Alcohol induces hepatocytes necroptosis through the LC3/RIPK1/RIPK3 pathway.

Excessive alcohol consumption leads to serious liver injury. Necroptosis is a programmed cell death form, which has been confirmed to be involved in alcoholic liver injury. However, the exact mechanism remains still unclear. In this study, we found that ethanol caused hepatocytes necroptosis by activating receptor-interacting serine/threonine-protein kinase 1 and 3 (RIPK1 and RIPK3). Meanwhile, autophagy was activated in ethanol-treated hepatocytes. Accumulative studies have demonstrated a possible link between autophagy and necroptosis. Microtubule-associated protein 1 light chain 3 (LC3), an autophagy marker protein, is essential for autophagosome biogenesis/maturation. But little attention has been paid to its functional role. In this study, we explored whether LC3 was involved in ethanol-induced necroptosis. The data showed that LC3 interacted with RIPK1 and RIPK3 in ethanol-treated AML12 cells and mice liver by co-immunoprecipitation (co-IP) and colocalization assay. Ethanol-induced necrosome formation and subsequent necroptosis were alleviated in hepatocytes by knockdown of LC3 or autophagy inhibitor 3-methyladenine (3-MA). These results demonstrated that LC3 accumulation facilitated the formation of necrosome by LC3-RIPK1 and LC3-RIPK3 interactions, eventually caused hepatocytes necroptosis after acute ethanol exposure. Our current research could potentially offer a new understanding of the intricate mechanisms involved in the development of acute alcoholic liver injury.

Elaidic acid induced hepatocyte pyroptosis via autophagy-CTSB-NLRP3 pathway.

Elaidic acid (EA, C18:1 trans) is a kind of principal Trans fatty acid (TFA) and is widely found in processed food. Pyroptosis is a form of programmed cell death, distinct from apoptosis and traditional necrosis. Excessive pyroptosis could induce body injury and serious inflammation. However, the effect of EA on pyroptosis has not been reported. In the study, we found that EA exposure caused liver damage and hepatocyte pyroptosis by testing GSDMD-N, Caspase 1, IL-18, and IL-1ß in mice and HepG2 cells. Further exploring the mechanisms, we found that EA-induced pyroptosis depended on Cathepsin B (CTSB)-mediated NLRP3 inflammasome activation. Cell autophagy was closely related to lysosomes. Our study revealed that EA promoted hepatocyte autophagy, and activated autophagy induced lysosomal membrane permeabilization (LMP) and CTSB leakage. Inhibition of autophagy by 3-MA mitigated the CTSB leak, reduced the activation of the NLRP3 inflammasome, and then attenuated the EA-induced pyroptosis. In summary, these results indicated that EA induced hepatocyte pyroptosis via autophagy-CTSB-NLRP3 inflammasome pathway. The study revealed new insights into the toxicity mechanism of EA.

3-MCPD Induced Mitochondrial Damage of Renal Cells Via the Rhythmic Protein BMAL1 Targeting SIRT3/SOD2.

Biorhythm regulates a variety of physiological functions and enables organisms to adapt to changing environments. 3-Monochloro-1,2-propanediol (3-MCPD) is a common food thermal processing contaminant, and the kidney is its toxic target organ. However, the nephrotoxicity mechanism of 3-MCPD has not been fully elucidated. In the study, we found that 3-MCPD caused mitochondrial damage in renal cells by inhibiting the SIRT3/SOD2 pathway. Further, we found that 3-MCPD could interfere with rhythm protein BMAL1 expression at protein and mRNA levels in mice kidney and NRK-52E cells. Simultaneously, the balance of the daily oscillation of SIRT3/SOD2 pathway proteins was impeded under 3-MCPD treatment. To determine the role of BAML1 in mitochondrial damage, we overexpressed the BMAL1 protein. The data showed that BMAL1 overexpression upregulated SIRT3 and SOD2 expression and attenuated mitochondrial damage caused by 3-MCPD. These results indicated that 3-MCPD inhibited the SIRT3/SOD2 pathway by affecting the expression of the rhythm protein BMAL1, thereby inducing mitochondrial damage in renal cells. Taken together, our work reveals that 3-MCPD may possess a toxic effect via circadian clock mechanisms.

Evaluation of the Potential Probiotic Yeast Characteristics with Anti-MRSA Abilities.

