3,4,5-Tri-O-caffeoylquinic acid methyl ester isolated from Lonicera japonica Thunb. Flower buds facilitates hepatitis B virus replication in HepG2.2.15 cells

doi:10.1016/j.fct.2020.111250

Food and Chemical Toxicology

Volume 138, April 2020, 111250

https://doi.org/10.1016/j.fct.2020.111250 Get rights and content

Highlights

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3,4,5-CQME increased intracellular and secreted HBsAg levels by more than two times.
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Levels of HBeAg, HBV DNA and RNA were enhanced by 3-day 3,4,5-CQME treatment.
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3,4,5-CQME induced HBx expression and activated HBV transcriptional regulation signals.
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Risk of intaking herbs contained 3,4,5-CQME for HBV treatment requires more evaluation.

Abstract

Caffeoylquinic acids are well known for their prominent antiviral activities. Beyond our expectations, we initially found 3,4,5-Tri-O-caffeoylquinic acid methyl ester (3,4,5-CQME) from L. japonica can facilitate HBV DNA and antigens secretion. This study aimed to investigate its underlying molecular mechanism. The results indicate that 3,4,5-CQME signally increased intracellular and secreted HBsAg levels by more than two times in HepG2.2.15 cells and HepAD38 cells. Furthermore, levels of HBeAg, HBV DNA and RNA were significantly enhanced by 3-day 3,4,5-CQME treatment; it didn't directly affect intracellular cccDNA amount, although it slightly increased cccDNA accumulation as a HBV DNA replication feedback. In addition, treatment with 3,4,5-CQME significantly induced HBx protein expression for viral replication. We utilized a phospho-antibody assay to profile the signal transduction change by 3,4,5-CQME to illuminate its molecular mechanism. The results indicate that treatment with 3,4,5-CQME activated AKT/mTOR, MAPK and NF-κB pathways verified by immunoblot. Moreover, 3,4,5-CQME upregulated the expression of nuclear transcriptional factors PGC1α and PPARα. In short, 3,4,5-CQME promotes HBV transcription and replication by upregulating HBx expression and activating HBV transcriptional regulation-related signals. As caffeoylquinic acids are widely present in traditional Chinese medicines, the risk of intaking caffeoylquinic acids-containing herbs for hepatitis B treatment requires more evaluation and further research.

Graphical abstract

Introduction

The hepatitis B virus (HBV), a partially double-stranded DNA virus, is a member of the orthohepadnavirus family. HBV infection is a serious health problem around the world, especially in China. About two billion people worldwide have been infected with HBV, including 350–400 million chronic carriers (Lai et al., 2003). Sustained infection with HBV can lead to hepatitis, cirrhosis, and hepatocellular carcinoma, which cause approximately one million deaths per year (Ott et al., 2012). Up to now, viral hepatitis B has not yet been completely cured in clinical practice. Although interferon and nucleoside analogues (NAs) have been widely used in the treatment of viral hepatitis, clinical therapeutic effects are far from satisfactory for their specificity, side effects, and drug resistance.

Traditional Chinese medicine (TCM) is composed of complex mixtures of compounds, and these natural, active compounds offer opportunities to identify novel effective antiviral agents. Recently, great efforts have been made to explore the active constitutions of TCM, which have the potential to be developed as candidates for antiviral drugs (Cui et al., 2010). Phyllanthus (Lam et al., 2006), Salvia miltiorrhiza (Zhou et al., 2007), Radix Astragali (Wang et al., 2009b), and Rheum palmatum L. (Kim et al., 2001) had been reported to exhibit activities against HBV. However, few studies about the toxicology and side effects of natural products used during the treatment of hepatitis B have been published. Exclusively, resveratrol, which is known to extend lifespan, impacts mitochondrial function and metabolic homeostasis and enhances HBV replication through activating the Sirt1-PGC-1α-PPARα pathway (Shi et al., 2016).

