茵陈五苓散调控AMPK/PGC-1α/TFAM通路改善线粒体功能减轻肝细胞性黄疸的机制研究OA

Objective:To investigate the effects of Yinchen Wuling(茵陈五苓)Powder on the HepaRG cell model of jaundice induced by bilirubin and probenecid and explore the underlying anti-jaundice mechanism.Methods:Hep-aRG cells were randomly allocated into the following groups:normal control,Yinchen Wuling Powder-containing serum(3.42,6.84,and 13.68 g/kg),0.10 g/kg ursodeoxycholic acid-containing serum,6.84 g/kg Yinchen Wuling Powder-containing serum,6.84 g/kg Yinchen Wuling Powder-containing serum+10 μmol/L dorsomorphin,and 6.84 g/kg Yinchen Wuling Powder-containing serum+0.095 mmol/L AICAR groups.Cell viability was assessed by the CCK-8 as-say.Except the normal control group,other groups of HepaRG cells were treated with bilirubin combined with probenecid for the modeling of jaundice.The cells were then exposed to different doses of corresponding drugs or drug-containing se-rum,followed by measurement of intracellular total bilirubin(TBIL)and indirect bilirubin(IBIL),as well as extracellu-lar direct bilirubin(DBIL).Reactive oxygen species(ROS),mitochondrial membrane potential,and adenosine triphos-phate(ATP)levels were assessed.The protein and mRNA levels of related targets in the adenosine monophosphate-acti-vated protein kinase(AMPK)/peroxisome proliferator-activated receptor λ coactivator 1α(PGC-1α)/transcription fac-tor A,mitochondrial(TFAM)signaling pathway were analyzed by Western blot and real-time quantitative PCR,respec-tively.Results:No significant differences were observed in cell viability between the normal control and various Yinchen Wuling Powder-containing serum(3.42,6.84,and 13.68 g/kg)or 0.10 g/kg ursodeoxycholic acid-containing serum groups(P>0.05).Compared with the normal control group,the model control group exhibited elevated intracellular TBIL and IBIL levels(P<0.01),down-regulated mRNA levels of Pgc1α and Tfam(P<0.01),down-regulated protein levels of phosphorylated AMPK(p-AMPK),PGC-1α,and TFAM(P<0.05),raised ROS level(P<0.01),reduced mi-tochondrial membrane potential(P<0.01),and decreased ATP content(P<0.01).Compared with the model control group,all Yinchen Wuling Powder(3.42,6.84,and 13.68 g/kg)groups and 0.10 g/kg ursodeoxycholic acid group ex-hibited declined intracellular TBIL and IBIL levels(P<0.01)and elevated extracellular DBIL level(P<0.01).Com-pared with the model control group,the 6.84 g/kg Yinchen Wuling Powder-containing serum group showed up-regulated mRNA levels of Pgc1α and Tfam(P<0.01),up-regulated protein levels of p-AMPK,PGC-1α,and TFAM(P<0.05),lowered ROS level(P<0.01),enhanced mitochondrial membrane potential(P<0.01),and increased ATP content(P<0.01).Compared with the 6.84 g/kg Yinchen Wuling Powder-containing serum group,the addition of 10 μmol/L dorso-morphin down-regulated the mRNA levels of Pgc1α and Tfam(P<0.01)and the protein levels of p-AMPK,PGC-1α,and TFAM(P<0.05),raised the ROS level(P<0.01),decreased the mitochondrial membrane potential(P<0.01),and reduced the ATP content(P<0.01).In contrast,no significant differences were observed in these parameters be-tween the 6.84 g/kg Yinchen Wuling Powder-containing serum group and the 6.84 g/kg Yinchen Wuling Powder-con-taining serum+0.095 mmol/L AICAR group.Conclusion:Yinchen Wuling Powder exerts its anti-jaundice effect by acti-vating the AMPK/PGC-1α/TFAM signaling pathway,thereby improving mitochondrial membrane potential,reducing the ROS level,and increasing the ATP content in the HepaRG cell model of jaundice.