Methicillin-resistant Staphylococcus aureus (MRSA) is a disreputable pathogenic bacterium that has been proven to colonize the intestinal tract. The goal of this study is to find anti-MRSA probiotic yeast from food and evaluate its probiotic characteristics and safety. Finally, 15 strains were isolated from fruit peel with anti-MRSA ability. Using DNA sequence analysis, they were identified as the genus Hanseniaspora (7 strains) and Starmerella (8 strains). Starmerella bacillaris CC-PT4 (CGMCC No. 23573) that was isolated from the grape peel has good auto-aggregation ability and hydrophobicity, and can tolerate 0.3% bile, pH 2, simulated gastric fluid (SGF), and simulated intestinal fluid (SIF). Strikingly, Starmerella bacillaris CC-PT4, like commercial probiotic Saccharomyces boulardii CNCM I-745 (Florastor ®), can adapt to the temperature of the human body (37 ℃). After safety assessment, this strain is sensitive to amphotericin B and cannot produced ß-hemolytic activities. Overall, this study provides a new candidate for probiotic yeast with anti-MRSA ability.

Ginsenoside Rb1 alleviates liver injury induced by 3-chloro-1,2-propanediol by stimulating autophagic flux.

In recent years, foodborne pollutants have become a hot issue in the field of food safety. 3-chloro-1,2-propanediol (3-MCPD) is a widely existing food contaminant. In our previous study, it was confirmed that 3-MCPD can block autophagic flux by inhibiting lysosomal function, thus causing liver injury. Ginseng is a traditional Chinese herbal medicine that contains a variety of bioactive ingredients, among which ginsenoside Rb1 (Gs-Rb1) is the most abundant. In this study, we aim to use Gs-Rb1 to improve 3-MCPD-induced autophagic flux blockage to alleviate liver injury. First, a nontoxic dose of Gs-Rb1 was identified by screening with the MTT method in which Gs-Rb1was added to HepG2 cells and co-treated with 3-MCPD. We found that Gs-Rb1 effectively enhanced the cell activity inhibited by 3-MCPD. Meanwhile, apoptosis data showed that Gs-Rb1 significantly alleviated the apoptosis of HepG2 cells induced by 3-MCPD. Subsequently, we found that Gs-Rb1 could alleviate autophagic flux blockage caused by 3-MCPD in a dose-dependent manner by detecting autophagy-related protein levels and transfecting mRFP-GFP-LC3 adenovirus. On this basis, we used Western blotting and qPCR to explore whether miR-128 was involved in the alleviation effect of Gs-Rb1 on autophagic flux blockade induced by 3-MCPD. The results showed that Gs-Rb1 inhibited the expression of miR-128 and promoted the nuclear expression and target gene transcription of TFEB. Finally, the findings were confirmed by using a hsa-miR-128 inhibitor and mimic. We found that hsa-miR-128 inhibitor alleviated the autophagic flux blockage and apoptosis caused by 3-MCPD and Gs-Rb1 also had a certain alleviation effect on the autophagic flux blockage and apoptosis caused by hsa-miR-128 mimic. This study elaborated the mechanism by which Gs-Rb1 alleviates hepatotoxicity induced by foodborne 3-MCPD by stimulating autophagic flux via miR-128-targeted TFEB, which provides a reliable theoretical basis and target for the use of natural substances to reduce the harm of food processing pollutants on the human body. PRACTICAL APPLICATION: We found that natural ginsenoside Rb1 can alleviate liver injury induced by 3-MCPD(a toxic substance found in foods such as refined vegetable oil, soy sauce, and baby milk powder), which is conducive to the development and utilization of ginseng and has practical significance for the prevention of foodborne liver injury.

Antibacterial Effect of Caprylic Acid and Potassium Sorbate in Combination against Listeria monocytogenes ATCC 7644.

