DBPF-net: dual-branch structural feature extraction reinforcement network for ocular surface disease image classification.

Pterygium and subconjunctival hemorrhage are two common types of ocular surface diseases that can cause distress and anxiety in patients. In this study, 2855 ocular surface images were collected in four categories: normal ocular surface, subconjunctival hemorrhage, pterygium to be observed, and pterygium requiring surgery. We propose a diagnostic classification model for ocular surface diseases, dual-branch network reinforced by PFM block (DBPF-Net), which adopts the conformer model with two-branch architectural properties as the backbone of a four-way classification model for ocular surface diseases. In addition, we propose a block composed of a patch merging layer and a FReLU layer (PFM block) for extracting spatial structure features to further strengthen the feature extraction capability of the model. In practice, only the ocular surface images need to be input into the model to discriminate automatically between the disease categories. We also trained the VGG16, ResNet50, EfficientNetB7, and Conformer models, and evaluated and analyzed the results of all models on the test set. The main evaluation indicators were sensitivity, specificity, F1-score, area under the receiver operating characteristics curve (AUC), kappa coefficient, and accuracy. The accuracy and kappa coefficient of the proposed diagnostic model in several experiments were averaged at 0.9789 and 0.9681, respectively. The sensitivity, specificity, F1-score, and AUC were, respectively, 0.9723, 0.9836, 0.9688, and 0.9869 for diagnosing pterygium to be observed, and, respectively, 0.9210, 0.9905, 0.9292, and 0.9776 for diagnosing pterygium requiring surgery. The proposed method has high clinical reference value for recognizing these four types of ocular surface images.

Dietary Supplementation With Lactobacillus plantarum Ameliorates Compromise of Growth Performance by Modulating Short-Chain Fatty Acids and Intestinal Dysbiosis in Broilers Under Clostridium perfringens Challenge.

Clostridium perfringens is an important zoonotic pathogen associated with food contamination and poisoning, gas gangrene, necrotizing enterocolitis or necrotic enteritis in humans and animals. Dysbacteriosis is supposedly associated with the development of C. perfringens infection induced necrotic enteritis, but the detailed relationship between intestinal health, microbiome, and C. perfringens infection-induced necrotic enteritis remains poorly understood. This research investigated the effect of probiotics on the growth performance and intestinal health of broilers, and the involved roles of intestinal microbiota and microbial metabolic functions under C. perfringens infection. Results showed that subclinical necrotic enteritis was successfully induced as evidenced by the significant lower body weight (BW), suppressed feed conversion ratio (FCR), decreased ileal villus height and mucosal barrier function, and increased ileal histopathological score and bursal weight index. Lactobacillus plantarum or Paenibacillus polymyxa significantly attenuated C. perfringens-induced compromise of growth performance (BW, FCR) and ileal mucosa damage as illustrated by the increased ileal villus height and villus/crypt ratio, the decreased ileal histopathological score and the enhanced ileal mucosal barrier function. L. plantarum also significantly alleviated C. perfringens-induced enlarged bursa of fabricius and the decreased levels of ileal total SCFAs, acetate, lactate, and butyrate. Furthermore, dietary L. plantarum improved C. perfringens infection-induced intestinal dysbiosis as evidenced by significantly enriched short-chain fatty acids-producing bacteria (Lachnospiraceae, Ruminococcaceae, Oscillospira, Faecalibacterium, Blautia), reduced drug-resistant bacteria (Bacteroides, Alistipes) and enteric pathogens (Escherichia coli, Bacteroides fragilis) and bacterial metabolic dysfunctions as illustrated by significantly increased bacterial fatty acid biosynthesis, decreased bacterial lipopolysaccharide biosynthesis, and antibiotic biosynthesis (streptomycin and vancomycin). Additionally, the BW and intestinal SCFAs were the principal factors affecting the bacterial communities and microbial metabolic functions. The above findings indicate that dietary with L. plantarum attenuates C. perfringens-induced compromise of growth performance and intestinal dysbiosis by increasing SCFAs and improving intestinal health in broilers.

