Hematopoietic Protein Tyrosine Phosphatase Mediates β2-Adrenergic Receptor-Induced Regulation of p38 Mitogen-Activated Protein Kinase in B Lymphocytes

Stimulation of the β₂-adrenergic receptor (β₂AR) on a CD40L/interleukin-4-activated B lymphocyte increases the level of immunoglobulin E (IgE) in a protein kinase A (PKA)- and p38 mitogen-activated protein kinase (MAPK)-dependent manner. However, the mechanism by which β₂AR stimulation mediates the increase in the level of p38 MAPK activation has remained unclear. Here we show that the β₂AR-induced increase in p38 MAPK activation occurred via a hematopoietic protein tyrosine phosphatase (HePTP)-mediated cross talk between PKA and p38 MAPK. β₂AR agonists, cAMP-elevating agents, and PKA inhibitors were used to show that β₂AR stimulation resulted in a PKA-dependent increase in p38 MAPK phosphorylation. Pharmacological agents and gene-deficient mice revealed that p38 MAPK phosphorylation was regulated by the G-stimulatory (Gs)/cAMP/PKA pathway independently of the G-inhibitory or β-arrestin-2 pathways. Coimmunoprecipitation and Western blot analysis showed that HePTP was phosphorylated in a PKA-dependent manner, which inactivated HePTP and allowed for increased free p38 MAPK to be phosphorylated by the MAPK cascade that was activated by CD40L. HePTP short hairpin RNA confirmed that HePTP played a role in regulating the level of p38 MAPK phosphorylation in a B cell. Thus, β₂AR stimulation on a B cell phosphorylates and inactivates HePTP in a Gs/cAMP/PKA-dependent manner to release bound p38 MAPK, making more available for phosphorylation and subsequent IgE regulation.     

The (2′S,7′S)-O-(2-methylbutanoyl)-columbianetin as a novel allergic rhinitis-control agent

Aims

The (2′S,7′S)-O-(2-methylbutanoyl)-columbianetin (OMC) is a novel secondary metabolite extracted from Corydalis heterocarpa, which has long been used as a folk medicine for various inflammatory diseases in Korea. We examined the effect of OMC on allergic rhinitis (AR).

Main methods

We assessed the therapeutic effects and regulatory mechanisms of OMC on the phorbol 12-myristate 13-acetate plus A23187-stimulated mast cell line, HMC-1 cells and ovalbumin (OVA)-induced AR models.

Key findings

OMC significantly decreased the releases of histamine and tryptase from stimulated HMC-1 cells. The degranulation process, characterized by morphological extension of the filopodia on the surface and membrane ruffling, was strongly induced in the stimulated-HMC-1 cell, however OMC suppressed the morphological changes in stimulated-HMC-1 cells. OMC reduced the production and mRNA expression of inflammatory cytokines. These inhibitory actions by OMC were dependent on the regulation of mitogen-activated protein kinases, nuclear factor-κB, and caspapase-1 signaling pathways. In the AR animal model, the increased rub scores and AR biomarkers (histamine and IgE) in ovalbumin (OVA)-sensitized mice were significantly reduced by the administration of OMC. Furthermore, eosinophils and mast cell infiltrations in nasal mucosa tissue were also blocked through the regulation of macrophage-inflammatory protein and intercellular adhesion molecule-1 levels.

Significance

OMC showed the possibility to regulate AR in activated mast cells and OVA-induced AR models. Hence, we suggest that OMC is a powerful and feasible new agent to suppress AR.     

