[Mechanism of Yuxuebi Tablets in treating synovial inflammation in rheumatoid arthritis based on transcriptomics].

This study investigated the mechanism of Yuxuebi Tablets(YXB) in the treatment of synovial inflammation in rheumatoid arthritis(RA) based on transcriptomic analysis. Transcriptome sequencing technology was employed to analyze the gene expression profiles of joint tissues from normal rats, collagen-induced arthritis(CIA) rats(an RA model), and YXB-treated rats. Common diffe-rentially expressed genes(DEGs) were subjected to Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses. RA synovial inflammation-related target genes were retrieved from the OMIM and GeneCards databases. Venny 2.1 software was used to identify the intersection of YXB target genes and RA synovial inflammation-related target genes, and GO and KEGG enrichment analyses were performed on the intersecting target genes. Immunohistochemistry was used to assess the protein expression levels of the inflammatory factors interleukin-1ß(IL-1ß) and tumor necrosis factor-α(TNF-α) in rat joint tissues. Western blot analysis was employed to measure the expression levels of key proteins in the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt) signaling pathway. A total of 2 058 DEGs were identified by intersecting the genes from the normal group vs model group and the model group vs YXB treatment group. A search in OMIM and GeneCards databases yielded 1 102 RA synovial inflammation-related target genes. After intersecting with the DEGs in the YXB treatment group, 204 intersecting target genes were identified, primarily involving biological processes such as immune response, signal transduction, and inflammatory response; cellular components including plasma membrane, extracellular space, and extracellular region; molecular functions like protein binding, identical protein binding, and receptor binding. These target genes were mainly enriched in signaling pathways such as PI3K/Akt, cytokine-cytokine receptor interaction, and Janus kinase/signal transducer and activator of transcription(JAK/STAT). Western blot results showed that YXB at low, medium, and high doses could significantly inhibit the expression levels of key proteins in the PI3K/Akt signaling pathway in rat joint tissues in a dose-dependent manner. Immunohistochemistry further confirmed these findings, showing that YXB not only suppressed the protein expression levels of the inflammatory factors IL-1ß and TNF-α in the joint synovial tissues of CIA rats, but also inhibited p-Akt protein expression. In conclusion, this study used transcriptomic analysis to uncover the key mechanisms of YXB in inhibiting synovial inflammation and alleviating the progression of RA, with a focus on its role in suppressing the PI3K/Akt signaling pathway.

Lactiplantibacillus plantarum P101 alleviates alcoholic liver injury by modulating the Nrf2/HO-1 pathway in mice.

AIMS: The occurrence of alcoholic liver injury is related to the oxidative stress. Bacteria for alleviating alcoholic related liver injury have received widespread attention. Study aims to investigate the alleviated efficacy of Lactiplantibacillus plantarum (L. plantarum) P101 on alcohol-induced liver injury and its potential mechanism. METHODS AND RESULTS: The model of alcoholic liver injury was obtained according to the NIAAA method and the mice were treated with L. plantarum P101 (108 CFU.mice-1). Results showed that treatment of L. plantarum P101 could significantly improve liver function and antioxidant capacity. Furthermore, L. plantarum P101 significantly up-regulated Nuclear factor erythroid 2-related factor (Nrf2) and its target molecule, Hemeoxygenase 1 (HO-1), by promoting nuclear translocation of Nrf2. Moreover, inflammatory factors and pro-apoptotic protein (Caspase3) levels were significantly decreased in mice treated with L. plantarum P101. CONCLUSIONS: This study confirmed that the beneficial effect of L. plantarum P101 supplement was achieved via regulating Nrf2/HO-1 antioxidant pathway, and alleviated alcoholic liver injury.

[Intervention effect of Qufeng Gutong Cataplasm on myofascial pain syndrome in rats and its mechanism].

