Identification and characterization of natural PR-1 protein as major allergen from Humulus japonicus pollen.

BACKGROUND: The Humulus japonicus pollen is one of the most common allergenic pollens in China. However, little is unveiled regarding the allergenic components in Humulus japonicus pollen. Our study aimed to purify and identify the pathogenesis-related 1 (PR-1) protein from Humulus japonicus pollen, and to characterize the molecular and immunochemical properties of this novel allergen. METHODS: The natural PR-1 protein (named as Hum j PR-1) was purified from Humulus japonicus pollen extracts with a combined strategy of chromatography, and identified by mass spectrometry. The coding sequence of Hum j PR-1 was confirmed by cDNA cloning. The recombinant Hum j PR-1 was expressed and purified from Escherichia coli. The allergenicity was assessed by immunoblot, enzyme-linked immunosorbent assay (ELISA), inhibition ELISA, and basophil activation test using Humulus japonicus allergic patients' whole blood. The physicochemical properties and 3-dimensional structure of it were comprehensively characterized by in silico methods. RESULTS: The allergenicity analysis revealed that 76.6 % (23/30) of the Humulus japonicus pollen allergic patients displayed specific IgE recognition of the natural Hum j PR-1. The cDNA sequence of Hum j PR-1 had a 516-bp open reading frame encoding 171 amino acids. Physicochemical analysis indicated that Hum j PR-1 was a stable and relatively thermostable protein. Hum j PR-1 shared a similar 3-dimensional folding pattern with other homologous allergens, which was a unique αßα sandwich structure containing 4 α-helices and 6 antiparallel ß-sheets, encompassing 4 conserved CAP domain. CONCLUSION: The natural PR-1 was firstly purified and characterized as a major allergenic allergen in Humulus japonicus pollen. These findings would contribute to developing diagnostic and therapeutic strategies for Humulus japonicus pollinosis.

Purification and characterization of enolase as a novel allergen in Platanus acerifolia pollen.

BACKGROUND: The pollen from Platanus acerifolia (P. acerifolia) is one of the main causes of allergic disorders. To date, only 4 allergens have been identified from this pollen. But previous studies showed that there still exist under-recognized allergens in it. The aim of this study was to comprehensively investigate the newly identified enolase (Pla a 6) as a novel allergen in the P. acerifolia pollen. METHODS: The natural (n) Pla a 6 was purified by combined chromatographic strategies. According to the identified internal peptides, the cDNA sequence encoding this allergen was matched from the mRNA-sequencing results of P. acerifolia pollen, which was further amplified and cloned. The recombinant (r) Pla a 6 was expressed and purified from E. coli. The allergenicity of this novel allergen was characterized by enzyme linked immunosorbent assay (ELISA), Western blot, inhibition ELISA, and basophil activation test (BAT). RESULTS: A novel allergen from P. acerifolia pollen, named as Pla a 6 was thoroughly studied, which contained an open reading frame of 1338 bp encoding 445 amino acids. The IgE-binding activity of nPla a 6 was initially proved by Western-blot, and a similar IgE-binding pattern to rPla a 6 was also exhibited. Moreover, the positivity for specific IgE against rPla a 6 was tested as 45.95% (17/37) by ELISA, and IgE binding to pollen extract could be inhibited up to 45.77% by 10 µg/ml of rPla a 6. The protein was also confirmed to activate patients' basophils. CONCLUSIONS: In this study, a novel allergen belonging to enolase family was comprehensively investigated and characterized through its natural and recombinant forms in P. acerifolia pollen. The study will contribute to the development of novel molecular-based diagnostic and therapeutic approaches for P. acerifolia pollen allergy.

Effects of Selenium Supplement on B Lymphocyte Activity in Experimental Autoimmune Thyroiditis Rats.

METHODS: 45 healthy and adult female SD rats were randomly divided into three groups: normal control group, EAT model group, and selenium yeast supplement EAT group. The EAT model rats were induced by subcutaneous injection of porcine thyroglobulin and fed with high iodine water. The concentrations of serum thyroid-stimulating hormone (TSH), TGAb, TPOAb, and B cell activating factor (BAFF) were detected in each group by enzyme-linked immunosorbent assay (ELISA), and the expression of interleukin-10 (IL-10) in thyroid tissue was detected by immunohistochemistry. B cells and regulatory B cells (Bregs) ratios in the spleen of rats were analyzed by flow cytometry. RESULTS: In contrast with the EAT model group, the levels of serum TSH, TGAB, TPOAb, and BAFF were decreased, while IL-10 expression was increased in thyroid tissue, and Bregs ratio was upregulated in the spleen (all p < 0.05) in the selenium yeast supplement EAT group. CONCLUSION: Selenium yeast supplement could partially attenuate immune imbalance in EAT rats, which may be related to the mechanism of modulating B lymphocyte activity.

