Inhibitory activities and mechanisms of free and bound phenolics on α-glucosidase in fresh fruits of Phyllanthus emblica Linn. using spectroscopy and molecular docking.

Phyllanthus emblica Linn. (PE) fresh fruits contain high concentrations of polyphenolics, of which free and bound phenolics are rich in biological activities. In this study, the inhibitory activity and mechanism of PEFP and PEBP on α-glucosidase (α-GLU) were investigated using spectroscopic techniques, kinetic analysis, and molecular docking. The results showed that 13 PEFP and 12 PEBP were identified by UPLC-MS/MS analysis, and Bis-HHDP-hexose and castalagin (vesgalagin) were found for the first time in PE fresh fruits. Kinetic analysis of enzyme inhibition showed that a mixture of free and bound phenolics inhibited α-GLU, and the effect of the conformational relationship of PEFP and PEBP with α-GLU on hypoglycemia was further explored by fluorescence quenching, circular dichroism (CD) spectroscopy, and molecular docking analysis. The findings demonstrated the inhibitory activity and mechanism of free and bound phenolics on α-GLU and provided a theoretical basis for PE polyphenolics as α-GLU inhibitors for hypoglycemia.

Genome-scale reconstruction of the metabolic network in Streptococcus thermophilus S-3 and assess urea metabolism.

BACKGROUND: Streptococcus thermophilus is an important strain widely used in dairy fermentation, with distinct urea metabolism characteristics compared to other lactic acid bacteria. The conversion of urea by S. thermophilus has been shown to affect the flavor and acidification characteristics of milk. Additionally, urea metabolism has been found to significantly increase the number of cells and reduce cell damage under acidic pH conditions, resulting in higher activity. However, the physiological role of urea metabolism in S. thermophilus has not been fully evaluated. A deep understanding of this metabolic feature is of great significance for its production and application. Genome-scale metabolic network models (GEMs) are effective tools for investigating the metabolic network of organisms using computational biology methods. Constructing an organism-specific GEM can assist us in comprehending its characteristic metabolism at a systemic level. RESULTS: In the present study, we reconstructed a high-quality GEM of S. thermophilus S-3 (iCH492), which contains 492 genes, 608 metabolites and 642 reactions. Growth phenotyping experiments were employed to validate the model both qualitatively and quantitatively, yielding satisfactory predictive accuracy (95.83%), sensitivity (93.33%) and specificity (100%). Subsequently, a systematic evaluation of urea metabolism in S. thermophilus was performed using iCH492. The results showed that urea metabolism reduces intracellular hydrogen ions and creates membrane potential by producing and transporting ammonium ions. This activation of glycolytic fluxes and ATP synthase produces more ATP for biomass synthesis. The regulation of fluxes of reactions involving NAD(P)H by urea metabolism improves redox balance. CONCLUSION: Model iCH492 represents the most comprehensive knowledge-base of S. thermophilus to date, serving as a potent tool. The evaluation of urea metabolism led to novel insights regarding the role of urease. © 2023 Society of Chemical Industry.

Complete Genome Sequence of Lactobacillus salivarius AR809, a Probiotic Strain with Oropharyngeal Tract Resistance and Adhesion to the Oral Epithelial Cells.

Lactobacillus salivarius AR809 was isolated from a healthy adult oral cavity with multiple probiotic properties, such as high antimicrobial activity, adhesion to the oral epithelium, resistance to acidic pH, bile, lysozyme, and H2O2. In this study, to investigate the genetic basis on probiotic potential and identify the functional genes in the strain, the complete genome of strain AR809 was sequenced by Illumina and PacBio platforms. Then comparative genome analysis on 11 strains of Lactobacillus salivarius was performed. The complete genome of AR809 consisted of a circular 1,747,224 bp chromosome with 33.00% GC content and four circular plasmids [pA (247,948 bp), pB (27,292 bp), pC (3349 bp), and pD (2898 bp), respectively]. From among the 1866 protein-coding genes, 130 carbohydrate metabolism-related genes, 18 bacteriocin biosynthesis-related genes, 74 environmental stress-related genes, and a series of adhesion-related genes were identified via clusters of orthologous genes, Koyto Encyclopedia of Genes and Genomes, and carbohydrate-active enzymes annotation. The comparative genome analysis indicated that genomic homology between AR809 and CICC23174 was the highest. In conclusion, the present work provided valuable insights into the gene's function prediction and understanding the genetic basis on adapting to host oropharyngeal-gastrointestinal tract in strain AR809.