Moreover, Lonicera japonica Thunb., a medicinal and edible plant, has been considered to possess effective antiviral activity and has been widely used in past centuries (Shang et al., 2011). Natural products purified from L. japonica Thunb. Have shown advantages in antiviral activity and the protection of the liver. Among them, caffeoylquinic acids and their derivatives are the main effective ingredients and have been widely reported to exhibit remarkable activities against HBV (Wang et al., 2009; Wu, 2016; Wu et al., 2012). In fact, 23 kinds of caffeoylquinic acid derivatives have been purified from L. japonica flower buds, and their effects on HBV replication were preliminarily explored in our previous study (Ge et al., 2018). Surprisingly, we initially found that treatment with 3,4,5-Tri-O-caffeoylquinic acid methyl ester (3,4,5-CQME) significantly upregulated the levels of secreted hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and intracellular HBV DNA, which prompted us to systematically reappraise the effects of caffeoylquinic acid derivatives on HBV replication and the toxicology of caffeoylquinic acid derivatives. In addition, the molecular mechanism remains largely unclear. Thus, the present study intends to systematically evaluate the effect of 3,4,5-CQME on HBV replication and explore the molecular mechanism behind it.

Section snippets

High-performance liquid chromatography (HPLC) analysis of 3,4,5-CQME

The purity of 3,4,5-CQME was directly analyzed by reversed-phase HPLC using a YMC-Pack C₁₈ analytical column (5 μm, 250 × 10 mm), which was eluted isocratically with 60% methanol in water at a flow rate of 2 ml/min. The injection volume was 10 μl, and the detection wave-length was 290 nm.

Reagents and antibodies

As previously reported, 3,4,5-CQME was purified from L. japonica flower buds (Ge et al., 2018). Cell Counting Kit-8 (CCK-8, E606335) was purchased from Sangon Biotech (Shanghai, China). The CytoTox 96

Chemical structure and purity of 3,4,5-CQME

The 3,4,5-CQME isolated from the L. japonica flower buds was analyzed on a HPLC system. The chemical structure is shown in Fig. 1, and the purity of 3,4,5-CQME was determined to 95.4% by HPLC (Fig. 1).

Effect of 3,4,5-CQME on HBV antigen expression in vitro

HepG2.2.15 cells are generated from the human hepatoblastoma cell line, HepG2, by stably transfecting the complete HBV genome (Sells et al., 1987). As a cell model that can persistently produce infectious HBV particles, HepG2.2.15 cells had been widely used in studies of HBV infection in vitro.

Discussion

In this study, we initially found that 3,4,5-CQME promotes HBV replication in HepG2.2.15 cells in a dose dependent manner without affecting cell viability or cytotoxicity. The molecular mechanism may be partially explained in three ways: (1) the 3,4,5-CQME treatment facilitated the expression of the HBx protein, which is a transcriptional activator essential for virus infection; (2) 3,4,5-CQME stimulation could activate AKT/mTOR/p70S6K, JNK, and the Ras/Raf/MEK/ERK signal axis, which can

Conclusion

In conclusion, the present study demonstrated that 3,4,5-CQME isolated from L. japonica flower buds can promote HBV replication and antigen expression in vitro. As caffeoylquinic acid derivatives are widely present in a variety of plant species, some of which are well known for their antiviral activities and widely used in TCM, healthy food, and cosmetics, the risk of intaking caffeoylquinic acid derivative-containing herbs for the treatment of hepatitis B should be reappraised.

