Linolenic Acid-Metronidazole: a Compound Relieving Drug Resistance and Inhibiting Helicobacter pylori.

Metronidazole (Met) is the first choice for treating Helicobacter pylori (Hp). However, Hp is easy to resistant, making Met unable to be widely used. How to overcome Hp's Met resistance is still an issue. In this study, Met was used as the primary raw material with linolenic acid to prepare a novel compound-linolenic acid-metronidazole (Lla-Met). The MIC, minimum bactericidal concentration (MBC), colonization amount of Hp in gastric mucosa, etc., were evaluated, respectively. Lla-Met was successfully prepared by the detection of nuclear magnetic resonance, etc., and its MIC and MBC to Hp were 2~4 µg/mL, 8~16 µg/mL. Moreover, in vivo experiments, Lla-Met significantly reduced the colonization of drug-resistant Hp in gastric mucosa. In the toxicity test, Lla-Met inhibited rate to GES-1 and BGC823 cells were 15% at 128 µg/mL; the mice were administered 10 times treatment Lla-Met treatment (240 mg/kg), have no difference significant injuries were found in their stomach, liver, spleen, kidney, and weight. In addition, Hp G27 continued for 18 days in vitro with sub-Lla-Met concentration, G27 did not show drug resistance to Lla-Met; Lla-Met did not exert an effect on non-Hp species with 128 µg/mL; Compared with a neutral environment, when the acid concentration is 3.0, Lla-Met is not decomposed and has better stability. Conclusion: Lla-Met, a newly prepared compound, has relatively well antibacterial of Met-resistant and sensitive Hp, with a capability of overcoming the metronidazole resistance of Hp.

Cinnamaldehyde: An effective component of Cinnamomum cassia inhibiting Helicobacter pylori.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinnamomum cassia Presl (Cinnamomum cassia) is a common traditional Chinese medicine, which can promote the secretion and digestion of gastric juice, improve the function of gastrointestinal tract. Cinnamaldehyde (CA) is a synthetic food flavoring in the Chinese Pharmacopoeia. AIM OF THE STUDY: This study aimed to search for the active ingredient (CA) of inhibiting H. pylori from Cinnamomum cassia, and elucidate mechanism of action, so as to provide the experimental basis for the treatment of H. pylori infection with Cinnamomum cassia. MATERIALS AND METHODS: It's in vitro and in vivo pharmacological properties were evaluated based on minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and an acute gastric inflammation model in mice infected with H. pylori. Drug safety was evaluated using the CCK8 method and high-dose administration in mice. The advantageous characteristics of CA in inhibiting H. pylori were confirmed using acidic conditions and in combination with the antibiotics. The mechanism underlying the action of CA on H. pylori was explored using scanning electron microscopy (SEM), adhesion experiments, biofilm inhibition tests, ATP and ROS release experiments, and drug affinity responsive target stability (DARTS) screening of target proteins. The protein function and target genes were verified by molecular docking and Real-Time quantitative reverse transcription PCR (qRT-PCR). RESULTS: The results demonstrated that CA was found to be the main active ingredient against H. pylori in Cinnamomum cassia in-vitro tests, with a MIC of 8-16 µg/mL. Moreover, CA effectively inhibited both sensitive and resistant H. pylori strains. The dual therapy of PPI + CA exhibited remarkable in vivo efficacy in the acute gastritis mouse model, superior to the standard triple therapy. DARTS, molecular docking, and qRT-PCR results suggested that the target sites of action were closely associated with GyrA, GyrB, AtpA, and TopA, which made DNA replication and transcription impossible, then leading to inhibition of bacterial adhesion and colonization, suppression of biofilm formation, and inhibition ATP and enhancing ROS. CONCLUSIONS: This study demonstrated the suitability of CA as a promising lead drug against H. pylori, The main mechanisms can target GyrA ect, leading to reduce ATP and produce ROS, which induces the apoptosis of bacterial.

Mechanistic research: Selenium regulates virulence factors, reducing adhesion ability and inflammatory damage of Helicobacter pylori.