The Arginine Repressor ArgR2 Controls Conjugated Linoleic Acid Biosynthesis by Activating the cla Operon in Lactiplantibacillus plantarum.

CLA (conjugated linoleic acid) has attracted substantial attention due to its physiological functions, including regulating immunity, reducing obesity, and contributing to cancer suppression. In Lactiplantibacillus plantarum, CLA oleate hydratase (CLA-HY), CLA short-chain dehydrogenase (CLA-DH), and CLA acetoacetate decarboxylase (CLA-DC) catalyze the biotransformation of linoleic acid (LA) to CLA. However, the underlying transcriptional regulation mechanism of this pathway remains largely unknown. In this study, the potential transcriptional regulators that might bind to the cla promoter of L. plantarum AR195 were investigated by DNA pulldown. Interestingly, ArgR2, the transcriptional regulator of arginine metabolism, was identified as a potential regulator involved in the regulation of CLA biotransformation. Electrophoretic mobility shift assay (EMSA) and molecular interaction results demonstrated the specific binding of ArgR2 to the regulatory region of the cla operon. The knockout of argR2 led to the downregulation of cla-dh and cla-dc by 91% and 34%, respectively, resulting in a decline in the CLA yield by 14%. A segmental EMSA revealed that ArgR2 bound to three distinct sites in the cla regulatory region, and these binding sites were highly conserved and rich in AT. The regulatory mechanism of ArgR2 on CLA biosynthesis further expanded our knowledge of the regulatory mechanism of CLA biosynthesis in L. plantarum and laid the theoretical foundation for the production and application of CLA. IMPORTANCE CLA (conjugated linoleic acid) has received extensive attention owing to its important physiological functions. CLA from natural sources is far from meeting people's demands. Lactic acid bacteria can efficiently synthesize cis-9,trans-11-CLA and trans-10,cis-12-CLA, which possess physiological activities. However, little is known about the regulatory mechanism. In this study, we identified that the biosynthesis of CLA in L. plantarum AR195 was transcriptionally regulated by the arginine biosynthesis regulatory protein ArgR2. The regulation mechanism of ArgR2 on CLA biosynthesis lays a theoretical foundation for the regulation of CLA synthesis and industrial production.

Probiotics Interact With Lipids Metabolism and Affect Gut Health.

Probiotics have attracted much attention due to their ability to modulate host intestinal microbe, participate in nutrient metabolism or immunomodulatory. Both inflammatory bowel disease (IBD) and bowel cancer are digestive system disease, which have become a global public health problem due to their unclear etiology, difficult to cure, and repeated attacks. Disturbed gut microbiota and abnormal lipid metabolism would increase the risk of intestinal inflammation. However, the link between lipid metabolism, probiotics, and IBD is unclear. In this review, we found that different lipids and their derivatives have different effects on IBD and gut microbes. ω-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid, eicosapentaenoic acid, and their derivatives resolvin E1, resolvin D can inhibit oxidative stress and reactive oxygen species activate NFκB and MAPk pathway. While ω-6 PUFAs linoleic acid and arachidonic acid can be derived into leukotrienes and prostaglandins, which will aggravate IBD. Cholesterol can be converted into bile acids to promote lipid absorption and affect microbial survival and colonization. At the same time, it is affected by microbial bile salt hydrolase to regulate blood lipids. Low denstiy lipoprotein (LDL) is easily converted into oxidized LDL, thereby promoting inflammation, while high denstiy lipoprotein (HDL) has the opposite effect. Probiotics compete with intestinal microorganisms for nutrients or ecological sites and thus affect the structure of intestinal microbiota. Moreover, microbial short chain fatty acids, bile salt hydrolase, superoxide dismutase, glutathione, etc. can affect lipid metabolism and IBD. In conclusion, probiotics are directly or indirectly involved in lipids metabolism and their impact on IBD, which provides the possibility to explore the role of probiotics in improving gut health.