Flt-3 ligand reverses late allergic response and airway hyper-responsiveness in a mouse model of allergic inflammation

Flt3 ligand (Flt3-L) is a growth factor for dendritic cells and induces type 1 T cell responses. We recently reported that Flt3-L prevented OVA-induced allergic airway inflammation and suppressed late allergic response and airway hyper-responsiveness (AHR). In the present study we examined whether Flt3-L reversed allergic airway inflammation in an established model of asthma. BALB/c mice were sensitized and challenged with OVA, and AHR to methacholine was established. Then mice with AHR were randomized and treated with PBS or 6 microg of Flt3-L i.p. for 10 days. Pulmonary functions and AHR to methacholine were examined after rechallenge with OVA. Treatment with Flt3-L of presensitized mice significantly suppressed (p < 0.001) the late allergic response, AHR, bronchoalveolar lavage fluid total cellularity, absolute eosinophil counts, and inflammation in the lung tissue. There was a significant decrease in proinflammatory cytokines (TNF-alpha, IL-4, and IL-5) in bronchoalveolar lavage fluid, with a significant increase in serum IL-12 and a decrease in serum IL-5 levels. There was no significant effect of Flt3-L treatment on serum IL-4 and serum total IgE levels. Sensitization with OVA significantly increased CD11b(+)CD11c(+) cells in the lung, and this phenomenon was not significantly affected by Flt3-L treatment. These data suggest that Flt3-L can reverse allergic airway inflammation and associated changes in pulmonary functions in murine asthma model.     

APOE2 promotes the development and progression of subretinal neovascularization in age-related macular degeneration via MAPKs signaling pathway

BackgroundNeovascular age-related macular degeneration (nvAMD) is one of the main pathological features of wet AMD. Apolipoprotein E2 is involved in the formation of nvAMD but the molecular mechanism has not been reported.MethodsThe APOE alleles in AMD patients were detected by genotyping. Mouse models were divided into 4 groups according to transfection different gene segments and laser-induced treatment. APOE2, VEGF, PDGF-BB, b-FGF and inflammatory cytokines (including p-NF-κB, TNF-α, IL-1β and IL-6) were tested by ELISA in mice retinal lysate. The formation of nvAMD in the indicated treatment groups at 3rd, 7th and 14th day after laser-induced damage were detected by FFA. Besides, qRT-PCR was used to determine the mRNA levels of p38, JNK and ERK in ARPE-19 cells. Finally, the inflammatory cytokines and MAPK proteins (including P38, p-P38, JNK, p-JNK, ERK and p-ERK) were detected by western blot.ResultsThe statistics of APOE alleles showed that APOE2 allele carriers were more likely to nvAMD. VEGF, PDGF-BB, b-FGF and related inflammatory cytokines were up-regulated significantly after treatment with APOE2, which were reduced after silencing the MAPK family genes, however. Further, the expression levels of neovascular growth factors and inflammatory cytokines were highly consistent between mouse models and ARPE-19 cells. Besides, the phosphorylation levels of p38, JNK and ERK were affected by APOE2.ConclusionnvAMD was affected directly by the overexpression of VEGF, PDGF-BB and b-FGF, which were regulated by APOE2 through activating MAPKs pathway.     

Ethyl pyruvate attenuates murine allergic rhinitis partly by decreasing high mobility group box 1 release

High-mobility group box 1 (HMGB1) protein, a pro-inflammatory DNA-binding protein, meditates inflammatory responses through Toll-like receptor-4 signals and amplifies allergic inflammation by interacting with the receptor for advanced glycation end products. Previous studies have shown that HMGB1 is elevated in the nasal lavage fluids (NLF) of children suffering from allergic rhinitis (AR) and is associated with the severity of this disease. Furthermore, HMGB1 has been implicated in the pathogenesis of lower airway allergic diseases, such as asthma. Ethyl pyruvate (EP) has proven to be an effective anti-inflammatory agent for numerous airway diseases. Moreover, EP can inhibit the secretion of HMGB1. However, few studies have examined the effect of EP on AR. We hypothesized that HMGB1 plays an important role in the pathogenesis of AR and studied it using an AR mouse model. Forty BALB/c mice were divided into four groups: the control group, AR group, 50 mg/kg EP group, and 100 mg/kg EP group. The mice in the AR and EP administration groups received ovalbumin (OVA) sensitization and challenge, whereas those in the control group were given sterile saline instead of OVA. The mice in the EP administration group were given an intraperitoneal injection of EP 30 min before each OVA treatment. The number of nasal rubbings and sneezes of each mouse was counted after final treatment. Hematoxylin–eosin staining, AB-PAS staining, interleukin-4 and 13 in NLF, IgE, and the protein expression of HMGB1 were measured. Various features of the allergic inflammation after OVA exposure, including airway eosinophilia, Th-2 cytokine production, total IgE, and goblet cell hyperplasia were significantly inhibited by treatment with EP and the expression and release of HMGB1 were reduced after EP administration in a dose-dependent manner. These results indicate that HMGB1 is a potential therapeutic target of AR and that EP attenuates AR by decreasing HMGB1 expression.     

