Recombinant Fusion Protein Vaccine Containing Clostridioides difficile FliC and FliD Protects Mice against C. difficile Infection.

Bacterial flagella are involved in infection through their roles in host cell adhesion, cell invasion, auto-agglutination, colonization, the formation of biofilms, and the regulation and secretion of nonflagellar bacterial proteins that are involved in the virulence process. In this study, we constructed a fusion protein vaccine (FliCD) containing the Clostridioides difficile flagellar proteins FliC and FliD. The immunization of mice with FliCD induced potent IgG and IgA antibody responses against FliCD, protected mice against C. difficile infection (CDI), and decreased the C. difficile spore and toxin levels in the feces after infection. Additionally, the anti-FliCD serum inhibited the binding of C. difficile vegetative cells to HCT8 cells. These results suggest that FliCD may represent an effective vaccine candidate against CDI.

Effects of oral florfenicol on intestinal structure, function and microbiota in mice.

Many kinds of antibiotics have effects on intestinal structure and function. In the current experimental study, we evaluate the effect of oral florfenicol on intestinal barrier in mice. Thirty adult male mice were randomly divided into two groups, the group none (N) and the group florfenicol (F), the mice in group F were orally administered florfenicol 100 mg/kg body weight (BW) for 7 days. At day 8, mice were euthanized and sampled for the analysis of alterations in genes and proteins from jejunum, jejunum morphology and microbiota analysis. Administration of florfenicol caused higher liver index (P < 0.05). In the jejunum, mucosa injury and villus rupture, compared with the group N, the villus length and V/C (villus length/crypt depth) in group F were marked decrease (P < 0.01). The transcription level of Muc2 and occludin in group F were significantly lower than those in group N (P < 0.01 or P < 0.05). The expression of APRIL, IL-17, IL-22, BAFF and sIgA on protein level were significantly down-regulated (P < 0.01 or P < 0.05), while the expression of IL-10, TGF-ß, IL-6, IL-4 were significantly up-regulated (P < 0.01) in group F. The abundances of bacteria in Firmicutes and Lactobacillus decreased significantly (P < 0.01) in group F. Our results indicated that oral administration of florfenicol might have a negative impact on functions of intestinal mucosal barrier, immune system and the intestinal microbiota.

A comparison of dynamic distributions of intestinal microbiota between Large White and Chinese Shanxi Black pigs.

Intestinal microbiota has been widely recognized to influence on their hosts with respect to digestion and absorption of nutrients, but little is known about the structure and composition of microbial communities at different growth periods of hosts as yet. In this case, 16S rRNA gene amplicon sequencing was applied to decode the microbiota architecture in four distinct intestinal compartments (duodenum, jejunum, ileum, and cecum) of both Large White pigs and Chinese Shanxi Black pigs at the weaning, nursery, and fast-growth developmental stages. In our study, the intestinal ecosystems were dynamically changing and influenced by host maturity and diets at different development stages. Species phylogenetically affiliated to phyla Firmicutes, Proteobacteria, and Bacteroidetes were abundant in both pig breeds; at the genus level, microbial communities were dominated by Prevotella, followed by Acinetobacter and Lactobacillus. Further inspection revealed that Lactobacillus was identified to be positively associated with villus height, whereas Acinetobacter and Prevotella were prone to reside in deep crypts. Furthermore, intestinal microbiota in Shanxi Black pigs had more metabolic and less infectious functions than that in Large White pigs. In short, our data present here indicated that microbiota with longitudinal diversity and lower infection in Shanxi Black pigs might contribute to the relatively stronger adaptability in comparison with Large White pigs.

Oral Immunization with Nontoxigenic Clostridium difficile Strains Expressing Chimeric Fragments of TcdA and TcdB Elicits Protective Immunity against C. difficile Infection in Both Mice and Hamsters.

