Shidong Zhang

Aqueous extract of Bai-Hu-Tang, a classical Chinese herb formula, prevents excessive immune response and liver injury induced by LPS in rabbits

Abstract Ethnopharmacological relevance Bai-Hu-Tang (BHT) was traditionally used to reduce fever heat and promote generation of body fluids. Aim of the study To investigate the effect and mechanism of BHT in the prevention of lipopolysaccharide (LPS) fever in manners of immune modulation. Materials and methods The model of fever syndrome of Chinese medicine pattern was imitated by LPS injection i.v. in rabbits, and BHT was gavaged. The serum levels of tumor necrosis factor-α (TNF-α), interleukin (IL-6, 10) and immunoglobulin (IgG, IgA, and IgM) were determined by enzyme-linked immunosorbent assay (ELISA); alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were tested by biochemical methods. Liver tissue damage was detected by hematoxylin–eosin (H&E) stain. Subpopulation of T cells was detected by Fluorescence Activated Cell Sorter (FACS). Genes expression of Toll-like receptor 4 (TLR4) and lipopolysaccharide binding protein (LBP) in liver tissue were assayed by real-time polymerase chain reaction (RT-PCR). Result The results demonstrated that BHT prevented sudden increase of IL-10, TNF-α, ALT and AST, and liver damage induced by LPS. BHT also prevented significant decrease of the percentage of CD8+ T cells since LPS injection. At the same time, BHT did not affect the gene expression of TLR4 and serum concentration of three immunoglobulins, which were increased by LPS, but made gene expression of LBP higher. Conclusion The results of this study indicated that BHT played an important role in immunity protection and anti-injury through preventing immunoinflammatory damage by LPS. The achievement thereby scientifically provided mechanism of BHT in the prevention of febrile disease, and supported its traditional use.

Palmatine inhibits TRIF-dependent NF-κB pathway against inflammation induced by LPS in goat endometrial epithelial cells

&NA; Palmatine, a natural pharmaceutical drug, possesses many biological activities. But its clinical application is rarely reported in the veterinary medicine. The aim of this study was to investigate the anti‐inflammatory effects of palmatine on lipopolysaccharide (LPS)‐induced inflammation in goat endometrial epithelial cells (gEECs), and the possible molecular mechanisms. Palmatine cell toxicity was determined by MTT assay, and the production of inflammatory cytokine in the cultured medium was measured with ELISA, qRT‐PCR and Western blotting. Our results showed that palmatine treatment inhibited the release of tumor necrosis factor (TNF)‐&agr;, interleukin (IL)‐1&bgr;, IL‐6, nitric oxide (NO), matrix metalloproteinase (MMP)‐9 and MMP‐2. Furthermore, palmatine enhanced the secretion of prostaglandins E2 (PGE2) and IL‐10. Palmatine significantly down‐regulated the expression of Toll‐like receptor 4 (TLR4), cluster of differentiation 14 (CD14), Toll/interleukin 1 receptor (TIR)‐domain‐containing adaptor protein inducing interferon‐&bgr; (TICAM, TRIF) and nuclear factor‐&kgr;B (NF‐&kgr;B) in LPS stimulated gEECs, but did not alter the production of MyD88. In conclusion, palmatine inhibits TRIF‐dependent NF‐&kgr;B pathway to reduce LPS‐induced inflammatory responses in goat endometrial epithelial cells. HighlightsWe identified that palmatine inhibits LPS‐induced inflammatory reaction in goat endometrial epithelial cells (gEECs).The 10–227 &mgr;g/mL palmatine promotes the proliferation of gEECs.Palmatine suppress TRIF‐dependent and TLR4 signaling pathways in gEECs.Palmatine can down‐regulate the expression of NF‐&kgr;B and suppress the action of NF‐&kgr;Bp65 in gEECs.In veterinary clinic, palmatine may be used to treat endometritis.

Short communication: N-Acetylcysteine-mediated modulation of antibiotic susceptibility of bovine mastitis pathogens

The aim of this study was to investigate the effects of N-acetylcysteine (NAC) on antibiotic susceptibility of bovine mastitis pathogens including Staphylococcus aureus, Streptococcus dysgalactiae, Escherichia coli, and Streptococcus agalactiae. Minimum inhibitory concentrations (MIC) were tested by the agar-based E-test method. The presence of 10mM NAC reduced the MIC of penicillin and ampicillin but enhanced the MIC of erythromycin and ciprofloxacin for all of the strains. In addition, NAC-mediated modulation of MIC of kanamycin, tetracycline, and vancomycin was diverse, depending on the target bacterial pathogen and antibiotic being used. The results suggest that NAC is an important modulator of antibiotic activity against the major bovine mastitis pathogens.

Differentially expressed genes of LPS febrile symptom in rabbits and that treated with Bai-Hu-tang, a classical anti-febrile Chinese herb formula.

