Phillygenin Inhibits Helicobacter pylori by Preventing Biofilm Formation and Inducing ATP Leakage.

With the widespread use and abuse of antibiotics, Helicobacter pylori (H. pylori) has become seriously drug resistant. The development of new antibiotics is an important way to solve H. pylori's drug resistance. Screening antibacterial ingredients from natural products is a convenient way to develop new antibiotics. Phillygenin, an effective antibacterial component, was selected from the natural product, forsythia, in this study. Its minimal inhibitory concentration (MIC) for 18 H. pylori strains was 16-32 µg/ml. The minimum bactericidal concentration (MBC) of H. pylori G27 was 128 µg/ml; the higher the drug concentration and the longer the time, the better the sterilization effect. It was non-toxic to gastric epithelial cell (GES)-1 and BGC823 cells at the concentration of 100 µg/ml. It presented a better antibacterial effect on H. pylori in an acidic environment, and after 24 days of induction on H. pylori with 1/4 MIC of phillygenin, no change was found in the MIC of H. pylori. In the mechanism of action, phillygenin could cause ATP leakage and inhibit the biofilm formation; the latter was associated with the regulation of spoT and Hp1174 genes. In addition, phillygenin could regulate the genes of Nhac, caggamma, MATE, MdoB, flagellinA, and lptB, leading to the weakening of H. pylori's acid resistance and virulence, the diminishing of H. pylori's capacity for drug efflux, H. pylori's DNA methylation, the initiation of human immune response, and the ATP leakage of H. pylori, thus accelerating the death of H. pylori. In conclusion, phillygenin was a main ingredient inhibiting H. pylori in Forsythia suspensa, with a good antibacterial activity, high safety, strong specificity, better antibacterial effect under acidic conditions, and low risk of resistance development by H. pylori. Its mechanism of action was mainly associated with inhibiting the biofilm formation and resulting in ATP leakage. In addition, phillygenin was shown to be able to reduce the acid resistance and virulence of H. pylori.

Improved method for inducing chronic atrophic gastritis in mice.

BACKGROUND: Chronic atrophic gastritis (CAG) is a common disease of the digestive system with pathological characteristics of a decreasing number, or disappearance, of inherent glands of the gastric mucosa. CAG has been defined as a precancerous condition of gastric cancer. Intestinal metaplasia or intraepithelial neoplasia accompanying atrophied glands of the stomach is regarded as one of the most important precancerous lesions of gastric cancer. As a common malignant tumour, gastric cancer remains without a satisfactory therapy and its pathogenesis remains unclear, seriously threatening human life. Therefore, some scholars have proposed to prevent the incidence of gastric cancer by avoiding precancerous lesions. If CAG can be reversed, the incidence of gastric cancer can be substantially reduced. To reverse and prevent CAG and study its pathogenesis and therapy, it is necessary to develop an ideal, safe, stable, animal model. AIM: To study a rapid, stable, and safe method of establishing a mouse model of human CAG. METHODS: Six-week-old Kunming mice were divided into a phosphate buffered solution control group, a Helicobacter pylori (H. pylori) group, an N-methyl-N'-nitroguanidine (MNNG) group, an ammonia water group, and a group combining H. pylori, MNNG, and ammonia water (hereinafter referred to as the combined group). The mice were administrated with drinking water containing ammonia or infected with H. pylori through gavage. At the 30th, 60th, 90th, and 120th day after the last H. pylori infection, mice were selected randomly to collect their gastric mucosa for hematoxylin eosin staining, terminal nick-end labelling staining detection, and immunohistochemical staining for Bax and Bcl-2. In addition, H. pylori was isolated, cultured, and identified, and its extent of colonisation calculated. Blood was collected to detect inflammatory factors interleukin (IL)-1ß, IL-8, and tumor necrosis factor (TNF)-α and immune function markers CD4 and CD8 to confirm successful establishment of the CAG model. RESULTS: The combined group showed slight CAG at the 90th day and moderate CAG at the 120th day, while other groups did not show CAG at that time. CONCLUSION: The combination of H. pylori, MNNG, and ammonia is an effective method of developing a mouse model of human CAG.