Glycyrrhizin Attenuates Salmonella Typhimurium-Induced Tissue Injury, Inflammatory Response, and Intestinal Dysbiosis in C57BL/6 Mice.

Salmonellae are one of the most important foodborne pathogens, which threaten the health of humans and animals severely. Glycyrrhizin (GL) has been proven to exhibit anti-inflammatory and tissue-protective properties. Here, we investigated the effects of GL on tissue injury, inflammatory response, and intestinal dysbiosis in Salmonella Typhimurium-infected mice. Results showed that GL or gentamicin (GM) significantly (P < 0.05) alleviated ST-induced splenomegaly indicated by the decreased spleen index, injury of liver and jejunum indicated by the decreased hepatocytic apoptosis, and the increased jejunal villous height. GL significantly (P < 0.05) increased secretion of inflammatory cytokines (IFN-γ, IL-12p70, IL-6, and IL-10) in spleen and IL-12p40 mRNA expression in liver. Meanwhile, GL or GM pre-infection treatments significantly (P < 0.05) decreased ST-induced pro-inflammatory cytokine (IFN-γ, TNF-α, and IL-6) expression in both spleen and liver and increased (P < 0.05) anti-inflammatory cytokine IL-10 secretion in spleen. Furthermore, GL or GM pre-infection treatment also regulates the diversities and compositions of intestinal microbiota and decreased the negative connection among the intestinal microbes in ST-infected mice. The above findings indicate that GL alleviates ST-induced splenomegaly, hepatocytic apoptosis, injury of jejunum and liver, inflammatory response of liver and spleen, and intestinal dysbacteriosis in mice.

Alkannin restrains oral squamous carcinoma cell growth, migration and invasion by regulating microRNA-9/RECK axis.

Alkannin (ALK) has anti-inflammatory and anti-tumour activities. We tried to probe the underlying functions of ALK in oral squamous carcinoma (OSCC) cells growth, migration and invasion. OSCC cells viability was investigated after treatment with ALK. Then, BrdU, flow cytometry, Western blot and Transwell assays were executed to appraise proliferation, apoptosis, migration and invasion in OSCC cells with ALK stimulation. The biological functions of miR-9 were explored after miR-9 mimic/inhibitor transfection. The relevance of RECK and miR-9 was predicated by dual luciferase activity assay. JAK1/STAT3 and PI3K/AKT pathways were estimated by Western blot. Tumour formation in vivo was executed by xenograft tumours assay. We found that ALK restrained cell proliferation, facilitated apoptosis, repressed migration and invasion also interdicted JAK1/STAT3 and PI3K/AKT pathways in CAL-27 and SCC-9 cells. miR-9 expression was upgraded in OSCC tissues but decreased in OSCC cells along with ALK administration; meanwhile, overexpressed miR-9 inverted the influences of ALK in OSCC cells growth, migration and invasion. RECK was predicated as a novel target gene of miR-9, and overexpressed RECK hindered JAK1/STAT3 and PI3K/AKT pathways in OSCC cells. ALK prohibited tumour formation in vivo. In conclusion, ALK restrained OSCC cells growth, migration and invasion via adjusting miR-9/RECK axis. Highlights ALK restrains cell growth, migration and invasion in OSCC cells; miR-9 is enhanced in OSCC tissues but is repressed by ALK in OSCC cells; miR-9 inverts the repressive effect of ALK on CAL-27 and SCC-9 cells; RECK is a novel target of miR-9; ALK or RECK hinders JAK1/STAT3 and PI3K/AKT pathways in CAL-27 and SCC-9 cells; ALK prohibits tumour formation in vivo.

Glycyrrhizin Attenuates Salmonella enterica Serovar Typhimurium Infection: New Insights Into Its Protective Mechanism.