Mechanisms of damage to sperm structure in mice on the zinc-deficient diet

BackgroundZinc (Zn)is an essential trace element for spermatogenesis and its deficiency causes abnormal spermatogenesis.ObjectiveThe present study was conducted to examine the mechanisms by which Zn-deficient diet impairs sperm morphology and its reversibility.Methods30 SPF grade male Kunming (KM) mice were randomly divided into three groups, 10 mice per group. Zn-normal diet group (ZN group) was given Zn-normal diet（Zn content= 30 mg/kg）for 8 weeks. Zn-deficienct diet group (ZD group) was given Zn-deficienct diet（Zn content< 1 mg/kg）for 8 weeks. Zn-deficient and Zn-normal diet group（ZDN group）was given 4 weeks Zn-deficienct diet followed by 4 weeks Zn-normal diet. After 8 weeks, the overnight fasted mice were sacrificed, and blood and organs were collected for further analysis.ResultsThe experimental results showed that Zn-deficienct diet leads to increased abnormal morphology sperm and testicular oxidative stress.The rate of abnormal morphology sperm, chromomycin A3(CMA3), DNA fragmentation index (DFI), malondialdehyde (MDA) were significantly increased, and a-kinase anchor protein 4(AKAP4), dynein axonemal heavy chain 1(DNAH1), sperm associated antigen 6(SPAG6), cilia and flagella associated protein 44(CFAP44), glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), nuclear factor erythroid 2-related factor (NRF2), NAD(P)H:quinone oxidoreductase 1(NQO1)and heme oxygenase 1(HO1) were significantly decreased in the ZD group mice. While the changes in above indicators caused by Zn-deficient diet were significantly alleviated in the ZDN group.ConclusionIt was concluded that Zn-deficient diet causes abnormal morphology sperm and testicular oxidative stress in male mice. Abnormal morphology sperm caused by Zn-deficient diet are reversible, and Zn-normal diet can alleviate them.     

Mesenchymal stem cells combined with traditional Chinese medicine (qi-fang-bi-min-tang) alleviates rodent allergic rhinitis

Mesenchymal stem cells (MSCs) have been proved to exert anti-inflammatory effects and regulate immune reactions. Traditional Chinese medicine (TCM), qi-fang-bi-min-tang, is effective for some patients with allergic diseases. However, it remains unclear whether MSCs combined with TCM could benefit the treatment of allergic rhinitis (AR). In this study, we reported an additional effect of TCM (qi-fang-bi-min-tang) on the therapy of AR under MSCs treatment. Intriguingly, we observed that TCM-treated MSCs significantly inhibited the symptoms of AR and reduced the pathological changes of nasal mucosa in ovalbumin (OVA)-induced rats. The expression levels of interferon γ (IFN-γ), interleukin-17 (IL-17), and IL-4 were significantly decreased in the plasma of AR rats after injection of TCM-treated MSCs. TCM-treated MSCs reduced the levels of histamine secreted by mast cells and immunoglobulin E (IgE) secreted by plasma cells. In addition, we found that MSCs combined with TCM had a better therapeutic effect than TCM alone on AR in an OVA-induced mouse model. After OVA induction, MSCs combined with TCM significantly reduced the ratio of T helper type 1 (Th1), Th2, and Th17, but increased the proportion of Treg in the spleen of mice. Consistently, the expression levels of IFN-γ, IL-4, and IL-17 were significantly decreased, but transforming growth factor-β1 was significantly increased in the plasma of AR mice after treated with TCM and MSCs. Our results from both rats and mice indicated that the effects of TCM combined with MSCs on the AR might be through regulating the secretion of Th1, Th2, and Th17 cytokines. This study suggested that TCM (qi-fang-bi-min-tang)-treated MSCs could be used in the clinical therapy of AR.     