CRediT authorship contribution statement

Haoqiang Wan: Data curation, Formal analysis, Investigation, Software, Visualization, Writing - original draft, Writing - review & editing. Lanlan Ge: Data curation, Investigation, Resources, Visualization, Writing - original draft. Lingyun Xiao: Investigation, Resources. Jiemei Li: Methodology, Software, Validation. Weigang Wu: Methodology, Validation. Shusong Peng: Project administration, Supervision. Jian Huang: Conceptualization, Funding acquisition, Project administration, Writing - review

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

This work was supported by grants from the Natural Science Foundation of China (81503221, 81703939), the Science and Technology Planning Project of Shenzhen Municipality (JCYJ20170307095556333, JCYJ20170413093108233, JCYJ20160427183814675), and the Natural Science Foundation of Guangdong Province (2017A030313659, 2014A030310365).

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Citation Excerpt :
Upregulated hepatitis B virus X protein (HBx) expression and activating HBx transcriptional regulation-related signals, 3,4,5-CQME (17, 100 μg mL−1) promoted HBV transcription and replication. Thus, Wan called attention to the intaking caffeoylquinic acids-containing herbs for HBV treatment, and the risk requires more evaluation (Wan et al., 2020). Also, phenolic acids are well-known for their beneficial health effects as potential antioxidants (Cai et al., 2004).
Lonicerae japonicae flos (flowers or flower buds of Lonicera japonica Thunberg, called Jinyinhua, JYH in Chinese), together with Lonicerae japonicae caulis (stems of L. japonica, called Rendongteng, RDT in Chinese), is representatives of two edible-medicinal herbs from the same medicinal plant. Leaves of L. japonica, non-medicinal parts in China, have already been used medically with RDT in Japan and Korea, namely Lonicerae folium cum caulis. For a long history, research and applications of L. japonica mainly concentrated on JYH, while RDT and leaves are generally thought to have less medicinal properties than JYH. To the best of our knowledge, there is no review systematically comparing the three parts of L. japonica. In contrast, a gap exists in recognising the intersubstitution of JYH, RDT, leaves, or at least, their differences.
The relevant information of flowers, stems and leaves was collected by scientific search engines including Elsevier, Google Scholar, Scifinder, CNKI (China national knowledge infrastructure), and Doctor's dissertations.
Based on our studies, the three showed remarkable homogeneity regarding major bioactive components, including caffeoylquinic and flavones. Differences in the content of chemical components may account for the discrepant intensities of anti-inflammatory, anti-microorganism, antioxidative activities of the three. In particular, leaves showed considerable activities, even more, active than JYH.
Neither comparative investigations among JYH, RDT, leaves nor their activity differences at a molecular level have been systematically reviewed. Our study gains insights into elucidating the similarities and differences of efficacy among them.
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Unlike the general research on anti-HBV activity of caffeoylquinic acids, there is no report that caffeoylquinic acids can promote HBV effect. Further mechanism studies confirm that 3,4,5-tri-O-caffeoylquinic acid methyl ester (341) promotes HBV transcription and replication by upregulating HBx expression and activating HBV transcriptional regulation-related signals (Wan et al., 2020). In addition, most terpenoids inhibit HBsAg and HBeAg secretion as well as HBV DNA replication.
Lonicera Linn. belonging to the family Caprifoliaceae, the largest genus in the plant family, includes about more than 200 species, which are mainly distributed in northern Africa, North America, Europe and Asia. Some species of this genus have been usually used in traditional Chinese medicine as well as functional foods, cosmetics and other applications, such as L. japonica Thunb. Bioactive components and pharmacological activities of the genus Lonicera plants have received an increasing interest from the scientific community. Thus, a comprehensive and systematic review on their traditional usage in China, chemical components, and their pharmacological properties of their whole plants, bioactive extracts, and bioactive isolates including partial structure-activity relationships from the genus is indispensable.
Information on genus Lonicera of this systematic electronic literature search was gathered via the published articles, patents, clinical trials website (https://clinicaltrials.gov/) and several online bibliographic databases (PubMed, Sci Finder, Research Gate, Science Direct, CNKI, Web of Science and Google Scholar). The following keywords were used for the online search: Lonicera, phytochemical composition, Lonicerae japonica, Lonicera review articles, bioactivities of Lonicera, anti-inflammatory, antiviral, antimicrobial, anticancer, hepatoprotective, antioxidant, neuroprotective, anti-diabetic, and clinical trials. This review paper consists of a total of 225 papers covering the Lonicera genus from 1800 to 2021, including research articles, reviews, patents, and book chapters.
In this review (1800s-2021), about 420 components from the genus of Lonicera Linn. including 87 flavonoids, 222 terpenoids, 51 organic acids, and other compounds, together with their pharmacological activities including anti-inflammatory, antiviral, antimicrobial, anticancer, hepatoprotective, antioxidant, neuroprotective, antidiabetic, anti-allergic, immunomodulatory effects, and toxicity were summarized.
The relationship is discussed among their traditional usage, their pharmacological properties, and their chemical components, which indicate the genus Lonicera have a large prospect in terms of new drug exploitation, especially in COVID-19 treatment.
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Two new chemical constituents, japopenoid D (1), and japopenoid E (2), were isolated and identified from the flower buds of Lonicera japonica Thunb. The structures of these compounds were elucidated based on spectroscopic analysis (HR-ESI-MS, NMR), and the absolute configurations of 1 and 2 were determined by comparison of their electronic circular dichroism (ECD) spectra with literature and theoretical calculation. The anti-inflammatory activities of the isolates were evaluated by measuring their inhibitory effects on PGE₂ and IL-6 production in LPS stimulated RAW 264.7 macrophages. As a result, compound 1 could reduce PGE₂ and IL-6 levels in LPS-activated RAW 264.7 macrophages with IC₅₀ values of 6.78 and 42.07 μM, respectively.
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¹: H. Wan and L. Ge contributed equally to this work.