Determination of the regulatory network and function of the lysR-type transcriptional regulator of Lactiplantibacillus plantarum, LpLttR.

BACKGROUND: Lactiplantibacillus plantarum has various healthcare functions including the regulation of immunity and inflammation, reduction of serum cholesterol levels, anti-tumor activity, and maintenance of the balance of intestinal flora. However, the underlying metabolic and regulatory mechanisms of these processes remain unclear. Our previous studies have shown that the LysR type transcriptional regulator of L. plantarum (LpLttR) regulates the biotransformation of conjugated linoleic acids (CLAs) through the transcriptional activation of cla-dh (coding gene for CLA short-chain dehydrogenase) and cla-dc (coding gene for CLA acetoacetate decarboxylase). However, the regulatory network and function of LpLttR have not yet been characterized in L. plantarum. RESULTS: In this study, the regulatory role of LpLttR in various cellular processes was assessed using transcriptome analysis. The deletion of LpLttR had no evident influence on the bacterial growth. The transcriptome data showed that the expression of nine genes were positively regulated by LpLttR, and the expression of only two genes were negatively regulated. Through binding motif analysis and molecular interaction, we demonstrated that the regulatory region of the directly regulated genes contained a highly conserved sequence, consisting of a 15-base long box and rich in AT. CONCLUSION: This study revealed that LpLttR of L. plantarum did not play a global regulatory role similar to that of the other transcriptional regulators in this family. This study broadens our knowledge of LpLttR and provides a theoretical basis for the utilization of L. plantarum.

Anti-Osteoporotic Effect of Lactobacillus brevis AR281 in an Ovariectomized Mouse Model Mediated by Inhibition of Osteoclast Differentiation.

Osteoporosis is a global disease characterized by weakened bone microarchitecture, leading to osteoporotic fractures. Estrogen replacement therapy is the traditional treatment for osteoporosis but carries with it an increased risk of cardiac events. In search of a safe and effective treatment, we used Lactobacillus brevis AR281, which has anti-inflammatory properties, to conduct a 7-week experiment, investigating its inhibitory effects on osteoporosis in an ovariectomized (ovx) mouse model. The results demonstrated that AR281 significantly improved bone microarchitecture and biomechanical strength in ovx mice by attenuating bone resorption. AR281 significantly decreased the critical osteoclast activator, the ratio of the receptor activator for nuclear factor kappa B (NF-κB) ligand (RANKL) to osteoprotegerin, and pro-inflammatory osteoclastogenic mediators, such as IL-1, IL-6, and IL-17, which can increase the RANKL expression. Moreover, AR281 modulated intestinal microbiota in ovx mice increased the abundance of Akkermansia, which is responsible for the improvement of gut epithelial barrier integrity. In an in vitro trial, AR281 suppressed the number of osteoclasts differentiated from the osteoclast precursor RAW264.7 cells caused by RANKL through the tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6)/NF-κB/nuclear factor of activated T cells c1 (NFATc1) pathway. Therefore, AR281 may be a natural alternative for combating osteoporosis.

Genetic evidence for the requirements of antroquinonol biosynthesis by Antrodia camphorata during liquid-state fermentation.

The solid-state fermentation of Antrodia camphorata could produce a variety of ubiquinone compounds, such as antroquinonol (AQ). However, AQ is hardly synthesized during liquid-state fermentation (LSF). To investigates the mechanism of AQ synthesis, three precursors (ubiquinone 0 UQ0, farnesol and farnesyl diphosphate FPP) were added in LSF. The results showed that UQ0 successfully induced AQ production; however, farnesol and FPP could not induce AQ synthesis. The precursor that restricts the synthesis of AQ is the quinone ring, not the isoprene side chain. Then, the Agrobacterium-mediated transformation system of A. camphorata was established and the genes for quinone ring modification (coq2-6) and isoprene synthesis (HMGR, fps) were overexpressed. The results showed that overexpression of genes for isoprene side chain synthesis could not increase the yield of AQ, but overexpression of coq2 and coq5 could significantly increase AQ production. This is consistent with the results of the experiment of precursors. It indicated that the A. camphorata lack the ability to modify the quinone ring of AQ during LSF. Of the modification steps, prenylation of UQ0 is the key step of AQ biosynthesis. The result will help us to understand the genetic evidence for the requirements of AQ biosynthesis in A. camphorata.