This paper aims to investigate the intervention effect of Qufeng Gutong Cataplasm(QFGT) on myofascial pain syndrome(MPS) in rats and to preliminarily explain its mechanism from the perspective of improving muscle inflammation and pain. Male SD rats were divided into 6 groups, namely normal group, model group, positive control drug(Huoxue Zhitong Ointment, HXZT) group, and low, medium, and high-dose QFGT groups(75, 150, and 300 mg·d~(-1)). The rat model of MPS was established by striking combined with centrifugation for 8 weeks, during which QFGT and HXZT were used for corresponding intervention. Standard VonFrey fiber was used to evaluate the mechanical pain threshold, and acetone was used to detect the cold pain threshold. The electrophysiological activity of muscle at trigger point was detected, and the electromuscular analysis of trigger point was performed. CatWalk gait analyzer was used to detect pain-induced gait adaptation changes. The hematoxylin-eosin(HE) staining was used to observe the pathological changes in muscle and skin tissues at the trigger point of rats. Immunohistochemistry was used to detect the expression of capsaicin receptor transient receptor potential vanilloid 1(TRPV1) in muscle tissues and interleukin(IL)-33 in skin tissues at the trigger point. The protein expression levels of TRPV1, protein kinase B(Akt), phosphorylated protein kinase B(p-Akt), IL-1ß, and tumor necrosis factor-α(TNF-α) in muscle tissues at the trigger point were detected by Western blot. The results showed that as compared with the model group, the mechanical pain threshold and cold pain threshold of rats in other groups were increased after treatment with QFGT. The spontaneous electromyography(EMG) activity was observed in the model group, but QFGT alleviated the EMG activity in a dose-dependent manner. Gait analysis showed that standing duration, average intensity, swing speed, maximum contact point, maximum contact area, paw print length, paw print width, and paw print area were significantly improved in all QFGT groups. Pathological results showed that the disorder of muscle arrangement at the trigger point was decreased, muscle fiber adhesion and atrophy were reduced, and inflammatory cell infiltration was alleviated after treatment with QFGT. In addition, QFGT and HXZT both inhibited the protein expression of TRPV1, PI3K, Akt, p-Akt, IL-1ß, and TNF-α in the muscle tissues of rats with MPS. However, there was no significant difference in the pathological structure and expression of IL-33 in the treated skin as compared with the normal group. The related results have proved that QFGT can inhibit the release of inflammatory factors by inhibiting the TRPV1/PI3K/Akt signaling pathway in the muscle trigger point of rats with MPS and finally attenuate the atrophy and adhesion of local muscles and inflammatory infiltration, thereby relieving the muscle pain of rats with MPS, and local administration has no skin irritation.

[Mechanism of artesunate on bone destruction in experimental rheumatoid arthritis based on transcriptomics and network pharmacology].

The present study investigated the mechanism of artesunate in the treatment of bone destruction in experimental rheumatoid arthritis(RA) based on transcriptomics and network pharmacology. The transcriptome sequencing data of artesunate in the inhibition of osteoclast differentiation were analyzed to obtain differentially expressed genes(DEGs). GraphPad Prism 8 software was used to plot volcano maps and heat maps were plotted through the website of bioinformatics. GeneCards and OMIM were used to collect information on key targets of bone destruction in RA. The DEGs of artesunate in inhibiting osteoclast differentiation and key target genes of bone destruction in RA were intersected by the Venny 2.1.0 platform, and the intersection target genes were analyzed by Gene Ontology(GO)/Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment. Finally, the receptor activator of nuclear factor-κB(RANKL)-induced osteoclast differentiation model and collagen-induced arthritis(CIA) model were established. Quantitative real time polymerase chain reaction(q-PCR), immunofluorescence, and immunohistochemistry were used to verify the pharmacological effect and molecular mechanism of artesunate in the treatment of bone destruction in RA. In this study, the RANKL-induced osteoclast differentiation model in vitro was established and intervened with artesunate, and transcriptome sequencing data were analyzed to obtain 744 DEGs of artesunate in inhibiting osteoclast differentiation. A total of 1 291 major target genes of bone destruction in RA were obtained from GeneCards and OMIM. The target genes of artesunate in inhibiting osteoclast differentiation and the target genes of bone destruction in RA were intersected to obtain 61 target genes of artesunate against bone destruction in RA. The intersected target genes were analyzed by GO/KEGG enrichment. According to the results previously reported, the cytokine-cytokine receptor interaction signaling pathway was selected for experimental verification. Artesunate intervention in the RANKL-induced osteoclast differentiation model showed that artesunate inhibited CC chemokine receptor 3(CCR3), CC chemokine receptor 1(CCR1) and leukemia inhibitory factor(LIF) mRNA expression in osteoclasts in a dose-dependent manner compared with the RANKL-induced group. Meanwhile, the results of immunofluorescence and immunohistochemistry showed that artesunate could dose-dependently reduce the expression of CCR3 in osteoclasts and joint tissues of the CIA rat model in vitro. This study indicated that artesunate regulated the CCR3 in the cytokine-cytokine receptor interaction signaling pathway in the treatment of bone destruction in RA and provided a new target gene for the treatment of bone destruction in RA.

Integrated Fabry-Perot filter with wideband noise suppression for satellite-based daytime quantum key distribution.

