α-Asarone alleviates allergic asthma by stabilizing mast cells through inhibition of ERK/JAK2-STAT3 pathway.

Asthma is a heterogeneous disease related to numerous inflammatory cells, among which mast cells play an important role in the early stages of asthma. Therefore, treatment of asthma targeting mast cells is of great research value. α-Asarone is an important anti-inflammatory component of the traditional Chinese medicine Acorus calamus L, which has a variety of medicinal values. To investigate whether α-asarone can alleviate asthma symptoms and its mechanism. In this study, we investigated the effect of α-asarone on mast cell activation in vivo and in vitro. The release of chemokines or cytokines, AHR (airway hyperresponsiveness), and mast cell activation were examined in a mast cell-dependent asthma model. Western blot was performed to determine the underlying pathway. α-Asarone inhibited the degranulation of LAD2 (laboratory allergic disease 2) cells and decreased IL-8, MCP-1, histamine, and TNF-α in vitro. α-Asarone reduced paw swelling and leakage of Evans blue, as well as serum histamine, CCL2, and TNF-α in vivo. In the asthma model, α-asarone showed an inhibitory effect on AHR, inflammation, mast cells activation, infiltration of inflammatory cells, and the release of IL-5 and IL-13 in lung tissue. α-Asarone decreased the levels of phosphorylated JAK2, phosphorylated ERK, and phosphorylated STAT3 induced by C48/80. Our findings suggest that α-asarone alleviates allergic asthma by inhibiting mast cell activation through the ERK/JAK2-STAT3 pathway.

Isoimperatorin reduces the effective dose of dexamethasone in a murine model of asthma by inhibiting mast cell activation.

Adverse effects that result from dexamethasone (DEX) use are common and serious in patients with asthma. Therefore, alternative anti-inflammatory treatments are being investigated. Isoimperatorin (ISO), an active natural furocoumarin, possesses multiple pharmacological properties, including an anti-inflammation effect. In this study, investigations were conducted on the effect of ISO on mast cell (MC) activation in vitro and whether ISO could reduce the effective dose of DEX in a mast cell-dependent murine model of asthma in vivo. Calcium imaging was used to assess intracellular Ca2+ mobilization. Enzyme-linked immunosorbent assay was used to measure the chemokines release. Western blot analysis was conducted to investigate the underlying pathway. Airway inflammation and hyperresponsiveness (AHR) were examined in an asthma model. ISO inhibited Ca2+ flux and MC degranulation via Lyn/PLCγ1/PKC, ERK, and P38 MAPK pathways. In the asthma model, ISO, in combination with DEX, showed an additive inhibitory effect on AHR, inflammation, and the number of activated MCs in the lungs and decreased the levels of interleukin (IL)-4, IL-5, IL-6, IL-13, tumor necrosis factor (TNF)-a, and C-C motif chemokine ligand (CCL)-2 in bronchoalveolar lavage fluid. A combination of DEX and ISO may be appropriate if a decrease in the steroid dose is desired owing to dose-dependent adverse effects.

Melatonin potentiates the antitumor effect of curcumin by inhibiting IKKß/NF-κB/COX-2 signaling pathway.

Curcumin, a natural polyphenolic compound, has commonly been used as a food additive or in many traditional medicine remedies for over 2,000 years in many Asian countries. Melatonin is a hormone secreted from pineal glands of mammals and possesses diverse physiological functions. Both curcumin and melatonin have the effective potential to inhibit proliferation of various types of cancers, but there is no report on their combination for bladder cancer treatment, and the underlying mechanism remains poorly understood. In the present study, we investigated whether the combination of curcumin and melatonin leads to an enhanced inhibition of cell proliferation in bladder cancer cells. Our results showed that the combinational treatment enhanced the repression of nuclear translocation of NF-κB and their binding on COX-2 promoter via inhibiting IKKß activity, resulting in inhibition of COX-2 expression. In addition, combined treatment with curcumin and melatonin induced cell apoptosis in bladder cancer through enhancing the release of cytochrome c from the mitochondrial intermembrane space into the cytosol. These results, therefore, indicated that melatonin synergized the inhibitory effect of curcumin against the growth of bladder cancer by enhancing the anti-proliferation, anti-migration, and pro-apoptotic activities, and provide strong evidence that combined treatment with curcumin and melatonin might exhibit an effective therapeutic option in bladder cancer therapy.