Molecular mechanisms of apoptosis in hepatocellular carcinoma cells induced by ethanol extracts of Solanum lyratum Thumb through the mitochondrial pathway.

AIM: To explore the induction effects and mechanism of Solanum lyratum Thumb (ST) on human hepatocellular carcinoma SMMC-7721 cells through the mitochondrial pathway. METHODS: The experiments were conducted on three groups: an experimental group (with ST ethanol extracts' concentration being 2.5, 5 and 10 mg/L), a negative control group (with only nutrient solution, 0 mg/L ST ethanol extracts), and a positive control group (2.5 mg/L DDP). The inhibition rate of cell proliferation was checked by using the methyl thiazolyl tetrazolium method, and cell apoptosis was tested by TUNEL method. Furthermore, RT-PCR was used to examine mRNA expression of Fas, FasL, caspase-8, caspase-3, p53 and Bcl-2 genes. RESULTS: Compared with the negative control group, the inhibition and apoptosis rates of the experimental group with different concentrations of ST extracts on human hepatocellular carcinoma SMMC-7721 cells significantly increased (P < 0.05). Besides, the mRNA expression of FasL and Bcl-2 significantly decreased (P < 0.05) while the mRNA expression of Fas, caspase-8, caspase-3 and p53 increased significantly. When compared with the positive control group, the experimental groups with 5 mg/L ST ethanol extracts showed effects similar to the positive control group. CONCLUSION: ST ethanol extracts induced the apoptosis of hepatocellular carcinoma SMMC-7721 cells through up-regulated Fas, caspase-8, caspse-3 and p53, and down-regulated FasL and Bcl-2 in the mitochondrial pathway.

Gene polymorphisms of pathogenic Helicobacter pylori in patients with different types of gastrointestinal diseases.

Helicobacter pylori (H. pylori) is a kind of chronic infectious pathogen which can cause chronic gastritis, peptic ulcer, gastric cancer and other diseases. The genetic structure of the pathogenic genes of H. pylori varies largely, which contributes to the differences in virulence among various strains, and in clinical symptoms. Virulence genes of H. pylori can be categorized into three main classes: those related to adhesion and colonization, those related to gastric mucosal injury, and others. This review focuses on the relationship between genetic polymorphisms of the three classes of virulence genes of H. pylori and diseases. Most of the genetic polymorphisms of the main virulence factors of H. pylori are summarized in this paper.

Inhibitory effects of emodin, baicalin, schizandrin and berberine on hefA gene: treatment of Helicobacter pylori-induced multidrug resistance.

AIM: To investigate the inhibitory effects of emodin, baicalin, etc. on the hefA gene of multidrug resistance (MDR) in Helicobacter pylori (H. pylori). METHODS: The double dilution method was used to screen MDR H. pylori strains and determine the minimum inhibitory concentrations (MICs) of emodin, baicalin, schizandrin, berberine, clarithromycin, metronidazole, tetracycline, amoxicillin and levofloxacin against H. pylori strains. After the screened MDR stains were treated with emodin, baicalin, schizandrin or berberine at a 1/2 MIC concentration for 48 h, changes in MICs of amoxicillin, tetracycline, levofloxacin, metronidazole and clarithromycin were determined. MDR strains with reduced MICs of amoxicillin were selected to detect the hefA mRNA expression by real-time quantitative PCR. RESULTS: A total of four MDR H. pylori strains were screened. Treatment with emodin, baicalin, schizandrin and berberine significantly decreased the MICs of amoxicillin and tetracycline against some strains, decreased by 1 to 2 times, but did not significantly change the MICs of clarithromycin, levofloxacin, and metronidazole against MDR strains. In the majority of strains with reduced MICs of amoxicillin, hefA mRNA expression was decreased; one-way ANOVA (SPSS 12.0) used for comparative analysis, P < 0.05. CONCLUSION: Emodin, baicalin, schizandrin and berberine significantly decreased the MICs of amoxicillin and tetracycline against some H. pylori strains, possibly by mechanisms associated with decreasing hefA mRNA expression.

