NLRP3 inflammasome sequential changes in Staphylococcus aureus-induced mouse model of acute rhinosinusitis.

The NLR pyrin domain containing 3 (NLRP3) inflammasome plays a crucial role in lung disease and may have a similar role in upper respiratory tract inflammation. We therefore constructed a C57BL/6 mouse model of acute rhinosinusitis induced by Staphylococcus aureus and investigated the role of the NLRP3 inflammasome in this model. Mice were classified as non-inoculated group (group A) and inoculated groups (groups B, C, D and E, sacrificed 1, 3, 7 and 14 days after inoculation, respectively). Hematoxylin-eosin staining showed that each group had inflammatory cell infiltration, except group A. The damage of the nasal mucosa was aggravated gradually over time. Western blot and immunofluorescence showed that the structural proteins of the NLRP3 inflammasome (NLRP3, ASC (apoptosis-associated speck-like protein containing CARD), procaspase-1) in groups B, C, D and E were increased gradually. But they were reduced in group B compared with group A, except for NLRP3. Western blot showed that the cleavage fragment of procaspase-1, p20 in groups B, C, D and E was increased gradually. Real-time PCR showed that the corresponding mRNAs of the structural proteins were changed the same as their proteins. IL-1ß mRNA and mature IL-1ß protein were increased gradually in groups A, B, C, D and E. These results indicate that NLRP3 inflammasome activation was associated with the acute rhinosinusitis, and that there was a positive correlation between the expression level of the NLRP3 inflammasome and the severity of acute rhinosinusitis.

Effects of IL-17 on expression of GRO-α and IL-8 in fibroblasts from nasal polyps.

Recent studies indicated that interleukin (IL)-17, growth-related oncogene (GRO)-α and IL-8 play an important role in the pathogenesis of nasal polyps. However, the effects of the increased amount of IL-17 and the production of GRO-α and IL-8 in human nasal polyp fibroblasts are not completely understood. This study aimed to determine the effects of the increased IL-17 on the changes of GRO-α and IL-8 expression in human nasal polyp fibroblasts and further investigate the mechanism of neutrophil infiltration in nasal polyps. Nasal polyp fibroblasts were isolated from six cases of human nasal polyps, and the cells were stimulated with five different concentrations of IL-17. Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of GRO-α and IL-8. The mRNA of GRO-α and IL-8 was expressed in unstimulated controls and remarkably increased by stimulation with IL-17. Moreover, the levels of GRO-α and IL-8 produced by fibroblasts were increased gradually with the increases in IL-17 concentrations. The present study showed that nasal fibroblasts can produce GRO-α and IL-8, and their production is remarkably enhanced by IL-17 stimulation, thereby clarifying the mechanism of the IL-17 mediated neutrophil infiltration in nasal polyps. These findings might provide a rationale for using IL-17 inhibitors as a treatment for nasal inflammatory diseases such as nasal polyps.

Design, synthesis and antithrombotic evaluation of novel dabigatran prodrugs containing methyl ferulate.

A novel series of prodrugs containing dabigatran and methyl (E)-3-(4-hydroxy-2-methoxyphenyl)propenoate (methyl ferulate) were synthesized. All of them reveal the effect of thrombin-induced anti-platelet aggregation in vitro. In addition, in vivo experiment shows that one of the target compounds, X-2 (ED50=3.7 ± 1.0 µmol/kg) possesses a more potent activity for inhibiting venous thrombosis than that of dabigatran etexilate (ED50=7.8 ± 1.5 µmol/kg).

Dropouts From Sublingual Immunotherapy and the Transition to Subcutaneous Immunotherapy in House Dust Mite-Sensitized Allergic Rhinitis Patients.

