This study aimed to investigate the mechanism by which Shegan Mahuang Decoction(SGMH) and its bitter Chinese herbs(BCHs) regulated the lung-gut axis through the bitter taste receptor 14(TAS2R14)/secretory immunoglobulin A(SIgA)/thymic stromal lymphopoietin(TSLP) to intervene in the epithelial cell barrier of cold asthma rats. Fifty SD rats were randomly divided into the following five groups: normal group, model group, dexamethasone group, SGMH group, and BCHs group. A 10% ovalbumin(OVA) solution was used to sensitize the rats via subcutaneous injection on both sides of the abdomen and groin, combined with 2% OVA atomization and cold(2-4 ℃) stimulation to induce a cold asthma model in rats. The SGMH, BCHs, and dexamethasone groups were given corresponding treatments by gavage and nebulization, while the normal and model groups received normal saline by gavage and nebulization. After the final stimulation, pathological changes in the lung and intestine tissues were observed using hematoxylin-eosin(HE) and periodic acid-Schiff(PAS) staining. Lung function was assessed by measuring the ratio of forced expiratory volume in the first second to forced vital capacity(FEV1/FVC), the ratio of the average flow rate at 25%-75% of forced vital capacity to foned vital capacity(FEV25%-75%/FVC), the peak expiratory flow(PEF), and pulmonary resistance(RL). The levels of IL-4, IL-5, IL-13, and TNF-α in serum, and sIgA in serum, intestinal, and bronchial mucosa were detected by enzyme-linked immunosorbent assay(ELISA). The expression of TAS2R14 protein in lung tissue was detected by Western blot(WB). The content of short-chain fatty acids(SCFAs) in rat feces was determined by gas chromatography-mass spectrometry(GC-MS). The effect of TAS2R14/TSLP on lipopolysaccharide(LPS)-induced inflammation in epithelial cells in the BCHs group was observed, and the expression of TAS2R14 and TSLP in cells was detected by WB. Compared with the normal group, the model group showed reduced water intake, diet, and body weight, increased infiltration of inflammatory cells in the lung and intestinal tissues, goblet cell hyperplasia, significantly decreased FEV1/FVC, FEV25%-75%/FVC, and PEF, and significantly increased RL. Moreover, serum levels of IL-4, IL-5, IL-13, and TNF-α were elevated, and sIgA levels in serum, intestine, and bronchial mucosa were significantly decreased. TAS2R14 expression in lung tissues was inhibited, and the content of acetic acid, propionic acid, and butyric acid in feces was significantly reduced. In the LPS group, TSLP expression increased, and TAS2R14 expression decreased. Compared with the model group, the general condition of rats in the SGMH and BCHs groups improved, with reduced infiltration of inflammatory cells and goblet cell hyperplasia in the lung and intestinal tissues. FEV1/FVC, FEV25%-75%/FVC, and PEF significantly increased, and RL significantly decreased. Serum levels of IL-4, IL-5, IL-13, and TNF-α decreased, while sIgA levels in serum, intestine, and bronchial mucosa significantly increased, and TAS2R14 expression was activated in lung and intestinal tissues. The content of acetic acid, propionic acid, and butyric acid in feces significantly increased. Compared with the model group, the BCHs group and the agonist group showed inhibited TSLP expression and increased TAS2R14 expression. The results showed that both SGMH and BCHs could reduce lung and intestinal inflammatory reactions, improve lung function, and regulate the content of intestinal SCFAs in asthmatic rats. There was no significant difference in TAS2R14 protein expression between the SGMH and BCHs groups, indicating that the clinical efficacy of BCHs may be related to the activation of the bitter receptor TAS2R14 and the regulation of immune inflammatory mediators in lung and intestinal epithelial cells.
Animals ; Drugs, Chinese Herbal/administration & dosage* ; Rats ; Rats, Sprague-Dawley ; Lung/metabolism* ; Asthma/metabolism* ; Cytokines/immunology* ; Male ; Receptors, G-Protein-Coupled/immunology* ; Epithelial Cells/metabolism* ; Thymic Stromal Lymphopoietin ; Immunoglobulin A, Secretory/genetics* ; Humans ; Cold Temperature