Spectral filtering is essential in daytime quantum key distribution (QKD), which can suppress the strong background noise caused by scattered solar irradiation. An integrated Fabry-Perot filter is implemented based on a scheme that combines a Fabry-Perot etalon and a dense-wavelength-division-multiplex filter for narrow linewidth filtering and broad-spectrum noise suppression, respectively. This filter is integrated into a butterfly package with single-mode fibers for optical input and output, thereby enhancing high robustness and ease of use. The measurement results show that the filter has a linewidth of 25.6 pm, a noise suppression of over 44.7 dB ranging between 1380-1760 nm, an optical efficiency of 74.5% with variation less than 0.9% in 120 min, and a polarization fidelity after compensation exceeding 99.9%. The ability of fine-tuning the central wavelength with 9.5 pm/°C makes it very suitable for satellite-based applications under the Doppler effect. Further analysis is also given to demonstrate the prospects of applying this filter in future satellite-based daytime QKD applications.

[Inhibitory effect of artesunate on bone destruction in rheumatoid arthritis: an exploration based on AhR/ARNT/NQO1 signaling pathway].

This study aimed to explore the effect of artesunate(ARS) on bone destruction in rheumatoid arthritis(RA) based on the aryl hydrocarbon receptor(AhR)/AhR nucleart ranslocator(ARNT)/NAD(P)H quinone dehydrogenase 1(NQO1) signaling pathway. Macrophage-colony stimulating factor(M-CSF) and receptor activator of nuclear factor-κB(RANKL) were used to induce the differentiation of primary bone marrow-derived mouse macrophages into osteoclasts. After intervention with ARS(0.2, 0.4, and 0.8 µmol·L~(-1)), the formation and differentiation of osteoclasts were observed by tartrate-resistant acid phosphatase(TRAP) and F-actin staining. The protein expression levels of AhR and NQO1 were detected by Western blot, and their distribution in osteoclasts was observed by immunofluorescence localization. Simultaneously, the collagen induced arthritis(CIA) rat model was established using type Ⅱ bovine collagen emulsion and then treated with ARS(7.5, 15, and 30 mg·kg~(-1)) by gavage for 30 days. Following the observation of spinal cord and bone destruction in CIA rats by Masson staining, the expression of AhR and ARNT in rat knee joint tissue was measured by immunohistochemistry and the NQO1 protein expression in the knee joint tissue by Western blot. The results showed that a large number of TRAP-positive cells were present in RANKL-induced rats. Compared with the RANKL-induced group, ARS(0.2, 0.4, and 0.8 µmol·L~(-1)) inhibited the number of TRAP-positive cells in a dose-dependent manner. F-actin staining results showed that the inhibition of F-actin formation was enhanced with the increase in ARS dose. As revealed by Western blot and immunofluorescence assay, ARS significantly promoted the expression of AhR and its transfer to the nucleus, thereby activating the protein expression of downstream ARNT and antioxidant enzyme NQO1. At the same time, the CIA rat model was successfully established. Masson staining revealed serious joint destruction in the model group, manifested by the failed staining of surface cartilage, disordered arrangement of collagen fibers, and unclear boundaries of cartilage and bone. The positive drug and ARS at different doses all improved cartilage and bone destruction to varying degrees, with the best efficacy detected in the high-dose ARS group. According to immunohistochemistry, ARS promoted AhR and ARNT protein expression in knee cartilage and bone of CIA rats and also NQO1 protein expression in rat knee and ankle joint tissues. In conclusion, ARS inhibited osteoclast differentiation by activating the AhR/ARNT/NQO1 signaling pathway, thus alleviating RA.

[Anti-rheumatoid arthritis mechanism of Sophorae Tonkinesis Radix et Rhizoma based on network pharmacology and experimental verification].

