H.pedunculosum),an annual climbing herb belonging to Cucurbitaceae family,is mainly distributed in Sichuan,Tibet,India,Nepal at the altitude ranging from 2300 m to 3500 m.Its dried mature seeds had the effects of purging liver and biliary fire,detoxification,and were frequently used in the treatment of “Chiba” diseases in Tibetan medicine.The clinical symptoms of “Chiba”diseases are similar to some clinical symptoms of intrahepatic cholestasis.In our previous study,the extract of the seeds of H.pedunculosum had showed a significant therapeutic effect on α-naphthylisothiocyanate(ANIT)-induced acute intrahepatic cholestasis in SD rats.But the active substances of the seeds of H.pedunculosum and their mechanism are still unclear.Therefore,this study was designed to investigate the pharmacodynamics of the bioactive extract and components of the seeds of H.pedunculosum against intrahepatic cholestasis,and explore its possible mechanism in regulating the FXR signaling pathway.This research was composed of the following four parts.1.Study on the mechanism of ethyl acetate extract of the seeds of H.pedunculosum(HPEAE)in ANIT-induced cholestasis rats.ANIT(60 mg/kg)induced acute cholestasis rats was used as animal model to study the anti-cholestasis mechanism of HPEAE.Rats were administrated with HPEAE(100,200,or 400 mg/kg)and UDCA(100 mg/kg)intragastrically once daily for 7 days and modeled with ANIT(60 mg/kg)on the fifth day.The in vivo pharmacodynamic study showed that HPEAE could significantly attenuate ANIT-induced cholestasis by restoring the liver function index levels,increasing bile flow as well as reversing liver tissue pathological damage and restoring the levels of proinflammatory mediators.Further researches revealed that HPEAE may could up-regulate the protein expressions of FXR,bile salt export pump(BSEP),multidrug resistance-associated protein 2(MRP2),Na+/taurocholate cotransporting polypeptide(NTCP).The nuclear factor erythroid 2-related factor 2(Nrf2),quinone oxidoreductase 1(NQO1),heme oxygenase1(HO-1)in the oxidative stress Keap1/Nrf2 signaling pathway,and inhibitor of NF-κB(IκB)in the inflammatory NF-κB signaling pathway were also up-regulated.Meanwhile,the down-regulation of the protein expressions of kelch ech associating protein 1(Keap1),P-glycoprotein 65(P65),phosphorylated P-glycoprotein 65(P-P65),and inhibitor of NF-κB kinase α(IKKα)were observed.All these results indicated that HPEAE could promote BAs transport by activating the FXR signaling pathway,inhibit oxidative stress by activating the Keap1/Nrf2 signaling pathway and inhibit the inflammatory response via activating the NF-κB/ARE signaling pathway,thereby alleviate ANIT-induced cholestatic liver injury.2.Screening of anti-cholestasis active components in vitro.(1)To further clarify the anti-cholestasis active substances of the seeds of H.pedunculosum,fifteen compounds isolated from HPEAE were screened for their FXR activating effect.L-02 cells were applied and treated with guggulsterone(50 μmol/L)to inhibit the expression of FXR,which were detected by immunofluorescence after treated with 100 μmol/L of compounds for 24 hours.The results showed that herpetotriol,spathulatd,lariciresinol,dehydrodiconiferyl alcohol(DA)and herpetrione(HPE)could significantly up-regulate the protein expression of FXR in guggulsterone-induced L-02 cells,and exhibited good agonistic actions.We speculated that these lignans may be the main anti-cholestasis bioactive components from HPEAE.(2)The Sybyl-X software was used to investigate the binding actions of 15 compounds with the FXR protein.As a result,DA and HPE got better docking scores closely to that of the agonist GW4064,and had good binding actions with FXR.Furthermore,the interaction between DA,HPE and FXR was simulated by Auto Dock software,respectively.The results showed that both DA and HPE could bind to the active site of FXR,and the binding pattern and action mode were similar to those of GW4064.The binding energies between DA,HPE with FXR was-6.51 kcal/mol and-6.80 kcal/mol,respectively(GW4064 was-8.57 kcal/mol).Hydrogen bonding and hydrophobic action were the main driving forces for the recognition of FXR protein with DA and HPE.The docking score of HPE was slightly higher than that of DA,which may be due to larger overlap between HPE with larger molecule and GW4064,occupying a larger space at the active site and having a stronger hydrophobic effect with surrounding amino acid residues.In addition,the hydrophobic force of HPE was stronger than that of DA,which is also the key driving force for the interaction between FXR and compound.3.Study on the protective effects of DA and HPE in ANIT-induced cholestasis rats.DA and HPE showed better binding action with FXR in the molecular docking experiments and were selected for the follow-up research to clarify their protective effects against ANIT-induced cholestasis.Forty-eight male SD rats were divided into 6groups,each group 8 rats: Control,Model(ANIT,60 mg/kg),Positive drug(UDCA,100 mg/kg),Low,Medium and High dose groups of DA or HPE(10,20 and 40 mg/kg for the three dose groups).Continuous administration with 0.5% CMC-Na solution,HPE or UDCA for 7 days.Except for that the rats in control group were given 0.5%CMC-Na solution,the rats in other groups were modeled with a dose of 60 mg/kg ANIT on the fifth day.The results showed that both DA and HPE could significantly attenuated ANIT-induced cholestasis in rats by decreasing liver function indicators in blood(ALT,AST,ALP,GGT,TBIL,DBIL and TBA),increasing of bile flow and recovery of liver histopathological injuries,as well as reversion the expressions of TNF-α,IL-1β,IL-6 and IL-10 in liver.In a word,DA and HPE could significantly reverse the hepatic cholestasis caused by ANIT in SD rats,and were the active substances of H.pedunculosum.DA exhibited better activity than HPE.4.Study on the mechanism of anti-cholestasis of DA and HPE based on FXR signaling pathways.