ABSTRACT: Listeria monocytogenes is a common foodborne pathogen that cause life-threatening infection with high mortality rates. Biofilm development of L. monocytogenes decreases its sensitivity to antibiotics, which has long attracted attention globally. Caprylic acid (CA) and potassium sorbate (PS) are both widely used food preservatives, but their synergistic effect against L. monocytogenes has not been described. This study explored the antibacterial activities of the CA-PS combination against L. monocytogenes ATCC 7644 grown in planktonic or biofilm cultures. The fractional inhibitory concentration index values, determined by the checkerboard microdilution method, were 0.37 ± 0.03 and 0.31 ± 0.04, showing their synergistic antimicrobial effects against L. monocytogenes ATCC 7644 in planktonic and biofilm cultures, respectively. CA-PS effectively eradicated the biofilm biomass to 10.8% by crystal violet assay and to 8.63% by fluorescence microscopic analysis compared with the control. The apoptosis rates of microbial cells embedded within biofilm significantly increased to 51.4%. Subsequent analysis revealed that the combination inhibited biofilm formation by affecting extracellular DNA release and polysaccharide intercellular adhesion expression, which was decreased from 8.93 to 1.04 ng of extracellular DNA per relative biomass and to 54.7% of the control, respectively. In addition, the combination inhibited the growth of L. monocytogenes ATCC 7644 by up to 0.67 ± 0.05 and 0.30 ± 0.03 log CFU/cm2 in planktonic and biofilm modes on a carrot surface, respectively. The synergistic antibacterial effects of CA-PS against L. monocytogenes ATCC 7644 were statistically significant, and the combination is an excellent candidate to be a novel food preservative.

Antimicrobial potential of myristic acid against Listeria monocytogenes in milk.

Listeria monocytogenes (L. monocytogenes), an important food-borne pathogenic microorganism, has resistance immune function to many commonly used drugs. Myristic acid is a traditional Chinese herbal medicine, but it has been rarely used as a food additive, limiting the development of natural food preservatives. In this study, the antibacterial activity and mechanism of myristic acid against L. monocytogenes were studied. The minimum inhibitory concentration (MIC) of myristic acid against 13 L. monocytogenes strains ranged from 64 to 256 µg ml-1. The time-kill assay demonstrated that when myristic acid was added to dairy products, flow cytometry confirmed that myristic acid influenced cell death and inhibited the growth of L. monocytogenes. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), and NPN uptake studies illustrated that myristic acid changed the bacterial morphology and membrane structure of L. monocytogenes, which led to rapid cell death. Myristic acid could bind to DNA and lead to changes in DNA conformation and structure, as identified by fluorescence spectroscopy. Our studies provide additional evidence to support myristic acid being used as a natural antibacterial agent and also further fundamental understanding of the modes of antibacterial action.

Phenotype and RNA-seq-Based transcriptome profiling of Staphylococcus aureus biofilms in response to tea tree oil.

Staphylococcus aureus (S. aureus) is a Gram-positive bacterium that causes a wide range of diseases, including food poisoning. Tea tree oil (TTO), an essential oil distilled from Melaleuca alternifolia, is well-known for its antibacterial activities. TTO effectively inhibited all 19 tested strains of S. aureus biofilm and planktonic cells. Phenotype analyses of S. aureus biofilm cells exposed to TTO were performed by biofilm adhesion assays, eDNA detection and PIA release. RNA sequencing (RNA-seq) was used in our study to elucidate the mechanism of TTO as a potential antibacterial agent to evaluate differentially expressed genes (DEGs) and the functional network in S. aureus ATCC 29213 biofilms. TTO significantly changed (greater than a 2- or less than a 2-fold change) the expression of 304 genes in S. aureus contained in biofilms. The levels of genes related to the glycine, serine and threonine metabolism pathway, purine metabolism pathway, pyrimidine metabolism pathway and amino acid biosynthesis pathway were dramatically changed in the biofilm exposed to TTO. Furthermore, the expression changes identified by RNA-seq analysis were verified by real-time RT-PCR. To the best of our knowledge, this research is the first study to report the phenotype and expression profiles of S. aureus in biofilms exposed to TTO.

Antibacterial activity and mode of action of ε-polylysine against Escherichia coli O157:H7.

Purpose. Gram-negative Escherichia coli O157:H7 were chosen as model bacteria to evaluate the antimicrobial mechanism of ε-polylysine (ε-PL).Methodology. The antibacterial activity of ε-PL was detected by measuring the minimum inhibitory concentration values as well as the time-kill curve. The membrane integrity was determined by ultraviolet (UV) absorption, membrane potential (MP) assay and flow cytometry (FCM) experiments. The permeability of the inner membrane was detected by ß-galactosidase activity assay. Furthermore, electron microscopy [scanning electron microscopy (SEM) and transmission electron microscopy (TEM)] was utilized to observe bacterial morphology.Key findings. These results demonstrated that ε-PL showed its antibacterial activity by changing the integrity and permeability of cell membranes, leading to rapid cell death. The electron microscopy analysis (SEM and TEM) results indicated that the bacterial cell morphology, membrane integrity and permeability were spoiled when the E. coli O157:H7 cells were exposed to minimum inhibitory concentrations of ε-PL (16 µg ml-1). In addition, the bacterial membrane was damaged more severely when the concentration of ε-PL was increased.Conclusion. The present study investigated the antimicrobial mechanism of ε-PL by measuring the content of cytoplasmic ß-galactosidase, proteins and DNA. In addition, SEM and TEM were carried out to assess the mechanism. These results show that ε-PL has the ability to decrease the content of large molecules, cellular soluble proteins and nucleic acids associated with increasing the content of cytoplasmic ß-galactosidase in supernatant by causing damage to the cell membranes. Consequently, the use of ε-PL as a natural antimicrobial agent should eventually become an appealing method in the field of food preservation.