The symptoms of Clostridium difficile infection (CDI) are attributed largely to two C. difficile toxins, TcdA and TcdB. Significant efforts have been devoted to developing vaccines targeting both toxins through parenteral immunization routes. However, C. difficile is an enteric pathogen, and mucosal/oral immunization would be particularly useful to protect the host against CDI, considering that the gut is the main site of disease onset and progression. Moreover, vaccines directed only against toxins do not target the cells and spores that transmit the disease. Previously, we constructed a chimeric vaccine candidate, mTcd138, comprised of the glucosyltransferase and cysteine proteinase domains of TcdB and the receptor binding domain of TcdA. In this study, to develop an oral vaccine that can target both C. difficile toxins and colonization/adhesion factors, we expressed mTcd138 in a nontoxigenic C. difficile (NTCD) strain, resulting in strain NTCD_mTcd138. Oral immunization with spores of NTCD_mTcd138 provided mice full protection against infection with a hypervirulent C. difficile strain, UK6 (ribotype 027). The protective strength and efficacy of NTCD_mTcd138 were further evaluated in the acute CDI hamster model. Oral immunization with spores of NTCD_mTcd138 also provided hamsters significant protection against infection with 2 × 104 UK6 spores, a dose 200-fold higher than the lethal dose of UK6 in hamsters. These results imply that the genetically modified, nontoxigenic C. difficile strain expressing mTcd138 may represent a novel mucosal vaccine candidate against CDI.

Oleanolic acid derivatives inhibit the Wnt/ß-catenin signaling pathway by promoting the phosphorylation of ß-catenin in human SMMC-7721 cells.

Oleanolic acid, isolated from privet, has shown antitumor effects in several cancers. However, the underlying molecular mechanism associated with these effects is largely unknown. In this study, we explored the effect of oleanolic acid derivatives on the Wnt/ß-catenin signaling pathway in human hepatocellular carcinoma SMMC-7721 cells. The mRNA and protein levels of related genes were determined by real-time quantitative PCR and Western blot, respectively. Treatment of SMMC-7721 cells with oleanolic acid derivatives led to the downregulation of the mRNA and protein levels of ß-catenin, c-myc, and cyclin D1. Treatment with oleanolic acid derivatives decreased the levels of ß-catenin in both the cytoplasm and the nucleus. Moreover, oleanolic acid derivatives promoted the phosphorylation of ß-catenin (Ser33/37/Thr41) in the cytoplasm. Our results suggest that oleanolic acid derivatives inhibit the Wnt/ß-catenin signaling pathway by stimulating the phosphorylation of ß-catenin (Ser33/37/Thr41) in human SMMC-7721 cells.

Transcriptomic Study on the Lungs of Broilers with Ascites Syndrome.

Although broiler ascites syndrome (AS) has been extensively studied, its pathogenesis remains unclear. The lack of cardiopulmonary function in broilers causes relative hypoxia in the body; hence, the lung is the main target organ of AS. However, the transcriptome of AS lung tissue in broilers has not been studied. In this study, an AS model was successfully constructed, and lung tissues of three AS broilers and three healthy broilers were obtained for RNA sequencing (RNA-seq) and pathological observation. The results showed that 614 genes were up-regulated and 828 genes were down-regulated in the AS group compared with the normal group. Gene Ontology (GO) functional annotation revealed the following up-regulated genes: FABP4, APLN, EIF2AK4, HMOX1, MMP9, THBS1, TLR4, BCL2; and down-regulated genes: APELA, FGF7, WNT5A, CDK6, IL7, IL7R, APLNR. These genes have attracted much attention in cardiovascular diseases such as pulmonary hypertension. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that multiple metabolic processes were enriched, indicating abnormal lung metabolism of AS in broilers. These findings elucidate the potential genes and signal pathways in the lungs of broilers with AS and provide a potential target for studying the pathogenesis and preventing AS.

Underlying mechanism of Qiling Jiaogulan Powder in the treatment of broiler ascites syndrome.

