Anti-Hepatitis B Virus Effect and Possible Mechanism of Action of 3,4-O-Dicaffeoylquinic Acid In Vitro and In Vivo

The anti-hepatitis B activity of 3,4-O-dicaffeoylquinic acid isolated from Laggera alata was studied using the D-galactosamine- (D-GalN-) induced hepatocyte damage model, HepG2.2.15 cells, and with HBV transgenic mice. In vitro results showed that 3,4-O-dicaffeoylquinic acid improved HL-7702 hepatocyte viability and markedly inhibited the production of HBsAg and HBeAg. At a concentration of 100 μg/mL, its inhibitory rates on the expression levels of HBsAg and HBeAg were 89.96% and 81.01%, respectively. The content of hepatitis B virus covalently closed circular DNA (HBV cccDNA) in HepG2.2.15 cells was significantly decreased after the cells were treated with the test compound. In addition, 3,4-O-dicaffeoylquinic acid significantly increased the expression of heme oxygenase-1 (HO-1) in HepG2.2.15 cells. In vivo results indicated that the test compound at concentrations of 100 μg/mL significantly inhibited HBsAg production and increased HO-1 expression in HBV transgenic mice. In conclusion, this study verifies the anti-hepatitis B activity of 3,4-O-dicaffeoylquinic acid. The upregulation of HO-1 may contribute to the anti-HBV effect of this compound by reducing the stability of the HBV core protein, which blocks the refill of nuclear HBV cccDNA. Furthermore, the hepatoprotective effect of this compound may be mediated through its antioxidative/anti-inflammatory properties and by the induction of HO-1 expression.


Introduction
Hepatitis B is an infectious illness caused by hepatitis B virus (HBV), which infects the liver of Hominoidea, including humans, and causes an inflammation reaction called hepatitis. Although there is an effective vaccine against HBV, chronic infection poses a huge health burden on the global community [1]. Its prevalence approaches 10% in hyperendemic areas such as Southeast Asia, China, and Africa [2]. Furthermore, approximately one-third of the world's population (more than 2 billion people) have been infected with the hepatitis B virus, which includes 350 million chronic carriers of the virus [3]. Some antiviral agents such as interferon-α and nucleosides (including lamivudine and adefovir dipivoxil) have been approved for the treatment of chronic HBV infection. However, a significant number of patients develop drug resistance after long-term use of these agents [4]. Therefore, there is a pressing need to continue developing safer and more effective anti-hepatitis B agents.
The development of natural substances as antiviral agents is thought to be a promising approach towards solving this public health concern [5].
Laggera alata belongs to the genus Laggera (Asteraceae) and is distributed mainly among the tropical areas of Africa, Southeast Asia, and China. This plant has been used as a folk medicine for over 300 years, especially for the treatment of some ailments associated with hepatitis [6]. Most of the previous studies examining L. alata have focused on its folk use and phytochemical analyses [7][8][9]. In previous investigations, we examined the anti-inflammatory and hepatoprotective activities of an L. alata extract containing dicaffeoylquinic acids and confirmed its potent effects [10,11]. In this study, we utilized the d-galactosamine-(d-GalN-) induced HL-7702 hepatocyte damage model, HBVtransfected HepG2.2.15 cells, and HBV transgenic mice to evaluate the anti-hepatitis B activity and possible hepatoprotective mechanisms of 3,4-O-dicaffeoylquinic acid isolated from L. alata (Figure 1). The study is the first to demonstrate  After the cells were incubated for 8 days, the MTT assay was carried out as described previously [11].