Objective To investigate the effect of salidroside on intestinal mucosal immune status in rats under compound stress of hypoxia and training (HTCS) and the mechanism. Methods SD rats were randomly divided into HTCS model group (model), placebo group (placebo) and salidroside group (salidro). Model group received no intervention, and placebo and salidro group received intraperitoneal injection of normal saline and salidroside, respectively. Then, ileum tissue of rats were collected and the intestinal damage was assayed by HE staining and Chiu scores. Intestinal permeability indices, including serum D-diamine oxidase (DAO), D-lactic acid (DLA) and endotoxin (END) and secretory immunoglobulin A (sIgA) of intestinal tissue were detected by ELISA. T lymphocyte subsets of intestinal tissue were detected by flow cytometry. Expression of tight junction molecules, including ZO-1, Claudin-3, occluding, were detected by PCR and western blot. Activation of TLR4/NF-κB signaling pathway was detected by Western blot analysis. Results Compared with model group and placebo group, salidro group had the decreased intestinal mucosal injury and low Chiu score, and the level of intestinal permeability indices including serum DAO, DLA and END fell off. CD4⁺ T cell percentage, CD4⁺/CD8⁺ ratio and sIgA level were went up, while CD8⁺ T cell percentage was went down. mRNA and the level of protein expressions of ZO-1, claudin-3 and occludin increased, while activation of TLR4/NF-κB signaling pathway was inhibited. Conclusion Salidroside can alleviate the intestinal barrier injury and improve intestinal mucosal immune status of rats under compound stress of hypoxia and training via inhibiting TLR4/NF-κB signalling pathway.
Animals ; Rats ; Rats, Sprague-Dawley ; NF-kappa B ; Toll-Like Receptor 4/genetics* ; Claudin-3 ; Hypoxia ; Immunoglobulin A, Secretory ; Signal Transduction

The effects of diclazuril on the bursa of Fabricius (BF) structure and secretory IgA (SIgA) expression in chickens infected with Eimeria tenella were examined. The morphology of the BF was observed by hematoxylin and eosin staining, while ultrastructural changes were monitored by transmission electron microscopy. E. tenella infection caused the BF cell volumes to decrease, irregularly arranged, as well as, enlargement of the intercellular space. Diclazuril treatment alleviated the physical signs of damages associated with E. tenella infection. The SIgA expression in BF was analyzed by immunohistochemistry technique. The SIgA expression increased significantly by 350.4% (P<0.01) after E. tenella infection compared to the normal control group. With the treatment of diclazuril, the SIgA was relatively fewer in the cortex, and the expression level was significantly decreased by 46.7% (P<0.01) compared with the infected and untreated group. In conclusion, E. tenella infection in chickens induced obvious harmful changes in BF morphological structure and stimulated the expression of SIgA in the BF. Diclazuril treatment effectively alleviated the morphological changes. This result demonstrates a method to develop an immunological strategy in coccidiosis control.
Animals ; Bursa of Fabricius/anatomy & histology/*parasitology ; Chickens ; Coccidiosis/drug therapy/metabolism/parasitology/*veterinary ; Coccidiostats/administration & dosage/*adverse effects ; Eimeria tenella/*physiology ; Female ; Immunoglobulin A, Secretory/*genetics/metabolism ; Male ; Nitriles/administration & dosage/*adverse effects ; Poultry Diseases/*drug therapy/genetics/metabolism/parasitology ; Triazines/administration & dosage/*adverse effects

Secretory IgA (SIgA) antibodies in external secretions play an important role in mucosal immune response. Polymeric SIgA was advantageous over monomeric IgA (mIgA) and IgG in several aspects. To express secretory IgA antibody against H5N1 virus, we constructed the secretory component and immunoglobulin J expressing plasmids and co-transfected the plasmids into the Chinese hamster ovary cells (CHO) stably expressing immunoglobulin A. Then we used Zeocin to select the positive clone cells, monoclonal cells stably secreting SIgA was screened through fold dilution method at last. The SIgA antibody secreted from the CHO cells was confirmed by Western blotting, which demonstrated that we had got the complete SIgA molecular. The successful expression of this polymeric anti-H5N1 SIgA in CHO cells will contribute to the production of recombinant SIgA as a preventive agent for infectious disease control.
Animals ; Antibodies, Viral ; biosynthesis ; genetics ; CHO Cells ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Genetic Vectors ; Immunoglobulin A ; immunology ; Immunoglobulin A, Secretory ; biosynthesis ; genetics ; immunology ; Influenza A Virus, H5N1 Subtype ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

OBJECTIVETo investigate the effect of total alkaloids of Sophora alopecuroides (TASA) on dextran sulfate sodium (DSS)-induced colitis in mice.

METHODSChronic experimental colitis was induced by administration of 4 cycles of 4% DSS. Fifty mice were randomly distributed into 4 groups (normal, DSS, DSS/high-dose TASA, and DSS/low-dose TASA groups) by a random number table with body weight stratification. Mice in the normal group (n=11) and DSS-induced colitis control group (n=15) received control treatment of 20 mL/kg distilled water; DSS plus TASA high- and low-dose groups (n=12 each) were treated with TASA solution (20 mL/kg) at the doses of 60 mg/kg and 30 mg/kg, respectively. The severity of colitis was assessed on the basis of clinical signs, colon length, and histology scores. Moreover, secretory immunoglobulin A (sIgA) and haptoglobin (HP) were analyzed by enzyme linked immunosorbent assay; intercellular adhesion molecule 1 (ICAM-1) and macrophage-migration inhibitory factor (MIF) gene expressions were analyzed by quantitative reverse transcriptase realtime polymerase chain reaction (qRT-PCR) using SYBA green I; and nuclear factor κ B (NF-κ B) expression and activation and p65 interaction with the promoter of ICAM-1 gene were assessed by Western blotting and chromatin immunoprecipitation assay.