Broiler ascites syndrome (AS), is a nutritional and metabolic disease that occurs in fast-growing commercial broiler chickens. AS can cause poor growth and a significant increase in the rate of broiler deaths, which has resulted in serious economic losses to the poultry industry. The classic traditional Chinese medicine Qiling Jiaogulan Powder (QLJP) has been demonstrated to have a certain therapeutic effect on broiler AS. However, its pharmacological mechanism remains to be elucidated. This study was performed to investigate the multitarget action mechanism of QLJP in the treatment of broiler AS based on network pharmacology analysis using a broiler AS model. First, all chemical components and targets of QLJP were obtained from the Traditional Chinese Medicine System Pharmacology Analysis Platform (TCMSP). Targets related to broiler AS were further obtained through the GeneCards database and the NCBI Gene sub-database. A protein-protein interaction (PPI) network was constructed. Then, enrichment analyses were performed to predict the potential mechanisms of QLJP in the treatment of broiler AS. Finally, the treatment effect of QLJP on AS was verified in a broiler AS model. Network pharmacology analysis generated 49 active ingredients and 167 core targets of QLJP, and a QLJP-single drug-target-disease network was successfully constructed. Gene enrichment analysis indicated that the core targets have played major roles in the Cell cycle, FOXO signaling pathways, etc. We demonstrated that QLJP improved clinical and organ damage symptoms and significantly reduced the ascites heart index in broilers with AS induced by administration of high-energy, high-protein diets and high-sodium drinking water in a low-temperature environment. QLJP may regulate lung oxidative stress, the cell cycle and apoptosis by activating the FOXO3a signaling pathway to interfere with the occurrence and development of AS in broilers. QLJP administration may be a good clinical strategy for the prevention and treatment of broiler AS.

Effect of Qiling Jiaogulan Powder on Pulmonary Fibrosis and Pulmonary Arteriole Remodeling in Low-Temperature-Exposed Broilers.

Chinese herbal medicine plays an important role in regulating the nutritional metabolism of poultry and maintaining or improving normal physiological functions and animal health. The present study investigated the effects of dietary supplementation with Qiling Jiaogulan Powder (QLJP) on pulmonary fibrosis and pulmonary arteriole remodeling in low temperature-exposed broilers. Seven-day-old Ross 308 broilers (n = 240) were reared adaptively to 14 days of age. The broilers were randomly divided into six groups: A control group (basal diet and normal feeding temperature); model group (basal diet); low-, medium- and high-dose QLJP groups (basal diet supplemented with 1 g/kg, 2 g/kg, 4 g/kg QLJP); and L-Arg group (basal diet supplemented with 10 g/kg L-arginine). Additionally, all the broilers, except the broilers in the control group, from the age of 14 days old, had a house temperature continuously lowered by 2 °C each day until it reached 12 °C at 21 days of age, and the low temperature was maintained until the end of the experiment. There were four replicates per group and 10 birds per replicate. The results showed that the structure of the lung tissue was clearer and basically intact in the broilers in the QLJP groups, with a small number of collagen fibers formed, and the content of hydroxyproline (HYP) was significantly reduced. QLJP improved pulmonary arteriole lesions, such as tunica media thickening, intimal hyperplasia, arterial wall hypertrophy, and lumen narrowing. QLJP reduced the relative media thickness (%) and relative medial area (%) of the pulmonary arteriole, and significantly decreased the expression level of the alpha-smooth muscle actin (α-SMA) protein in pulmonary arteriole, which alleviated pulmonary arteriole remodeling. The quantitative real-time PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) results showed that QLJP treatment significantly reduced the gene and protein levels of transforming growth factor-beta l (TGF-ß1) and Smad2 in the lung and downregulated the gene and protein levels of collagen type I alpha 1 (COL1A1) and matrix metalloproteinase 2 (MMP2). In conclusion, the results of our study suggested that dietary supplementation with QLJP improved pulmonary fibrosis and pulmonary arteriole remodeling by inhibiting the expression of genes related to the TGF-ß1/Smad2 signaling pathway and inhibited the occurrence and development of pulmonary arterial hypertension in low-temperature-exposed broilers.

BHBA regulates the expressions of lipid synthesis and oxidation genes in sheep hepatocytes through the AMPK pathway.