Purpose: Both subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT) are effective in reducing symptoms and medication scores and inducing long-term efficacy in patients with allergic rhinitis (AR). However, SLIT has been associated with poor patient adherence. This study investigates the factors impacting dropout rates from SLIT in house dust mite (HDM)-sensitized AR patients. Methods: A retrospective study was performed to analyze dropout rates and reasons in AR patients receiving Dermatophagoides farinae (Der f) SLIT with a follow-up period of 2 years. Results: A total of 719 HDM-sensitized AR patients received Der f-SLIT. Dropout rates increased with time and most occurred after 1 year of SLIT. By month 24, 654 (91%) patients had discontinued SLIT. The dropout rates by month 24 were 100, 90.1, and 91.1% in children <5 years old, children aged 5-18 years old, and adults ≥ 18 years old, respectively. Combination with allergic asthma and mono- or multi-sensitization to other aeroallergens did not affect the dropout rates. The most common self-reported reasons for dropouts were refusal of continuation, dissatisfaction with the efficacy, transition to SCIT, and adverse effects. Refusal of continuation increased with age, whereas transition to SCIT decreased with age. Ninety-seven cases transitioned from SLIT to SCIT, and the transition rates increased with time. Comorbid allergic asthma did not affect the transition rates. However, multi-sensitization was associated with a slightly higher rate of transition to SCIT. The most common reason for the transition was dissatisfaction with the efficacy (54.6%), which was only reported by patients older than 5 years. For children who began SLIT at younger than 5 years old, the most common reason (81.2%) for transition was age reaching 5 years. Conclusions: HDM-SLIT has a very high dropout rate, which is mainly due to refusal of continuation and dissatisfaction with the efficacy. Transitioning from SLIT to SCIT may help keep these patients on AIT and thus increase adherence and long-term efficacy.

Dual inhibition of HY023016 based on binding properties of platelet membrane receptor subunit glycoprotein Ibα and thrombin exosites.

Thrombin has long been suggested as a desirable antithrombotic target, but anti-thrombin therapy without anti-platelet thereby has never achieved the ideal effect. HY023016 is a novel compound, in our previous study, it exerted better anti-thrombotic than dabigatran etexilate. The present study aims to illustrate the excess anti-thrombotic molecular mechanisms of HY023016 through thrombin anion exosites and the platelet membrane receptor subunit glycoprotein Ibα (GPIbα). HY023016 strongly inhibited the conversion of fibrinogen to fibrous may via blocking thrombin exosite I. We also discovered that HY023016 remarkably inhibited exosite II by a loss of affinity for the γ'-peptide of fibrinogen and for heparin. Furthermore, a solid phase binding assay revealed that HY023016 inhibited ristocetin-induced washed platelets bind to von Willebrand factor (vWF). In GST pull-down assay, HY023016 decreased the binding of recombinant vWF-A1 to GPIbα N-terminal. Thus, HY023016 provides an innovative idea for designing multi-targeted anti-thrombotic drugs and laying a scientific foundation for reducing "total thrombosis risk" in a clinical drug treatment.

Discovery of novel limonin derivatives as potent anti-inflammatory and analgesic agents.

Novel series of limonin derivatives (V-A-1-V-A-8, V-B-1-V-B-8) were synthesized by adding various tertiary amines onto the C (7)-position of limonin. The synthesized compounds possessed favorable physicochemical property, and the intrinsic solubility of the novel compounds were significantly improved, compared with limonin. Different pharmacological models were used to evaluate the analgesic and anti-inflammatory activities of the target compounds. Compound V-A-8 exhibited the strongest in vivo activity among the novel limonin analogs; its analgesic activity was more potent than aspirin and its anti-inflammatory activity was stronger than naproxen under our testing conditions.

Expression of IL-17 and syndecan-1 in nasal polyps and their correlation with nasal polyps.