Respiratory exposure to PM2.5 soluble extract disrupts mucosal barrier function and promotes the development of experimental asthma.

BACKGROUND: Air pollutants are important factors that contribute to the development and exacerbation of asthma, but experimental evidence still needs to be collected and the mechanisms still need to be addressed. In this study, we aimed to assess the association between PM2.5 exposure and asthma development. The effects of PM2.5 exposure on the barrier functions of airway epithelial cells were also determined. METHODS: PM2.5 was collected from Nanjing, China, and its soluble extract was prepared. Human lung epithelial cells (BEAS-2B) were treated with different concentrations of soluble PM2.5 extract, and cell viability was detected by FACS using Annexin V-FITC staining. PM2.5-induced oxidative stress and inflammatory events were assessed by DCF-DA staining and qPCR. PM2.5-induced dysfunction of the airway epithelial barrier was assessed by measuring the expression of tight junction molecules. In vivo, BALB/c mice were treated with OVA in the presence or absence of PM2.5 solution, followed by exposure to OVA aerosols. Allergy-induced airway inflammation and lung injury were assessed by histopathological analyses. RESULTS: Soluble PM2.5 extract exposure in vitro decreased the viability and increased apoptosis of airway epithelial cells. Soluble PM2.5 extract induced oxidative stress and enhanced pro-inflammatory factor expression by activating the NF-κB and MAPK signalling pathways, which were accompanied by reduced airway barrier function. The in vivo data demonstrated that PM2.5 exposure increased the effects of allergy sensitization after respiratory exposure to allergens, which led to the development of asthma. CONCLUSION: This study suggests that exposure to soluble PM2.5 extract contributes to airway barrier dysfunction. The soluble mediators generated by airway epithelial cells in response to PM2.5 exposure orchestrate the breaking of inhalational tolerance and sensitization to allergic antigens, leading to the exacerbated development of asthma.

Ginsenoside Rg1 alleviates corticosterone-induced dysfunction of gap junctions in astrocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginsenoside Rg1 (Rg1), one of the major bioactive ingredients of Panax ginseng C. A. Mey, has neuroprotective effects in animal models of depression, but the mechanism underlying these effects is still largely unknown AIM OF THE STUDY: Gap junction intercellular communication (GJIC) dysfunction is a potentially novel pathogenic mechanism for depression. Thus, we investigated that whether antidepressant-like effects of Rg1 were related to GJIC. MATERIALS AND METHODS: Primary rat prefrontal cortical and hippocampal astrocytes cultures were treated with 50µM CORT for 24h to induce gap junction damage. Rg1 (0.1, 1, or 10µM) or fluoxetine (1µM) was added 1h prior to CORT treatment. A scrape loading and dye transfer assay was performed to identify the functional capacity of gap junctions. Western blot was used to detect the expression and phosphorylation of connexin43 (Cx43), the major component of gap junctions. RESULTS: Treatment of primary astrocytes with CORT for 24h inhibited GJIC, decreased total Cx43 expression, and increased the phosphorylation of Cx43 at serine368 in a dose-dependent manner. Pre-treatment with 1µM and 10µM Rg1 significantly improved GJIC in CORT-treated astrocytes from the prefrontal cortex and hippocampus, respectively, and this was accompanied by upregulation of Cx43 expression and downregulation of Cx43 phosphorylation. CONCLUSION: These findings provide the first evidence indicating that Rg1 can alleviate CORT-induced gap junction dysfunction, which may have clinical significance in the treatment of depression.

Electroacupuncture for Functional Constipation: A Multicenter, Randomized, Control Trial.