Materials and Methods
To measure the effect of 3,4-O-dicaffeoylquinic acid on the expression of HBV antigens and HBV DNA, HepG2.2.15 cells were treated with various concentrations of the test compound in the 96-well plates. The medium with the compound was replaced every 4 days. On the fourth day, the replaced medium was assayed for HBsAg and HBeAg. On the eighth day, the replaced medium was measured for HBsAg, HBeAg and HBV DNA. Lamivudine was used as the reference drug. The levels of HBsAg and HBeAg in the replaced culture supernatants were determined by HBsAg and HBeAg enzyme-immunoassay kits, respectively. The results were read at 450 nm by a multiwell plate reader (MULTISKAN MK3, Thermo Fisher Scientific Inc., USA). The HBV viral load in the replaced culture supernatants was detected with a HBV DNA PCR-fluorescence quantitation kit as follows: HBV DNA was extracted and amplified with a Bio-Rad iQ5 real-time PCR system. The forward primer was 5-CCG TCT GTG CCT TCT CAT CTG-3, the reverse primer was 5-AGT CCA AGA GTA CTC TTA TAG AAG ACC TT-3, and the Taqman probe was FAM-CCG TGT GCA CTT CGC TTC ACC TCT GC. The thermal program comprised of an initial  25, and 10 μg/mL) was added. Three parallel controls were performed, including positive controls with oxymatrine (50 μg/mL), a vehicle control of 0.1% DMSO, and a normal control with no antiviral drug. The medium with the compound was replaced every 3 days. On the sixth day, the cells of each well were harvested. Based on the similarity of cccDNA and plasmid structures, the cell pellet containing 1.0 × 10 6 cells was extracted with the Mini Plasmid Extraction Kit. The extracted plasmid was further purified by plasmid safe ATP-dependent DNase to remove the residual HBV relaxed circular DNA. Hepatitis B virus covalently closed circular DNA (HBV cccDNA) was detected by selective realtime fluorescent quantitative PCR with specific primers and a Taqman MGB probe [13]. The forward primer was 5-TGA ATC CTG CGG ACG ACC-3, the reverse primer was 5-ACA GCT TGG AGG CTT GAA CAG-3 and the Taqman probe was 5-FAM-CCT AAT CAT CTC TTG TTC ATG TC-MGB-3. According to the structural differences between cccDNA and rcDNA, only the cccDNA should have been amplified with the designed primers and probe. The serum was separated for the measurements of HBsAg, and HO-1. The serum HBsAg and HO-1 levels were determined using the HBsAg and HO-1 detection kits according to the respective protocols provided with the ELISA kits. For histopathological analysis, formalin-fixed, paraffinembedded liver specimens were routinely stained with hematoxylin and eosin (HE). The liver HBsAg expression level was determined using the HBsAg immunohistochemical detection kit according to the manufacturer's instructions. The pathological and immunohistochemical changes were evaluated and photographed under the microscope.

Statistical
Analysis. Data were expressed as the mean ± standard deviation. Statistical analyses were carried out by the application of one-way analysis of variance (ANOVA) and student's t-test. P < 0.05 was chosen as the criterion for statistical significance.  Table 3). The HBsAg and HBeAg levels in culture supernatants were assayed after the cells were   incubated with the test compound for 4 days ( Table 4). The results showed that the test compound significantly inhibited HBsAg expression at concentrations of 50-100 μg/mL and markedly repressed HBeAg expression at a concentration of 100 μg/mL. The HBsAg, HBeAg and HBV DNA levels in culture supernatants were measured after the cells were treated with the test compound for 8 days (Table 5). At concentrations of 50-100 μg/mL, the test compound significantly inhibited the expression of HBsAg and HBeAg. At a concentration of 100 μg/mL, the test compound inhibited the expression rates of HBsAg and HBeAg by 89.96% and 81.01%, respectively. Table 6. The results indicated that 3,4-O-dicaffeoylquinic acid significantly reduced the HBV cccDNA content of HepG2.2.15 cells at a concentration of 50 μg/mL. Furthermore, the test compound exhibited a larger effect than the reference drug oxymatrine.  (Table 7). At concentrations of 10-50 μg/mL, 3,4-Odicaffeoylquinic acid significantly increased HO-1 expression. Oxymatrine, which was the reference drug, showed a similar effect.

Anti-HBV Activity of 3,4-O-Dicaffeoylquinic Acid in HBV Transgenic
Mice. The anti-HBV activity of 3,4-Odicaffeoylquinic acid was determined in HBV transgenic mice (Table 8). These results show that the test compound significantly reduced the serum HBsAg level at concentrations of 50-100 μg/mL. Meanwhile, the test compound markedly induced HO-1 expression at a concentration of 100 μg/mL. Histological analysis revealed almost normal lobule architecture and slight swelling of liver cells, but no obvious pathological changes were observed in the control and drug-treated mice ( Figure 2). Immunohistochemical detection indicated that the strongest HBsAg-positive signals were detected in the control group, and the different concentrations of test compound clearly repressed the expression of liver HBsAg (Figure 3).