Glycyrrhizin (GL), a triterpenoid glycoside, serves important functions in various biological activities, including antiviral and antitumor immune responses. However, the anti-inflammatory effects of GL on Salmonella enterica serovar Typhimurium (ST)-induced injury in mice and the mechanisms underlying the protection of GL are poorly understood. Here, we investigated the effects of GL on host immune responses against ST infection in mice. A phenotypic analysis using hematoxylin and eosin (H&E) staining and transmission electron microscopy showed that GL relieved ST-induced weight loss and intestinal mucosal injury. A colonization assay showed that GL significantly reduced ST colonization in the ileum and colon and translocation to the liver and spleen. An antibacterial activity assay and real-time PCR revealed that GL had no direct inhibitory impact on ST growth or virulence gene expression. ELISA showed that GL pretreatment significantly decreased proinflammatory cytokine (IFN-γ, TNF-α, IL-6) secretion and increased anti-inflammatory cytokine (IL-10) secretion in the ileum, colon and serum of ST-infected mice. Moreover, flora analysis showed that GL reduced Akkermansia, Sutterella, Prevotella and Coprococcus but enriched Parabacteroides and Anaerotruncus in the cecum of ST-infected mice. These results suggest that GL promotes the secretion of immune factors and modulates intestinal flora to prevent further ST infection. We also analyzed the effect of GL on immunocytes and found that GL promoted the phenotypic and functional maturation of murine bone marrow-derived dendritic cells (BMDCs). Flow cytometry and western blotting demonstrated that NF-κB, ERK, and p38 MAPK were required for GL-induced BMDC maturation. The above findings indicate that GL attenuates ST infection by modulating immune function and intestinal flora. This study enriches our current knowledge of GL-mediated immunological function and provides a new perspective on the prevention of Salmonella infection in animals and humans.

Glycyrrhizic acid activates chicken macrophages and enhances their Salmonella-killing capacity in vitro.

OBJECTIVE: Salmonella enterica remains a major cause of food-borne disease in humans, and Salmonella Typhimurium (ST) contamination of poultry products is a worldwide problem. Since macrophages play an essential role in controlling Salmonella infection, the aim of this study was to evaluate the effect of glycyrrhizic acid (GA) on immune function of chicken HD11 macrophages. METHODS: Chicken HD11 macrophages were treated with GA (0, 12.5, 25, 50, 100, 200, 400, or 800 µg/ml) and lipopolysaccharide (LPS, 500 ng/ml) for 3, 6, 12, 24, or 48 h. Evaluated responses included phagocytosis, bacteria-killing, gene expression of cell surface molecules (cluster of differentiation 40 (CD40), CD80, CD83, and CD197) and antimicrobial effectors (inducible nitric oxide synthase (iNOS), NADPH oxidase-1 (NOX-1), interferon-γ (IFN-γ), LPS-induced tumor necrosis factor (TNF)-α factor (LITAF), interleukin-6 (IL-6), and IL-10), and production of nitric oxide (NO) and hydrogen peroxide (H2O2). RESULTS: GA increased the internalization of both fluorescein isothiocyanate (FITC)-dextran and ST by HD11 cells and markedly decreased the intracellular survival of ST. We found that the messenger RNA (mRNA) expression of cell surface molecules (CD40, CD80, CD83, and CD197) and cytokines (IFN-γ, IL-6, and IL-10) of HD11 cells was up-regulated following GA exposure. The expression of iNOS and NOX-1 was induced by GA and thereby the productions of NO and H2O2 in HD11 cells were enhanced. Notably, it was verified that nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) pathways were responsible for GA-induced synthesis of NO and IFN-γ gene expression. CONCLUSIONS: Taken together, these results suggested that GA exhibits a potent immune regulatory effect to activate chicken macrophages and enhances Salmonella-killing capacity.