Probiotic yeast BR14 ameliorates DSS-induced colitis by restoring the gut barrier and adjusting the intestinal microbiota.

The probiotic Saccharomyces boulardii has been widely used in colitis treatment; however, the beneficial effects of other yeast species are rarely studied. Saccharomyces cerevisiae with great stress tolerance and potential in colitis treatment was investigated in this study. Among 16 yeast strains, BR14, BR54, and BR174 strains showed good stress-resistant capacity, anti-inflammatory activity, and little toxicity to macrophages. As for the colitis mice, BR14 inhibited weight loss the most, as well as the disease activity index and colon shortening. After treatment with BR14, the expression levels of genes related to histological damage were all upregulated. BR14 significantly attenuated the expression levels of TNF-α and IL-6, while the expression of IL-10 was upregulated. Additionally, BR14 rebalanced the intestinal microbial composition of colitis mice by increasing the abundance of Muribaculaceae, Lactobacillus and Rikenellaceae and decreasing the abundance of Turicibacter, Escherichia-Shigella, Desulfovibrio, and Lachnospiraceae. In summary, BR14 exhibited great potential in alleviating colitis through restoring the gut barrier and adjusting the intestinal microbiota.

Enhancement of antroquinonol production via the overexpression of 4-hydroxybenzoate polyprenyltransferase biosynthesis-related genes in Antrodia cinnamomea.

Antroquinonol (AQ) as one of the most potent bioactive components in Antrodia cinnamomea (Fomitopsidaceae) shows a broad spectrum of anticancer effects. The lower yield of AQ has hampered its possible clinical application. AQ production may potentially be improved by genetic engineering. In this study, the protoplast-polyethylene glycol method combined with hygromycin as a selection marker was used in the genetic engineering of A. cinnamomea S-29. The optimization of several crucial parameters revealed that the optimal condition for generating maximal viable protoplasts was digestion of 4-day-old germlings with a mixture of enzymes (lysing enzyme, snailase, and cellulase) and 1.0 M MgSO4 for 4 h. The ubiA and CoQ2 genes, which are involved in the synthesis of 4-hydroxybenzoate polyprenyltransferase, were cloned and overexpressed in A. cinnamomea. The results showed that ubiA and CoQ2 overexpression significantly increased AQ production in submerged fermentation. The overexpressing strain produced maximum AQ concentrations of 14.75 ± 0.41 mg/L and 19.25 ± 0.29 mg/L in pCT74-gpd-ubiA and pCT74-gpd-CoQ2 transformants, respectively. These concentrations were 2.00 and 2.61 times greater than those produced by the control, respectively. This research exemplifies how the production of metabolites may be increased by genetic manipulation, and will be invaluable to guide the genetic engineering of other mushrooms that produce medically useful compounds.

Fractionation, chemical characterization and immunostimulatory activity of ß-glucan and galactoglucan from Russula vinosa Lindblad.

Water-extracted polysaccharides from Russula vinosa Lindblad (WRP) were separated into three fractions (WRP-1, WRP-2 and WRP-3) by gradient ethanol precipitation and gel chromatography. Structural characterization indicated that WRP-1 was a branched ß-(1→3)-glucan and exhibited rigid helical conformation in aqueous solution with Mw of 2,180 kDa and radius of gyration (Rg) of 123.4 nm. The galactoglucan of WRP-2 and WRP-3 were mainly composed of →6)-Galp-(1→ and →4)-Glcp-(1→ terminated by glucose and mannose, presenting much lower Mw (392 and 93.6 kDa) and Rg (57.6 and 42.6 nm), and more incompact flexible conformation than WRP-1. All fractions showed potential immunostimulatory activity by promoting macrophage proliferation, phagocytosis, as well as the release of nitric oxide and cytokines (TNF-α and IL-1ß). WRP-1 with unique structure and conformation showed the best immunostimulatory effects among them. This study suggests that WRP could be explored as natural immunostimulator used in the food and pharmaceutical industry.