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3,4,5-Tri-O-caffeoylquinic acid methyl ester isolated from Lonicera japonica Thunb. Flower buds facilitates hepatitis B virus replication in HepG2.2.15 cells

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

High-performance liquid chromatography (HPLC) analysis of 3,4,5-CQME

Reagents and antibodies

Chemical structure and purity of 3,4,5-CQME

Effect of 3,4,5-CQME on HBV antigen expression in vitro

Discussion

Conclusion

CRediT authorship contribution statement

Declaration of competing interest

Acknowledgment

Life Sci.

Food Chem.

J. Hepatol.

Int. J. Biochem. Cell Biol.

Viral hepatitis B. Lancet.

J. Hepatol.

Vaccine

J. Ethnopharmacol.

Curr. Opin. Genet. Dev.

J. Hepatol.

Antivir. Res.

Micromachines

J. Hepatol.

Phytomedicine

J. Hepatol.

Antivir. Res.

J. Biol. Chem.

J. Ethnopharmacol.

Quantification of pregenomic RNA and covalently closed circular DNA in hepatitis B virus-related hepatocellular carcinoma

Int. J. Hepatol.

Hepatitis B viral HBx induces matrix metalloproteinase-9 gene expression through activation of ERK and PI-3K/AKT pathways: involvement of invasive potential

Faseb. J.

Suppression of p38 mitogen-activated protein kinase inhibits hepatitis B virus replication in human hepatoma cell: the antiviral role of nitric oxide

J. Viral Hepat.

Traditional Chinese medicine and related active compounds against hepatitis B virus infection

Biosci. Trends.

A nationwide study of the incidence rate of herb-induced liver injury in Korea

Arch. Toxicol.

Peroxiredoxin 1, a novel HBx-interacting protein, interacts with exosome component 5 and negatively regulates hepatitis B virus (HBV) propagation through degradation of HBV RNA

J. Virol.

Antioxidant activity of food constituents: an overview

Arch. Toxicol.

Novel caffeoylquinic acid derivatives from Lonicera japonica Thunb. flower buds exert pronounced anti-HBV activities

RSC Adv.

Hostile takeovers: viral appropriation of the NF-kappaB pathway

J. Clin. Invest.