Glycyrrhizic Acid Promotes M1 Macrophage Polarization in Murine Bone Marrow-Derived Macrophages Associated with the Activation of JNK and NF-κB.

The roots and rhizomes of Glycyrrhiza species (licorice) have been widely used as natural sweeteners and herbal medicines. The aim of this study is to investigate the effect of glycyrrhizic acid (GA) from licorice on macrophage polarization. Both phenotypic and functional activities of murine bone marrow-derived macrophages (BMDMs) treated by GA were assessed. Our results showed that GA obviously increased the cell surface expression of CD80, CD86, and MHCII molecules. Meanwhile, GA upregulated the expression of CCR7 and the production of TNF-α, IL-12, IL-6, and NO (the markers of classically activated (M1) macrophages), whereas it downregulated the expression of MR, Ym1, and Arg1 (the markers of alternatively activated (M2) macrophage). The functional tests showed that GA dramatically enhanced the uptake of FITC-dextran and E. coli K88 by BMDMs and decreased the intracellular survival of E. coli K88 and S. typhimurium. Moreover, we demonstrated that JNK and NF-κB activation are required for GA-induced NO and M1-related cytokines production, while ERK1/2 pathway exhibits a regulatory effect via induction of IL-10. Together, these findings indicated that GA promoted polarization of M1 macrophages and enhanced its phagocytosis and bactericidal capacity. The results expanded our knowledge about the role of GA in macrophage polarization.

Effects of Bacillus subtilis†B10 spores on viability and biological functions of murine macrophages.

This study was conducted to investigate the effects of Bacillus subtilis B10 spores on the viability and biological functions of murine macrophage. RAW 264.7 cells were stimulated both with and without B. subtilis B10 spores for 12 h. Then cell viability was determined to evaluate the cytotoxic effect of B. subtilis B10 spores to the cells, and the activities of acid phosphatase (ACP) and lactate dehydrogenase (LDH), the production of nitric oxide (NO) and inducible nitric oxide synthase (iNOS), and the secretion of inflammatory cytokines were measured to analyze the functions of macrophages. The results showed that B. subtilis B10 spores were not harmful to RAW 264.7 cells and they also strongly enhanced the activities of ACP and LDH (P < 0.01), remarkably increased NO and iNOS production (P < 0.01), and significantly stimulated the secretion of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-1 beta (IL-1ß), IL-6, IL-8 and IL-12 (P < 0.01) while they reduced anti-inflammatory cytokine IL-10 (P < 0.01). The outcomes suggest that B. subtilis B10 spores are not only safe for murine macrophages, but also can activate these cells and enhance their immune function. The above findings suggest that B. subtilis B10 spores are potentially probiotic.

Immunomodulatory effects of Bacillus subtilis (natto) B4 spores on murine macrophages.

To investigate the immunomodulatory effects of Bacillus subtilis (B. subtilis) (natto) B4 spores on murine macrophage, RAW 264.7 cells were cultured alone or with B subtilis (natto) B4 spores at 37°C for 12 hrs, then both cells and culture supernatants were collected for analyses. Exposure of RAW 264.7 cells to B. subtilis (natto) B4 spores had no significant effects on macrophage viability and amounts of extracellular lactate dehydrogenase (LDH). However, it remarkably increased the activities of acid phosphatase (ACP), lactate dehydrogenase (LDH) and inducible nitric oxide synthase (iNOS) in cells and the amounts of nitric oxide (NO) and cytokines (tumor necrosis factor-alpha, interferon-gamma, interleukin [IL]-1 beta, IL-6, IL-12, IL-10 and macrophage inflammatory protein-2) in culture supernatants. These results demonstrate that B. subtilis (natto) B4 spores are harmless to murine macrophages and can stimulate their activation through up-regulation of ACP and LDH activities and enhance their immune function by increasing iNOS activity and stimulating NO and cytokine production. The above findings suggest that B. subtilis (natto) B4 spores have immunomodulatory effects on macrophages.