Expression, purification and identification of Pla a1 in a codon-optimized Platanus pollen allergen.

The present study aimed to express, purify and identify the major allergen gene, Pla a1, in Platanus pollen. According to previous studies, the major gene sequences of the Pla a1 allergen were obtained and codon optimization and synthesis of the genome were performed using DNAStar software. Following binding of the target gene fragment and the pET-44a vector, the JM109 cells were transfected to produce positive clones. The vectors were then transformed into Escherichia coli Rosetta cells to induce the expression of the target protein. The exogenous protein was purified using affinity chromatography and was identified by western blot analysis. Pla a1, the major allergen protein in Platanus pollen, was successfully isolated and this exogenous protein was purified using affinity chromatography. The present study was the first, to the best of our knowledge, to obtain expression of the allergen recombinant protein, Pla a1, fused with a Strep-TagII via codon optimization and provided the basis for the preparation of allergens with high purity, recombinant hypoallergenic allergens and allergen nucleic acid vaccines.

[Immunological characteristics of the recombinant major pollen allergen pTSX2 of Humulus scandens].

OBJECTIVE: To identify the immunological characteristics of the recombinant major pollen allergen pTSX2 of Humulus scandens and evaluate its safety in immunotherapy of allergic asthma in mice. METHODS: Western blotting was used to characterize the immunological properties of pTSX2, and its immunogenicity in normal mice was evaluated by detecting sIgG and sIgE levels. The mouse models of allergic asthma were immunized with pTSX2 and examined for sIgE and sIgG levels, total cells and eosinophils percentage in BALF, interleukin-4 (IL-4) and interferon-γ (IFN-γ) levels in BALF and spleen homogenate, and changes in lung pathologies. RESULTS: Western blotting showed that pTSX2 reacted with the majority (about 70%) of sera from patients allergic to Humulus pollen. In normal mice, pTSX2 mainly induced the production of sIgG. In mouse models of allergic asthma, intervention with pTSX2 caused a significant reduction of sIgE and an increase of sIgG (P<0.05), significantly decreased the total cells and eosinophils in BALF (P<0.05), obviously lowered IL-4 but increased IFN-γ in BALF and spleen homogenate (P<0.05), and diminished inflammatory cell infiltration and percentage of eosinophils in the lung tissues. CONCLUSIONS: pTSX2 shows a definite therapeutic effect and safety in the treatment of allergic asthma in mice possibly by inhibiting sIgE and inducing sIgG production, suppressing airway allergic inflammation and regulating the balance between Thl and Th2.

[Construction and identification of the expression library of album pollen allergens cDNA].

AIM: To construct and identify the express library of album pollen allergens cDNA. METHODS: Total RNA were extracted from the album pollen with TRIzol reagent and the mRNA was isolate for the amplify followed. A double stranded cDNA (ds cDNA) was synthesized using primers containing Xho I and Poly(dT) sequence by ZAP Express®cDNA synthesis kit. The ds cDNA was modified and purified by gel chromatography, and then the cDNA fragment with the length of more than 400 bp containing sticky ends was obtained. The cDNA fragment was ligated with Uni-ZAP XR vector and subsequently treated with in vitro packaging using phage by ZAP-cDNA express GigapackIII Gold cloning kit. The express library of album pollen cDNA was constructed by in vitro packaging. The recombination rate and the lengths of fragments inserted of the cDNA library were detected by polymerase chain reaction. RESULTS: The titer and the recombination rate of cDNA expression library constructed were 9.7×10(5) and 100%, respectively. The capacity of the library was 4.85 Pfu. The average length of cDNA fragments inserted was about 1.0 kb. CONCLUSION: Based on the capacity of cDNA expression library constructed and the length of cDNA insertion fragments, the cDNA expression library constructed is qualified to screening target cDNA clone, laying the foundation for preparation of gene recombinant allergen pollen vaccine.

