Preliminary Study On The Molecular Mechanism Of Jaundice Removing And The Potential Bioactive Compounds Screening Of Yin-zhi-huang

Aims1.To establish a HepaRG cell model with stable hyperbilirubin;2.To investigate the effect and the underlying molecular mechanism of Yin-zhi-huang on jaundice removing in hyperbilirubin HepaRG cell model;3.To screen the potential bioactive compounds from the compounds of Yin-zhi-huang by HPLC-MS/MS method.Methods1.The hyperbilirubin HepaRG cell model was established by bilirubin and probenecid co-incubation HepaRG cells.The intracellular total bilirubin(TBIL)and indirect bilirubin(IBIL)were used to evaluate the hyperbilirubin cell model.2.Aqueous extract of Yin-zhi-huang was added and incubated in the hyperbilirubin HepaRG cell model,and the jaundice removing effect of Yin-zhi-huang was evaluated by evaluating the intracellular total bilirubin,indirect bilirubin and the extracellular direct bilirubin(DBIL)content.To investigate the molecular mechanism of Yin-zhi-huang on “jaundice removing” effect,RT-q PCR and western-blot techniques were also used to evaluate the expression of transporters,metabolic enzymes and nuclear receptors in hyperbilirubin HepaRG cell model.3.Phenylhydrazine hydrochloride-induced hyperbilirubin rat model was treated with Yin-zhi-huang to prepare the serum containing Yin-zhi-huang.Then,the hyperbilirubin HepaRG cells were incubated with Yin-zhi-huang-containing serum.The aqueous extract of Yin-zhi-huang,Yin-zhi-haung-containing serum and the intracellular content of Yin-zhi-huang were determined by HPLC-MS/MS to screen the potential bioactive compounds of Yin-zhi-huang.Results1.Establishment of hyperbilirubin cell model: HepaRG cells were incubated with 40 μg/mL bilirubin and 50 μg/mL probenecid for 12 h,the intracellular total bilirubin and indirect bilirubin content reached the highest point,and then incubated the cellls in the medium with 50 μg/mL of probenecid canstably keep the high bilirubin level within 48 h.2.Study onthejaundiceremoving effect of Yin-zhi-huang and its molecular mechanism: The hyperbilirubin cell model was incubated with Yin-zhi-huang(720μg/mL)for 24 h,the intracellular total bilirubin and indirect bilirubin in Yin-zhi-huang-treated group were significantly decreased when compared to that of themodel group(P<0.01),and the content of extracellular direct bilirubin in Yin-zhi-huang treated group was significantly increased(P<0.01).The findingsin Yin-zhi-huang treated groupwere as sameasthe results from positive control group(phenobarbital-treatedgroup).The m RNA and protein expression of cell transporter Multi-drug resistant associate protein 2(MRP2),metabolic enzyme UDP glucuronosyltransferase family 1 member A1(UGT1A1)and nuclear receptor constitutive androstane receptor(CAR)in Yin-zhi-huang-treated group were allsignificantly increased(P<0.05)when compared to thatin the model group.3.Preliminary screening forthe bioactive compounds in Yin-zhi-huang: Compared to the model group,the serum levels of total bilirubin,direct bilirubin and indirect bilirubin in hyerbilirubin rat model were significantly decreased(P<0.05)after 7 days’ Yin-zhi-huang treatment.In Yin-zhi-huang-containing serum,10 compounds were discovered from the total of 31 compounds in Yin-zhi-huang,and 7 compounds were found in hyperbilirubin cells treated with Yin-zhi-huang-containing serum,they are Wogoni,Baicali,6,8-Dimethoxy-7-hydroxycoumarin,Genipin 1-β-gentiobioside,Safflor yellow A and two unidentified compounds.Conclusions1.In this study,a stable hyperbilirubin cell model was established by incubating Heap RG cells with bilirubin and probenecid.2.The aqueos extract of Yin-zhi-huang can reduce the content of TBIL and IBIL in hyperbilirubin HepaRG cellmodel,indicating the significant jaundice-removing effect.The efficacy of Yin-zhi-huangon jaundice-removingis as similar as the effect of positive drug phenobarbital.The jaundice-removing effect of Yin-zhi-huang may have correlation with the increased m RNA and protein expression of bilirubin-related metabolism enzyme UGT1A1 and efflux transporter MRP2 via regulating the expression of nuclear receptor CAR.3.In this study,7 compounds were discovered in the cells-treated with Yin-zhi-huang containing serum,which may be the potential bioactive compounds that responsible for the jaundice-removing effect of Yin-zhi-huang.