This study aims to develop a method to detect bovine multi-cytokines based on flow cytometry. Previously we have prepared and screened monoclonal antibodies against bovine cytokines IFN-γ, IL-2, TNF-α, IP-10 and MCP-1. These bovine cytokine monoclonal antibodies were fluorescently labeled, and the combination of antibody and cell surface molecules were used to develop the method for detecting bovine multi-cytokines. Subsequently, the developed method was used to determine the cytokine expression profile of Mycobacterium bovis BCG infected bovine peripheral blood mononuclear cells in vitro, and evaluate the cytokine expression level of peripheral blood CD4⁺ T cells of tuberculosis-positive cattle. The bovine multi-cytokine flow cytometry detection method can effectively determine the cytokine expression of BCG-infected bovine peripheral blood T lymphocytes. Among them, the expression levels of IFN-γ, IL-2, and TNF-α continue to increase after 40 hours of infection, while the expression levels of IP-10 and MCP-1 decreased. The combined detection of IFN-γ, IL-2, and TNF-α on CD4⁺ T lymphocytes in peripheral blood of cattle can effectively distinguish tuberculosis-positive and tuberculosis-negative samples. This method may facilitate evaluating the level of cellular immune response after bovine pathogen infection and vaccine injection.
Cattle ; Animals ; Cytokines ; BCG Vaccine/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-2 ; Flow Cytometry/methods* ; Chemokine CXCL10/metabolism* ; Leukocytes, Mononuclear ; CD4-Positive T-Lymphocytes/metabolism* ; Tuberculosis ; Antibodies, Monoclonal/metabolism*

17α hydroxylase is a key enzyme for the conversion of progesterone to prepare various progestational drug intermediates. To improve the specific hydroxylation capability of this enzyme in steroid biocatalysis, the CYP260A1 derived from cellulose-mucilaginous bacteria Sorangium cellulosum Soce56 and the Fpr and bovine adrenal-derived Adx_4-108 derived from Escherichia coli str. K-12 were used to construct a new electron transfer system for the conversion of progesterone. Selective mutation of CYP260A1 resulted in a mutant S276I with significantly enhanced 17α hydroxylase activity, and the yield of 17α-OH progesterone reached 58% after optimization of the catalytic system in vitro. In addition, the effect of phosphorylation of the ferredoxin Adx_4-108 on 17α hydroxyl activity was evaluated using a targeted mutation technique, and the results showed that the mutation Adx_4-108T69E transferred electrons to S276I more efficiently, which further enhanced the catalytic specificity in the C17 position of progesterone, and the yield of 17α-OH progesterone was eventually increased to 74%. This study provides a new option for the production of 17α-OH progesterone by specific transformation of bacterial-derived 17α hydroxylase, and lays a theoretical foundation for the industrial production of progesterone analogs using biotransformation method.
Animals ; Cattle ; Progesterone/metabolism* ; Hydroxylation ; Biocatalysis ; Electron Transport ; Mixed Function Oxygenases/metabolism*

In order to investigate the enzyme production mechanism of yak rumen-derived anaerobic fungus Orpinomyces sp. YF3 under the induction of different carbon sources, anaerobic culture tubes were used for in vitro fermentation. 8 g/L of glucose (Glu), filter paper (Flp) and avicel (Avi) were respectively added to 10 mL of basic culture medium as the sole carbon source. The activity of fiber-degrading enzyme and the concentration of volatile fatty acid in the fermentation liquid were detected, and the enzyme producing mechanism of Orpinomyces sp. YF3 was explored by transcriptomics. It was found that, in glucose-induced fermentation solution, the activities of carboxymethyl cellulase, microcrystalline cellulase, filter paper enzyme, xylanase and the proportion of acetate were significantly increased (P < 0.05), the proportion of propionate, butyrate, isobutyrate were significantly decreased (P < 0.05). The results of transcriptome analysis showed that there were 5 949 differentially expressed genes (DEGs) between the Glu group and the Flp group, 10 970 DEGs between the Glu group and the Avi group, and 6 057 DEGs between the Flp group and the Avi group. It was found that the DEGs associated with fiber degrading enzymes were significantly up-regulated in the Glu group. Gene ontology (GO) function enrichment analysis identified that DEGs were mainly associated with the xylan catabolic process, hemicellulose metabolic process, β-glucan metabolic process, cellulase activity, endo-1,4-β-xylanase activity, cell wall polysaccharide metabolic process, carbohydrate catabolic process, glucan catabolic process and carbohydrate metabolic process. Moreover, the differentially expressed pathways associated with fiber degrading enzymes enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were mainly starch and sucrose metabolic pathways and other glycan degradation pathways. In conclusion, Orpinomyces sp. YF3 with glucose as carbon source substrate significantly increased the activity of cellulose degrading enzyme and the proportion of acetate, decreased the proportion of propionate, butyrate and isobutyrate. Furthermore, the degradation ability and energy utilization efficiency of fungus in the presence of glucose were improved by means of regulating the expression of cellulose degrading enzyme gene and participating in starch and sucrose metabolism pathway, and other glycan degradation pathways, which provides a theoretical basis for the application of Orpinomyces sp. YF3 in practical production and facilitates the application of Orpinomyces sp. YF3 in the future.
Animals ; Cattle ; Neocallimastigales/metabolism* ; Anaerobiosis ; Rumen/microbiology* ; Propionates/metabolism* ; Isobutyrates/metabolism* ; Cellulose/metabolism* ; Fungi ; Starch/metabolism* ; Glucose/metabolism* ; Acetates ; Sucrose/metabolism* ; Cellulases ; Cellulase