Dietary zinc supplementation attenuates hyperglycemia in db/db mice

Although zinc (Zn) deficiency has been associated with insulin resistance, and altered Zn metabolism (e.g., hyperzincuria, low-normal plasma Zn concentrations) may be present in diabetes, the potential effects of Zn on modulation of insulin action in Type II diabetes have not been established. The objective of this study was to compare the effects of dietary Zn deficiency and Zn supplementation on glycemic control in db/db mice. Weanling db/db mice and lean littermate controls were fed Zn-deficient (3 ppm Zn; dbZD and InZD groups), Zn-adequate control (30 ppm Zn; dbC and InC groups) or Zn-supplemented (300 ppm Zn; dbZS and InZS groups) diets for 6 weeks. Mice were assessed for Zn status, serum and urinary indices of diabetes, and gastrocnemius insulin receptor concentration and tyrosine kinase activity. Fasting serum glucose concentrations were significantly lower in the dbZS group compared with the dbZD group (19.3 +/- 2.9 and 27.9 +/- 4.1 mM, respectively), whereas the dbC mice had an intermediate value. There was a negative correlation between femur Zn and serum glucose concentrations (r = -0.59 for lean mice, P = 0.007). The dbZS group had higher pancreatic Zn and lower circulating insulin concentrations than dbZC mice. Insulin-stimulated tyrosine kinase activity in gastrocnemius muscle was higher in the db/db genotype, and insulin receptor concentration was not altered. In summary, dietary Zn supplementation attenuated hyperglycemia and hyperinsulinemia in db/db mice, suggesting that the roles of Zn in pancreatic function and peripheral tissue glucose uptake need to be further investigated.     

Leuconostoc citreum HJ-P4 (KACC 91035) regulates immunoglobulin E in an ovalbumin-induced allergy model and induces interleukin-12 through nuclear factor-kappa B and p38/c-Jun N-terminal kinases signaling in macrophages

Leuconostoc citreum ( L. citreum ) HJ-P4 (KACC 91035) is one of the major predominant species in kimchi fermentation in Korea. The purpose of the present study was to test the immunomodulatory capacity of L. citreum to modulate the IgE-mediated allergic response and to examine the involvement of NF-κB and MAPK in IL-12 production in macrophages. Balb/c mice were sensitized with OVA/alum and oral administration of L. citreum to the mice began before or after the OVA sensitization. Protein and mRNA expression of Th1 cytokines in splenocytes by L. citreum in vitro was measured. The role of NF-κB and MAPK such as p38, ERK1/2 and JNK in L. citreum -induced IL-12 was investigated in peritoneal macrophages and RAW264.7 cell lines. L. citreum inhibited the serum levels of total IgE, IgG1 and IgG2a altogether and increased OVA-specific IFN-γ production in splenocytes from pre- and post-sensitized animals. However, the downregulation of IL-4 and IL-5 production was observed only in the pre-sensitization group. The ability of L. citreum to stimulate IFN-γ was dependent on its induction of IL-12. NF-κB, p38 and JNK were mainly involved in L. citreum -induced IL-12 production. In conclusion, the current study demonstrated that L. citreum is able to regulate serum IgE generation at the induction and effector phases of allergic response through overall control over antibody production and that its involvement of IL-12 production was mediated through NF-κB and p38/JNK. Taken together, the use of L. citreum can be useful in preventing the development and progression of IgE production.     

