中药药理与临床

目的:研究茵陈五苓散对胆红素联合丙磺舒诱导的HepaRG黄疸细胞模型的影响，探讨其退黄作用机制。方法:将HepaRG细胞随机分为正常对照血清组、3.42、6.84、13.68 g/kg茵陈五苓散10%含药血清组、0.10 g/kg熊去氧胆酸10%含药血清组、6.84 g/kg茵陈五苓散含药血清+10μmol/L多索吗啡组、6.84 g/kg茵陈五苓散含药血清+0.095 mmol/L阿卡地新组，CCK-8法检测细胞活力；除正常对照血清组外，均采用胆红素联合丙磺舒构建HepaRG黄疸细胞模型，加入相应药物或含药血清，检测细胞内总胆红素(TBIL)、间接胆红素(IBIL)含量和细胞外直接胆红素(DBIL)含量；检测细胞活性氧(ROS)、线粒体膜电位水平和三磷酸腺苷含量(ATP);实时荧光定量聚合酶链式反应(Real-time qPCR)和蛋白免疫印迹法(Western blot)分别检测腺苷活化蛋白激酶(Ampk)/过氧化物酶体增殖物激活受体γ辅激活因子1α(Pgc1α)/线粒体转录因子A(Tfam)通路相关靶点mRNA和蛋白表达。结果:与正常对照血清组相比，3.42、6.84、13.68 g/kg茵陈五苓散10%含药血清组及熊去氧胆酸10%含药血清组对细胞活力无明显影响(P>0.05);与正常对照血清组相比，模型对照组细胞TBIL、IBIL含量和ROS水平显著升高(P<0.01),Pgc1α、Tfam的mRNA表达显著下调(P<0.01),p-AMPK、PGC-1α、TFAM蛋白表达明显下调(P<0.05),线粒体膜电位和ATP含量显著降低(P<0.01);与模型对照组相比，3.42、6.84、13.68 g/kg茵陈五苓散10%含药血清组及熊去氧胆酸10%含药血清组细胞内TBIL、IBIL含量显著降低(P<0.01),细胞外DBIL含量显著升高(P<0.01),6.84 g/kg茵陈五苓散10%含药血清组Pgc1α和Tfam的mRNA表达显著上调(P<0.01),p-AMPK、PGC-1α、TFAM蛋白表达明显上调(P<0.05),ROS水平显著降低(P<0.01),线粒体膜电位水平、ATP含量显著升高(P<0.01);与6.84 g/kg茵陈五苓散10%含药血清组相比，6.84 g/kg茵陈五苓散含药血清+10μmol/L多索吗啡组Pgc1α、Tfam的mRNA表达显著下调(P<0.01),p-AMPK、PGC-1α、TFAM蛋白表达明显下调(P<0.05),ROS水平显著升高(P<0.01),线粒体膜电位水平、ATP含量显著降低(P<0.01)。结论:茵陈五苓散通过激活AMPK/PGC-1α/TFAM通路，改善HepaRG黄疸细胞模型中线粒体膜电位，降低ROS水平，提高ATP含量，进而发挥退黄作用。

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:HepaRG 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 assay. 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 serum, followed by measurement of intracellular total bilirubin(TBIL) and indirect bilirubin(IBIL),as well as extracellular direct bilirubin(DBIL). Reactive oxygen species(ROS),mitochondrial membrane potential, and adenosine triphosphate(ATP) levels were assessed. The protein and mRNA levels of related targets in the adenosine monophosphate-activated protein kinase(AMPK)/peroxisome proliferator-activated receptor λ coactivator 1α(PGC-1α)/transcription factor A,mitochondrial(TFAM) signaling pathway were analyzed by Western blot and real-time quantitative PCR,respectively. 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 mitochondrial 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 exhibited declined intracellular TBIL and IBIL levels(P<0.01) and elevated extracellular DBIL level(P<0.01). Compared 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 dorsomorphin 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 between the 6.84 g/kg Yinchen Wuling Powder-containing serum group and the 6.84 g/kg Yinchen Wuling Powder-containing serum+0.095 mmol/L AICAR group. Conclusion:Yinchen Wuling Powder exerts its anti-jaundice effect by activating 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.