Phellodendron amurense, exhibits antifungal activity mainly by bioactive components including berberine hydrochloride and palmatine hydrochloride. This study was conducted to evaluate the antifungal effects of berberine hydrochloride, palmatine hydrochloride, and a mixture of both substances against Microsporum canis in vivo and in vitro. The minimal inhibitory concentrations (MICs) of monomers and clotrimazole were determined using 1.5 % tryptic soy agar. The effects of these drugs on Microsporum canis growth was detected by determining dry weight. Transmission electron microscopy (TEM) was performed to observe the effect of chemicals on cell ultrastructure. Differential mRNA expressions of eight genes of M. canis treated with berberine or palmatine or their combination at different time points were determined by real-time PCR. NADH enzyme concentration was also detected. Clinical evaluation via in-vivo antifungal assay was also performed. Skin histology PAS staining was also carried out. Results showed that MICs of berberine, palmatine and clotrimazole were 1, 1, and 0.015 mg/mL, respectively. No significant difference was observed among the growth curves of the three groups before 18 h was reached. TEM showed that these drugs could destroy the cell membrane and organelles of M. canis at different time points. After 30 h of incubation, relative mRNA expressions of the genes in the combined group were significantly higher than those in the other groups including the clotrimazole group (P < 0.05); Palmatine initially induced the mRNA up-regulation of PGAL4, FSH1, PQ-LRP, NADH1 and NDR in M. canis; by contrast, berberine maintained a high expression level of these genes to shorten fungal life cycle and eradicate M. canis. Clinical results showed that combined treatment was more effective than single administration of each monomer or clotrimazole. Hence, berberine mixed with palmatine significantly elicited antifungal activities and could be used to treat M. canis in rabbits. These results provide a comprehensive view of the mechanism of berberine and palmatine in anti-M. canis activity.

Background: Rehmannia glutinosa polysaccharide is the main reason that contributes to the immunological function of R. glutinosa. Due to its disadvantages in clinical use, here we designed the PEGylation nano-RGP (pRL) to improve the drug-targeting effect and the immunological function. Our present work aims to establish the optimum condition of preparing the pRL and to investigate its immunological function on macrophages. Methods: pRL was prepared by thin film hydration method combined with ultra-sonication technique. And its preparation conditions were optimized with response surface methodology. Also, the lyophilization method was optimized. The characteristics of the pRL were evaluated, including particle size, drug loading, encapsulation efficiency and morphology. The immunological function of pRL on macrophage was investigated through CCK-8 test, ELISA and flow cytometry. Results: The lipid-to-cholesterol molar ratio of 8:1, the addition of DSPE-PEG₂₀₀₀ of 9% and the lipid-to-drug ratio of 5.4:1 were the optimum preparation technology for pRL. The encapsulation efficiency (EE) of pRL under this preparation technology was 95.81±1.58%, with a particle size of 31.98 ± 2.6 nm. The lactose-to-lipid ratio (2:1) was the optimal lyophilization method. pRL promoted macrophage proliferation, which is significantly better than that of nano-RGP without PEGylation (RL). pRL-stimulated RAW264.7 cells showed a high secretion of pro-inflammatory cytokines, which is the characteristic indicator of M1 polarization. Enhanced cellular uptake through macropinocytosis-dependent and caveolae-mediated endocytosis was observed in pRL-treated RAW264.7 cells. Conclusion: Our study concluded that PEGylation effectively overcame the poor targeting effect of Rehmannia glutinosa polysaccharide (RGP) and significantly improved the immunological profile of its nano-formulation, which suggested that pRL could serve as an immune adjuvant in clinical application.

The objective of this study was to assess the effects of dietary supplementation with Clostridium butyricum (CB) and a bacteriophage cocktail (BP) on growth performance, serum biochemical parameters, intestinal digestive and oxidase enzymes, intestinal morphology, immune responses, and the cecum microbiota in rabbits. In total, 108 New Zealand rabbits (5 weeks old) were randomly and equally allotted into three dietary treatment groups (four replicates per treatment, n = 36/treatment): (1) the control (CN) group—rabbits fed the basal diet; (2) CB group—rabbits fed the basal diet supplemented with 100 mg/kg diet Clostridium butyricum; and (3) BP group—rabbits fed the basal diet supplemented with 200 mg/kg diet BP cocktail, respectively, for 6 weeks. Compared with the CN diet, dietary CB and BP inclusion increased the average daily gain (ADG) and average daily feed intake (ADFI) and decreased the feed/gain (F/G) ratio of rabbits. Furthermore, CB increased the digestive enzyme activity (α-amylase and trypsin in the ileum); the chymotrypsin activity was also significantly increased in the duodenum and jejunum. Supplementation with CB significantly enhanced antioxidant capacity (SOD and GSH-Px) in the jejunum and ileum and reduced MDA levels. Additionally, rabbits fed CB had significantly elevated villus height (V) and (V/C) ratios but reduced crypt depth (C). Moreover, dietary CB supplementation markedly increased the ileal expression of tight junction proteins (occludin, ZO-1, and claudin-1) and increased secretory immunoglobulin A (sIgA) production. High-throughput sequencing indicated that the microbiota in the rabbit intestine was altered by CB and BP. Venn diagrams and heatmap plots revealed that the gut microbial community composition varied obviously among rabbits fed different diets. Specifically, CB increased the relative abundance of beneficial bacteria to maintain intestinal barrier homeostasis, whereas BP decreased the relative abundance of Gammaproteobacteria, which included a plenty of pathogenic bacteria.

