ADT-OH improves intestinal barrier function and remodels the gut microbiota in DSS-induced colitis.

Owing to the increasing incidence and prevalence of inflammatory bowel disease (IBD) worldwide, effective and safe treatments for IBD are urgently needed. Hydrogen sulfide (H2S) is an endogenous gasotransmitter and plays an important role in inflammation. To date, H2S-releasing agents are viewed as potential anti-inflammatory drugs. The slow-releasing H2S donor 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), known as a potent therapeutic with chemopreventive and cytoprotective properties, has received attention recently. Here, we reported its anti-inflammatory effects on dextran sodium sulfate (DSS)-induced acute (7 days) and chronic (30 days) colitis. We found that ADT-OH effectively reduced the DSS-colitis clinical score and reversed the inflammation-induced shortening of colon length. Moreover, ADT-OH reduced intestinal inflammation by suppressing the nuclear factor kappa-B pathway. In vivo and in vitro results showed that ADT-OH decreased intestinal permeability by increasing the expression of zonula occludens-1 and occludin and blocking increases in myosin II regulatory light chain phosphorylation and epithelial myosin light chain kinase protein expression levels. In addition, ADT-OH restored intestinal microbiota dysbiosis characterized by the significantly increased abundance of Muribaculaceae and Alistipes and markedly decreased abundance of Helicobacter, Mucispirillum, Parasutterella, and Desulfovibrio. Transplanting ADT-OH-modulated microbiota can alleviate DSS-induced colitis and negatively regulate the expression of local and systemic proinflammatory cytokines. Collectively, ADT-OH is safe without any short-term (5 days) or long-term (30 days) toxicological adverse effects and can be used as an alternative therapeutic agent for IBD treatment.

Recombinant Bifidobacterium longum Carrying Endostatin Protein Alleviates Dextran Sodium Sulfate-Induced Colitis and Colon Cancer in Rats.

Bifidobacterium has been widely administrated orally as probiotics to prevent pathogen colonization and modulate the gut microbiome balance. Endostatin is an endogenous inhibitor of angiogenesis and has been shown to inhibit tumor growth, invasion, and metastasis. At present, the combination of endostatin and chemotherapeutic drugs has been regarded as a promising antitumor treatment strategy. In this study, we selected a safe strain of Bifidobacterium longum as a delivery system to transport endostatin to the gastrointestinal tract and explored their combined effect on inflammatory bowel disease (IBD) and colitis-associated cancer. The results indicated that B. longum-Endo relieved dextran sulfate sodium-induced body weight loss, diarrhea, colon shortening, and epithelium damage. Long-term oral administration of B. longum-Endo significantly decreased tumor formation rate, tumor number, and tumor size. Moreover, the effect of B. longum-Endo on gut microbiota dysbiosis was also confirmed by 16S rRNA sequencing analysis. The levels of potentially beneficial bacteria, such as Lactobacillus, Bifidobacterium, Allobaculum, and Parabateroides, were increased in the B. longum-Endo group compared to the model and B. longum groups. Meanwhile, levels of potentially pathogenic bacteria including Desulfovibrio, Helicobacter, and Enterorhabdus were decreased. Taken together, these results suggested that oral administration of recombinant B. longum-Endo strain may be a promising therapeutic strategy for IBD and colitis-associated cancer.

ADT-OH inhibits malignant melanoma metastasis in mice via suppressing CSE/CBS and FAK/Paxillin signaling pathway.

