BABL/c mice (8–10 weeks of age) were obtained from the Animal Center, Kunming Medial University (Yunnan, China). The animals were housed at 21–22°C, with a 12-h light/dark cycle of 12:12 h. The protocol of the animal experiments was approved by the Animal Care and Use Committee of Kunming Medical University. All animal experiments complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication no. 8523, revised 1985).

The mice received an intragastric administration of 0.1 ml of a suspension of H. pylori ATCC 43504 (a gift from Professor Lu Hong, Shanghai Jiao Tong University School of Medicine, Shanghai, China) in brain-heart infusion broth (BHI broth; 10⁸ CFU/ml) once daily for 4 consecutive days. A total of 192 mice was divided into 4 groups as follows: mice in group A wre treated intragastrically with saline; mice in group B were treated intragastrically with 1.5 g of rhLf derived from the milk of transgenic goats (Shanghai Jielong Bioengineering Co., Ltd., Shanghai, China); mice in group C were treated intragastrically with the standard triple regimen [clarithromycin (Goldensun Pharmaceutical Co., Ltd., Xiamen, China), amoxicillin (Baker Norton Pharmaceutical Co., Ltd., Kunming, China) and a proton pump inhibitor (omeprazole; Harbin Pharmaceutical Co., Ltd., Harbin, China)]; and mice in group D were treated intragastrically with the triple therapy regimen plus 1.5 g of rhLf daily. Following 7 days of treatment, the mice were sacrificed by cervical dislocation, and a part of the stomach tissue of each mouse was immediately removed and quickly frozen in liquid nitrogen. The remaining parts of the stomach tissue were fixed in 10% formalin, dehydrated in graded ethyl alcohol and embedded in paraffin.

The paraffin-embedded sections were examined for the presence of H. pylori by silver staining. The bacteria were characterized as a spiral rod of 3.0–0.5 mm, located adjacent to the gastric epithelium. The sections were also stained with hematoxylin and eosin (H&E), and were scored for their degrees of inflammation based on the intensity of neutrophilic and/or lymphocytic infiltration.

H. pylori ATCC 43504 was inoculated onto a Columbia Agar base (CAB; Qingdao Hope Bio-Technology Co., Ltd., Qingdao, China) supplemented with 10% (v/v) fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), 1% (v/v) mixed antibiotics [trimethoprim, 5 g/l; polymyxin B sulfate, 3.85 g/l; amphotericin B, 10 g/l; and vancomycin, 10 g/l (all obtained from Solarbio Co., Beijing, China)] in a jar with an AnaeroPack Campylo sachet (Mitsubishi Gas Chemical Co., Tokyo, Japan) at 37°C for 3–5 days. For liquid culture, the plate-grown bacteria were inoculated into liquid BHI broth (Qingdao Hope Bio-Technology) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) mixed antibiotics, with an initial optical density at 600 nm (OD600) of 0.1.

After the H. pylori bacteria were allowed to grow for 18–20 h in the BHI liquid medium, the bacterial cultures were diluted in 10 ml of BHI liquid medium with rhLf (0–0.5 mg/ml) or desferrioxamine (DFO; Chengdu Best Reagent Co., Ltd., Chengdu, China) (0–50 µM), to an initial OD600 of 0.1. The H. pylori broth cultures were then incubated at 37°C. Every 8 h, over a 64-h time period, a 200-µl sample of each bacterial culture was isolated and the OD600 measured. The effect of ferric ammonium citrate (FAC; Chengdu Best Reagent Co., Ltd.) (0–50 µM) in the presence of 0.5 mg/ml rhLf on bacterial growth was examined following the same procedure.

Total RNA from the H. pylori cultures was isolated using TRIzol reagent (Takara, Dalian, China). cDNA was generated by the reverse transcription of total RNA using a PrimeScript™ RT reagent kit with a gDNA Eraser (Takara). Quantitative PCR (qPCR) was conducted using SYBR^®Premix Ex Taq™ II (Takara) on a LightCycler^® 480 system (Roche Applied Science, Penzberg, Germany). 16S rRNA was used as an internal control. The primers used for PCR were as follows: cytotoxin-associated gene A (CagA) forward, 5′-GGGCGTG TTTGATGAGTCCT-3′ and reverse, 5′-TGT ATG TCG GTG GTG GTA GTG-3′; vacuolating cytotoxin A (VacA) forward, 5′-CCA ACT TAC CCA CAA ACA CC-3′ and reverse, 5′-TAG CCA ATT CAA ACA CGC TC-3′; urease (Ure) forward, 5′-TTC TTC TGC CTG GAG TGA TAG T-3′ and reverse, 5′-TTC TTC TGC CTG GAG TGA TAG T-3′; and 16S rRNA forward, 5′-TGT GGG AGA GGT AGG TGG AA-3′ and reverse, 5′-CAT CGT TTA GGG CGT GGA CT-3′.

The bacteria were collected and washed with PBS. Total proteins were extracted using a radioimmu-noprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Beijing, China). The lysates (25 µg) of total protein were loaded and run on a 10% polyacrylamide gel and transferred onto a polyvinylidene fluoride (PVDF) membrane.