RESULTSTASA administration significantly attenuated the damage and substantially reduced HP elevation and maintained the level of cecum sIgA. TASA inhibited the ICAM-1 gene expression and had no effect on MIF gene expression. Also, TASA was able to reduce phospho-I κ B α (p-I κ B α) protein expression; however, it had no effect on the activation of I κ B kinase α (IKK α) and inhibitor of NF-κ B α (I κ B α). Moreover, TASA inhibited the p65 recruitment to the ICAM-1 gene promoter.

CONCLUSIONSTASA had a protective effect on DSS-induced colitis. Such effect may be associated with its inhibition of NF-κ B activation and blockade of NF-κ B-regulated transcription activation of proinflammatory mediator gene.

Alkaloids ; pharmacology ; therapeutic use ; Animals ; Cecum ; drug effects ; metabolism ; pathology ; Colitis ; chemically induced ; drug therapy ; pathology ; prevention & control ; Colon ; pathology ; ultrastructure ; Dextran Sulfate ; Down-Regulation ; drug effects ; Female ; Haptoglobins ; metabolism ; I-kappa B Proteins ; metabolism ; Immunoglobulin A, Secretory ; metabolism ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Intestinal Mucosa ; drug effects ; pathology ; ultrastructure ; Macrophage Migration-Inhibitory Factors ; genetics ; metabolism ; Mice ; Mice, Inbred C57BL ; NF-KappaB Inhibitor alpha ; Phosphorylation ; drug effects ; Phytotherapy ; Promoter Regions, Genetic ; genetics ; Protective Agents ; pharmacology ; therapeutic use ; Protein Binding ; drug effects ; Signal Transduction ; drug effects ; Sophora ; chemistry ; Transcription Factor RelA ; metabolism

OBJECTIVETo valuate the intestinal mucosal secretary IgA (sIgA) responses to the ovalbumin-induced allergy in mice, to provide some clues for the exploration of mechanisms and therapeutic methods in the children's food allergy.

METHODSFemale BALB/c mice aged 6 weeks fed on the ovalbulmin-free diet, were randomly divided into 2 groups with 8 mice in each. The mice in group Ch were sensitized with ovalbumin (OVA) intraperitoneal injection two times and challenged intragastrically 3 times. Two days after the last challenge with oral OVA, the mice were sacrificed and the samples were collected. The mice in group Ns were given intraperitoneal and intragastrical normal saline as control. The levels of total IgA and OVA-specific IgA in the intestinal mucus of the mice were determined by ELISA; the immunohistochemical methods were adopted to observe IgA(+) plasmacytes in lamina propria (LP) and surface membrane IgA (smIgA)(+) lymphocytes in peyer's patch (PP); the IL-4 mRNA expression in LP was assessed by RT-PCR. The IL-4 mRNA expression in PP was evaluated by in situ hybridization.

RESULTSAfter the mice in Ch group were sensitized and challenged with OVA, the levels of the total IgA and the OVA-specific IgA in mucus remarkably increased (P < 0.01 respectively), the amounts of the IgA(+) plasmacytes in LP and the smIgA(+) lymphocytes in PP significantly increased (P < 0.01 respectively); a significantly positive correlation was found among the total IgA levels, the OVA-specific IgA levels, the IgA(+) plasmacyte counts in LP and the smIgA(+) lymphocyte counts in PP (P < 0.01 respectively); the mRNA expressions of IL-4 in LP and in PP were significantly augmented (P < 0.01 respectively); significantly positive correlations were found either between the IL-4 mRNA expression and the IgA(+) plasmacyte counts in LP (P < 0.01) or between the IL-4 mRNA expression and the smIgA(+) lymphocyte counts in PP (P < 0.01).

CONCLUSIONSThe intestinal mucosal sIgA responses are abnormally augmented in the ovalbumine-induced allergic mice, which may be partly due to the increased expression of IL-4 mRNA in gut.