Nasal polyp (NP) is a common chronic inflammatory disease of the nasal cavity and sinuses. Although some authors have suggested that NP is related to inflammatory factors such as interleukin (IL)-1ß, IL-5, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, and IL-17, the mechanisms underlying the pathogenesis and progression of NP remain obscure. This study investigated the expression and distribution of IL-17 and syndecan-1 in NP, and explored the roles of these two molecules in the pathogenesis of eosinophilic chronic rhinosinusitis with nasal polyps (Eos CRSwNP) and non-Eos CRSwNP. Real-time PCR and immunohistochemistry were used to detect the expression of IL-17 and syndecan-1 in samples [NP, unciform process (UP) from patients with CRS, and middle turbinate (MT) from healthy controls undergoing pituitary tumor surgery]. The results showed that the expression levels of IL-17 and syndecan-1 were upregulated in both NP and UP tissues, but both factors were higher in NP tissues than in UP tissues. There was no significant difference in IL-17 levels between the Eos CRSwNP and non-Eos CRSwNP samples, and syndecan-1 levels were increased in the non-Eos CRSwNP tissues as compared with those in Eos CRSwNP tissues. In all of the groups, there was a close correlation between the expression of IL-17 and syndecan-1 in nasal mucosa epithelial cells, glandular epithelial cells, and inflammatory cells, suggesting that IL-17 and syndecan-1 may play a role, and interact with each other, in the pathogenesis of non-Eos CRSwNP.

Evaluating antithrombotic activity of HY023016 on rat hypercoagulable model.

The generation of thrombus is not considered as an isolated progression without other pathologic processes, which may also enhance procoagulant state. The purpose of this study was to assess whether HY023016, a novel dabigatran prodrug and an oral direct thrombin inhibitor, or dabigatran etexilate, another thrombin inhibitor can improve the state of whole blood hypercoagulability in vitro/vivo. By using whole blood flow cytometry we explored the effects of HY023016 and dabigatran etexilate on thrombin and ADP-induced human platelet-leukocyte aggregation generated in vitro. With the method of continuous infusion of thrombin intravenous, we successfully established a rat hypercoagulable model and evaluated the effect of HY023016 or dabigatran etexilate in vivo. HY023016 was able to inhibit thrombin- or ADP-induced platelet P-selectin or CD40L expression, leukocyte CD11b expression and formation of platelet-leukocyte aggregates in dose-dependent manner. Dabigatran etexilate was unable to affect ADP-induced platelet P-selectin or CD40L expression, leukocyte CD11b expression and formation of platelet-leukocyte aggregates. Based on rat hypercoagulable model, dabigatran etexilate could reverse thrombin-induced circulatory system hypercoagulable state in a concentration-dependent manner. Dabigatran etexilate also inhibited electrical stimulation induced formation of arterial thrombus in rat under hypercoagulable state, and extracorporal circulation-induced formation of thrombus in dose-dependent manner. Compared with dabigatran etexilate, HY023016 showed nearly equal or even better antithrombotic activity, regardless of reversing the cycle of rat hypercoagulable state or inhibiting platelet-leukocyte aggregation. In surrmary, HY023016 could effectively improve hypercoagulable state of circulatory system.

Antidepressant-like effect of evodiamine on chronic unpredictable mild stress rats.

Evodiamine is a major alkaloid compound extracted from the dry unripened fruit Evodia fructus (Evodia rutaecarpa Benth., Rutaceae), which has a variety of pharmacological activities. The present study aims to determine the antidepressant-like effect of evodiamine in a rat model of chronic unpredictable mild stress (CUMS). We identified that evodiamine could reverse the following CUMS-induced behavioural deficits and biochemical changes in rats: the decreases of sucrose preference, number of crossings, 5-HT and NA levels, as well as the increase of immobility time. Evodiamine treatments also ameliorated the corticosterone hypersecretion induced by CUMS. Furthermore, we found that evodiamine was able to up-regulate the expression of brain-derived neurotrophic factor (BDNF) and phosphorylated tropomyosin-related kinase B (pTrkB) without altering TrkB. This study suggests potential antidepressant-like effect of evodiamine on CUMS rats, and its underlying mechanisms can be potentially linked to their modulating effects on the monoamine transmitters and BDNF-TrkB signaling in the hippocampus.

Antithrombotic activity of HY023016, a novel Dabigatran prodrug evaluated in animal thrombosis models.