BACKGROUND: The pathogenicity of Helicobacter pylori is dependent on factors including the environment and the host. Although selenium is closely related to pathogenicity as an environmental factor, the specific correlation between them remains unclear. AIM: To investigate how selenium acts on virulence factors and reduces their toxicity. METHODS: H. pylori strains were induced by sodium selenite. The expression of cytotoxin-associated protein A (CagA) and vacuolating cytotoxin gene A (VacA) was determined by quantitative PCR and Western blotting. Transcriptomics was used to analyze CagA, CagM, CagE, Cag1, Cag3, and CagT. C57BL/6A mice were infected with the attenuated strains subjected to sodium selenite induction, and H. pylori colonization, inflammatory reactions, and the cell adhesion ability of H. pylori were assessed. RESULTS: CagA and VacA expression was upregulated at first and then downregulated in the H. pylori strains after sodium selenite treatment. Their expression was significantly and steadily downregulated after the 5th cycle (10 d). Transcriptome analysis revealed that sodium selenite altered the levels affect H. pylori virulence factors such as CagA, CagM, CagE, Cag1, Cag3, and CagT. Of these factors, CagM and CagE expression was continuously downregulated and further downregulated after 2 h of induction with sodium selenite. Moreover, CagT expression was upregulated before the 3rd cycle (6 d) and significantly downregulated after the 5th cycle. Cag1 and Cag3 expression was upregulated and downregulated, respectively, but no significant change was observed by the 5th cycle. C57BL/6A mice were infected with the attenuated strains subjected to sodium selenite induction. The extent of H. pylori colonization in the stomach increased; however, sodium selenite also induced a mild inflammatory reaction in the gastric mucosa of H. pylori-infected mice, and the cell adhesion ability of H. pylori was significantly weakened. CONCLUSION: These results demonstrate that H. pylori displayed virulence attenuation after the 10th d of sodium selenite treatment. Sodium selenite is a low toxicity compound with strong stability that can reduce the cell adhesion ability of H. pylori, thus mitigating the inflammatory damage to the gastric mucosa.

Therapeutic effect of demethylated hydroxylated phillygenin derivative on Helicobacter pylori infection.

Modifying and transforming natural antibacterial products is a novel idea for developing new efficacious compounds. Phillygenin has an inhibitory effect on H. pylori. The aim of the present study was to prepare a phillygenin derivative (PHI-Der) through demethylation and hydroxylation. The minimum inhibitory concentration of 18 strains of H. pylori from different sources was 8-32 µg/mL in vitro, and the activity increased 2-8 times than that of phillygenin. PHI-Der could significantly inhibit the colonization of H. pylori in vivo, reduce the inflammatory response, and promote the repair of inflammatory damage. Further, we used SwissTargetPrediction to predict that its main targets are ALOX5, MCL1, and SLC6A4, and find that it can inhibit bacterial biofilm formation and reduce bacterial infection of cells. It can enhance the intracellular oxidative capacity of H. pylori to inhibit H. pylori growth. Further, it could prevent the oxidation of H. pylori-infected cells and reduce the inflammatory response, which plays a role in protection. In conclusion, compared to phillygenin, PHI-Der had better antibacterial activity and was more effective in treating H. pylori infection. It has characteristics of high safety, specificity, resistance to drug resistance and better antibacterial activity than phillygenin, it's a good antioxidant for host cells.

Preparation and hypoglycemic effects of chromium- and zinc-rich Acetobacter aceti.