Animals ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Female ; Food Hypersensitivity ; immunology ; Immunoglobulin A, Secretory ; metabolism ; Immunohistochemistry ; Interleukin-4 ; genetics ; metabolism ; Intestinal Mucosa ; immunology ; metabolism ; Mice ; Mice, Inbred BALB C ; Ovalbumin ; administration & dosage ; immunology ; Peyer's Patches ; immunology ; RNA, Messenger ; metabolism ; Random Allocation

Oral vaccination may be the most efficient way of inducing an immune response at the remote mucosal site through the common mucosal immune network. Antigenspecific secretory IgA (sIgA) is the major immunoglobulin type generally detected in the secretions of experimental animals following an effective oral immunization. Actinobacillus pleuropneumoniae causing disease in the lung of pig initially interacts, colonizes, and infects the host tissues at the mucosal surface of the respiratory tract. Also, importantly for A. pleuropneumoniae protection, the quantity of sIgA in the lung had merits associated with the mucosal immunity. However, there is no simple method to monitor the level of sIgA as an indicator for the induction of local immune responses by an oral vaccination in the target tissue. Therefore, the relationship between sIgA and IgG was analyzed to evaluate the induction of local immune responses by an oral immunization with Saccharomyces cerevisiae expressing the apxIA and apxIIA genes of A. pleuropneumoniae in this study. The correlation coefficient of determination (r2 x 100) for paired samples in both vaccinated and control groups showed a significant positive-relationship between IgG in sera and sIgA in the lung or intestine. These results indicated that IgG antibody titers in sera could be useful to indirectly predict local immune response, and sIgA, in the lung or intestine to evaluate the efficacy of an oral vaccination.
Actinobacillus pleuropneumoniae ; Administration, Oral ; Animals ; Antigens, Fungal/*immunology ; Bacterial Proteins/genetics/immunology ; Bacterial Vaccines/*immunology ; Disease Models, Animal ; Female ; Hemolysin Proteins ; Immunity, Mucosal/*immunology ; Immunoglobulin A, Secretory/*analysis ; Immunoglobulin G/*blood ; Intestine, Small/immunology ; Lung/immunology ; Mice ; Mice, Inbred BALB C ; Saccharomyces cerevisiae/*immunology

OBJECTIVETo observe the protection against periodontal bone loss in the Sprague-Dawley (SD) rats periodontitis model, with the recombined plasmid pcDNA3.1+/kgpcd as DNA gene vaccine.

METHODSPcDNA3.1+/kgpcd was delivered into rats by submandibular gland-targeted injection. The anti-KGPcd sIgA in saliva was measured by indirect ELISA method. Immunohistochemistry staining was used to assess the protection in the animal model.

RESULTSThe level of specific anti-KGPcd sIgA in saliva of the experimental group was significantly higher than that of control group. HE staining showed that immunization with recombined plasmid pcDNA3.1+/kgpcd could protect or minimize tissue destruction caused by subsequent P. gingivalis challenge in the rat model.

CONCLUSIONSThe results indicate that pcDNA3.1+/kgpcd was a good candidate for anti-periodontitis gene vaccine and could provide protection against Porphyromonas gingivalis-caused periodontitis in rat lesion model.

Animals ; Bacterial Vaccines ; immunology ; therapeutic use ; Immunoglobulin A, Secretory ; analysis ; Periodontitis ; immunology ; microbiology ; prevention & control ; Porphyromonas gingivalis ; genetics ; immunology ; Rats ; Rats, Sprague-Dawley ; Vaccines, DNA ; immunology ; therapeutic use

OBJECTIVEGlucosyltransferase-B (GTF-B) of Streptococcus mutans has been implicated as a principal virulent factor in the development of dental caries. The objective was to use recombined plasmid pcDNA-gtfB expressing multiple antigen of glucosyltransferase-B as gene vaccine to immunize rats through different route, and to investigate the immunization effects of immunization routes.

METHODSA total of 18 Wistar rats were divided into 3 groups, including the quadriceps injection group, the intransal irrigation group and the submandibular gland-targeted injection group. The serum IgG and salivary IgA were assayed by using ELISA after pcDNA3-gtfB immunization. The serum IgG and salivary IgA in different groups were compared using statistical one-way ANOVA.

RESULTSCompared these 3 groups, the serum IgG in the quadriceps injection group was much higher than those of other two groups (P < 0.01), while the salivary IgA of the submandibular gland-targeted injection was much higher than those of other two groups (P < 0.01).

CONCLUSIONIt is indicated pcDNA3-gtfB is good candidate for anticarious gene vaccine, and submandibular gland-targeted injection is an effective immunization route for stimulating salivary IgA.

Animals ; Antibodies, Bacterial ; biosynthesis ; Bacterial Vaccines ; administration & dosage ; immunology ; Cloning, Molecular ; Dental Caries ; prevention & control ; Glucosyltransferases ; genetics ; immunology ; Immunization ; Immunoglobulin A, Secretory ; analysis ; Immunoglobulin G ; blood ; Male ; Plasmids ; genetics ; immunology ; Random Allocation ; Rats ; Rats, Wistar ; Recombination, Genetic ; Saliva ; immunology ; Streptococcus mutans ; genetics ; immunology ; Vaccines, DNA ; administration & dosage ; immunology