Dai Xiao-Qing, Cai Wen-Tao, Wu Xiao, Chen Yong, Han Feng-Mei
Hubei Province Key Laboratory of Biotechnology of Chinese Traditional Medicine, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei University, Wuhan, Hubei, China.
Hubei Province Key Laboratory of Biotechnology of Chinese Traditional Medicine, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei University, Wuhan, Hubei, China.
Life Sci. 2017 Jul 1;180:68-74. doi: 10.1016/j.lfs.2017.05.015. Epub 2017 May 12.
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.
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.
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.
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.
原儿茶酸（PCA）是一种存在于多种抗病毒中草药中的酚类化合物。肝细胞核因子（HNF）家族成员HNF4α和HNF1α在乙型肝炎病毒（HBV）的基因转录中起重要调节作用。以往研究发现PCA可抑制HepG2.2.15细胞中HBV抗原分泌和HBV DNA复制，但其抗HBV机制尚未完全明确。我们旨在阐明PCA的抗HBV机制。
采用MTT法评估细胞毒性。使用酶联免疫吸附测定试剂盒检测HBsAg或HBeAg含量。通过PCR试剂盒检测无细胞培养基中的HBV DNA。采用实时PCR检测HNF1α和HNF4α mRNA表达。通过蛋白质免疫印迹法检测HNF1α、HNF4α和ERK1/2蛋白表达，并通过荧光素酶报告基因测定法检测HBV启动子活性。
我们的结果表明，PCA抑制Huh7和HepG2.2.15细胞中HNF1α和HNF4α的基因转录和蛋白质翻译，以及在转染了HBV启动子荧光素酶报告质粒的Huh7细胞中HBV X和preS1的启动子活性。进一步研究表明，PCA诱导细胞外信号调节激酶（ERK）1/2磷酸化，从而抑制HepG2.2.15细胞中HNF4α和HNF1α表达以发挥其抗病毒活性。
据我们所知，本研究首次揭示了PCA的抗HBV机制。我们的结果表明，PCA通过激活ERK1/2通路并随后下调HepG2.2.15细胞中的HNF4α和HNF1α来抑制HBV复制。