LysR Family Regulator LttR Controls Production of Conjugated Linoleic Acid in Lactobacillus plantarum by Directly Activating the cla Operon.

Conjugated linoleic acids (CLAs) have attracted more attention as functional lipids due to their potential physiological activities, including anticancer, anti-inflammatory, anti-cardiovascular disease, and antidiabetes activities. Microbiological synthesis of CLA has become a compelling method due to its high isomer selectivity and convenient separation and purification processes. In Lactobacillus plantarum, the generation of CLA from linoleic acids (LAs) requires the combination of CLA oleate hydratase (CLA-HY), CLA short-chain dehydrogenase (CLA-DH), and CLA acetoacetate decarboxylase (CLA-DC), which are separately encoded by cla-hy, cla-dh, and cla-dc. However, the regulatory mechanisms of CLA synthesis remain unknown. In this study, we found that a LysR family transcriptional regulator, LTTR, directly bound to the promoter region of the cla operon and activated the transcription of cla-dh and cla-dc. The binding motif was also predicted by bioinformatics analysis and verified by electrophoretic mobility shift assays (EMSAs) and DNase I footprinting assays. The lttR overexpression strain showed a 5-fold increase in CLA production. Moreover, we uncovered that the transcription of lttR is activated by LA. These results indicate that LttR senses LA and promotes CLA production by activating the transcription of cla-dh and cla-dc. This study reveals a new regulatory mechanism in CLA biotransformation and provides a new potential metabolic engineering strategy to increase the yield of CLA.IMPORTANCE Our work has identified a novel transcriptional regulator, LTTR, that regulates the production of CLA by activating the transcription of cla-dh and cla-dc, essential genes participating in CLA synthesis in Lactobacillus plantarum This study provides insight into the regulatory mechanism of CLA synthesis and broadens our understanding of the synthesis and regulatory mechanisms of the biosynthesis of CLA.

Effect of D-Ala-Ended Peptidoglycan Precursors on the Immune Regulation of Lactobacillus plantarum Strains.

The resistance of Lactobacillus plantarum to vancomycin depends on its peptidoglycan composition. Vancomycin has poor binding affinity with peptidoglycan precursors ending in D-alanyl-D-lactate (D-Ala-D-Lac) but binds strongly to peptidoglycan precursors ending in D-alanyl-D-alanine (D-Ala-D-Ala), resulting in resistance and sensitivity, respectively. The ligase Ddl, which generates D-Ala-D-Lac or D-Ala-D-Ala incorporated into the peptidoglycan precursor chain, is responsible for this specificity. To study the effect of peptidoglycan precursors on immunity, we constructed several strains of L. plantarum expressing the ddl gene of Lactococcus lactis to change their peptidoglycan precursors. The change in the termini of the peptidoglycan precursors was determined by the sensitivity of the strains to vancomycin. The overexpression of ddl increased the susceptibility of the strains to vancomycin. We further explored the regulation of the macrophage inflammatory response pathway by the wild-type and constructed strains, and found that these strains induced the MyD88-dependent TRAF6/MAPK pathway, and the increase in D-Ala L. plantarum peptidoglycan precursors increased the secretion of the inflammatory factors IL-6, IL-1ß and TNF-α. These results indicate that D-Ala-ended peptidoglycan precursors play a central role in the variable immunomodulatory ability of L. plantarum.