Based on the network pharmacology, molecular docking, and animal experiment, this study explored the anti-rheumatoid arthritis(RA) mechanism of Sophorae Tonkinesis Radix et Rhizoma(STRR). The active components of STRR were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), Traditional Chinese Medicine Integrative Database(TCMID), and previous research, main targets of STRR from TCMSP and SwissTargetPrediction, and targets of RA from GeneCards, DrugBank, Online Mendelian Inheritance in Man(OMIM), and Therapeutic Target Database(TTD). The common targets of the two were screened by Venny 2.1.0. Cytoscape 3.6.0 was used to generate the "component-target" network, and STRING and Cytoscape were used to construct the protein-protein interaction(PPI) network. DAVID 6.8 was employed for Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment, and AutoDock Vina for molecular docking. Finally, collagen-induced rheumatoid arthritis(CIA) mouse model was constructed, and the expression of core target proteins was detected by Western blot. A total of 27 active components, including quercetin, genistein, kaempferol, subprogenin C, and daidzein, and 154 anti-RA targets, such as signal transducer and activator of transcription 3(STAT3), tumor necrosis factor(TNF), mitogen-activated protein kinase 1(MAPK1), AP-1 transcription factor subunit(JUN), and interleukin 6(IL6), of STRR were screened out. It was preliminarily indicated that STRR may regulate phosphatidylinositol-3-kinase-protein kinase B(PI3 K-AKT) signaling pathway and TNF signaling pathway to modulate the positive regulation of RNA polymerase Ⅱ promoter transcription, inflammatory response, and other biological processes, thus exerting the anti-RA effect. The results of molecular docking showed that the main active components in STRR had high binding affinity to the core targets. Animal experiment suggested that the water extract of STRR can significantly reduce the levels of p-STAT3, p-MAPK1, and TNF. This study demonstrated the multi-component, multi-target and multi-pathway synergistic effect of STRR in the treatment of RA, laying an experimental basis for clinical application of this medicine.

Protection of surface layer protein from Enterococcus faecium WEFA23 against Listeria monocytogenes CMCC54007 infection by modulating intestinal permeability and immunity.

Enterococcus faecium WEFA23 was previously found effectively against adherence and colonization of Listeria monocytogenes CMCC54007, which might be closely related to its surface layer protein (SLP). In this study, the protective of SLP of E. faecium WEFA23 against infection of L. monocytogenes CMCC54007 was systemically investigated. In vitro assay showed that SLP actively inhibited L. monocytogenes internalization into Caco-2 cell line, with decreasing mRNA level of pro-inflammation cytokines and virulence factors and restoring destroyed intestinal barrier. In vivo assay through excluding SLP of E. faecium WEFA23 by 5 M LiCl represented that SLP increased body weight, reduced mortality and cell counts of L. monocytogenes CMCC54007 in tissues of mice. Further researches showed that SLP protected against L. monocytogenes CMCC54007 infection by modulation of intestinal permeability and immunity, namely, it decreased fluorescein isothiocyanate (FITC)-Dextran in serum, ameliorated destroyed colon structure, and increased number of goblet cells and protein level of TJ protein (Claudin-1, Occludin, and ZO-1) in colon. For immunity, SLP decreased number of CD4+ and CD8+ T cells in liver, mRNA level, and content of pro-inflammatory factors IL-6, IL-1ß, IFN-γ ,TNF-α, and NO, and restored the structure of liver and spleen. Key Points•SLP of E. faecium inhibited L. monocytogenes internalization and colonization•SLP of E. faecium ameliorated host intestinal barrier dysfunction•SLP of E. faecium decreased pro-inflammatory cytokines and cells.

Probiotic Enterococcus faecalis Symbioflor 1 ameliorates pathobiont-induced miscarriage through bacterial antagonism and Th1-Th2 modulation in pregnant mice.

The bacterium-bacterium interaction between pathogenic and probiotic Enterococcus as well as the bacterium-host interaction between Enterococcus and intestinal epithelium has drawn increasing attentions, but the influence of those interactions on host pregnancy remains largely unexplored. In the present study, we evaluated the effects of probiotic E. faecalis Symbioflor 1 or/and pathogenic E. faecalis OG1RF on the miscarriage of pregnant mice. Using in vitro assays of competition and exclusion and displacement, antagonistic property of E. faecalis Symbioflor 1 against E. faecalis OG1RF was observed, and the former inhibited the translocation of the later in vivo. The rate of miscarriage induced by E. faecalis OG1RF challenge was significantly reduced by 28% with E. faecalis Symbioflor 1 intervention; and the tissue integrity of ileum, colon, uterus, and placenta and placental blood cell density in pregnant mice were drastically improved by such probiotic intervention. Compared with the controls, probiotic intervention significantly upregulated the level of IL-10 and TGF-ß, downregulated levels of IFN-γ, and increased progesterone level that reversed the trend of being Th1 predominance state reported for adverse pregnancy outcome at early pregnancy stage. In conclusion, E. faecalis Symbioflor 1 decreased the translocation of E. faecalis OG1RF, prevented pathogen-induced tissue damage, and changed Th1-Th2 homeostasis toward Th2 predominance during early pregnancy resulting in decreased miscarriage. KEY POINTS: •The mechanism of how probiotic E. faecalis Symbioflor 1 improves pregnancy of mice • Influence of interactions of pathogenic and probiotic Enterococcus on host pregnancy • E. faecalis Symbioflor 1 change Th1-Th2 homeostasis toward Th2 predominance.