Bacillus cereus belongs to Gram-positive bacteria, which is widely distributed in nature and shows certain pathogenicity. Different B. cereus strains carry different subsets of virulence factors, which directly determine the difference in their pathogenicity. It is therefore important to study the distribution of virulence factors and the biological activity of specific toxins for precise prevention and control of B. cereus infection. In this study, the hemolysin BL triayl was expressed, purified, and characterized. The results showed that the bovine pathogenic B. cereus hemolysin BL could be expressed and purified in the prokaryotic expression system, and the bovine pathogenic B. cereus hemolysin BL showed hemolysis, cytotoxicity, good immunogenicity and certain immune protection in mice. In this study, the recombinant expression of hemolysin BL triayl was achieved, and the biological activity of hemolysin BL of bovine pathogenic ceroid spore was investigated. This study may facilitate further investigating the pathogenic mechanism of B. cereus hemolysin BL and developing a detection method for bovine pathogenic B. cereus disease.
Cattle ; Animals ; Mice ; Bacterial Proteins/metabolism* ; Bacillus cereus/metabolism* ; Hemolysin Proteins/metabolism* ; Virulence Factors/metabolism* ; Enterotoxins/metabolism*

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic autoimmune disease. MicroRNA has been shown to play an important role in RA. MicroRNA-124a (miR-124a) has anti-proliferative and anti-inflammatory effects in RA fibroblast synovial cells. This study aims to explore the effects of miR-124a overexpression on arthritis in collagen-induced arthritis (CIA) mice and the underlying mechanisms. METHODS: Bovine type II collagen and complete Ferris adjuvant were used to induce CIA model from DBA/1 mice. Twenty-eight days after initial immunization (D28), CIA mice were randomly divided into a model group, a miR-124a treatment group, and a negative control (NC) group. Physiological saline, miR-124a agomir, and miR-124a agomir NC were injected into the skin at the tail root of mice every 3 days for 4 times, respectively. The degree of joint swelling and arthritis index of mice were recorded accordingly. Sixty-three days after initial immunization (D63), the mice were sacrificed to obtain the synovial tissue of ankle joint. HE staining was used to observe the proliferation of synovial cell, infiltration of inflammatory cell, pannus, and bone erosion of synovial tissues; TUNEL staining was used to detect cell apoptosis; qRT-PCR was used to detect the mRNA expression of miR-124a, phosphatidylinositol-3-kinase catalytic subunit alpha (PIK3CA) and its downstream genes Bcl-2 and Bax. Immunohistochemistry was used to detect the protein expression of PIK3CA, Bcl-2, and Bax protein in synovial tissues of each group. RESULTS: Different degrees of swelling presented in the paws of DBA/1 mice at D28, which indicated the CIA model was constructed successfully. Forty-eight days after initial immunization (D48), the paws of mice in the miR-124a treatment group were only slightly red and swollen, while the paws of mice in the model group and the NC group were obviously red and swollen. The arthritis index of mice in the miR-124a treatment group were decreased significantly compared to the NC group at D51, D53, D59, and D62 (51, 53, 59, 62 days after initial immunization) (all P<0.05). Sixty-three days after initial immunization (D63), HE staining indicated that the scores of synovial cell proliferation, inflammatory cell infiltration, synovial pannus, and bone erosion were significantly reduced in the miR-124a treatment group (P<0.05 or P<0.01), while cell apoptosis was increased in the miR-124a treatment group compared with the model group and NC group (P<0.01 or P<0.001). Besides, the expression of miR-124a and Bax in the synovial tissue in miR-124a treatment group was significantly higher than those in the model group and NC group (P<0.01 or P<0.001), while the expressions of PIK3CA and Bcl-2 were decreased (P<0.05 or P<0.01 or P<0.001), and the ratio of Bcl-2 to Bax was significantly decreased (P<0.01 or P<0.001). CONCLUSIONS Overexpression of miR-124a can reduce arthritis in CIA mice bacause it could promote synovial cell apoptosis and inhibit synovial cell proliferation via targeting PIK3CA and regulating its downstream pathways.
Animals ; Arthritis, Experimental/metabolism* ; Arthritis, Rheumatoid/genetics* ; Cattle ; Cell Proliferation ; Class I Phosphatidylinositol 3-Kinases/metabolism* ; Mice ; Mice, Inbred DBA ; MicroRNAs/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Synovial Membrane ; bcl-2-Associated X Protein/metabolism*