Bile salt hydrolase-overexpressing Lactobacillus strains can improve hepatic lipid accumulation in vitro in an NAFLD cell model.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) includes a range of liver diseases that occur in the absence of significant alcohol consumption. The probiotic bacterial strains Lactobacillus casei LC2W, which overexpresses the bile salt hydrolase (BSH) gene (referred to as pWQH01), and Lactobacillus plantarum AR113, which exhibits high BSH activity, have been shown to improve hepatic lipid accumulation and may lower cholesterol levels in vivo. These effects may be BSH-dependent, as L. casei LC2W without BSH activity did not exert these beneficial effects. OBJECTIVE: This study aimed to investigate the effects of Lactobacillus with high BSH activity on cholesterol accumulation and lipid metabolism abnormalities in oleic acid (OA)- and cholesterol-induced HepG2 cell models, and to determine the mechanism underlying the effects. DESIGN: A HepG2 cell model of OA-induced steatosis and cholesterol-induced cholesterol accumulation was developed. OA- and cholesterol-treated HepG2 cells were incubated with L. plantarum AR113, L. casei LC2W or L. casei pWQH01 for 6 h at 37°C with 5% CO2. Subsequently, a series of indicators and gene expressions were analysed. RESULTS: Both L. plantarum AR113 and L. casei pWQH01 significantly reduced lipid accumulation, total cholesterol (TC) levels and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) mRNA expression relative to the control group, whereas L. casei LC2W had no similar effect. Additionally, exposure to L. plantarum AR113 or L. casei pWQH01 significantly reduced the expression of sterol regulatory element-binding protein 1c (SREBP-1c), Acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and tumour necrosis factor-α (TNF-α) andsignificantly increased the expression of 5' adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor alpha (PPARα). CONCLUSION: Both L. plantarum AR113 and L. casei pWQH01 appear to improve steatosis in vitro in a BSH-dependent manner.

The second messenger c-di-AMP mediates bacterial exopolysaccharide biosynthesis: a review.

Cyclic dimeric adenosine 3'-5'-monophosphate (c-di-AMP) is a recently discovered nucleotide messenger in bacteria. It plays an important role in signaling, transcription, and cell physiology, such as in bacterial growth, potassium transport, fatty acid synthesis, the metabolic balance of cell wall components, and biofilm formation. Exopolysaccharides (EPSs) have distinct physico-chemical properties and diverse bioactivities including antibacterial, hypolipidemic, and antioxidative activities, and they are widely used in the food, pharmaceutical, and cosmetic industries. Although c-di-AMP has been demonstrated to regulate the biosynthesis of bacterial EPSs, only a single c-di-AMP receptor, CabpA, has been identified in EPS synthesis. With the aim of describing current understanding of the regulation of microbial EPSs, this review summarizes c-di-AMP biosynthesis and degradation as well as the mechanism through which c-di-AMP regulates bacterial EPSs.

Comprehensive transcriptomic and proteomic analyses of antroquinonol biosynthetic genes and enzymes in Antrodia camphorata.

Antroquinonol (AQ) has several remarkable bioactivities in acute myeloid leukaemia and pancreatic cancer, but difficulties in the mass production of AQ hamper its applications. Currently, molecular biotechnology methods, such as gene overexpression, have been widely used to increase the production of metabolites. However, AQ biosynthetic genes and enzymes are poorly understood. In this study, an integrated study coupling RNA-Seq and isobaric tags for relative and absolute quantitation (iTRAQ) were used to identify AQ synthesis-related genes and enzymes in Antrodia camphorata during coenzyme Q0-induced fermentation (FM). The upregulated genes related to acetyl-CoA synthesis indicated that acetyl-CoA enters the mevalonate pathway to form the farnesyl tail precursor of AQ. The metE gene for an enzyme with methyl transfer activity provided sufficient methyl groups for AQ structure formation. The CoQ2 and ubiA genes encode p-hydroxybenzoate polyprenyl transferase, linking coenzyme Q0 and the polyisoprene side chain to form coenzyme Q3. NADH is transformed into NAD+ and releases two electrons, which may be beneficial for the conversion of coenzyme Q3 to AQ. Understanding the biosynthetic genes and enzymes of AQ is important for improving its production by genetic means in the future.