Pharmacokinetics and tissue distribution study of herpetotriol in rats by ultrahigh-performance liquid chromatography with tandem mass spectrometry.

Herpetotriol, a typical lignan in Herpetospermum pedunculosum Wall's seeds that has long been used to treat icterhepatitis and indigestion and other related diseases in Tibet, is of potential hepatoprotection. This study aims to study the pharmacokinetics features of herpetotriol, including the blood drug concentration - time curve and tissue distribution. The ultrahigh-performance liquid chromatography with tandem mass spectrometry method was established to detect herpetotriol concentration in plasma and tissues, and the method showed good linearity from 10 to 2000 ng/mL (r ≥ 0.9972) and sensitivity (≥10 ng/mL). Our blood drug concentration - time curve indicated that herpetotriol was distributed quickly in rats with a Tmax value at about 0.083 h and eliminated rapidly with a clearance rate at 98.13 ± 8.05 and 137.04 ± 9.48 L·h-1·kg-1 with doses of 5 and 2.5 mg/kg, respectively. Although herpetotriol was detectable in all tested tissues, it has a higher concentration in liver than in heart, lung, spleen, and kidney, which is in line with its hepatoprotection. The pharmacokinetics features uncovered by the present study could provide more information for future pharmacological and toxicological study of herpetotriol.

Enterococcus hirae WEHI01 isolated from a healthy Chinese infant ameliorates the symptoms of type 2 diabetes by elevating the abundance of Lactobacillales in rats.

Enterococcus hirae WEHI01 is a potential probiotic strain isolated from a healthy Chinese infant. This strain has previously been characterized as having cholesterol-lowering potential and good dairy fermentation performance. In this study, we used rat models with obesity and type 2 diabetes mellitus (T2DM) induced by a high fat and sucrose diet and low-dose streptozotocin, respectively, and we evaluated the effect of E. hirae WEHI01 on glycolipid metabolism, glycolipid-related gene expression, organ histopathology, and intestinal flora changes in the 2 models. Our results showed that administration of 5.0 × 109 cfu of E. hirae WEHI01 for 4 wk decreased serum lipid levels and regulated glycolipid metabolism in the liver of obese rats. Following continuous administration of the same concentration of E. hirae WEHI01 to a T2DM rat model for another 5 wk, E. hirae WEHI01 improved glucose tolerance, recovered body weight loss, and led to significant decreases in tumor necrosis factor-α, IL-6, IL-10, and total bile acid in serum. We also found that E. hirae WEHI01 restored the morphology of the pancreas, kidney, and liver, and changed the composition of the gut microbiota (i.e., decreased the Shannon index, increased the Simpson index, and substantially increased the abundance of Lactobacillales). Combining the results for the obese model and the T2DM model, we speculated that beneficial effects of E. hirae WEHI01 on T2DM could be due to (1) a significant increase in PPARA expression and a tendency for increased CYP7A1 expression in the liver of obese rats, promoting the conversion of cholesterol into bile acid and reducing serum total bile acid levels in T2DM model rats; or (2) a change in gut microbial diversity, especially elevated Lactobacillales abundance, which reduced the total bile acid in T2DM model rats. These results demonstrated that E. hirae WEHI01 has the potential to ameliorate type 2 diabetes in rats and provide a promising rationale for further research into the prevention and treatment of T2DM.

Anti-adhesion of probiotic Enterococcus faecium WEFA23 against five pathogens and the beneficial effect of its S-layer proteins against Listeria monocytogenes.

Enterococcus faecium WEFA23 is a potential probiotic strain isolated from Chinese infant feces. In this study, the antagonistic activity of E. faecium WEFA23 on adhesion to pathogens was investigated. Enterococcus faecium WEFA23 was able to compete, exclude, and displace the adhesion of Escherichia coli O157:H7, Salmonella Typhimurium ATCC 13311, Listeria monocytogenes CMCC54007, Staphylococcus aureus CMCC26003, and Shigella sonnei ATCC 25931 to Caco-2 cells. Among them, L. monocytogenes achieved the strongest inhibition rate in both competition and displacement assays. Those anti-adhesion capacities were related to the bacterial physicochemical properties (hydrophobicity, auto-aggregation, and co-aggregation) of the bacterial surface. For L. monocytogenes, the anti-adhesion capacity was affected by the heat treatment, cell density, and growth phase of E. faecium WEFA23; 108 colony-forming units of viable cells per millilitre at the stationary phase exhibited the strongest anti-adhesion activity. In addition, removal of S-layer proteins of E. faecium WEFA23 by treatment with 5 mol/L LiCl significantly decreased its adhesion capacity, and those S-layer proteins were able to compete, displace, and exclude L. monocytogenes at different levels. Both cells and S-layer proteins of E. faecium WEFA23 significantly reduced the apoptosis of Caco-2 cells induced by L. monocytogenes, which was mediated by caspase-3 activation. This study might be helpful in understanding the anti-adhesion mechanism of probiotics against pathogens.