This paper aims to explore the activity of Codonopsis canescens extract against rheumatoid arthritis(RA) based on the Toll-like receptors(TLRs)/mitogen-activated protein kinases(MAPKs)/nuclear factor kappa B(NF-κB) signaling pathways and its mechanism. The ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry(UPLC-Q-TOF-MS) was used to identify the components of C. canescens extract. Forty-eight male SD rats were randomly divided into six groups, namely the normal group, the model group, the methotrexate(MTX) tablet group, and the low, medium, and high-dose C. canescens extract(ZDS-L, ZDS-M, and ZDS-H) groups, with 8 rats in each group. The model of collagen-induced arthritis in rats was induced by injection of bovine type Ⅱ collagen emulsion. MTX(2.5 mg·kg~(-1)), ZDS-L, ZDS-M, and ZDS-H(0.3 g·kg~(-1), 0.6 g·kg~(-1), and 1.2 g·kg~(-1)) were administrated by gavage. Rats in the normal group and the model group received distilled water. MTX was given once every three days for 28 days, and the rest medicines were given once daily for 28 days. Body weight, degree of foot swelling, arthritis index, immune organ index, synovial histopathological changes, and serum levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and interleukin-6(IL-6) were observed. Protein expressions of TLR2, TLR4, NF-κB p65, p38 MAPK, and p-p38 MAPK in rats were determined by Western blot. Thirty-four main components were identified by UPLC-Q-TOF-MS, including 15 flavonoids, 7 phenylpropanoids, 4 terpenoids, 4 organic acids, 2 esters, and 2 polyalkynes. As compared with the normal group, the body weight of the model group was significantly decreased(P<0.01), and foot swelling(P<0.05, P<0.01), arthritis index(P<0.01), and the immune organ index(P<0.01) were significantly increased. The synovial histopathological injury was obviously observed in the model group. The serum levels of inflammatory factors TNF-α, IL-1β, and IL-6 were significantly increased(P<0.01), and the protein expression levels of TLR2, TLR4, NF-κB p65, p-p38 MAPK/p38 MAPK in the synovial tissue were significantly increased(P<0.01) in the model group. As compared with the model group, the body weights of the ZDS dose groups were increased(P<0.01), and the degree of foot swelling(P<0.01) and the arthritis index were decreased(P<0.05, P<0.01). The immune organ index was decreased(P<0.01) in the ZDS dose groups, and the synovial tissue hyperplasia and inflammatory cell infiltration were alleviated. The serum levels of TNF-α, IL-1β, and IL-6 were significantly decreased(P<0.05, P<0.01), and the protein expression levels of TLR2, TLR4, NF-κB p65, p-p38 MAPK/p38 MAPK were decreased(P<0.05, P<0.01) in the ZDS dose groups. C. canescens extract containing apigenin, tricin, chlorogenic acid, aesculin, ferulic acid, caffeic acid, and oleanolic acid has a good anti-RA effect, and the mechanism may be related to the inhibition of TLRs/MAPKs/NF-κB signaling pathways.
Animals ; Cattle ; Male ; Rats ; Arthritis, Experimental/drug therapy* ; Arthritis, Rheumatoid/drug therapy* ; Body Weight ; Codonopsis/chemistry* ; Interleukin-6/blood* ; NF-kappa B/genetics* ; p38 Mitogen-Activated Protein Kinases/metabolism* ; Plant Extracts/therapeutic use* ; Rats, Sprague-Dawley ; Signal Transduction ; Toll-Like Receptor 2/metabolism* ; Toll-Like Receptor 4/metabolism* ; Tumor Necrosis Factor-alpha/pharmacology*