Background and Aim. To investigate the efficacy and safety of electroacupuncture (EA) with different current intensities for functional constipation (FC) and to assess whether the effects of EA with different current intensities are superior to the mosapride. Methods. Patients with FC were randomly divided into low current intensity group (LCI), high current intensity group (HCI), and mosapride group (MC). The primary outcome was three or more spontaneous bowel movements (SBMs) per week and an increase of one or more SBMs from baseline during at least 3 of the 4 weeks. Results. The primary outcome was reached by 53.45%, 66.15%, and 52.24% of the patients who received LCI, HCI, and mosapride, respectively. EA can significantly improve the weekly SBMs and stool consistency and reduce straining severity (p < 0.0001, all). HCI improved the quality of life better than mosapride (p < 0.05) and reduced the proportion of severe constipation more than LCI and mosapride (p < 0.05, both). Conclusions. EA is effective and safe at both current intensities for FC; therapeutic effects of LCI and HCI are not superior to mosapride. EA is superior to mosapride in improving patients' life quality and satisfaction level of treatment; EA has fewer adverse events than mosapride.

[Metabonomics of Syndrome Markers in Infantile Bronchial Asthma Episode].

Objective To analyze urinary metabolites of bronchial asthma children patients with phlegm-heat obstructing Fei syndrome (PHOFS) and non-PHOFS using gas chromatography-mass spec- trometry/mass spectrometer ( GC-MS/MS) , thus performing research on syndrome markers. Methods Totally 44 bronchial asthma children patients with PHOFS and non-PHOFS in onset of asthma were recrui- ted. Another 29 healthy children were also recruited. Their urine samples were analyzed by GC-MS/MS. The profiles were analyzed using orthogonal partial least squares-discriminant analysis (OPLS-DA) , vari- able importance in the projection (VIP) , and non-parametric test to determine intergroup differential metabolites. Abnormal metabolic pathways were determined by Metaboanalyst. Results Compared with the health control group, contents of fourteen substances like inositol, uric acid, stearic acid, and so on de- creased, and mino-malonic acid content increased in asthma episode children (P <0. 05). The process was mainly involved in 5 metabolic pathways such as lysine degradation and biosynthesis, pyruvate me- tabolism, and so on. Compared with the non-PHOFS group in bronchial asthma episode, contents of nine substances like oxalic acid, L-threonine, pyrimidine, and so on decreased in the PHOFS group (P < 0. 05). The process was mainly involved in 5 metabolic pathways such as pentose phosphate pathway, inositol phosphate metabolism, and so on. Conclusions Urinary metabolites are different in infantile bronchial asthma episode and healthy children. Metabolic biomarkers and pathways exist in different syn- dromes in bronchial asthma episode.

Clinical study of electro-acupuncture treatment with different intensities for functional constipation patients.

Functional constipation (FC) is a common functional bowel disorder disease that affects life quality of a large number of people. This study aimed to explore the impact of different intensities of electro-acupuncture (EA) treatment for FC patients. Totally, 111 patients with FC meeting the Rome III criteria were randomly assigned to different intensities of EA groups (low and high intensity of EA groups) and medicine-controlled (MC) group. In EA groups, patients were treated with EA at quchi (LI11) and shangjuxu (ST37) bilaterally for 4 weeks, 5 times/week in the first 2 weeks, and 3 times/week in the last 2 weeks. In MC group, 5 mg mosapride citrate was administered orally 3 times/day for 4 weeks. Spontaneous bowel movement frequency each day was recorded using a constipation diary. Self-rating anxiety scale (SAS) and self-rating depression scale (SDS) were used to assess the patients' psychological state. Cortisol (CORT), substance P (SP), and vasoactive intestinal polypeptide (VIP) were evaluated at baseline and at the end of 4 weeks after treatment. As compared with the baseline, there was statistically significant increase in stool frequency every week (P<0.01), but there was no statistically significant difference among the three groups. As compared with the baseline, after 4 weeks of EA therapy, the scores of SDS and serum levels of CORT were decreased significantly in low intensity of EA group (P<0.01), and the serum levels of SP and VIP were increased significantly (P<0.05); the scores of SAS and SDS and serum levels of CORT were decreased significantly in high intensity of EA group (P<0.05), and the serum levels of SP and VIP were increased significantly (P<0.05); the serum levels of CORT and VIP were increased significantly in MC group (P<0.05). As compared with MC group, after 4 weeks of treatment, the serum levels of SP were signifcicantly increased in low intensity of EA group (P<0.01). Low and high intensities of EA could increase the stool frequency, improve the FC patient's anxiety and depression, reduce the serum levels of CORT, and increase the serum levels of SP and VIP effectively. It is concluded that both low and high intensities of EA are effective for FC patients, but there is no significant difference between the low and high intensities of EA.