INTRODUCTION: Thrombin is a multifunctional trypsin-like serine protease that plays key roles in coagulation and thrombogenesis. HY023016, a novel Dabigatran prodrug, is an oral direct thrombin inhibitor. The purpose of this study was to compare the anti-thrombotic activities and haemorrhagic effects of HY023016 with Dabigatran etexilate and tetramethylpyrazine in several animal thrombosis models. METHODS: To investigate drug exposure, liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to determine the pharmacokinetic profile of HY023016. After single intragastric administrations of HY023016, Dabigatran etexilate or tetramethylpyrazine, the anti-thrombotic activities were evaluated through rabbit jugular vein thrombosis model, rat inferior vena cava thrombosis model, ex vivo rabbit platelet aggregation assay, in vivo rabbit coagulation assay, and direct thrombin binding assay. Meanwhile, we evaluated the effect of HY023016 on expression of tissue factor (TF) by RT-PCR. Rabbit cuticle bleeding assay and mouse tail bleeding assay were applied to evaluate the effects of HY023016 on haemorrhage. RESULTS: Pharmacokinetic parameters indicated that HY023016 can convert to Dabigatran and tetramethylpyrazine. Our studies showed that HY023016 was able to significantly inhibit thrombus formation in a dose-dependent manner in rabbit and rat models (P<0.05). Similarly, it was able to dose-dependently inhibit thrombin- or ADP-induced platelet aggregation, prolonging the activated partial thromboplastin time (APTT) and prothrombin time (PT), inhibiting the activity of thrombin and inhibiting thrombin- or ADP-induced expression of TF (P<0.05 or 0.01). Dabigatran etexilate was also able to dose-dependently and significantly inhibit thrombus formation (P<0.01) but was unable to affect ADP-induced platelet aggregation and expression of TF. In contrast, tetramethylpyrazine could only exhibit mild antithrombotic activity compared with HY023016 and Dabigatran etexilate (P<0.05). HY023016 could prolong bleeding time (P<0.001), but the prolongations were significantly less than Dabigatran etexilate (P<0.05). CONCLUSION: HY023016 showed thrombosis-inhibition activities comparable to those of Dabigatran etexilate, but better than those of tetramethylpyrazine. The attendant bleeding risk of HY023016 was lower than Dabigatran etexilate in rabbits and mice.

Synthesis and antithrombotic evaluation of novel dabigatran prodrugs containing a cleavable moiety with anti-platelet activity.

A novel series of prodrugs consisting of dabigatran and 2-hydroxymethyl-3,5,6-trimethylpyrazine (HTMP) were synthesized. The pharmacological results show that all of them possess the effect of anti-platelet aggregation induced by thrombin in vitro. Moreover, one of those compounds, Y-2 (ED(50) = 2.1 ± 1.3 mg/kg) shows more potent activity for inhibiting thrombosis in vivo than that of dabigatran etexilate (ED(50) = 4.4 ± 2.2 mg/kg).

Synthesis and Na+/H+ exchanger inhibitory activity of benzoylguanidine derivatives.

Twenty-two compounds of substituted benzoylguanidine derivatives were designed and synthesized as potent NHE1 inhibitors. Twelve compounds showed more potent NHE1 inhibitory activity than cariporide. The activities of compounds 7e, 7h and 7j (IC(50) = 0.073 ± 0.021, 0.084 ± 0.012 and 0.068 ± 0.021 nmol/L, respectively) were two orders of magnitude higher than that of cariporide (30.7 ± 2.5 nmol/L). Myocardial cells in vitro screening showed 7j had highlighted protective effect on cardiomyocytes subjected to hypoxia/reoxygenation. Thus it is valuable for further investigation.

Benzimidazol-2-yl or benzimidazol-2-ylthiomethyl benzoylguanidines as novel Na+/H+ exchanger inhibitors, synthesis and protection against ischemic-reperfusion injury.

A novel series of benzimidazol-2-yl or benzimidazol-2-ylthiomethyl benzoylguanidines were designed and synthesized as Na(+)/H(+)exchanger inhibitors. Most of them were found to inhibit NHE1-mediated platelet swelling in a concentration-dependent manner, and to have significant cardioprotective effect against myocardial ischemic-reperfusion injury, among which compounds 10a and 34 were more potent than cariporide in both in vivo and in vitro tests.