Effect of iron saturation of bovine lactoferrin on the inhibition of hepatitis B virus in vitro

Abstract

Background: Hepatitis B virus (HBV) infection poses a major public health problem worldwide. Bovine lactoferrin (bLf) is a natural product that can inhibit HBV, but the effect of iron saturation on its resistance to HBV is unknown.

Aims: The purpose of this study is to investigate the impact of iron saturation of bLf against HBV.

Methods: HepG2 cells were cultured in DMEM high glucose containing 10% inactivated fetal calf serum, at 37 °C, in 5% CO₂. MTT method was used to detect the cytotoxicity of bLf to HepG2 cells. Apo-bLf and holo-bLf were prepared from bLf. Iron saturation of these proteins was determined by atomic absorption spectrophotometry. Non-cytotoxic concentrations of candidate proteins were used in anti-HBV tests. Fluorescent quantitative polymerase chain reaction was used to detect HBV-DNA.

Results: The TC₅₀ and TC₀of bLf were 54.570 mg/ml and 1.997 mg/ml, respectively. The iron saturation of bLf, apo-bLf and holo-bLf were 10.29%, 8.42% and 85.32%, respectively. In this study, four non-cytotoxic concentrations of candidate proteins (1.5, 1.0, 0.5, and 0.1 mg/ml, respectively) were used to inhibit HBV in HepG2 cells. The results showed that 1.5 mg/ml bLf and 0.1 mg/ml holo-bLf effectively impaired the HBV-DNA amplification in HBV-infected HepG2 cells (P < 0.05). However, apo-bLf, and Fe³⁺ did not show the anti-HBV effects.

Conclusion: A total of 1.5 mg/ml bLf and 0.1 mg/ml holo-bLf could inhibit HBV-DNA in HepG2 cells. Complete bLf structure, appropriate concentration and iron saturation of bLf are necessary conditions for anti-HBV effects.

Keywords: Bovine lactoferrin; HepG2 cell; Hepatitis B virus; Iron saturation; Polymerase chain reaction.

Figure 1. (A) Electrophoretogram of lactoferrin with different iron saturation. (B) The standard curve of iron saturation of lactoferrin. (C) Fluorescence spectra of lactoferrin with different iron saturation.

Figure 2. (A) Standard curve of HBV DNA detected by FQ-PCR. (B) FQ-PCR amplification curves of standard, the concentrations of standards are 1 ×10⁸ copies/ml, 1 ×10⁷ copies/ml, 1 ×10⁶ copies/ml, 1 ×10⁵ copies/ml, 1 ×10⁴ copies/ml, respectively.

Figure 3. The effect of candidate agents inhibits HBV.

Non-toxic concentrations of each protein are 1.5, 1.0, 0.5 and 0.1 mg/ml, respectively. 2,500 IU/ml IFN-alpha and 6 μg/ml FeCl₃ were involved in this test. The infected cells without any treatment are defined as positive groups. Y-axis represents the natural logarithm of HBV copies/ml. (A) Inhibition of bLf on HBV virus. (B) Inhibition of apo-bLf on HBV virus. (C) Inhibition of holo-bLf on HBV virus. (D) Inhibition of Fe³⁺ on HBV virus.