Yinzhihuang Oral Liquid Ameliorates Hyperbilirubinemia Induced by δ‐Aminolevulinic Acid and Novobiocin in Neonatal Rats

Abstract Yinzhihuang oral liquid (YZH) is a traditional Chinese medicine that has been widely used in Asia to prevent and treat neonatal hyperbilirubinemia, but the published preclinical studies on its anti‐hyperbilirubinemia effect are conducted in adult animals, partly due to the lack of preclinical neonatal hyperbilirubinemia animal models. In the present study, we tested six reagents to induce hyperbilirubinemia in neonatal rats, and established two appropriate neonatal hyperbilirubinemia rat models by subcutaneous injection of δ‐Aminolevulinic acid (ALA, 200 mg/kg) or novobiocin (NOVO, 200 mg/kg). Oral treatment of YZH (80, 160 and 320 mg/kg) significantly decreased serum conjugated bilirubin levels in ALA‐treated neonatal rats and serum unconjugated bilirubin levels in NOVO‐treated neonatal rats, respectively. Additionally, pre‐treatment of YZH also prevented the increase of serum bilirubin levels in both ALA‐ and NOVO‐treated rats. Mechanistically, YZH significantly up‐regulated the mRNA expression of genes involved in hepatic bilirubin disposition (organic anion‐transporting polypeptide 1b2, Oatp1b2 ; multidrug resistance‐associated protein 2, Mrp2 ) and bilirubin conjugation (UDP‐glucuronosyltransferase 1a1, Ugt1a1 ). Additionally, YZH up‐regulated the mRNA expression of cytochrome P450 1A1 ( Cyp1a1 ), the target gene of aryl hydrocarbon receptor (AhR), and increased the nuclear protein levels of AhR in livers of neonatal rats. YZH and its two active ingredients, namely baicalin (BCL) and 4’‐hydroxyacetophenone (4‐HT), up‐regulated the mRNA expression of AhR target genes ( CYP1A1 and UGT1A1 ) and increased nuclear protein levels of AhR in HepG2 cells. In conclusion, the present study provides two neonatal hyperbilirubinemia animal models and evaluates the anti‐hyperbilirubinemia effect and mechanisms of YZH in neonatal animals.