Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Feb;41(2):278-285.
doi: 10.1038/s41401-019-0302-0. Epub 2019 Sep 25.

ERK1/2-HNF4α axis is involved in epigallocatechin-3-gallate inhibition of HBV replication

Zi-Yu Wang  1 , Yu-Qi Li  1 , Zhi-Wei Guo  1 , Xing-Hao Zhou  2   3 , Mu-Dan Lu  4 , Tong-Chun Xue  5   6 , Bo Gao  7
Affiliations

ERK1/2-HNF4α axis is involved in epigallocatechin-3-gallate inhibition of HBV replication

Zi-Yu Wang et al. Acta Pharmacol Sin. 2020 Feb.

Abstract

Epigallocatechin gallate (EGCG), a major polyphenol in green tea, exhibits diverse biological activities. Previous studies show that EGCG could effectively suppress HBV gene expression and replication, but the role of EGCG in HBV replication and its underlying mechanisms, especially the signaling pathways involved, remain unclear. In this study we investigated the mechanisms underlying EGCG inhibition on HBV replication with a focus on the signaling pathways. We showed that EGCG (12.5-50 μM) dose-dependently inhibited HBV gene expression and replication in HepG2.2.15 cells. Similar results were observed in HBV mice receiving EGCG (25 mg· kg-1· d-1, ip) for 5 days. In HepG2.2.15 cells, we showed that EGCG (12.5-50 μM) significantly activate ERK1/2 MAPK signaling, slightly activate p38 MAPK and JAK2/STAT3 signaling, while had no significant effect on the activation of JNK MAPK, PI3K/AKT/mTOR and NF-κB signaling. By using specific inhibitors of these signaling pathways, we demonstrated that ERK1/2 signaling pathway, but not other signaling pathways, was involved in EGCG-mediated inhibition of HBV transcription and replication. Furthermore, we showed that EGCG treatment dose-dependently decreased the expression of hepatocyte nuclear factor 4α (HNF4α) both at the mRNA and protein levels, which could be reversed by pretreatment with the ERK1/2 inhibitor PD98059 (20 μM). Moreover, we revealed that EGCG treatment dose-dependently inhibited the activity of HBV core promoter and the following HBV replication. In summary, our results demonstrate that EGCG inhibits HBV gene expression and replication, which involves ERK1/2-mediated downregulation of HNF4α.These data reveal a novel mechanism for EGCG to inhibit HBV gene expression and replication.