Zinc deficiency induces enhanced depression-like behaviour and altered limbic activation reversed by antidepressant treatment in mice

A relationship between zinc (Zn)-deficiency and mood disorders has been suspected. Here we examined for the first time whether experimentally-induced Zn-deficiency in mice would alter depression- and anxiety-related behaviour assessed in established tests and whether these alterations would be sensitive to antidepressant treatment. Mice receiving a Zn-deficient diet (40% of daily requirement) had similar homecage and open field activity compared to normally fed mice, but displayed enhanced depression-like behaviour in both the forced swim and tail suspension tests which was reversed by chronic desipramine treatment. An anxiogenic effect of Zn-deficiency prevented by chronic desipramine and Hypericum perforatum treatment was observed in the novelty suppressed feeding test, but not in other anxiety tests performed. Zn-deficient mice showed exaggerated stress-evoked immediate-early gene expression in the amygdala which was normalised following DMI treatment. Taken together these data support the link between low Zn levels and depression-like behaviour and suggest experimentally-induced Zn deficiency as a putative model of depression in mice.     

Bulleyaconitine A inhibits the lung inflammation and airway remodeling through restoring Th1/Th2 balance in asthmatic model mice

ABSTRACT

The current study aimed to study the effects of Bulleyaconitine A (BLA) on asthma. Asthmatic mice model was established by ovalbumin (OVA) stimulation, and the model mice were treated by BLA. After BLA treatment, the changes in lung and airway resistances, total and differential leukocytes in the bronchoalveolar lavage fluid (BALF) were detected, and the changes in lung inflammation and airway remodeling were observed. Moreover, the secretion of IgE, Th1/Th2-type and IL-17A cytokines in BALF and serum of the asthmatic mice were determined. The resuts showed that BLA attenuated OVA-induced lung and airway resistances, inhibited the inflammatory cell recruitment in BALF and the inflammation and airway remodeling of the asthmatic mice. In addition, BLA suppressed the secretion of IgE, Th2-type cytokines, and IL-17A, but enhanced secretions of Th1-type cytokines in BALF and serum. The current study discovered that BLA inhibited the lung inflammation and airway remodeling via restoring the Th1/Th2 balance in asthmatic mice.     

Anti-inflammatory effects of mitogen-activated protein kinase kinase inhibitor U0126 in an asthma mouse model

Mitogen-activated protein kinase (MAPK) signaling cascade plays a pivotal role in the activation of inflammatory cells. Recent findings revealed that the activity of p42/44 MAPK (also known as extracellular signal-regulated kinase (ERK)) in the lungs was significantly higher in asthmatic mice than in normal controls. We hypothesized that inhibition of ERK activity may have anti-inflammatory effects in allergic asthma. BALB/c mice were sensitized with OVA and, upon OVA aerosol challenge, developed airway eosinophilia, mucus hypersecretion, elevation in cytokine and chemokine levels, up-regulation of VCAM-1 expression, and airway hyperresponsiveness. Intraperitoneal administration of U0126, a specific MAPK/ERK kinase inhibitor, significantly (p < 0.05) inhibited OVA-induced increases in total cell counts, eosinophil counts, and IL-4, IL-5, IL-13, and eotaxin levels recovered in bronchoalveolar lavage fluid in a dose-dependent manner. U0126 also substantially (p < 0.05) reduced the serum levels of total IgE and OVA-specific IgE and IgG1. Histological studies show that U0126 dramatically inhibited OVA-induced lung tissue eosinophilia, airway mucus production, and expression of VCAM-1 in lung tissues. In addition, U0126 significantly (p < 0.05) suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine in a dose-dependent manner. Western blot analysis of whole lung lysates shows that U0126 markedly attenuated OVA-induced tyrosine phosphorylation of ERK1/2. Taken together, our findings implicate that inhibition of ERK signaling pathway may have therapeutic potential for the treatment of allergic airway inflammation.     

