RESUMEN
AIMS: Protocatechuic acid (PCA) is a phenolic compound found in many antiviral Chinese herbal medicines. HNF4α and HNF1α, the members of hepatocyte nuclear factor (HNF) family, play an important regulatory role in the gene transcription of hepatitis B virus (HBV). Previous studies found that PCA inhibited HBV antigen secretion and HBV DNA replication in HepG2.2.15 cells, but its anti-HBV mechanism has not been fully understood. We aim to illustrate the anti-HBV mechanism of PCA. MATERIALS AND METHODS: MTT was used to estimate cytotoxicity. The content of HBsAg or HBeAg was detected using an enzyme-linked immunosorbent assay kit. HBV DNA in cell-free culture media was detected by PCR kit. HNF1α and HNF4α mRNA expression was detected by real-time PCR. HNF1α, HNF4α and ERK1/2 protein expression was detected by western blotting and HBV promoter activity was tested by luciferase reporter assay. KEY FINDINGS: Our results demonstrated that PCA inhibited the gene transcription and protein translation of HNF1α and HNF4α in Huh7 and HepG2.2.15 cells, as well as the promoter activities of HBV X and preS1 in Huh7 cells transfected with the luciferase reporter plasmid of HBV promoter. Further study suggested that PCA induced the phosphorylation of extracellular-signal-related kinase (ERK) 1/2, and thereby inhibited HNF4α and HNF1α expression in HepG2.2.15 cells to exert its antiviral activity. SIGNIFICANCE: To our knowledge, this study is the first to reveal the anti-HBV mechanism of PCA. Our results demonstrate that PCA inhibits HBV replication by activating ERK1/2 pathway and subsequently down-regulating HNF4α and HNF1α in HepG2.2.15 cells.
Asunto(s)
Antivirales/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Hidroxibenzoatos/farmacología , Replicación Viral/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , ADN Viral , Regulación hacia Abajo/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Células Hep G2 , Antígenos de Superficie de la Hepatitis B/metabolismo , Antígenos e de la Hepatitis B/metabolismo , Factor Nuclear 1-alfa del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/genética , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , TransfecciónRESUMEN
The Chinese herb Sparganum stoloniferum Buch.-Ham. (SBH) is frequently used to improve blood circulation and to rehabilitate vascular obstruction in traditional Chinese medicine. It was recently reported that SBH reduces the proliferation of renal epithelial cells stimulated by epidermal growth factor (EGF), and inhibits the phosphorylation of the EGF receptor. SBH has also been used as a trial drug to treat polycystic kidney disease (PKD) patients in China. The potential molecular actions of SBH on PKD remain unknown. Autosomal dominant PKD (ADPKD) is associated with mutations in polycystin-1 or polycystin-2 (PC2). PC2 and its homologue, polycystin-L (PCL), are nonselective cation channels permeable to potassium, sodium, and calcium. Here, we examine the effects of SBH on the human PCL channel expressed in Xenopus oocytes, using 2-microelectrode voltage-clamp electrophysiology and radiotracer uptake measurements. In PCL-expressing oocytes, with or without preincubation with SBH, the PCL channel was inhibited by SBH in a dose-dependent and reversible manner; a concentration of 2% SBH completely abolished the channel activation. The IC50 value for SBH was 0.48% +/- 0.03%, with a 10-min preincubation period. SBH was also found to inhibit the PCL-mediated 45Ca tracer uptake in oocytes. Our study suggests that SBH contains 1 or more yet-to-be determined components that are inhibitors of PCL channel. The therapeutic potential of SBH for ADPKD and its chemical composition remain to be investigated.