Keywords: EGCG; ERK1/2; HBV; HBV mice; HNF4α; HepG2.2.15 cells; PD98059; core promoter.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
EGCG inhibited HBV gene expression and replication in HepG2.2.15 cells. HepG2.2.15 cells were treated with increasing amounts of EGCG (12.5, 25, 50 μM) for 24 h. The levels of HBsAg (a) and HBeAg (b) in culture supernatants were determined by ELISA; the level of HBcAg (c) in the cell lysates was determined by Western blotting; the levels of HBV pgRNA and HBV RNAs (d) were determined by qRT-PCR; the levels of intracellular core particle-associated HBV-DNA (e, f) were determined by quantitative real-time PCR and Southern blotting, respectively; and the cell viability (g) was determined by CCK8 assay. The values are expressed as the means ± SEM of three independent experiments
Fig. 2
Fig. 2
The effect of EGCG on HBV gene expression and replication in mice. To evaluate the effect of EGCG on HBV replication, HBV mice were treated with or without EGCG (25 mg/kg every day, ip) for 5 days (n = 10 per group). The serum concentrations of HBsAg (a), HBeAg (b), and HBV-DNA (c) were determined by ELISA and quantitative real-time PCR; the level of HBcAg (d) in liver tissue was determined by immunohistochemical staining; the levels of HBV pgRNA and HBV RNAs (e) in liver tissues were determined by qRT-PCR; the levels of intracellular core particle-associated HBV-DNA (f) in liver tissues were determined by Southern blotting; and the levels of ALT (g) in mouse sera were determined by an ALT/GPT ELISA kit. The values were expressed as the means ± SEM of three independent experiments; *P < 0.05 (unpaired, two-tailed Student’s t tests)
Fig. 3
Fig. 3
ERK1/2 signaling pathway was involved in the EGCG-mediated inhibition of HBV. a The effect of EGCG on the activation of the MAPK (p38, ERK, JNK), AKT/mTOR, and JAK2/STAT3 pathways. HepG2.2.15 cells were treated with increasing doses of EGCG (25 μM and 50 μM) for 24 h, and the cells were then subjected to Western blotting with the indicated antibodies. b The effect of pharmacological inhibitors on the activation of p38, ERK MAPK, or JAK2/STAT3 signaling pathways. HepG2.2.15 cells were pretreated with the ERK inhibitor PD98059 (20 μM), the p38 inhibitor 203580 (10 μM) or the JAK2/STAT3 inhibitor AG490 (50 μM) for 1 h and then treated with 25 μM of EGCG for 24 h. The levels of p-p38, p-ERK, p-JAK2, or p-STAT3 were determined by Western blotting. c The role of p38, ERK MAPKs, or JAK2/STAT3 signaling pathways in the EGCG-mediated inhibition of HBV. HepG2.2.15 cells were treated as in b, and the levels of HBeAg or HBV-DNA were determined by ELISA or qPCR, respectively. The values were expressed as the means ± SEM of three independent experiments; *P < 0.05 (unpaired, two-tailed Student’s t tests)
Fig. 4
Fig. 4
The ERK1/2 signaling pathway was involved in EGCG-mediated inhibition of HNF4α. a The effect of EGCG on the mRNA level of HNF4α in HepG2.2.15 cells. HepG2.2.15 cells were treated with increasing doses of EGCG (25 and 50 μM) for 24 h. The mRNA levels of HNF4α were then determined by quantitative RT-PCR. b The effect of EGCG on the protein level of HNF4α in HepG2.2.15 cells. HepG2.2.15 cells were treated as in a, and the protein levels of HNF4α were then determined by Western blotting. c HepG2.2.15 cells were treated with 50 μM of EGCG for the indicated time periods, and Western blotting was then performed using antibodies against p-ERK1/2, HNF4α and GAPDH. The effect of ERK signaling on the EGCG-mediated inhibition of HNF4α. HepG2.2.15 cells were pretreated with ERK inhibitor PD98059 for 1 h and then treated with 50 μM of EGCG for 24 h, and the mRNA levels (d) and protein levels (e) of HNF4α were determined by qRT-PCR and Western blotting, respectively. The values were expressed as the means ± SEM of three independent experiments; *P < 0.05; (unpaired, two-tailed Student’s t tests)
Fig. 5
Fig. 5
The effect of EGCG on the activity of HBV promoters. HepG2.2.15 cells were transfected with SpLUC (a), CpLUC (b), pS(1) LUC (c) or XpLUC (d), respectively. Twenty-four hours post transfection, the cells were further treated with increasing doses of EGCG (25 µM and 50 µM) for another 24 h. The luciferase activity in cell lysates was then determined. The luciferase activity of EGCG-untreated cells was set to 100%. In all transfection assays, pCMV-β-gal was cotransfected to normalize the transfection efficiency. The values are expressed as the means ± SEM of three independent experiments
Fig. 6
Fig. 6
ERK1/2-HNF4α axis contributed to EGCG inhibition of HBV core promoter activity. a The effect of ERK signaling on EGCG-mediated inhibition of HBV core promoter activity. The cells were transfected with a core promoter-dependent reporter plasmid (CpLUC). Twenty-four hours post transfection, the cells were treated with EGCG in the absence or presence of the ERK inhibitor PD98059 (20 μM), the p38 inhibitor 203580 (10 μM) or the JAK2/STAT3 inhibitor AG490 (50 μM) for 24 h, followed by the detection of luciferase activity in the cell lysates. b The effect of ectopic HNF4α expression on the EGCG-mediated inhibition of HBV promoter activity. The HNF4α expression plasmid (pHNF4α-HA) was transfected into HepG2.2.15 cells together with CpLUC. Twenty-four hours post transfection, the cells were further stimulated with EGCG for another 24 h, followed by the detection of luciferase activity. Below: The whole cell lysate was subjected to Western blotting using antibodies against HA and GAPDH. c The effect of HNF4α expression on EGCG-mediated inhibition of HBV gene expression and replication. HepG2.2.15 cells were transfected with control empty vector or an increasing dose of pHNF4α for 24 h and then treated with EGCG for another 24 h. HBeAg and HBV-DNA levels were determined by ELISA and qPCR, respectively. The values were expressed as the means ± SEM of three independent experiments; *P < 0.05; (unpaired, two-tailed Student’s t tests)
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Tsai KN, Kuo CF, Ou JJ. Mechanisms of hepatitis B virus persistence. Trends Microbiol. 2018;26:33–42. doi: 10.1016/j.tim.2017.07.006. - DOI - PMC - PubMed
    1. Schweitzer A, Horn J, Mikolajczyk RT, Krause G, Ott JJ. Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013. Lancet. 2015;386:1546–55. doi: 10.1016/S0140-6736(15)61412-X. - DOI - PubMed
    1. Wong GL, Wong VW, Chan HL. Virus and host testing to manage chronic hepatitis B. Clin Infect Dis. 2016;62(Suppl 4):298–305. doi: 10.1093/cid/ciw024. - DOI - PMC - PubMed
    1. Ward H, Tang L, Poonia B, Kottilil S. Treatment of hepatitis B virus: an update. Future Microbiol. 2016;11:1581–97. doi: 10.2217/fmb-2016-0128. - DOI - PMC - PubMed
    1. Narotzki B, Reznick AZ, Aizenbud D, Levy Y. Green tea: a promising natural product in oral health. Arch Oral Biol. 2012;57:429–35. doi: 10.1016/j.archoralbio.2011.11.017. - DOI - PubMed

MeSH terms

-