OBJECTIVE: To compare the therapeutic effect of different animal bile powders on lipid metabolism disorders induced by high-fat diet in rats, and analyze the bioactive components of each animal bile powder. METHODS: Sixty Sprague-Dawley rats were randomly divided into 6 groups (n=10): normal diet control group, high-fat diet model group, high-fat diet groups orally treated with bear, pig, cow and chicken bile powders, respectively. Serum biochemical markers from the abdominal aorta in each group were analyzed. Changes in the body weight and liver weight were recorded. Pathohistological changes in the livers were examined. High performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry was used to determine the composition of bioactive components in each animal bile powder. RESULTS: Treatment with different types of animal bile powders had different inhibitory effects on high-fat diet-induced increase of body weight and/or liver weight in rats, most notably in bear and pig bile powders (P<0.05). High-fat diet induced lipid metabolism disorder in rats, which could be reversed by treatment with all kinds of bile powders. Bear bile and chicken bile showed the most potent therapeutic effect against lipid metabolism disorder. Cow and bear bile effectively alleviated high-fat diet induced liver enlargement and discoloration, hepatocyte swelling, infiltration of inflammatory cells and formation of lipid vacuoles. Bioactive component analysis revealed that there were significant differences in the relative content of taurocholic acid, taurodeoxycholic acid and ursodeoxycholic acid among different types of animal bile. Interestingly, a unique component with molecular weight of 496.2738 Da, whose function has not yet been reported, was identified only in bear bile powder. CONCLUSIONS Different animal bile powders had varying therapeutic effect against lipid metabolism disorders induced by high-fat diet, and bear bile powder demonstrated the most effective benefits. Bioactive compositions were different in different types of animal bile with a novel compound identified only in bear bile powder.
Animals ; Bile/metabolism* ; Biomarkers/metabolism* ; Body Weight ; Cattle ; Diet, High-Fat ; Female ; Lipid Metabolism ; Lipid Metabolism Disorders/metabolism* ; Lipids/analysis* ; Liver/metabolism* ; Powders ; Rats ; Rats, Sprague-Dawley ; Swine ; Taurodeoxycholic Acid/metabolism* ; Ursidae/metabolism* ; Ursodeoxycholic Acid/metabolism*

The aim of this paper was to investigate the molecular mechanism of Calculus Bovis Sativus( CBS) in alleviating lipid accumulation in vitro by serum pharmacology. The CBS-containing serum of mice was obtained by serum pharmacology method to evaluate its effect on the proliferation of LO2 hepatocytes. The lipid reducing effects of CBS-containing serum through Nrf2 was evaluated by fructose-induced LO2 hepatocyte steatosis model,nuclear factor erythroid 2 related factor 2( Nrf2) agonist oltipraz combined intervention,cell oil red O staining and intracellular triglyceride( TG) content. The effects of CBS-containing serum on lipid peroxidation and hepatocytes apoptosis were evaluated by reactive oxygen species( ROS) and apoptosis assay,respectively. Real-time quantitative polymerase chain reaction( PCR) was used to detect the relative expression of lipid synthesis-related genes and apoptosis-related genes.RESULTS:: showed that CBS drug-containing serum had no significant effect on LO2 hepatocyte proliferation. As compared with the model group,CBS-containing serum could effectively reduce the formation of lipid droplets in fructose-induced LO2 hepatocytes,significantly reduce intracellular TG and ROS levels,and significantly reduce hepatocyte apoptosis rate( P < 0. 05). As compared with the model group,carbohydrate responsive element binding protein( ChREBP),sterol regulatory element binding protein-1 c( SREBP-1 c),fatty acid synthase( FAS),acetyl-CoA carboxylase 1( ACC1),stearoyl-CoA desaturase 1( SCD1),Bax and caspase-3 mRNA levels were significantly reduced in CBS drug-containing serum treatment group( P<0. 05). All of the above effects could be reversed by oltipraz.In conclusion,CBS-containing serum can significantly inhibit the fructose-induced LO2 liver fat deposition,and the mechanism may be related to reducing intracellular ROS level through the Nrf2 pathway and improving intracellular peroxidation state to reduce apoptosis.
Animals ; Apoptosis ; Cattle ; Cells, Cultured ; Fatty Liver ; Fructose ; Gallstones ; chemistry ; Hepatocytes ; cytology ; metabolism ; Lipid Metabolism ; Lipid Peroxidation ; Liver ; Medicine, Chinese Traditional ; Mice ; Reactive Oxygen Species ; metabolism ; Serum ; chemistry ; Sterol Regulatory Element Binding Protein 1 ; metabolism ; Triglycerides

