Secretion expression of human neutrophil peptide 1 (HNP1) in Pichia pastoris and its functional analysis against antibiotic-resistant Helicobacter pylori.

Human neutrophil peptide 1 (HNP1) is a small (3.44 kDa) cationic peptide that is a distinct member of the defensin family. HNP1 plays a crucial role in controlling bacterial infections, particularly by antibiotic-resistant bacteria, through membrane perforation patterns. The structural characteristics of HNP1's three intramolecular disulfide bridges cause difficulty in its synthesis via chemical methods. In this study, bioactive recombinant HNP1 was produced using the Pichia pastoris (P. Pichia) expression system. HNP1 was fused with the polyhedrin of Bombyx mori and enhanced green fluorescent protein (EGFP) to prevent HNP1 toxicity in yeast host cells under direct expression. An enterokinase protease cleavage site (amino acid sequence DDDDK) was designed upstream of the HNP1 peptide to obtain the antibacterial peptide HNP1 with native structure after it was cleaved by the enterokinase. The fusion HNP1 protein (FHNP1) was successfully expressed and had a molecular mass of approximately 62.6 kDa, as determined using SDS-PAGE and Western blot. Then, the recovered FHNP1 was digested and purified; Tricine-SDS-PAGE results showed that HNP1 was successfully released from FHNP1. Functional analysis of induction against antibiotic-resistant Helicobacter pylori (H. pylori) showed that it was challenging for HNP1 to acquire resistance to the antibiotic-resistant H. pylori. Moreover, in vitro studies showed that HNP1 exerted a strong effect against antibiotic-resistant H. pylori activity. Furthermore, the animal model of H. pylori infection established in vivo showed that HNP1 significantly reduced the colonization of antibiotic-resistant H. pylori in the stomach. Our study indicated that this could be a new potential avenue for large-scale production of HNP1 for therapeutic application against the antibiotic-resistant H. pylori infection in humans.

Fusion expression of the PGLa-AM1 with native structure and evaluation of its anti-Helicobacter pylori activity.

Helicobacter pylori (H. pylori) shows increasingly enhanced resistance to various antibiotics, and its eradication has become a major problem in medicine. The antimicrobial peptide PGLa-AM1 is a short peptide with 22 amino acids and exhibits strong antibacterial activity. In this study, we investigated whether it has anti-H. pylori activity for the further development of anti-H. pylori drugs to replace existing antibiotics. However, the natural antimicrobial peptide PGLa-AM1 shows a low yield and is difficult to separate, limiting its application. A good strategy to solve this problem is to express the antimicrobial peptide PGLa-AM1 using gene engineering at a high level and low cost. For getting PGLa-AM1 with native structure, in this study, a specific protease cleavage site of tobacco etch virus (TEV) was designed before the PGLa-AM1 peptide. For convenience to purify and identify high-efficiency expression PGLa-AM1, the PGLa-AM1 gene was fused with the polyhedrin gene of Bombyx mori (B. mori), and a 6 × His tag was designed to insert before the amino terminus of the fusion protein. The fusion antibacterial peptide PGLa-AM1 (FAMP) gene codon was optimized, and the gene was synthesized and cloned into the Escherichia coli (E. coli) pET-30a (+) expression vector. The results showed that the FAMP was successfully expressed in E. coli. Its molecular weight was approximately 34 kDa, and its expression level was approximately 30 mg/L. After the FAMP was purified, it was further digested with TEV protease. The acquired recombinant antimicrobial peptide PGLa-AM1 exerted strong anti-H. pylori activity and therapeutic effect in vitro and in vivo.

Resveratrol Protects against Helicobacter pylori-Associated Gastritis by Combating Oxidative Stress.

Helicobacter pylori (H. pylori)-induced oxidative stress has been shown to play a very important role in the inflammation of the gastric mucosa and increases the risk of developing gastric cancer. Resveratrol has many biological functions and activities, including antioxidant and anti-inflammatory effect. The purpose of this study was to probe whether resveratrol inhibits H. pylori-induced gastric inflammation and to elucidate the underlying mechanisms of any effect in mice. A mouse model of H. pylori infection was established via oral inoculation with H. pylori. After one week, mice were administered resveratrol (100 mg/kg body weight/day) orally for six weeks. The mRNA and protein levels of iNOS and IL-8 were assessed using RT-PCR, Western blot and ELISA. The expression levels of IκBα and phosphorylated IκBα (which embodies the level and activation of NF-κB), Heme Oxygenase-1 (HO-1; a potent antioxidant enzyme) and nuclear factor-erythroid 2 related factor 2 (Nrf2) were determined using Western blot, and lipid peroxide (LPO) level and myeloperoxidase (MPO) activity were examined using an MPO colorimetric activity assay, thiobarbituric acid reaction, and histological-grade using HE staining of the gastric mucosa. The results showed that resveratrol improved the histological infiltration score and decreased LPO level and MPO activity in the gastric mucosa. Resveratrol down-regulated the H. pylori-induced mRNA transcription and protein expression levels of IL-8 and iNOS, suppressed H. pylori-induced phosphorylation of IκBα, and increased the levels of HO-1 and Nrf2. In conclusion, resveratrol treatment exerted significant effects against oxidative stress and inflammation in H. pylori-infected mucosa through the suppression of IL-8, iNOS, and NF-κB, and moreover through the activation of the Nrf2/HO-1 pathway.