ETHNOPHARMACOLOGICAL RELEVANCE Bai-Hu-Tang (BHT) has been traditionally used to clear heat and engender fluids. AIM OF THE STUDY To reveal the alteration of differentially expressed genes (DEGs) between lipopolysaccharide (LPS) febrile syndrome in rabbits and treatment with BHT which is a classical anti-febrile formula in traditional Chinese medicine. MATERIALS AND METHODS Febrile model was induced by LPS injection (i.v.) in rabbits, and BHT was gavaged to another group of febrile rabbits. After sacrifice of animals, total RNA of liver tissue was isolated, processed, and hybridized to rabbit cDNA microarrays obtained from Agilent Co. The data of DEGs were obtained by lazer scanning and analyzed with Cluster program 3.0. Then bioinformatic analysis of DEGs was conducted through gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In addition, expression levels of four relative genes were detected by quantitative real time ployenzyme chain reaction (qRT-PCR) to validate the accuracy of microarrays. RESULT The results demonstrated that genes expression pattern could be clustered into three groups significantly, and there were 606 up-regulated genes and 859 down-regulated genes in the model group, and 106 up-regulated genes and 429 down-regulated genes in BHT treated group. There were 286 DEGs existed as the common in two experimental groups. Enrichment analysis of GO annotations indicated that DEGs in model and BHT treated animals mainly referred catalytic activity and oxidoreductase activity for metabolic processes located in the membrane system at intracellular part, and binding activities increased significantly in treatment with BHT. Enrichment of KEGG analysis showed that the pathways of phagosome and protein processing in endoplasmic reticulum contained the most altered genes in the LPS group, but the percentage of phagosome pathway almost doubled in BHT group. Most DEGs involved in the LPS signal recognition system was up-regulated in LPS group, but partly decreased in BHT group. RT-PCR results of eight relative genes were consistent with the results of microarrays. CONCLUSION DEGs of LPS febrile syndrome mainly involved oxidoreductase and catalytic activity of the metabolic processes, and pathways of processing protein for pyrotoxin recognition; BHT mostly regulated the DEGs in the phagosome pathway to clear LPS in the liver, and partly interfered with gene expression in LPS recognition system. The study provided an important pioneering result on gene expression profiling research, and will facilitate the clinical care or further studies of the formula.

Comparative proteomics analysis provide novel insight into laminitis in Chinese Holstein cows

BackgroundLaminitis is considered as the most important cause of hoof lameness in dairy cows, which causes abundant economic losses in husbandry. Through intense efforts in past decades, the etiology of laminitis is preliminarily considered to be subacute ruminal acidosis; however, the pathogenesis of laminitis needs further research. The differentially expressed proteins (DEP) were detected in plasma of healthy cows and clinical laminitis cows by two-dimensional gel electrophoresis (2-DE) and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.ResultsNineteen protein spots were differentially expressed, and 16 kinds of proteins were identified after peptide mass fingerprint search and bioinformatics analysis. Of these, 12 proteins were differentially up-regulated and 4 down-regulated. Overall, these differential proteins were involved in carbohydrate metabolism, lipids metabolism, molecular transport, immune regulation, inflammatory response, oxidative stress and so on.ConclusionsThe DEPs were closely related to the occurrence and development of laminitis and the lipid metabolic disturbance may be a new pathway to cause laminitis in dairy cows. The results provide the theory foundation for further revealing the mechanism of laminitis and screening the early diagnostic proteins and therapeutic target.

iTRAQ-based quantitative proteomic analysis reveals Bai-Hu-Tang enhances phagocytosis and cross-presentation against LPS fever in rabbit

ETHNOPHARMACOLOGICAL RELEVANCE Bai-Hu-Tang (BHT), a classical anti-febrile Chinese formula comprising of liquorice, anemarrhena rhizome, gypsum and rice, has been traditionally used to anti-febrile treatment and promote the production of body fluid to relieve thirst. In this paper, we aim to explore anti-febrile mechanism of BHT at protein level through analyzing alteration of differentially expressed proteins (DEPs) both lipopolysaccharide (LPS) fever syndrome and that was treated with BHT in rabbits. MATERIALS AND METHODS Febrile model was induced by LPS injection (i.v.) in rabbits, and BHT (750mg dry extract/kg body weight) was gavaged to another group of LPS fever rabbits. After sacrifice of animals, total protein of liver tissue was isolated, and two-dimensional liquid chromatography (LC) - tandem mass spectrometry (MS) coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling analysis was employed to quantitatively identify differentially expressed proteins in two group animals, which were compared with control group. Then bioinformatic analysis of DEPs was conducted through hierarchical Clustering, Venn analysis, gene ontology (GO) annotation enrichment, and kyoto encyclopedia of genes and genomes (KEGG) pathways enrichment. RESULT The results demonstrated there were 63 and 109 DEPs in LPS fever group and BHT-treated group, respectively. Enrichment analysis of GO annotations indicated that BHT mainly regulated expression of some extracellular structural proteins for response to stimulus and stress. KEGG analysis showed that ribosome and phagosome were the most significant pathways. Thereinto, several proteins in phagosome pathway were significantly up-regulated by BHT, including F-actin, coronin, Rac, and major histocompatibility complex class I (MHC I), which work in phagocytosis and cross-presentation CONCLUSION: BHT may contribute to pyrogen clearance by boosting antigenic phagocytosis, degradation, and cross presentation in the liver.