Intratracheal administration of mitochondrial DNA directly provokes lung inflammation through the TLR9–p38 MAPK pathway

An increasing number of studies have focused on the phenomenon that mitochondrial DNA (mtDNA) activates innate immunity responses. However, the specific role of mtDNA in inflammatory lung disease remains elusive. This study was designed to examine the proinflammatory effects of mtDNA in lungs and to investigate the putative mechanisms. C57BL/6 mice were challenged intratracheally with mtDNA with or without pretreatment with chloroquine. Changes in pulmonary histopathology, cytokine concentrations, and phosphorylation levels of p38 MAPK were assayed at four time points. In in vitro experiments, THP-1 macrophages were pretreated or not pretreated with chloroquine, TLR9 siRNA, p38 MAPK siRNA, or SB203580 and then incubated with mtDNA. The levels of cytokines and p-p38 MAPK were detected by ELISA and Western blot, respectively. The intratracheal administration of mtDNA induced infiltration of inflammatory cells, production of proinflammatory cytokines (including IL-1β, IL-6, and TNF-α), and activation of p38 MAPK. The chloroquine pretreatment resulted in an abatement of mtDNA-induced local lung inflammation. In vitro experiments showed that the exposure of THP-1 macrophages to mtDNA also led to a significant upregulation of IL-1β, IL-6, and TNF-α and the activation of p38 MAPK. And these responses were inhibited either by chloroquine and TLR9 siRNA or by SB203580 and p38 MAPK siRNA pretreatment. The intratracheal administration of mtDNA induced a local inflammatory response in the mouse lung that depended on the interactions of mtDNA with TLR9 and may be correlated with infiltrating macrophages that could be activated by mtDNA exposure via the TLR9–p38 MAPK signal transduction pathway.     

Involvements of p38 MAPK and oxidative stress in the ozone-induced enhancement of AHR and pulmonary inflammation in an allergic asthma model

Background

Exposure to ambient ozone (O₃) increases the susceptivity to allergens and triggers exacerbations in patients with asthma. However, the detailed mechanisms of action for O₃ to trigger asthma exacerbations are still unclear.

Methods

An ovalbumin (OVA)-established asthmatic mouse model was selected to expose to filtered air (OVA-model) or 1.0 ppm O₃ (OVA-O₃ model) during the process of OVA challenge. Next, the possible involvements of p38 MAPK and oxidative stress in the ozone actions on the asthma exacerbations were investigated on the mice of OVA-O₃ model by treating them with SB239063 (a p38 MAPK inhibitor), and/or the α-tocopherol (antioxidant). Biological measurements were conducted including airway hyperresponsiveness (AHR), airway resistance (Raw), lung compliance (CL), inflammation in the airway lumen and lung parenchyma, the phosphorylation of p38 MAPK and heat shock protein (HSP) 27 in the tracheal tissues, and the malondialdehyde (MDA) content and the glutathione peroxidase (GSH-Px) activity in lung tissues.

Results

In OVA-allergic mice, O3 exposure deteriorated airway hyperresponsiveness (AHR), airway resistance (Raw), lung compliance (CL) and pulmonary inflammation, accompanied by the increased oxidative stress in lung tissues and promoted p38 MAPK and HSP27 phosphorylation in tracheal tissues. Administration of SB239063 (a p38 MAPK inhibitor) on OVA-O3 model exclusively mitigated the Raw, the CL, and the BAL IL-13 content, while α-tocopherol (antioxidant) differentially reduced the BAL number of eosinophils and macrophages, the content of BAL hyaluronan, the peribronchial inflammation, as well as the mRNA expression of TNF-α and IL-5 in the lung tissues of OVA-O3 model. Administration of these two chemical inhibitors similarly inhibited the AHR, the BAL IFN-γ and IL-6 production, the perivascular lung inflammation and the lung IL-17 mRNA expression of OVA-O3 model. Interestingly, the combined treatment of both compounds together synergistically inhibited neutrophil counts in the BALF and CXCL-1 gene expression in the lung.