Pregnancy toxemia (PT) is the most frequent metabolic disease of sheep during late pregnancy, which can lead to enormous economic losses in sheep farm industry. However, the underlying mechanism of PT in sheep has not been fully elucidated. High levels of ß-hydroxy butyric acid (BHBA) exist in PT sheep. The AMP-activated protein kinase (AMPK) pathway plays a major role in regulating liver function. The aim of this study was to explore the effects of gradient concentrations of BHBA on lipid metabolism of sheep hepatocytes and the underlying molecular mechanism in vitro. The results showed that 0.6, 1.2 mmol/L BHBA could activate AMPKα, promoted the expressions of peroxisome proliferator-activated receptor alpha (PPARα) and its target genes, and inhibited the expressions of sterol regulatory element binding protein-1c (SREBP-1c) as well as its downstream genes. When the concentration of BHBA was beyond 1.2 mmol/L, the expressions of the above-mentioned proteins and genes were just the opposite. However, the expressions of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) did not change significantly. The levels of very low density lipoprotein (VLDL), triglyceride (TG) and cholesterol (T-CHOL) showed a gradually increasing trend with the increase of BHBA concentration. According to the results above, it demonstrates that high levels of BHBA can inhibit the expression of the AMPK pathway and cause lipid metabolism disorders in sheep hepatocytes, which may lead to the occurrence of PT.

Composition of the Fecal Microbiota of Piglets at Various Growth Stages.

Gastrointestinal (GI) microbiota play an important role in promoting growth in piglets. However, studies on microbiota composition at various growth stages are lacking. We measured body weights of Jinfen White and Mashen piglets every 7 days and collected their fecal samples by rectal swabbing at nine time points during suckling (1-28 days) and nursery (35-70 days) stages to gain insight into microbiota variability during piglet growth. The fecal microbiota were characterized via 16S rRNA gene sequencing to analyze the effects of microbial diversity on piglet growth and development preliminarily. The results showed that although the two breeds of piglets have similar body weights at birth, weaned Jinfen White piglets demonstrated a significantly greater body weight and daily weight gain than weaned Mashen piglets (P < 0.01). A total of 1,976 operational taxonomic units (OTUs) belonging to 27 phyla and 489 genera were uncovered, in which the highest numbers of OTUs belong to the phyla Firmicutes and Bacteroidetes. Lactobacillus, Bacteroides, and Prevotellaceae NK3B31 groups accounting for 12.4, 8.8, and 5.8% of OTUs, respectively, showed relatively high abundance at the genus level. Nine sampling time points were divided into three growth stages, namely, immediate postfarrowing (1 day old), suckling (7, 14, and 21 days old), and nursery (28, 35, 49, 63, and 70 days old), on the basis of the results of microbial diversity, principal coordinate, and co-occurrence network analyses. In addition, it identified 54 discriminative features in the microbiota between two breeds of piglets by LEfSe analysis, in which 17 genera enriched the microbiota community of Jinfen White piglets. Finally, abundances of 29 genera showed significant positive correlations with body weights and daily weight gain of piglets. Conversely, abundances of 12 genera demonstrated significant negative correlations with body weights of piglets. The results of our study will provide a theoretical basis for succession patterns in fecal microbiota of piglets and suggest the need for meticulous management of piglets in pig production.

A new ATP-sensitive potassium channel opener protects endothelial function in cultured aortic endothelial cells.

OBJECTIVE: Endothelial dysfunction is an early risk factor for cardiovascular disease and hypertension. Mechanisms that participate in endothelial dysfunction include reduced nitric oxide (NO) generation and increased endothelin-1 (ET-1) generation. Endothelial ATP-sensitive potassium (K(ATP)) channels are responsible for maintaining the resting potential of endothelial cells and modulating the release of vasoactive compounds. We hypothesized that activation of endothelial K(ATP) channels might result in the protection against endothelial dysfunction. METHODS: Using cultured bovine or rat aortic endothelial cells, we examined the effects of a new K(ATP) channels opener, iptakalim, on the secretion of vasoactive substances. We also investigated its effects on the expression of adhesion molecules in metabolically disturbed cultured endothelial cells. RESULTS: In cultured aortic endothelial cells, iptakalim caused a concentration-dependent inhibition of ET-1 release and synthesis that correlated with reduced levels of mRNA for ET-1 and endothelin-converting enzyme. These effects of iptakalim were significantly inhibited by pretreatment with glibenclamide (a K(ATP) channel blocker) for 1 h. Similarly, iptakalim enhanced the release of NO in a concentration-dependent manner and increased basal levels of free intracellular calcium. Iptakalim at the concentrations of 100 and 1000 microM increased the activities of NO synthase (NOS) significantly. After the activity of NOS was blocked by L-N(omega)-nitro-arginine methyl ester (L-NAME), the inhibition of iptakalim on ET-1 release was abolished. In endothelial cell models of metabolic disturbance induced by low-density lipoprotein, homocysteine, or hyperglycemia, treatment with iptakalim could inhibit the overexpression of monocyte chemoattractant protein-1 (MCP-1), Intercellular adhesive molecule-1 (ICAM-1), and vascular cell adhesive molecule-1 (VCAM-1) mRNA. CONCLUSION: Iptakalim is a promising drug that could protect against endothelial dysfunction through activating K(ATP) channels in endothelial cells.