Phage therapy is a valid weapon that we could use to fight against pathogens. Bacteriophages kill bacteria and self-proliferate in the digestive tract. Furthermore, it was assumed that phage therapy could preserve the existing gut microbiota. In this study, 45 rabbits were equally divided into three groups after they were orally inoculated with pathogenic Escherichia coli to induce gut infection. Each group was treated with bacteriophage ZRP1 (Group P), ciprofloxacin lactate (Group A), or phosphate-buffered solution (PBS) (Group N). Another 15 healthy rabbits composed the control group (Group C). The body weight gain decreased significantly, but the white blood cell (WBC) count, especially the percentage of large WBCs, and the serum endotoxin levels increased significantly after infection. The result of microscopic examination of the ileum showed that E. coli ZR1 adhered to villi and caused hemorrhage inside the villi. Groups P and A rabbits recovered after treatments, and both bacteriophage and antibiotic treatment significantly decreased the eaeA gene concentration in cecal contents. The microbiota in cecal contents changed in infected rabbits that were treated with PBS. The relative abundance of Clostridiales and YS2 decreased but the relative abundance of Enterobacteriales increased significantly. According to the principal components analysis, the microbiota of Groups P and C rabbits were similar to one another in type and relative abundance but different from those of Groups N and A rabbits. The results demonstrated that oral administration of bacteriophage can cure gut infection with minimal impact on the cecal microbiota.

With the coming of the post‐antibiotic era, there is an increasingly urgent need for safe and efficient antibacterial vaccines. Bacterial outer membrane vesicles (OMVs) have received increased attention recently as a potential subunit vaccine. OMVs are non‐replicative and contain the principle immunogenic bacterial antigen, which circumvents the safety concerns of live‐attenuated vaccines. Here, we developed a novel nano‐vaccine by coating OMVs onto PEGylated nano‐Rehmannia glutinosa polysaccharide (pRL) in a structure consisting of concentric circles, resulting in a more stable vaccine with improved immunogenicity. The immunological function of the pRL‐OMV formulation was evaluated in vivo and in vitro, and the underlying mechanism was studied though transcriptomic analysis. The pRL‐OMV formulation significantly increased dendritic cell (DC) proliferation and cytokine secretion. Efficient phagocytosis of the formulation by DCs was accompanied by DC maturation. Further, the formulation demonstrated superior lymph node targeting, contributing to a potent mixed cellular response and bacterial‐specific antibody response against Bordetella bronchiseptica infection. Specifically, transcriptomic analysis revealed that the immune protection function correlated with T‐cell receptor signalling and Th1/Th2/Th17 differentiation, among other markers of enhanced immunological activity. These findings have implications for the future application of OMV‐coated nano‐carriers in antimicrobial immunotherapy.

Purpose: Outer membrane vesicles (OMVs) are spherical nano-sized proteolipids secreted by numerous pathogenic Gram-negative bacteria. Due to the immunostimulatory properties and protective efficacy, OMVs have received increasing attention as a candidate for the vaccine to prevent and treat bacterial infections. However, the immune response remains elusive due to the low structural stability and poor size homogeneity of the vesicles. In this study, OMVs were used to coat self-assembled glycyrrhizic acid nanoparticles (GANs) and obtain a stable OMV vaccine. The immunoprotective effects and anti-infection efficacy were evaluated in vivo and in vitro. Methods: The OMVs were prepared by ultrafiltration method and fused with GAN through mechanical extrusion. The characteristics, including morphology, hydrodynamic size, zeta potential, and stability were evaluated. The in vitro immunological function of GAN-OMV on the macrophages and in vivo immune efficacy and anti-infection effect were examined and compared. Results: The results showed that the GAN-OMV were homogenous with a size of 130 nm and a stable core-shell structure. Micropinocytosis-dependent and clathrin-mediated endocytotic pathways effectively internalized the GAN-OMV into the macrophages and promoted cell proliferation, cytokine secretion, and M1 polarization. Furthermore, subcutaneous GAN-OMV vaccination contributed to significantly higher Borderella bronchiseptica (Bb)-specific antibody production and lymphocyte proliferation. The splenic lymphocytes of mice immunized with GAN-OMVs displayed a higher ratio of CD4+/CD8+ T cells and CD19+ B cells and produced significantly higher levels of Th1/Th2/Th17 cytokines. GAN-OMV also effectively prevented Bb reinfection. Conclusion: In this study, GAN-OMV was developed successfully to stimulate Th1/Th2/Th17 immune responses against Bb and provide a promising strategy for novel vaccine development against the microbial pathogen.