OBJECTIVE: To investigate the expression of EGR1 gene and the localization of EGR1 protein in bovine skeletal muscle-derived satellite cells (MDSCs), as well as to investigate the mechanism that EGR1 protein enters the nucleus. METHODS: Bovine MDSCs were cultured in differentiation medium for 1 day, 3 days and 5 days, respectively, and each group was triplicate. The expression of EGR1 gene and the localization of EGR1 protein were studied at different differentiation period in MDSCs by qRT-PC and Western blot. Moreover, the changes on the expression of endogenous EGR1 gene and EGR1 proteins were explored by CRISPRi, site-directed mutagenesis and laser confocal method. RESULTS: The results from the qRT-PCR and Western blot showed that the expressions of EGR1 gene on transcription level and translation level were significantly higher in differentiated cells than those in undifferentiated cells. The highest expression was found on the third day after the differentiation, and then began to decline. Immunofluorescence assays showed that EGR1 proteins were preferentially expressed in differentiated MDSCs, and increased along with the increase of number of myotubes. Confocal observation revealed that some EGR1 proteins were transferred into the nucleus in the differentiation of cells, however, the EGR1 proteins would not be detected in the differentiated MDSCs nuclei if a site directed mutagenesis (serine) on EGR1 protein occurred. CONCLUSION During the differentiation of bovine skeletal muscle satellite cells, the transcriptional level of EGR1 gene is increased, and some EGR1 proteins are transferred into the nucleus. The serine phosphorylation at position 533 of the C terminal of EGR1 protein is necessary for the nucleus transfer.
Animals ; Cattle ; Cell Differentiation ; Cell Nucleus ; Cells, Cultured ; Early Growth Response Protein 1 ; genetics ; metabolism ; Muscle Fibers, Skeletal ; Satellite Cells, Skeletal Muscle ; metabolism

Interferon-γ (IFN-γ) has been used to control cancers in clinical treatment. However, an increasing number of reports have suggested that in some cases effectiveness declines after a long treatment period, the reason being unclear. We have reported previously that long-term IFN-γ treatment induces malignant transformation of healthy lactating bovine mammary epithelial cells (BMECs) in vitro. In this study, we investigated the mechanisms underlying the malignant proliferation of BMECs under IFN-γ treatment. The primary BMECs used in this study were stimulated by IFN-γ (10 ng/mL) for a long term to promote malignancy. We observed that IFN-γ could promote malignant cell proliferation, increase the expression of cyclin D1/cyclin-dependent kinase 4 (CDK4), decrease the expression of p21, and upregulate the expression of cellular-abelsongene (c-Abl) and histone deacetylase 2 (HDAC2). The HDAC2 inhibitor, valproate (VPA) and the c-Abl inhibitor, imatinib, lowered the expression level of cyclin D1/CDK4, and increased the expression level of p21, leading to an inhibitory effect on IFN-γ-induced malignant cell growth. When c-Abl was downregulated, the HDAC2 level was also decreased by promoted proteasome degradation. These data suggest that IFN-γ promotes the growth of malignant BMECs through the c-Abl/HDAC2 signaling pathway. Our findings suggest that long-term application of IFN-γ may be closely associated with the promotion of cell growth and even the carcinogenesis of breast cancer.
Animals ; Carcinogenesis/pathology* ; Cattle ; Cell Cycle Proteins/metabolism* ; Cell Proliferation/drug effects* ; Cell Transformation, Neoplastic/pathology* ; Cells, Cultured ; Epithelial Cells/pathology* ; Female ; Histone Deacetylase 2/metabolism* ; Imatinib Mesylate/pharmacology* ; Interferon-gamma/pharmacology* ; Mammary Glands, Animal/pathology* ; Mammary Neoplasms, Experimental/pathology* ; Proto-Oncogene Proteins c-abl/metabolism* ; Signal Transduction ; Valproic Acid/pharmacology*