Conclusions

O₃ exposure during the OVA challenge process promoted exacerbation in asthma. Both p38 MAPK and oxidative stress were found to play a critical role in this process and simultaneous inhibition of these two pathways significantly reduced the O₃-elicited detrimental effects on the asthma exacerbation.

     

β3-adrenergic receptor induction of adipocyte inflammation requires lipolytic activation of stress kinases p38 and JNK

Activation of β-adrenergic receptors (AR) in adipocytes triggers acute changes in metabolism that can alter patterns of gene expression. This work examined the mechanisms by which activation of hormone sensitive lipase (HSL) induces expression of inflammatory cytokines in adipocytes in vivo and model adipocytes in vitro. β3-AR activation in mice triggered expression of inflammatory genes CCL2, IL-6, and PAI-1, as well as endoplasmic reticulum (ER) stress markers GRP78 and CHOP. Pharmacological inhibition of HSL blocked induction of inflammatory genes, but not ER stress markers. Promoting intracellular accumulation of free fatty acids (FFAs) in 3T3-L1 adipocytes increased expression of inflammatory cytokines, whereas inhibiting ceramide synthesis partly blocked PAI-1 expression, but not IL-6. Induction of inflammatory markers in vivo and in vitro was preceded by phosphorylation of p38 and JNK, and inhibition of HSL prevented activation of these kinases. Experiments with pharmacological inhibitors of specific MAP kinases demonstrated the importance of p38 MAPK as a mediator of lipolysis-induced inflammation in vivo and in vitro. Together, these results demonstrate that FFAs liberated by HSL activate p38 and JNK, and p38 mediates pro-inflammatory cytokine expression in adipose tissue.     

Higenamine alleviates allergic rhinitis by activating AKT1 and suppressing the EGFR/JAK2/c-JUN signaling

BackgroundAllergic rhinitis (AR) is an inflammatory, immunoglobulin E (IgE)-mediated disease characterized by the typical symptoms of sneezing, rhinorrhea, nasal itching, and congestion. Higenamine (HG) is a plant-based alkaloid, possesses a wide range of activities, including vascular and tracheal relaxation, antioxidative, antiapoptotic, anti-inflammatory, and immunomodulatory activities. So far, the effect and the underlying mechanism of HG on AR have not been studied.Hypothesis/PurposeThe purpose of this study was to evaluate the effects of HG on AR and investigate its underlying mechanism.MethodsThe effects of HG on AR were evaluated in an ovalbumin-induced AR mouse model. Network pharmacology-based methods such as target prediction, protein-protein interaction (PPI) network analysis, pathway analysis, and molecular docking were used to identify the likely HG targets. Finally, we validated the mechanism of action of HG through its effects on these targets in human nasal epithelial cells (HNEpCs).ResultsOral administration of 30, 60, and 120 mg/kg HG significantly alleviated rubbing and sneezing in AR mice and attenuated histopathological changes in the lung and nasal tissues. Additionally, HG reduced the levels of IgE, histamine, and IL-4 in the serum of AR mice, and regulated imbalance in Th1/Th2 cells. Using network pharmacology-based methods, we identified 29 HG targets related to AR. These targets are mainly involved in the PD-L1, relaxin, estrogen, HIF-1, Th1 and Th2 cell differentiation, T cell receptor, and the Th17 cell differentiation signaling pathways. Molecular docking showed that HG may well be suited to the receptor binding pockets of key target AKT1, EGFR, c-Jun, NOS2, and JAK2. In HNEpCs, HG inhibited the histamine-induced mRNA expression and secretion of interleukin (IL)-6, and IL-8, as well as the expression of MUC5AC and the phosphorylation of NF-κB. Moreover, HG affected the changes of AKT1, EGFR, c-Jun, iNOS, and JAK2 induced by histamine.ConclusionOverall, our results suggest that HG may alleviate AR by activating AKT1 and suppressing the EGFR/JAK2/c-JUN signaling. HG, therefore, has great potential as a therapeutic agent for the treatment of AR.     