Hydrogen sulfide (H2S) is widely recognized as the third endogenous gas signaling molecule and may play a key role in cancer biological processes. ADT-OH (5-(4-hydroxyphenyl)-3H-1,2-dithiocyclopentene-3-thione) is one of the most widely used organic donors for the slow release of H2S and considered to be a potential anticancer compound. In this study, we investigated the antimetastatic effects of ADT-OH in highly metastatic melanoma cells. A tail-vein-metastasis model was established by injecting B16F10 and A375 cells into the tail veins of mice, whereas a mouse footpad-injection model was established by injecting B16F10 cells into mouse footpads. We showed that administration of ADT-OH significantly inhibited the migration and invasion of melanoma cells in the three different animal models. We further showed that ADT-OH dose-dependently inhibited the migration and invasion of B16F10, B16F1 and A375 melanoma cells as evaluated by wound healing and Transwell assays in vitro. LC-MS/MS and bioinformatics analyses revealed that ADT-OH treatment inhibited the EMT process in B16F10 and A375 cells by reducing the expression of FAK and the downstream response protein Paxillin. Overexpression of FAK reversed the inhibitory effects of ADT-OH on melanoma cell migration. Moreover, after ADT-OH treatment, melanoma cells showed abnormal expression of the H2S-producing enzymes CSE/CBS and the AKT signaling pathways. In addition, ADT-OH significantly suppressed the proliferation of melanoma cells. Collectively, these results demonstrate that ADT-OH inhibits the EMT process in melanoma cells by suppressing the CSE/CBS and FAK signaling pathways, thereby exerting its antimetastatic activity. ADT-OH may be used as an antimetastatic agent in the future.

Characterization and Analysis of the Temporal and Spatial Dynamic of Several Enteritis Modeling Methodologies.

Inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis, is a complex disease involving genetic, immune, and microbiological factors. A variety of animal models of IBD have been developed to study the pathogenesis of human IBD, but there is no model that can fully represent the complexity of IBD. In this study, we established two acute enteritis models by oral 3% DSS or intraperitoneal injection of anti-CD3 antibody, and two chronic enteritis models by feeding 3 cycles of 1.5% DSS or 3 months of the high-fat diet, respectively, and then examined the clinical parameters, histological changes, and cytokine expression profiles after the successful establishment of the models. Our results indicated that in 3% DSS-induced acute enteritis, the colorectal injury was significantly higher than that of the small intestine, while in anti-CD3 antibody-induced acute enteritis, the small intestine injury was significantly higher than that of colorectal damage. Besides, in the 1.5% DSS-induced chronic enteritis, the damage was mainly concentrated in the colorectal, while the damage caused by long-term HFD-induced chronic enteritis was more focused on the small intestine. Therefore, our work provides a reference for selecting appropriate models when conducting research on factors related to the pathogenesis of IBD or evaluating the potential diagnosis and treatment possibilities of pharmaceuticals.

Transcriptome and gene expression analysis of docosahexaenoic acid producer Schizochytrium sp. under different oxygen supply conditions.

BACKGROUND: Schizochytrium sp. is a promising strain for the production of docosahexaenoic acid (DHA)-rich oil and biodiesel, and has been widely used in the food additive and bioenergy industries. Oxygen is a particularly important environmental factor for cell growth and DHA synthesis. In general, higher oxygen supply favors lipid accumulation, but could lead to a reduction of the DHA percentage in total fatty acids in Schizochytrium sp. To tackle this problem, it is essential to understand the mechanisms regulating the response of Schizochytrium sp. to oxygen. In this study, we aimed to explore the acclimatization of this DHA producer to different oxygen supply conditions by examining the transcriptome changes. RESULTS: Two different fermentation processes, namely normal oxygen supply condition (shift agitation speeds from 400 rpm to 300 rpm) and high oxygen supply condition (constant agitation speeds: 400 rpm), were designed to study how the fermentation characteristics of Schizochytrium sp. HX-308 were affected by different oxygen supply conditions. The results indicated that high oxygen supply condition resulted in 49% and 37.5% improvement in the maximum cell dry weight (CDW) and total lipid concentration, respectively. However, the DHA percentage in total fatty acids decreased to 35%, which was 31.4% lower than that produced by normal oxygen supply condition. Moreover, transcriptome analysis was performed to explore the effect of the oxygen supply condition on genetic expression and metabolism. The results showed that glycolysis and pentose phosphate pathway metabolism-associated genes (hexokinase, phosphofructokinase, fructose-bisphosphate aldolase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase) were substantially upregulated in response to high oxygen supply, resulting in more NADPH was available for Schizochytrium. Specially, high oxygen supply condition also led to genes (Δ6 desaturase, Δ12 desaturase, FAS, ORFA, ORFB, and ORFC) involved in fatty acid biosynthesis upregulation. In addition, a transcriptional upregulation of catalase (CAT) became apparent under high oxygen supply condition, while superoxide dismutase (SOD) and ascorbate peroxidase (APX) were found to be down-regulated. CONCLUSIONS: This study is the first to investigate the differences of gene expression at different levels of oxygen availability in the DHA producer Schizochytrium. The results of transcriptome analyses indicated that high oxygen supply condition resulting in more NADPH and acetyl-CoA production for cell growth and lipid synthesis in Schizochytrium. Δ12 desaturase and ORFC showed higher expression levels at high oxygen supply condition, which might be the key regulators for enhancing fatty acid biosynthesis in the future. These results enrich the current knowledge regarding genetic expression and provide important information to enhance DHA production in Schizochytrium sp.