Penicillin-resistant characterization of Staphylococcus aureus isolated from bovine mastitis in Gansu, China

Abstract Bovine mastitis caused by Staphylococcus aureus is difficult to treat because of increasing resistance against antibiotics, especially penicillin. β-Lactamase and biofilm are responsible for penicillin resistance of S. aureus. The aim of this study was to investigate the β-lactamase activity and biofilm formation capacity of 37 penicillin-resistant S. aureus strains (35 were blaZ positive and 2 were blaZ negative) from bovine mastitis in Gansu Province, China, as well as to measure the intercellular adhesion genes icaA and icaD of these strains. β-Lactamase Test Kit was used to determine the β-lactamase activity, biofilm formation was tested by semi-quantitative adherence assay method. Moreover, the presence of icaA and icaD were measured by PCR. A total of 32 penicillin-resistant S. aureus strains, including the two blaZ-negative strains, were identified as β-lactamase producers. All tested S. aureus isolates produced biofilm in the microtiter plate assay. Meanwhile, all these strains were PCR-positive for the ica locus, icaA and icaD. The study indicated high prevalence of β-lactamase activity, biofilm-forming capacity, and the ica genes among the penicillin-resistant S. aureus isolates, and implied that S. aureus resistant to penicillin was attributed to multiple mechanisms.

Differential proteomic profiling of endometrium and plasma indicate the importance of hydrolysis in bovine endometritis

Endometritis is an important disease of dairy cows that leads to significant economic losses in the dairy cattle industry. To investigate the alteration of proteins associated with endometritis in the dairy cow, the isobaric tags for relative and absolute quantification (iTRAQ) technique was applied to quantitatively identify differentially expressed proteins (DEP) in the endometrium and peripheral plasma of Chinese Holstein cows with endometritis. Compared with the normal (control) group, 159 DEP in the endometrium and 137 DEP in the plasma were identified in cows with endometritis. Gene ontology analysis demonstrated that the predominant endometrial DEP were primarily involved in responses to stimulus and stress processes and mainly played a role in hydrolysis in the extracellular region. The predominant plasma DEP were mainly components of the cytosol and non-membrane-bound organelles, and they were involved in the response to stress and regulation of enzyme activity. Protein-protein interaction of tissue DEP revealed that some core seed proteins, such as RAC2, ITGB2, and CDH1 in the same network as CD14, MMP3, and MMP9, had important functions in the cross-talk of pathways related to extracellular proteolysis. In summary, significant enzymatic hydrolase activity in the extracellular region is proposed as a molecular mechanism by which altered proteins may promote inflammation and hence endometritis.

Genetic characterization of antimicrobial resistance in Staphylococcus aureus isolated from bovine mastitis cases in Northwest China

Abstract Staphylococcus aureus is the most common etiological pathogen of bovine mastitis. The resistant strains make the disease difficult to cure. The aim of this study was to characterize the genetic nature of the antimicrobial resistance in S. aureus cultured from bovine mastitis in Northwest China in 2014. A total of 44 S. aureus were isolated for antimicrobial resistance and resistance-related genes. Antimicrobial resistance was determined by disc diffusion and the corresponding resistance genes were detected by PCR. Phenotype indicated that S. aureus isolates were resistant to penicillin (84.09%), erythromycin (20.45%), tetracycline (15.91%), gentamicin (9.09%), tobramycin (6.82%), kanamycin (6.82%) and methicillin (2.27%). 9.09% of the S. aureus isolates were classified as multidrug resistant. In addition, genotypes showed that the isolates were resistant to rifampicin (100%, rpoB), penicillin (95.45%, blaZ), tetracycline (22.73%, tetK, tetM, alone or in combination), erythromycin (22.73%, ermB or ermC), gentamicin/tobramycin/kanamycin (2.27%, aacA-aphD), methicillin (2.27%, mecA) and vancomycin (2.27%, vanA). Resistance to tetracycline was attributed to the genes tetK and tetM (r=0.558, P

Draft Genome Sequence of Streptococcus agalactiae Serotype Ia Strain M19, a Multidrug-Resistant Isolate from a Cow with Bovine Mastitis

ABSTRACT Streptococcus agalactiae is a major contagious pathogen causing bovine mastitis worldwide. We report here the draft sequence of S. agalactiae Ia strain M19, a multidrug-resistant isolate from a bovine mastitis case in Ningxia Hui autonomous region, China.