RNA-Seq transcriptomic analyses of Antrodia camphorata to determine antroquinonol and antrodin C biosynthetic mechanisms in the in situ extractive fermentation.

BACKGROUND: In situ extractive fermentation (ISEF) is an important technique for improving metabolite productivity. The different extractants can induce the synthesis of different bioactive metabolites of Antrodia camphorata during ISEF. However, a lack of research on the molecular genetics of A. camphorata during ISEF currently hinders such studies on metabolite biosynthetic mechanisms. RESULTS: To clarify the differentially expressed genes during ISEF, the gene transcriptional expression features of A. camphorata S-29 were analysed. The addition of n-tetradecane as an extractant during ISEF showed more pronounced up-regulation of ubiquinone and other terpenoid-quinone biosynthesis pathway genes (CoQ2, wrbA and ARO8). When oleic acid was used as an extractant, the terpenoid backbone biosynthesis and ubiquinone and other terpenoid-quinone biosynthesis pathways were significantly enriched, and genes (IDI, E2.3.3.10, HMGCR atoB, and CoQ2) related to these two pathways were also significantly up-regulated. The CoQ2 genes encode puru-hydroxybenzoate:polyprenyltransferase, playing an important role in antroquinonol synthesis. The IDI, E2.3.3.10, HMGCR and atoB genes of the terpenoid backbone biosynthesis pathway might play an important role in the synthesis of the triquine-type sesquiterpene antrodin C. CONCLUSION: This investigation advances our understanding of how two different extractants of n-tetradecane and oleic acid affect the biosynthesis of metabolites in A. camphorata. It is beneficial to provide potential strategies for improving antrodin C and antroquinonol production by genetic means. © 2020 Society of Chemical Industry.

Antrodin A from mycelium of Antrodia camphorata alleviates acute alcoholic liver injury and modulates intestinal flora dysbiosis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Antrodia camphorata (A. camphorata) is a rare functional fungus in Taiwan and contains a variety of biologically active ingredients. Antrodin A (AdA) is one of the main active ingredients in the solid-state fermented A. camphorata mycelium. It protects the liver from alcohol damage by improving the antioxidant and anti-inflammatory capacity of the liver and maintaining the stability of the intestinal flora. AIM OF THE STUDY: The aim of this study was to evaluate the hepatoprotective activities of ethyl acetate layer extract (EALE), AdA, and Antroquinonol (Aq) from mycelium of A. camphorata on alcoholic liver injury. MATERIALS AND METHODS: Mice were given with intragastrically vehicle (NC, 2% CMC-Na), alcohol (AL, 12 mL/kg bw), or different A. camphorata samples (EALE, AdA, Aq) at low (100 mg/kg bw) or high (200 mg/kg bw) dosages. The positive control (PC) group was given with silymarin (200 mg/kg bw). Except the NC group, each group of mice was fasted for 4 h after the last treatment and was intragastrically administrated with 50% alcohol (12 mL/kg bw). At the end of experiment, mouse serum was collected and the liver was excised. A portion of the liver was fixed in formalin and used for histopathological analysis, whereas the rest was used for biochemical analysis and real-time PCR analysis. The intestinal flora structure of feces was analyzed by determining the v3-v4 region sequence in 16S rDNA. RESULTS: The high-dose groups of the three samples (EALEH, AdAH, and AqH) significantly alleviated the alcohol-induced increases in liver index, serum ALT, AST, and AKP activities. Serum TG level was significantly reduced in all treatment groups. The increase of HDL-C content indicated that active ingredients of A. camphorata could reduce the lipid content in serum. Furthermore, MDA contents of the AdAH and AqH groups in liver were significantly reduced, accompanying with the levels of SOD, CAT, and GSH elevated to various extents. Antioxidant and anti-inflammatory capabilities in the liver were increased in the AdAH group, as evidenced by the mRNA expression levels of Nrf-2 and HO-1 were significantly increased; while those of CYP2e1, TNF-α, and TLR-4 were significantly decreased. Analysis of intestinal flora of feces showed that alcohol treatment significantly changed the composition of intestinal flora. Supplementation with AdA could mitigate dysbiosis of intestinal flora induced by alcohol. Flora of Faecalibaculum, Lactobacillus, and Coriobacteriaceae_UCG-002 showed significantly negative correlations with ALT, AST, AKP, and MDA levels. CONCLUSION: Antrodin A could improve the antioxidant and anti-inflammatory capacities of the liver and maintain the stability of intestinal flora. It is potentially a good candidate compound against acute alcoholic liver injury.