Helicobacter pylori isolates from ethnic minority patients in Guangxi: resistance rates, mechanisms, and genotype.

AIM: To investigate the rate of Helicobacter pylori (H. pylori) resistance to clarithromycin among ethnic minority patients in Guangxi, explore the underlying mechanisms, and analyze factors influencing genotype distribution of H. pylori isolates. METHODS: H. pylori strains were isolated, cultured and subjected to drug sensitivity testing. The 23S rRNA gene of H. pylori isolates was amplified by PCR and analyzed by PCR-RFLP and direct sequencing to detect point mutations. REP-PCR was used for genotyping of H. pylori isolates, and NTsys_2 software was used for clustering analysis based on REP-PCR DNA fingerprints. Factors potentially influencing genotype distribution of H. pylori isolates were analyzed. RESULTS: The rate of clarithromycin resistance was 31.3%. A2143G and A2144G mutations were detected in the 23S rRNA gene of all clarithromycin-resistant H. pylori isolates. At a genetic distance of 78%, clarithromycin-resistant H. pylori isolates could be divided into six groups. Significant clustering was noted among H. pylori isolates from patients with peptic ulcer or gastritis. CONCLUSION: The rate of clarithromycin resistance is relatively high in ethnic minority patients in Guangxi. Main mechanisms of clarithromycin resistance are A2143G and A2144G mutations in the 23S rRNA gene. Clarithromycin-resistant H. pylori isolates can be divided into six groups based on REP-PCR DNA fingerprints. Several factors such as disease type may influence the genotype distribution of H. pylori isolates.

Genetic polymorphisms in DNA repair genes XRCC4 and XRCC5 and aflatoxin B1-related hepatocellular carcinoma.

BACKGROUND: Genetic polymorphisms in DNA repair genes may influence individual variation in DNA repair capacity and may play an important role in carcinogenesis. We investigated the role of genetic polymorphisms at XRCC4 codon 247 (rs3734091, XRCC4P) and XRCC5 codon 180 (rs80309960, XRCC5P) in liver cancer (hepatocellular carcinoma) caused by aflatoxin B1 (AFB1). METHODS: A hospital-based case-control study, including 1499 liver cancer cases and 2045 controls without any liver disease, was conducted in a high aflatoxin exposure area in the Guangxi region of China to assess the relationship between these two polymorphisms and aflatoxin-related liver cancer risk and prognosis. Genotypes, mRNA levels, and the hot-spot mutation of TP53 gene (TP53M) related to AFB1 exposure was tested using TaqMan-PCR technique. XRCC4 protein level was analyzed by immunohistochemistry. RESULTS: For XRCC4P and XRCC5P, only XRCC4P modified liver cancer risk. Compared with the homozygote of XRCC4 codon 247 Ala alleles (XRCC4-AA), the genotypes of XRCC4 codon 247 Ser alleles (namely XRCC4-AS or -SS) increased liver cancer risk (odds ratio [OR] = 1.35 and 2.02, respectively). Significant interactive effects between risk genotypes (OR > 1) and aflatoxin exposure status were also observed in the joint effects analysis. Moreover, this polymorphism was associated not only with lower XRCC4 expression levels but also with higher AFB1-DNA adduct levels and increasing TP53M and portal vein tumor risk. Additionally, XRCC4P modified the recurrence-free survival and overall survival of cases, especially under conditions of high aflatoxin exposure. CONCLUSION: XRCC4P may be a genetic modifier for the risk and outcome of hepatocellular carcinoma induced by AFB1 exposure.