The primary antibodies used were anti-CagA (sc-25766, rabbit polyclonal antibody; 1:3,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-VacA (sc-25790, rabbit polyclonal antibody; 1:3,000 dilution; Santa Cruz Biotechnology), anti-Ure (sc-21016, rabbit polyclonal antibody; 1:3,000 dilution; Santa Cruz Biotechnology), and anti-GAPDH antibodies (GA1R, mouse monoclonal antibody; 1:5,000 dilution; Thermo Fisher Scientific, Waltham, MA, USA). The secondary antibody was a horseradish peroxidase-coupled anti-rabbit or mouse IgG (1:5,000 dilution; Santa Cruz Biotechnology). The membrane was exposed to Kodak X-Omat film (Kodak, Xiamen, China), and the film was then developed.

After the stomach tissues were homogenized in liquid nitrogen, the homogenate was lysed on ice for 60 min in lysis buffer (BioTeke, Beijing, China). The protein concentration was determined in the supernatant by a Bradford protein assay kit (BioTeke) using bovine serum albumin as the standard. The levels of TNF-α and IL-8 in the supernatant were measured using commercially available ELISA kits (Xinbosheng Biotechnology Co., Ltd., Shenzhen, China). The values of TNF-α and IL-8 in the stomach tissues were normalized to the protein concentration.

The statistical significance of the differences in gene and protein expression and in the levels of inflammatory factors were assessed by one-way analysis of variance (ANOVA), followed by a Student-Newman-Keuls test. The differences in bacterial eradication and inflammation were analyzed using the Wilcoxon rank-sum test. Data were analyzed using SPSS 11.0 software (SPSS, Inc., Chicago, IL, USA).

It has been shown that rhLf obtained from bacteria, yeast, or rice is an effective adjuvant for use in the treatment of H. pylori (18,22). In order to determine the efficacy of rhLf derived from goat milk in the treatment of H. pylori, we first examined the effect of rhLf on the growth of H. pylori. As shown in Fig. 1A, treatment with rhLf suppressed the growth of H. pylori in a concentration-dependent manner. The growth of H. pylori was almost completely inhibited by rhLf at a concentration of 0.5 mg/ml. By contrast, H. pylori in the control group, which was not treated with rhLf, reached a growth peak after 48 h.

Lactoferrin has been shown to exhibit bacteriostatic activities, probably due to its ability to chelate iron (10). We found that similar to rhLf, DFO, a well-known chelating agent, also effectively suppressed the growth of H. pylori in a dose-dependent manner (Fig. 1B). By contrast, the addition of FAC significantly restored the growth of H. pylori (Fig. 1C). However, the growth of H. pylori was not restored to the same level as the control group even with a saturated iron supplement, suggesting that other mechanisms, aside from the chelation of iron, also participate in the bacteriostatic activity of rhLF.

We then determined whether rhLf affects the expression of 3 major virulence factors (CagA, VacA and Ure) by RT-qPCR. As shown in Fig. 2, a significant decrease in the mRNA levels of 2 of these virulence factors (CagA and VacA) following treatment with 0.4 mg/ml of rhLf was noted. The results were further confirmed by western blot analysis (Fig. 3). The protein levels of CagA and VacA were suppressed following treatment with 0.4 mg/ml of rhLf (Fig. 3A and B). By contrast, rhLf did not affect the expression of Ure (Fig. 3C). These results suggest that lactoferrin suppresses the expression of 2 major pathogenic virulence factors of H. pylori.

In the present study, the infected mice were divided into 4 groups: those treated with saline, rhLf, the standard triple therapy regimen, or the standard triple therapy regimen plus rhLf (quadruple treatment). To determine the efficiency of rhLf in the eradication of H. pylori in vivo, we performed semi-qualitative analysis of the distribution of H. pylori in the stomach tissues; silver staining was applied to the formalin-embedded tissue sections. As shown in Fig. 4, the number of H. pylori bacteria was significantly higher in the rhLf group than in the group which received the standard triple therapy regimen. However, quadruple treatment exerted a profound protective effect against the bacteria, as demonstrated by the complete absence of H. pylori bacteria in the silver-stained histological sections.

To determine the extent of inflammation, the stomach tissues from the mice in the 4 groups were harvested and stained with H&E. The group treated with rhLf alone exhibited a similar degree of gastric inflammation to the group treated with saline (control group), and the standard triple therapy regimen significantly reduced inflammation compared with these 2 groups. Notably, quadruple treatment reduced inflammation to a greater extent than the triple therapy regimen (Fig. 5A). Subsequently, we determined the levels of the inflammatory cytokines TNF-α and IL-8 in the stomach tissues (Fig. 5B). Unlike the standard triple therapy regimen, which markedly inhibited the increase in the TNF-α and IL-8 levels induced by the bacterium, treatment with rhLf alone resulted in only a slight decrease in the TNF-α and IL-8 levels (Fig. 5B). However, both the TNF-α and IL-8 levels markedly decreased in the group that received the quadruple treatment, compared to the group treated with the the triple therapy regimen alone. These results suggest that the addition of rhLf to the triple therapy regimen further inhibits the inflammatory response.