[Screening specific allergens of allergic asthma from DNA expression library of Humulus pollen].

OBJECTIVE: To screen the allergic asthma-specific allergen from complementary DNA (cDNA) expression library of Humulus pollen. METHODS: Screened the Humulus pollen lambdaTripIEx2 cDNA expression library which had been constructed using the method of immunological screening with patients' blood serum of Humulus pollen allergic asthma, extracted the positive phage DNA and the positive phage DNA, and identified by double restriction enzyme with EcoRI, Hind III and agarose gel electrophoresis. Then the DNA fragments were sequenced and the repeats and sequence homology were analyzed. RESULTS: After 3 rounds of screening, three specific allergic cDNA clone of Humulus pollen were obtained and the sequences were 868, 550 and 592 base pairs respectively. The results of sequencing showed that these three clones represented different cDNA sequences. The repeats analysis found no repeats and these clones had no high homology with any known gene. CONCLUSIONS: Three specific allergic cDNA clones of Humulus pollen obtained may be new genes. It provides a rational basis for constructing a recombinant allergen or nuclear acid vaccine.

[Purification and analysis of the immunoactivity of humulus pollen allergen].

OBJECTIVE: To purify the humulus pollen allergen and study the allergenicity and immunogenicity of it. METHODS: Crude humulus pollen extracts were purified by gel filtration with Sephadex G-75 and Sephacryl S-200HR. Various fractions of the allergen protein were collected respectively. The molecular weights of protein were confirmed by SDS-PAGE. The inhibition rate and reaction rate with sIgG and sIgE of patient's serum were determined by ELISA inhibition test and western blotting. RESULTS: Two peaks were obtained from crude humulus pollen extracts by gel filtration of Sephadex G-75. The first peak contained most of protein while the second peak contained little protein and lots of pigments. So the second peak was thrown away. P solution which contained the first peak and valley fraction were purified by gel filtration of Sephacryl S-200HR and four components that included the 1st peak, the valley, the 2nd peak and the end fraction were obtained. The results of electrophoresis demonstrated that purified humulus pollen contained more than 20 kinds of protein with the molecular weights ranged from 5.0 x 10(3) to 97.4 x 10(3). The fraction of the 1st peak contained protein with the molecular weights ranged from 43 x 10(3) to 97.4 x 10(3), and the fraction of the valley and the 2nd peak contained protein with the molecular weights ranged from 5.0 x 10(3) to 43 x 10(3). The fraction of the end contained protein with the molecular weights lower than 5.0 x 10(3). The results of ELISA inhibition test showed that the inhibition rate of the 1st peak, the valley, the 2nd peak and the end fraction to sIgG were 68%, 70%, 95%, 5% respectively, and those to sIgE were 25%, 64%, 71%, 11% respectively. The results of western blotting demonstrated that the reaction rate of the 1st peak, the valley, the 2nd peak and the end fraction with sIgG of patients' serum were 65.63%, 78.13%, 87.50%, 6.25% respectively, and those with patients' sIgE were 25.00%, 71. 88%, 84.38%, 15.63% respectively. CONCLUSION: Humulus pollen contained more than 20 kinds of protein. The proteins with molecular weights ranged from 5.0 x 10(3) to 43 x 10(3) were the major allergen with strong allergenicity and immunogenicity. The proteins with molecular weights ranged from 43 x 10(3) to 97.4 x 10(3) were subordinated allergen with strong immunogenicity and weak allergenicity.