(1)In vivo experiment: To further investigate the possible anti-cholestasis mechanisms of DA and HPE.Western blot experiments were carried out to determine the expressions of FXR,BSEP,MRP2,NTCP,small heterodimer chaperone 1/2(SHP1/2),cholesterol 7α-hydroxylase(CYP7A1)and cholesterol 27α-hydroxylase(CYP27A1)in the liver tissues preserved in the previous study.Results showed that both DA and HPE could up-regulate the protein expression of FXR,SHP1,SHP2,BSEP,MRP2 and NTCP,and down-regulate the protein expression of CYP7A1 and CYP27A1.All these results sugguested that DA and HPE may could activate FXR expression,and thereby up or down regulation of its downstream target proteins,resulting in inhibiting the synthesis of BAs and promoting the transport of toxic BAs.(2)In vitro experiment: To further investigate whether DA and HPE exert anti-cholestasis effects by activating FXR,human normal L-02 cells were used to study the mechanism in vitro.(1)Different concentrations of DA and HPE(6.25,12.5,25,50,100,200,400 and 800 μmol/L)were treated to normal L-02 cells to investigate the safeties of DA and HPE by MTT method.The results showed that DA and HPE had no significant inhibitory effects on L-02 cells and there was no significant damage to normal cells at a dosage of 100 μmol/L.(2)The antagonist of FXR guggulsterone(50μmol/L)was selected to co-culture with L-02 cells.After interfering with the expression of FXR in L-02 cells,the agonists GW4064(6 μmol/L),DA and HPE(25,50 and 100μmol/L as low,medium and high doses)were added to co-culture for 24 h,respectively.The m RNA and protein expressions of FXR,SHP1/2,CYP7A1,CYP27A1,BSEP,MRP2 and NTCP were detected by RT-PCR and Western blot.The results showed that the expressions of FXR,SHP1,SHP2,BESP,MRP2 and NTCP were significantly up-regulated and the expressions of CYP7A1 and CYP27A1 were significantly down-regulated in a dose-dependent manner after the treatment with DA and HPE.(3)To further demonstrate the effect of DA and HPE on FXR,fluorescent labeled Si-RNA(20 μmol/L)were constructed and transfected into L-02 cells to interfere with the expression of FXR.After 48 h,the cells were treated with DA and HPE(25,50 and 100μmol/L),and co-cultured for further 24 h.RT-PCR and Western blot results showed that the decreased gene and protein expressions of FXR、SHP、BESP、MRP2 and NTCP caused by Si-FXR were significantly reversed by DA and HPE.The increased gene and protein expressions of CYP7A1 and CYP27A1 were down-regulated.These data suggested that DA and HPE,similar with GW4064,had FXR-activating effect and showed better regulating effect than GW4064 at a dosage of 100 μmol/L.(4)To further confirm the FXR activating effects of DA and HPE,fluorescent labeled FXR overexpressed plasmids(1 μg/μL)was constructed and transfected into L-02 cells.After48 h,the cells were treated with DA and HPE(25,50 and 100 μmol/L),and co-cultured for further 24 h.RT-PCR and Western blot results also showed that the gene and protein expressions of FXR、SHP、BESP、MRP2 and NTCP increased and the gene and protein expressions of CYP7A1 and CYP27A1 were decreased after treatment with DA and HPE.DA and HPE showed better FXR-activating effect than GW4064 at a dosage of100 μmol/L.Therefore,it was further confirmed that DA and HPE exhibited anti-cholestasis effects acting as FXR agonist.In summary,the anti-cholestasis bioactive extract of the seeds of H.pedunculosum,HPEAE,may exhibited improving effect on BAs transport and protecting effect against cholestasis caused liver injury by activating FXR,thereby regulating the expression of its downstream transporters BSEP,MRP2 and NTCP,reducing oxidative stress through Keap1/Nrf2 signaling pathway,and inhibiting the release of inflammatory factors through NF-κB signaling pathway.High content screening and molecular docking results showed that two compounds,DA and HPE,exhibited strong FXR activating effect,and may be the important active components of the seeds of H.pedunculosum.In vivo pharmacodynamics studies showed that both of the two compounds had protecting effect in ANIT-induced cholestasis rats and DA showed better bioactivity.Combined with the results of in vitro interfering and overexpression experiments on FXR,it was suggested that DA and HPE could act as agonist of FXR to regulate the expression of its downstream signal factors and inhibit the synthesis,promote the transport of BAs,ameliorate cholestasis caused liver injury.Our research preliminarily elucidated the pharmacodynamic action and mechanism of bioactive components from the seeds of H.pedunculosum in the treatment of "Chiba disease" by activating FXR and regulating the downstream gene expressions.This study laid a foundation for the modern development and utilization of H.pedunculosum and provided scientific basis for the innovative drug discovery which targeting FXR and for the treatment of cholestasis diseases.