Sulfonation of Lactobacillus plantarum WLPL04 exopolysaccharide amplifies its antioxidant activities in vitro and in a Caco-2 cell model.

Exopolysaccharide (EPS) of Lactobacillus plantarum WLPL04 and its sulfated EPS were systematically investigated for their antioxidant activities and effects on protecting the oxidative damage of Caco-2 cells from H2O2. Exopolysaccharide was successfully sulfonated from purified EPS as confirmed by Fourier-transform infrared spectroscopy, and the degree of sulfonation was 0.30. Both EPS and sulfated EPS showed antioxidant activities in vitro determined by 1,1-diphenyl-2-picrylhydrazyl, superoxide, and hydroxyl radical scavenging tests, and those activities of sulfated EPS were significantly enhanced at 1,000 µg/mL. Cell viabilities of Caco-2 in the range of 1 to 100 µg/mL of EPS and sulfated EPS showed no significant difference. In H2O2-damaged Caco-2 cells models, EPS and sulfated EPS significantly inhibited the enhancement of reactive oxygen species and malondialdehyde levels, and sulfated EPS enhanced the effects by 40.86% and 61.11% when compared with the purified EPS at the same concentration of 100 µg/mL, respectively. For the activities of antioxidant-related enzymes (superoxide dismutase, catalase, and glutathione peroxidase) and expression of genes (SOD2, GPX2, MT1M) on Caco-2 cells, strong protection abilities against the oxidative stress were displayed from both EPS and sulfated EPS, and sulfated EPS exhibited significant enhancement as compared with either EPS or control groups. In summary, sulfonation is an effective strategy for improving the antioxidant activities of EPS from L. plantarum WLPL04 in vitro and on Caco-2 cells.

Enterococcus faecium WEFA23 from infants lessens high-fat-diet-induced hyperlipidemia via cholesterol 7-alpha-hydroxylase gene by altering the composition of gut microbiota in rats.

Enterococcus faecium WEFA23 is a potential probiotic strain from Chinese infants with the ability to decrease cholesterol levels. Aiming to explore the mechanism of E. faecium WEFA23 in lowering cholesterol in vivo, we examined the gene transcriptions related to cholesterol metabolism, the composition of bile acids in feces, the synthesis of trimethylamine N-oxide (TMAO) in liver, and the composition of the gut microbiota of rats. We found that E. faecium WEFA23 enhanced the synthesis of bile acids by promoting cholesterol excretion, upregulating the genes transcript level relevant to cholesterol decomposition and transportation, and downregulating the genes involved in cholesterol synthesis. In addition, E. faecium WEFA23 not only downregulated the transcript levels of farnesoid X receptor and fibroblast growth factor 15 as well as flavin-containing monooxygenase 3, but also decreased the TMAO production followed by increasing the CYP7A1 transcript level. Furthermore, when orally administered to rats for 35 d, E. faecium WEFA23 improved the gut microbiota diversity of rats fed a high-fat diet. Therein, the ratio of Bacteroidetes to Firmicutes and the abundance of Rikenellaceae increased, whereas the number of Veillonellaceae decreased. These results suggest that reduction of cholesterol level by E. faecium WEFA23 might be related to the changes in the gut microbiota. Our finding provides important information on lowering cholesterol by E. faecium and reveals that Enterococcus spp. might have the potential to decrease the TMAO level.

Characterization and bioactivities of the exopolysaccharide from a probiotic strain of Lactobacillus plantarum WLPL04.

Exopolysaccharide (EPS) was extracted and purified from Lactobacillus plantarum WLPL04, which has been confirmed previously as a potential probiotic for its antagonistic and immune-modulating activity. It has a molecular weight of 6.61 × 104 Da, consisting of xylose, glucose, and galactose in an approximate molar ratio of 3.4:1.8:1. Microstructural studies demonstrated that the EPS appeared as a smooth sheet structure with many homogeneous rod-shaped lumps. The preliminary in vitro assays indicated that the EPS could significantly inhibit the adhesion of Escherichia coli O157:H7 to HT-29 cells in competition, replacement, and inhibition assays at a dose of 1.0 mg/mL, with an inhibition rate of 20.24 ± 2.23, 29.71 ± 1.21, and 30.57 ± 1.73%, respectively. Additionally, the EPS exhibited strong inhibition against biofilm formation by pathogenic bacteria, including Pseudomonas aeruginosa CMCC10104, E. coli O157:H7, Salmonella Typhimurium ATCC13311, and Staphylococcus aureus CMCC26003. Furthermore, the EPS showed good inhibitory activity against the proliferation of HT-29 cells. The characteristics and bioactivities of this EPS may make it a promising candidate in developing functional food.