Discussion
Patients with hepatitis B are often treated with antiviral agents, hepatoprotective drugs, and immunomodulatory drugs. Typically, the beneficial role of hepatoprotectors in viral hepatitis is achieved through their inhibitory action on the inflammatory and cytotoxic cascades induced by viral infection. In addition, these agents can also improve the regeneration process and normalize liver enzymes through their effects on protein synthesis [14]. Among the numerous models of experimental hepatitis, d-GalN-induced liver damage is very similar to human viral hepatitis in its morphological and functional features [15]. d-GalN reduces the intracellular pool of uracil nucleotides in hepatocytes,  thereby inhibiting the synthesis of RNA and proteins [16]. Oxygen-derived free radicals released from activated hepatic macrophages are the primary cause of d-GalN-induced liver damage [17]. In previous studies, the potent antiinflammatory and hepatoprotective activities of an L. alata extract containing dicaffeoylquinic acids were confirmed [10,11]. Furthermore, dicaffeoylquinic acids exhibit a variety of pharmacological activities, such as antioxidative, antiinflammatory, and antiviral effects [18][19][20]. In the current study, 3,4-O-dicaffeoylquinic acid protected d-GalN-injured Evidence-Based Complementary and Alternative Medicine   hepatocytes, thereby implying that its antioxidative and anti-inflammatory properties may have contributed to the amelioration of hepatocyte damage. HepG2.2.15 cells are derived from human hepatoblastoma HepG2 cells that were transfected with a plasmid containing HBV DNA. These cells can stably secrete viral particles in culture medium [21]. The presence of HBsAg is the Oxymatrine was used as the positive control. 0.1% DMSO was used as the vehicle control. Data are expressed as the means ± SD of three independent experiments. * P < 0.05 and * * P < 0.01 compared with the vehicle group.
most common marker of HBV infection, whereas HBeAg is used as an ancillary marker primarily to indicate active HBV replication and associated progressive liver disease [22].  Lamivudine was used as the positive control in the anti-HBV assay. Normal saline solution was used as the vehicle control. P/N (positive-to-negative) ratios were determined as the mean absorbency value of the test compounds divided by that of the negative control. Data are expressed as the means ± SD of four samples. * P < 0.05 and * * P < 0.01 compared with the vehicle group.
Hepadnaviruses have a relaxed circular DNA genome. Following infection of hepatocytes, this DNA is transported to the nucleus and converted to a covalently closed form (cccDNA) that serves as a transcriptional template. Viral DNA is synthesized within nucleocapsids via reverse transcription of a viral RNA known as the pregenome [23]. Nucleocapsids containing mature forms of viral DNA are packaged into viral envelopes and secreted from the cell. cccDNA does not replicate; however, additional copies (up to 50 per cell) may be formed from viral DNA synthesized in the cytoplasm [24]. The cccDNA plays a key role in the life cycle of the virus and permits the persistence of infection. The formation of cccDNA is inhibited by viral envelope proteins [25]. cells and its effect was stronger than the reference drug oxymatrine, thereby indicating the anti-HBV effect of this compound was probably related to inhibiting the formation of cccDNA. HBV transgenic mice with a known genetic background and a well-characterized HBV isolate have been employed as an animal model of the HBV-carrier state and are thought to be a good model to evaluate the anti-HBV efficacy of candidate compounds in vivo [12,26]. Based on in vitro results, we further studied the anti-hepatitis B activity of 3,4-O-dicaffeoylquinic acid in HBV transgenic mice. The results indicated that 3,4-O-dicaffeoylquinic acid significantly inhibited the serum and liver HBsAg levels and significantly increased HO-1 expression in these transgenic mice, which was in good agreement with the results of in vitro research. Upon histopathological analysis, no obvious pathological changes were found in both the control and experimental groups of mice, which is probably related to the immunotolerance of HBV transgenic mice to HBV. Because these transgenic mice are immunotolerant to HBV, they did not present with any of the disease signs that would normally be associated with immunopathological responses [27].
Heme oxygenases catalyze the initial and rate-limiting step in the oxidative degradation of heme. Among the three known heme oxygenases, HO-1 is the only inducible form of these enzymes [28]. Overexpression of HO-1 protects organs and/or tissues from immune-mediated organ injury, which can occur either through the prevention of oxidative damage or via local immunomodulatory influence on inflammatory cells [29]. Induction of HO-1 has been shown to be beneficial in immune-mediated liver damage. In addition, liver injury was significantly reduced after HO-1 induction in an acute hepatitis B model [30]. In addition to its hepatoprotective effect, HO-1 exhibited a pronounced antiviral effect, which was confirmed in stably HBV-transfected hepatoma cells and in persistently HBV replicating transgenic mice. HO-1 induction repressed HBV replication directly in hepatocytes at a posttranscriptional step by reducing the stability of the HBV core protein, which resulted in blocking the refill of nuclear HBV cccDNA. Small interfering RNAs directed against HO-1 have demonstrated that this effect is dependent on the expression level of HO-1 [30]. Therefore, the induction of HO-1 might be a novel therapeutic option for inflammatory flares of hepatitis B. In this study, 3,4-Odicaffeoylquinic acid significantly increased the expression of HO-1 in vitro and in vivo, thereby suggesting that the hepatoprotective and anti-HBV effects of 3,4-O-dicaffeoylquinic acid were achieved by HO-1 induction.
In conclusion, this study verifies the in vitro and in vivo anti-hepatitis B effects of 3,4-O-dicaffeoylquinic acid isolated from L. alata. The upregulation of HO-1 may contribute to the anti-HBV effect of this compound by reducing the stability of the HBV core protein and by blocking the refill of nuclear HBV cccDNA. Additionally, the hepatoprotective effect of this compound was mediated by its antioxidative/ anti-inflammatory properties and the induction of HO-1. Therefore, 3,4-O-dicaffeoylquinic acid should be considered a potential candidate or lead compound for the development of novel antiviral agents.