Influences of PON1 on airway inflammation and remodeling in bronchial asthma

This study aims to explore the influences of Paraoxonase‐1 (PON1) involved in airway inflammation and remodeling in asthma. Mice were divided into control, asthma, asthma + PON1 and asthma + NC groups, and asthma models were established via aerosol inhalation of ovalbumin (OVA). HE, Masson, and PAS stains were used to observe airway inflammation and remodeling, Giemsa staining to assess inflammatory cells in bronchoalveolar lavage fluid (BALF), qRT‐PCR and Western blot to detect PON1 expression, lipid peroxidation and glutathione assays to quantify malondialdehyde (MDA) activity and glutathione peroxidase (GSH) levels, ELISA to determine inflammatory cytokines and immunoglobulin, and colorimetry to detect PON1 activities. Additionally, mice lung macrophages and fibroblasts were transfected with PON1 plasmid in vitro; ELISA and qRT‐PCR were performed to understand the effects of PON1 on inflammatory cytokines secreted by lung macrophages, MTT assay for lung fibroblasts proliferation and qRT‐PCR and Western blot for the expressions of PON1, COL1A1, and fibronectin. After overexpression of PON1, the asthma mice had decreased inflammatory cell infiltration, fibrosis degree, and airway wall thickness; inflammatory cells and inflammatory cytokines in BALF were also reduced, expressions of OVA‐IgE and IgG1, and MDA activity were decreased, but the expressions of OVA‐IgG2a and INF‐γ and GSH levels were increased. Besides, PON1 significantly inhibited microphage expression of LPS‐induced inflammatory cytokines, lung fibroblast proliferation, and COL1A1 and fibronectin expression. Thus, PON1 could relieve airway inflammation and airway remodeling in asthmatic mice and inhibit the secretion of LPS‐induced macrophage inflammatory cytokines and the proliferation of lung fibroblasts.     

The PDZ-Binding Motif of Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Is a Determinant of Viral Pathogenesis

A recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) lacking the envelope (E) protein is attenuated in vivo. Here we report that E protein PDZ-binding motif (PBM), a domain involved in protein-protein interactions, is a major determinant of virulence. Elimination of SARS-CoV E protein PBM by using reverse genetics caused a reduction in the deleterious exacerbation of the immune response triggered during infection with the parental virus and virus attenuation. Cellular protein syntenin was identified to bind the E protein PBM during SARS-CoV infection by using three complementary strategies, yeast two-hybrid, reciprocal coimmunoprecipitation and confocal microscopy assays. Syntenin redistributed from the nucleus to the cell cytoplasm during infection with viruses containing the E protein PBM, activating p38 MAPK and leading to the overexpression of inflammatory cytokines. Silencing of syntenin using siRNAs led to a decrease in p38 MAPK activation in SARS-CoV infected cells, further reinforcing their functional relationship. Active p38 MAPK was reduced in lungs of mice infected with SARS-CoVs lacking E protein PBM as compared with the parental virus, leading to a decreased expression of inflammatory cytokines and to virus attenuation. Interestingly, administration of a p38 MAPK inhibitor led to an increase in mice survival after infection with SARS-CoV, confirming the relevance of this pathway in SARS-CoV virulence. Therefore, the E protein PBM is a virulence domain that activates immunopathology most likely by using syntenin as a mediator of p38 MAPK induced inflammation.