BACKGROUND: At present, there is no ideal method to cure diabetes, and there are few reports on the treatment of diabetes with probiotics. AIM: To propose a method for preparing a new type of chromium- and zinc-rich Acetobacter aceti (A. aceti) and explore its ability to enhance the hypoglycemic effects of probiotics in the treatment of diabetes. METHODS: A. aceti was cultured in a liquid medium that contained chromium trichloride and zinc chloride, both at a concentration of 64 mg/mL, with the initial concentration of the bacterial solution 1 × 104 CFU/mL. After the bacterial solution had been inducted for 48 h, the culture media was changed and the induction was repeated once. The levels of chromium and zinc in the bacteria were detected by inductively coupled plasma mass spectrometry, and the contents of NADH and glucose dehydrogenase were determined using an NAD/NADH kit and glucose dehydrogenase kit, respectively. Streptozotocin was used to establish a mouse model to evaluate the hypoglycemic effects of the proposed chromium- and zinc-rich A. aceti. Ten-times the therapeutic dose was administered to evaluate its biological safety. The effect on MIN6 islet cells was also assessed in vitro. RESULTS: The levels of chromium metal, metallic zinc, NADH coenzyme, and glucose dehydrogenase in A. aceti prepared by this method were 28.58-34.34 mg/kg, 5.35-7.52 mg/kg, 5.13-7.26 µM, and 446.812-567.138 U/g, respectively. The use of these bacteria resulted in a better hypoglycemic effect than metformin, promoting the repair of tissues and cells of pancreatic islets in vivo and facilitating the growth of MIN6 pancreatic islet cells and increasing insulin secretion in vitro. Ten-times the therapeutic dose of treatment was non-toxic to mice. CONCLUSION: Chromium trichloride and zinc chloride can be employed to induce the preparation of chromium- and zinc-rich A. aceti, which can then promote the hypoglycemic effect found in normal A. aceti. The bacteria biotransforms the chromium and zinc in a way that could increase their safety as a treatment for diabetes.

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.

Application of traditional Chinese medicine in treatment of Helicobacter pylori infection.

Helicobacter pylori (H. pylori) has a high rate of infection and antibiotic resistance and poses a serious threat to human life. One of the main strategies to overcome drug resistance is to develop new treatment plans. Traditional Chinese medicine (TCM) that is commonly used to treat many diseases in China can reduce drug resistance and increase the eradication rate of H. pylori. In this paper, we review the research progress on TCM in the treatment of H. pylori infection. The mechanism of action of TCM is reviewed and research and applications of TCM in the treatment of H. pylori are demonstrated. Finally, we discuss problems confronting the use of TCM for the treatment of H. pylori infection and propose possible solutions. In addition, the plans of TCM in H. pylori treatment were also screened: Dampness-heat syndrome in the spleen and stomach, deficiency of spleen and stomach, and cold-heat complicated syndrome, and the effective components therein are studied. The antibacterial effect of TCM is relatively slow; for rapid improvement of the treatment effect of refractory H. pylori gastritis, we provide an appropriate treatment regime combining TCM and Western medicine with immune-regulatory and synergistic antibacterial effects.

Mechanism of combined use of vitamin D and puerarin in anti-hepatic fibrosis by regulating the Wnt/ß-catenin signalling pathway.

AIM: To reveal the protective mechanism of the combined use of vitamin D and puerarin in the progression of hepatic fibrosis induced by carbon tetrachloride (CCl4). METHODS: Eight-week-old male Wistar rats were randomly divided into a normal control group (C group), a CCl4 group (CCl4 group), a vitamin D group (V group), a puerarin group (P group), and a combined group of vitamin D and puerarin (V + P group), each of which contained ten rats. In this way, we built a rat model of CCl4-induced hepatic fibrosis with intervention by vitamin D, puerarin, or a combination of the two. After eight weeks, the mice were sacrificed to collect serum and liver specimens. Blood was collected to detect the hyaluronic acid (HA). We also measured hydroxyproline (Hyp) and prepared paraffin sections of liver. After Sirius red staining, the liver specimens were observed under a microscope. RT-PCR and western blot analysis were adopted to detect the mRNA and the protein levels of Collagen I, Collagen III, Wnt1, and ß-catenin in the liver tissues, respectively. RESULTS: Hepatic fibrosis was observed in the CCl4 group. In comparison, hepatic fibrosis was attenuated in the V, P, and V + P groups: the HA level in blood and the Hyp level in liver were reduced, and the mRNA levels of Collagen I, Collagen III, Wnt, and ß-catenin in liver were also decreased, as well as the protein levels of Wnt1 and ß-catenin. Among these groups, the V + P group demonstrated the greatest amelioration of hepatic fibrosis. CONCLUSION: The combined application of vitamin D and puerarin is capable of alleviating CCl4-induced hepatic fibrosis of rats. As to the mechanism, it is probably because the combined use is able to silence the Wnt1/ß-catenin pathway, suppress the activation of hepatic stellate cells, and reduce the secretion of collagen fibers, therefore improving the anti-hepatic fibrosis effect.

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.