Beneficial effects of probiotic cholesterol-lowering strain of Enterococcus faecium WEFA23 from infants on diet-induced metabolic syndrome in rats.

The aim of this study was to select probiotic Enterococcus strains that have the potential to improve metabolic syndrome (MS). Ten Enterococcus strains isolated from healthy infants were evaluated for their probiotic properties in vitro, and Enterococcus faecium WEFA23 was selected due to its cholesterol removal ability (1.89 ± 0.07 mg/1010 cfu), highest glycodeoxycholic acid-hydrolase activity (1.86 ± 0.01 U/mg), and strong adhesion capacity to Caco-2 cells (17.90 ± 0.19%). The safety of E. faecium WEFA23 was verified by acute oral administration in mice, and it was found to have no adverse effects on general health status, bacterial translocation, and gut mucosal histology. Moreover, the beneficial effects of E. faecium WEFA23 on high-fat diet-induced MS in rats were investigated, and we found WEFA23 significantly decreased body weight, serum lipid levels (total cholesterol, triacylglycerols, and low-density lipoprotein cholesterol), blood glucose level, and insulin resistance in rats fed with a high-fat diet. This indicated that administration of E. faecium WEFA23 improved almost all key markers of MS, including obesity, hyperlipidemia, hyperglycemia, and insulin resistance. Our results supported E. faecium WEFA23 as a candidate for cholesterol-lowering dairy products and improvement of MS. Our research provided novel insights on Enterococcus as a strategy to combat MS.

Antagonistics against pathogenic Bacillus cereus in milk fermentation by Lactobacillus plantarum ZDY2013 and its anti-adhesion effect on Caco-2 cells against pathogens.

Lactobacillus plantarum ZDY2013 is a potential probiotic isolated from fermented bean acid. In this study, we aimed to evaluate the in vitro antimicrobial activity of this organism against Bacillus cereus in milk fermentation, the antiadhesion ability on intestinal epithelial cells, as well as its ability to abrogate the cytotoxic effect and expression levels of genes. We found no antimicrobial activity produced by L. plantarum once the pH was adjusted to 6.0 and 7.0. The pH decreased continuously when L. plantarum and B. cereus were co-incubated during milk fermentation, which caused a decrease in the B. cereus counts. Antiadhesion assays showed that L. plantarum can significantly inhibit the adhesion of enterotoxin-producing B. cereus ATCC14579 and pathogenic B. cereus HN001 by inhibition, competition, and displacement. The supernatants of B. cereus, either alone or in conjunction with L. plantarum, caused damage to the membrane integrity of Caco-2 cells to release lactate dehydrogenase. In addition, L. plantarum tended to attenuate proinflammatory cytokine and oxidative stress gene expression on Caco-2 cells, inducing with B. cereus HN001 supernatants. This study provided systematic insights into the antagonistic effect of L. plantarum ZDY2013, and the information may be helpful to explore potential control measures for preventing food poisoning by lactic acid bacteria.

Physiological and transcriptional responses and cross protection of Lactobacillus plantarum ZDY2013 under acid stress.

Acid tolerance responses (ATR) in Lactobacillus plantarum ZDY2013 were investigated at physiological and molecular levels. A comparison of composition of cell membrane fatty acids (CMFA) between acid-challenged and unchallenged cells showed that acid adaptation evoked a significantly higher percentage of saturated fatty acids and cyclopropane fatty acids in acid-challenged than in unchallenged cells. In addition, reverse transcription-quantitative PCR analysis in acid-adapted cells at different pH values (ranging from 3.0 to 4.0) indicated that several genes were differently regulated, including those related to proton pumps, amino acid metabolism, sugar metabolism, and class I and class III stress response pathways. Expression of genes involved in fatty acid synthesis and production of alkali was significantly upregulated. Upon exposure to pH 4.5 for 2 h, a higher survival rate (higher viable cell count) of Lactobacillus plantarum ZDY2013 was achieved following an additional challenge to 40 mM hydrogen peroxide for 60 min, but no difference in survival rate of cells was found with further challenge to heat, ethanol, or salt. Therefore, we concluded that the physiological and metabolic changes of acid-treated cells of Lactobacillus plantarum ZDY2013 help the cells resist damage caused by acid, and further initiated global response signals to bring the whole cell into a state of defense to other stress factors, especially hydrogen peroxide.