Antibacterial activity and mode of action of totarol against Staphylococcus aureus in carrot juice.

Food contaminated with pathogenic bacteria such as Staphylococcus aureus (S. aureus), represents a serious health risk to human beings. Totarol is an antibacterial novel phenolic diterpenes. In present study, the antibacterial activity of totarol against S. aureus was investigated in a food system. The antibacterial activity of totarol was determined by measuring the zones of inhibition and minimum inhibitory concentrations (MICs). The MICs for S. aureus strains were in the range of 2-4 µg/ml. The probable antibacterial mechanism of totarol was the alteration in cell membranes integrity and permeability, which leading to the leakage of cellular materials. The electric conductivity showed a time- and dose-dependent increasing manner, and we utilized totarol to induce the production of cytoplasmic ß-galactosidase in S. aureus. Scanning electron microscopy and transmission electron microscopy analysis further confirmed that S. aureus cell membranes were damaged by totarol. The time-kill assay and detection of the kinetics of S. aureus deactivation in situ indicated that totarol has good preservative activities in a food model. Totarol successfully inhibited S. aureus development in carrot juice, at room temperature (25 °C) and in refrigerator (4 °C) respectively. Our works provided not only additional evidences in support of totarol being regarded as a natural antibacterial food preservative but also fundamental understanding on the mode of antibacterial action. It is necessary to consider that totarol will become a promising antibacterial additive for food preservative.

Antibacterial Effect of Gallic Acid against Aeromonas hydrophila and Aeromonas sobria Through Damaging Membrane Integrity.

In the study, we investigated the antibacterial activity and mechanism of gallic acid against Aeromonas hydrophila and Aeromonas sobria. Gallic acid showed strong antimicrobial activity against the two bacteria. Furthermore, the antibacterial mechanism of gallic acid (0, 3, 6, 12 mM) was performed by membrane integrity assay and scanning electron microscopy (SEM) assay. The results showed that gallic acid notably increased the released material absorption value at 260, 280 nm and electric conductivity in a dose-dependent manner. Moreover, the SEM assay showed that gallic acid induced severe shrink of bacterial intima and irregular morphology in a dose-dependent manner. The SDS-PAGE profiles further confirmed that gallic acid could damage bacterial cells. These results indicated gallic acid exhibited antibacterial effect by destroying membrane integrity of A. hydrophila and A. sobria. Hence, gallic acid has great potential as a new natural food preservative in food fresh-keeping and storage.

Marrow mesenchymal stromal cells reduce methicillin-resistant Staphylococcus aureus infection in rat models.

BACKGROUND AIMS: Staphylococci account for a large proportion of hospital-acquired infections, especially among patients with indwelling devices. These infections are often caused by biofilm-producing strains, which are difficult to eradicate and may eventually cause bacteremia and metastatic infections. Recent evidence suggests that mesenchymal stem cells can enhance bacterial clearance in vivo. METHODS: In this study, a rat model with carboxymethyl cellulose pouch infection was used to analyze the efficacy of bone marrow-derived mesenchymal stromal cells (BMSCs) against the methicillin-resistant Staphylococcus aureus. RESULTS: The results showed that the administration of BMSCs effectively reduced the number of bacterial colonies and the expression of many cytokines and chemokines (such as interleukin [IL]-6, IL-1ß, IL-10 and CCL5). Unlike the fibroblast control groups, the pouch tissues from the BMSC-treated rats showed the formation of granulations, suggesting that the healing of the wound was in progress. CONCLUSIONS: The results indicate that the treatment of BMSCs can reduce methicillin-resistant S aureus infection in vivo, thereby reducing the inflammatory response.

A novel type of N-formylation and related reactions of amines via cyanides and esters as formylating agents.

A novel N-formylation and related reactions proceed from cyanides promoted by esters.