RESUMEN
BACKGROUND: Vitamin K3 (VK3), a fat-soluble synthetic analog of the vitamin K family, has coagulant, anti-inflammatory, antibacterial, and anticancer properties. Pseudo allergy is a IgE-independent immune response associated with mast cells. This study investigated the role of VK3 in IgE-independent mast cell activation. METHODS: Substance P (SP) was used to induce LAD2-cell activation in order to analyze the effects of VK3 in vitro. Cutaneous allergy and systemic allergy mouse models were used to analyze the anti-pseudo-allergic effects of VK3. Proteome microarray assays were used to analyze VK3-binding protein. Biolayer interferometry and immunoprecipitation were used to verify interaction between VK3 and its key targets. RNA interference was used to determine the role of GAB1 in LAD2cell activation. RESULTS: VK3 inhibited SP-induced LAD2-cell activation, and resulted in the release of ß-hexosaminidase, histamine and cytokines; VK3 inhibited SP-induced pseudo allergic reactions in mice, and serum histamine and TNF-α levels decreased. Degranulation of skin mast cells was reduced; GAB1 in mast cells was stably bound to VK3. GAB1 participated in SP-induced LAD2-cell activation. GAB1 knockdown in LAD2 cells prevented SP-induced ß-hexosaminidase release, calcium mobilization and cell skeletal remodeling. VK3 directly binds to GAB1 and reduces its expression to inhibited SP-induced LAD2 cell activation. CONCLUSION: The anti-pseudo-allergic activity of VK3 was confirmed in vitro and in vivo. VK3 can inhibit SP-induced mast cell activation by directly targeting GAB1. This study provides new insights on the activity of VK3 and the mechanism of pseudoallergic reaction.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Mastocitos , Mastocitos/inmunología , Mastocitos/efectos de los fármacos , Animales , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Ratones , Humanos , Sustancia P/metabolismo , Degranulación de la Célula/efectos de los fármacos , Ratones Endogámicos BALB C , Hipersensibilidad/inmunología , Hipersensibilidad/tratamiento farmacológico , Antialérgicos/farmacología , Antialérgicos/uso terapéutico , Femenino , Línea Celular , beta-N-Acetilhexosaminidasas/metabolismo , Modelos Animales de EnfermedadRESUMEN
BACKGROUND: Mast cells (MCs) are important effector cells in anaphylaxis and anaphylactic disease. 3',4',5,7-tetrahydroxyflavone (THF) presents in many medicinal plants and exerts a variety of pharmacological effects. In this study, we evaluated the effect of THF on C48/80-induced anaphylaxis and the mechanisms underlying its effects, including the role of secreted phosphoprotein 1 (SPP1), which has not been reported to IgE-independent MC activation. RESULTS: THF inhibited C48/80-induced Ca2+ flow and degranulation via the PLCγ/PKC/IP3 pathway in vitro. RNA-seq showed that THF inhibited the expression of SPP1 and downstream molecules. SPP1 is involved in pseudo-anaphylaxis reactions. Silencing SPP1 affects the phosphorylation of AKT and P38. THF suppressed C48/80-induced paw edema, hypothermia and serum histamine, and chemokines release in vivo. CONCLUSIONS: Our results validated SPP1 is involved in IgE-independent MC activation anaphylactoid reactions. THF inhibited C48/80-mediated anaphylactoid reactions both in vivo and in vitro, suppressed calcium mobilization and inhibited SPP1-related pathways.
Asunto(s)
Anafilaxia , Humanos , Anafilaxia/inducido químicamente , Anafilaxia/tratamiento farmacológico , Luteolina/farmacología , Osteopontina/metabolismo , Osteopontina/farmacología , Mastocitos , Inflamación/metabolismo , Degranulación de la Célula , Inmunoglobulina E/metabolismoRESUMEN
Fusion with host cell membrane is the main mechanism of infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we propose that a new strategy to screen small-molecule antagonists blocking SARS-CoV-2 membrane fusion. Using cell membrane chromatography (CMC), we found that harringtonine (HT) simultaneously targeted SARS-CoV-2 S protein and host cell surface TMPRSS2 expressed by the host cell, and subsequently confirmed that HT can inhibit membrane fusion. HT effectively blocked SARS-CoV-2 original strain entry with the IC50 of 0.217 µM, while the IC50 in delta variant decreased to 0.101 µM, the IC50 in Omicron BA.1 variant was 0.042 µM. Due to high transmissibility and immune escape, Omicron subvariant BA.5 has become the dominant strain of the SARS-CoV-2 virus and led to escalating COVID-19 cases, however, against BA.5, HT showed a surprising effectiveness. The IC50 in Omicron BA.5 was even lower than 0.0019 µM. The above results revealed the effect of HT on Omicron is very significant. In summary, we characterize HT as a small-molecule antagonist by direct targeting on the Spike protein and TMPRSS2.
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COVID-19 , Harringtoninas , Humanos , SARS-CoV-2RESUMEN
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.