Short communication: Modulation of the small intestinal microbial community composition over short-term or long-term administration with Lactobacillus plantarum ZDY2013.

The small intestinal (SI) microbiota has an essential role in the maintenance of human health. However, data about the indigenous bacteria in SI as affected by probiotics are limited. In our study, the short-term and long-term effects of a probiotic candidate, Lactobacillus plantarum ZDY2013, on the SI microbiota of C57BL/6J mice were investigated by the Illumina HiSeq (Novogene Bioinformatics Technology Co., Ltd., Tianjin, China) platform targeting the V4 region of the 16S rDNA. A total of 858,011 sequences in 15 samples were read. The α diversity analysis revealed that oral administration with L. plantarum ZDY2013 for 3 wk led to a significant increase in the richness and diversity of the SI bacterial community. Principal coordinate analysis and unweighted pair-group method with arithmetic means analysis showed a clear alteration in the SI microbiota composition after 3 wk of L. plantarum ZDY2013 treatment, although these changes were not found 6 wk after ceasing L. plantarum ZDY2013 administration. Species annotation showed that the dominant phyla in SI microbiota were Firmicutes, Bacteroidetes, Proteobacteria, and Verrucomicrobia. Interestingly, operational taxonomic unit cluster analysis showed that administration with L. plantarum ZDY2013 for 3 wk significantly increased the abundance of Proteobacteria, but decreased that of Bacteroidetes. Linear discriminant analysis coupled with effect size identified 18 bacterial taxa (e.g., Ruminococcus spp. and Clostridium spp.) that overgrew in the SI microbiota of the mice administered with L. plantarum ZDY2013 for 3 wk, and most of them belonged to the phyla Bacteroidetes and Proteobacteria. However, only one bacterial taxon (e.g., Nocardioides spp.) was over-represented in the SI microbiota of mice 6 wk after L. plantarum ZDY2013 administration. Overall, this study shows that oral administration with probiotic results in an important but transient alteration in the microbiota of SI.

Changes in gastric microbiota induced by Helicobacter pylori infection and preventive effects of Lactobacillus plantarum ZDY 2013 against such infection.

Helicobacter pylori is a gram-negative pathogen linked to gastric ulcers and stomach cancer. Gastric microbiota might play an essential role in the pathogenesis of these stomach diseases. In this study, we investigated the preventive effect of a probiotic candidate Lactobacillus plantarum ZDY 2013 as a protective agent against the gastric mucosal inflammation and alteration of gastric microbiota induced by H. pylori infection in a mouse model. Prior to infection, mice were pretreated with or without 400 µL of L. plantarum ZDY 2013 at a concentration of 10(9) cfu/mL per mouse. At 6 wk postinfection, gastric mucosal immune response and alteration in gastric microbiota mice were examined by quantitative real-time PCR and high-throughput 16S rRNA gene amplicon sequencing, respectively. The results showed that L. plantarum ZDY 2013 pretreatment prevented increase in inflammatory cytokines (e.g., IL-1ß and IFN-γ) and inflammatory cell infiltration in gastric lamina propria induced by H. pylori infection. Weighted UniFrac principal coordinate analysis showed that L. plantarum ZDY 2013 pretreatment prevented the alteration in gastric microbiota post-H. pylori infection. Linear discriminant analysis coupled with effect size identified 22 bacterial taxa (e.g., Pasteurellaceae, Erysipelotrichaceae, Halomonadaceae, Helicobacteraceae, and Spirochaetaceae) that overgrew in the gastric microbiota of H. pylori-infected mice, and most of them belonged to the Proteobacteria phylum. Lactobacillus plantarum ZDY 2013 pretreatment prevented this alteration; only 6 taxa (e.g., Lachnospiraceae, Ruminococcaceae, and Clostridiaceae), mainly from the taxa of Firmicutes and Bacteroidetes, were dominant in the gastric microbiota of the L. plantarum ZDY 2013 pretreated mice. Administration of L. plantarum ZDY 2013 for 3 wk led to increase in several bacterial taxa (e.g., Rikenella, Staphylococcus, Bifidobacterium), although a nonsignificant alteration was found in the gastric microbiota. Overall, this study demonstrated that L. plantarum ZDY 2013 pretreatment played an important role in preventing gastric mucosal inflammation and gastric microbiota alteration induced by H. pylori infection, and the selective modulation in gastric microbiota posed by this intervention suggested that targeting gastric microbiota through oral administration of probiotics might be an alternative strategy to prevent H. pylori infection.