Induction of apoptosis by an oleanolic acid derivative in SMMC-7721 human hepatocellular carcinoma cells is associated with mitochondrial dysfunction.

The aim of the present study was to investigate the effects of an oleanolic acid derivative, a novel antitumor drug, on the growth of SMMC-7721 human hepatocellular carcinoma cells and the underlying mechanism. An MTT assay was performed to determine the cytotoxicity of the oleanolic acid derivative. Cell membrane integrity was assessed using fluorescence microscopy to assess the uptake of annexin V-FITC/propidium iodide (PI). Western blotting was used to detect the apoptosis-associated proteins B cell lymphoma-2 (Bcl-2), Bax, caspase-9 and caspase-3. A spectrophotometer was used to analyze the intracellular adenosine triphosphate (ATP) expression level. The loss of mitochondrial membrane potential was detected by performing the JC-1 assay. ELISA was used to evaluate the content of cytochrome c (Cyt-C). The oleanolic acid derivative reduced the cell viability of SMMC-7721 cells in a dose- and time-dependent manner. The half maximal inhibitory concentration values of the oleanolic acid derivative in SMMC-7721 cells at 24, 48 and 72 h were 26.80, 11.85, and 6.66 µM, respectively. The antiapoptotic-protein Bcl-2 was downregulated, and the proapoptotic protein Bax was upregulated following treatment with the oleanolic acid derivative for 48 h. The oleanolic acid derivative induced the cleavage of caspase-9 and caspase-3 as well as promoted annexin V-FITC/PI uptake in SMMC-7721 cells. Furthermore, treatment of SMMC-7721 cells with the oleanolic acid derivative induced a reduction of the intracellular ATP expression level, loss of ΔΨm and Cyt-C release from the mitochondria. The oleanolic acid derivative induced apoptosis in SMMC-7721 human cells. Mitochondrial dysfunction was involved in the anticancer effects of this derivative on SMMC-7721 human cells.

Sodium tanshinone IIA sulfonate inhibits the meq, ul49 and VP22 expression of Marek's disease virus.

BACKGROUND: Our previous studies have demonstrated that sodium tanshinone IIA sulfonate (STS), a natural compound derived from Salviae Miltiorrhizae Radix et Rhizoma (Danshen), could effectively inhibit Marek's disease virus (MDV) infection both in vitro and in vivo, but the underlying mechanisms remain unclear. The main objective of the study was to explore the effect of STS on the meq, ul49 and VP22 expression of MDV in vitro. METHODS: Quantitative real-time PCR (qRT-PCR) was used to analyse the effect of STS on meq and ul49 expression at both the DNA and messenger RNA (mRNA) level, and the effect of STS on VP22 was assessed by immunofluorescence assay and western blotting. RESULTS: The DNA and mRNA copy numbers of meq and ul49 significantly decreased in the groups treated with STS compared with MDV control (P<0.05), which indicated that STS could inhibit the expression of meq and ul49 at both the DNA and mRNA level. Moreover, the expression of VP22 encoded by ul49 was also significantly inhibited (P<0.05). CONCLUSIONS: STS possessed anti-MDV activity in chicken embryo fibroblasts. Its antiviral mechanisms may be ascribed to inactivating MDV directly, disturbing meq and ul49 replication and inhibiting the expression of VP22 encoded by ul49. These results suggested that STS is a promising natural compound to be further developed as an antiviral agent against MDV infection.