Human-origin Lactobacillus salivarius AR809 protects against immunosuppression in S. aureus-induced pharyngitis via Akt-mediated NF-κB and autophagy signaling pathways.

Lactobacillus salivarius AR809 is a newly discovered probiotic strain from a healthy human pharynx and has potential ability to adhere to the pharyngeal epithelium and inhibit Staphylococcus aureus (S. aureus)-induced inflammatory response. Pharyngeal spray administration of AR809 exhibited protective effects in a S. aureus-induced mouse model of pharyngitis. The inhibitory effect and underlying molecular mechanism of AR809 on S. aureus-stimulated pharyngitis were further investigated. AR809 significantly increased phagocytosis and bactericidal activity, reduced the production of inflammatory mediators (intracellular reactive oxygen species (ROS), prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), nitric oxide (NO), inducible NOS (iNOS)) and the expression of inflammatory cytokines (tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß)), and induced macrophages to adopt the M2 phenotype. AR809 also attenuated S. aureus-induced phosphorylations of protein kinase B (Akt) and rapamycin (mTOR), and elevated the autophagic protein (light chain 3 from II (LC3-II) and Beclin-1) level. Furthermore, AR809 inhibited nuclear transcription factor kappa-B (NF-κB) activation by suppressing the nuclear translocation of NF-κB p65. Likewise, 740Y-P (a PI3K activator) decreased the anti-inflammatory effect of AR809 against S. aureus-induced inflammatory response, while AR809 treatments with wortmannin (a PI3K inhibitor) markedly reversed this inflammatory response. AR809 prevents S. aureus-induced pharyngeal inflammatory response, possibly by regulating TLR/PI3K/Akt/mTOR signalling pathway-related autophagy and TLR/PI3K/Akt/IκB/NF-κB pathway activity, and therefore has potential for use in preventing pharyngitis and other inflammatory diseases.

The ameliorative effect of a Lactobacillus strain with good adhesion ability against dextran sulfate sodium-induced murine colitis.

The objective of this study was to effectively screen out a Lactobacillus strain with excellent adhesion ability and ameliorative effect on the disease symptoms of a murine ulcerative colitis model. The auto-aggregation rate (RAA), co-aggregation rate with Escherichia coli O157 (RCA), and cell surface hydrophobicity (AHC) of 17 Lactobacillus strains were measured for primary selection. The results indicate that Lactobacillus plantarum AR326 displayed the topmost overall aggregation performance among all strains examined. A positive and linear relationship between RCA and RAA values was observed for 15 Lactobacillus strains with RAA = 16-46% and RCA = 15-27%. For adhesion ability to human adenocarcinoma HT-29 cells, five representative Lactobacillus strains showed a good, positive dependence on RAA, where L. plantarum AR326 and L. fermentum AR184 showed a higher adhesion ability than the others. Using the 5 (6)-carboxyfluorescein diacetate N-succinimidyl ester (cFDA SE)-labeling technique, L. plantarum AR326 was confirmed to adhere to and colonize well in the intestinal mucosa of mice mainly in the ileum and colon. Finally, L. plantarum AR326 at the dosage applied (daily 2 × 109 cfu per mouse) could attenuate murine dextran sulfate sodium (DSS)-induced colitis by effectively decreasing the body weight loss, disease activity index, colon length shortening, myeloperoxidase activity, and colon epithelial damage of experimental animals. The protective effects involved the restoration of the tight junction protein expression and reduction of the abnormal expression of pro-inflammatory cytokines. In conclusion, L. plantarum AR326 can be used as promising probiotics to ameliorate DSS-induced colitis.