Adaptive evolution of microalgae Schizochytrium sp. under high salinity stress to alleviate oxidative damage and improve lipid biosynthesis.

Lipid accumulation of Schizochytrium sp. can be induced by stress condition, but this stress-induction usually reduce cell growth and cause oxidative damage, which can eventually lower the lipid yield. Here, adaptive laboratory evolution (ALE) combined high salinity was performed to enhance the antioxidant system and lipid accumulation. The final strain ALE150, which was obtained after 150 days, showed a maximal cell dry weight (CDW) of 134.5 g/L and lipid yield of 80.14 g/L, representing a 32.7 and 53.31% increase over the starting strain, respectively. Moreover, ALE150 exhibited an overall higher total antioxidant capacity (T-AOC) and lower reactive oxygen species (ROS) levels than the starting strain. Furthermore, the regulatory mechanisms responsible for the improved performance of ALE150 were analyzed by transcriptomic analysis. Genes related to the antioxidant enzymes and central carbon metabolism were up-regulation. Moreover, the metabolic fluxes towards the fatty acid synthase (FAS) and polyketide synthase (PKS) pathways were also changed.

Development of a cooperative two-factor adaptive-evolution method to enhance lipid production and prevent lipid peroxidation in Schizochytrium sp.

BACKGROUND: Schizochytrium sp. is a marine microalga with great potential as a promising sustainable source of lipids rich in docosahexaenoic acid (DHA). This organism's lipid accumulation machinery can be induced by various stress conditions, but this stress induction usually comes at the expense of lower biomass in industrial fermentations. Moreover, oxidative damage induced by various environmental stresses can result in the peroxidation of lipids, and especially polyunsaturated fatty acids, which causes unstable DHA production, but is often ignored in fermentation processes. Therefore, it is urgent to develop new production strains that not only have a high DHA production capacity, but also possess strong antioxidant defenses. RESULTS: Adaptive laboratory evolution (ALE) is an effective method for the development of beneficial phenotypes in industrial microorganisms. Here, a novel cooperative two-factor ALE strategy based on concomitant low temperature and high salinity was applied to improve the production capacity of Schizochytrium sp. Low-temperature conditions were used to improve the DHA content, and high salinity was applied to stimulate lipid accumulation and enhance the antioxidative defense systems of Schizochytrium sp. After 30 adaptation cycles, a maximal cell dry weight of 126.4 g/L and DHA yield of 38.12 g/L were obtained in the endpoint strain ALE-TF30, which was 27.42 and 57.52% higher than parental strain, respectively. Moreover, the fact that ALE-TF30 had the lowest concentrations of reactive oxygen species and malondialdehyde among all strains indicated that lipid peroxidation was greatly suppressed by the evolutionary process. Accordingly, the ALE-TF30 strain exhibited an overall increase of gene expression levels of antioxidant enzymes and polyketide synthases compared to the parental strain. CONCLUSION: This study provides important clues on how to overcome the negative effects of lipid peroxidation on DHA production in Schizochytrium sp. Taken together, the cooperative two-factor ALE process can not only increase the accumulation of lipids rich in DHA, but also prevent the loss of produced lipid caused by lipid peroxidation. The strategy proposed here may provide a new and alternative direction for the industrial cultivation of oil-producing microalgae.