Asunto(s)
Asma , Mastocitos , Humanos , Asma/inducido químicamente , Asma/metabolismo , Citocinas/metabolismo , Histamina/metabolismo , Histamina/farmacología , Janus Quinasa 2/efectos adversos , Janus Quinasa 2/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Sistema de Señalización de MAP QuinasasRESUMEN
The outbreak of coronavirus disease 2019 (COVID-19) has induced a large number of deaths worldwide. Angiotensin-converting enzyme 2 (ACE2) is the entry receptor for the 2019 novel coronavirus (2019-nCoV) to infect the host cells. Therefore, ACE2 may be an important target for the prevention and treatment of COVID-19. The aim of this study was to investigate the inhibition effect of valaciclovir hydrochloride (VACV), zidovudine (ZDV), saquinavir (SQV), and efavirenz (EFV) on 2019-nCoV infection. The results of molecule docking and surface plasmon resonance showed that VACV, ZDV, SQV, and EFV could bind to ACE2 protein, with the KD value of (4.33 ± 0.09) e-8 , (6.29 ± 1.12) e-6 , (2.37 ± 0.59) e-5 , and (4.85 ± 1.57) e-5 M, respectively. But only ZDV and EFV prevent the 2019-nCoV spike pseudotyped virus to enter ACE2-HEK293T cells with an EC50 value of 4.30 ± 1.46 and 3.92 ± 1.36 µM, respectively. ZDV and EFV also have a synergistic effect on preventing entry of virus into cells. In conclusion, ZDV and EFV suppress 2019-nCoV infection of ACE2-HEK293T cells by interacting with ACE2.
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Antivirales/farmacología , Peptidil-Dipeptidasa A/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , Pseudotipado Viral , Sitio Alostérico , Antivirales/metabolismo , COVID-19/prevención & control , COVID-19/virología , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Peptidil-Dipeptidasa A/metabolismo , Unión Proteica , Resonancia por Plasmón de Superficie , Tratamiento Farmacológico de COVID-19RESUMEN
Licochalcone A (Lico A) is a natural flavonoid belonging to the class of substituted chalcone that has various biological effects. Mast cells (MCs) are innate immune cells that mediate hypersensitivity and pseudo-allergic reactions. MAS-related GPR family member X2 (MRGPRX2) on MCs has been recognized as the main receptor for pseudo-allergic reactions. In this study, we investigated the anti-pseudo-allergy effect of Lico A and its underlying mechanism. Substance P (SP), as an MC activator, was used to establish an in vitro and in vivo model of pseudo-allergy. The in vivo effect of Lico A was investigated using passive cutaneous anaphylaxis (PCA) and active systemic allergy, along with degranulation, Ca2+ influx in vitro. SP-induced laboratory of allergic disease 2 (LAD2) cell mRNA expression was explored using RNA-seq, and Lico A inhibited LAD2 cell activation by reverse transcription polymerase chain reaction (RT-PCR), western blotting, and immunofluorescence staining. Lico A showed an inhibitory effect on SP-induced MC activation and pseudo-allergy both in vitro and in vivo. The nuclear factor (NF)-κB pathway is involved in MRGPRX2 induced MC activation, which is inhibited by Lico A. In conclusion, Lico A inhibited the pseudo-allergic reaction mediated by MRGPRX2 by blocking NF-κB nuclear migration.
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Chalconas , Hipersensibilidad , Degranulación de la Célula , Chalconas/farmacología , Humanos , Hipersensibilidad/tratamiento farmacológico , Mastocitos , FN-kappa B , Proteínas del Tejido Nervioso , Receptores Acoplados a Proteínas G/genética , Receptores de NeuropéptidoRESUMEN
Chrysin, one of the most pharmacologically active natural flavonoids, has been extracted from various plants. Mast cells are an important part of innate immunity-mediating anaphylaxis. Pseudo-allergic reactions are currently believed to be associated with the MAS-related GPR family member X2 (MrgX2). In this study, the anti-pseudo allergy effect of chrysin and its underlying mechanisms were studied in vitro and in vivo. Chrysin inhibited passive cutaneous anaphylaxis and systemic pseudo-allergy in vivo. LAD2 cell degranulation, calcium ion (Ca2+) influx, and adenosine 5'-triphosphate (ATP) content were significantly suppressed in a dose-dependent manner. Chrysin suppressed pseudo-allergic reactions through the PLC/IP3/Ca2+ and ERK/STAT3 serine 727 pathways downstream of MrgX2. Therefore, mitochondrial ATP, but not glycolysis, is vital for pseudo-allergic reactions mediated by MrgX2. This study provides new insights for the treatment of pseudo-allergy.
Asunto(s)
Anafilaxia , Receptores Acoplados a Proteínas G , Degranulación de la Célula , Flavonoides , Humanos , Mastocitos , Receptores Acoplados a Proteínas G/genéticaRESUMEN
The SARS-CoV-2 outbreak, began in late 2019, has caused a worldwide pandemic and shows no signs of slowing. Glucocorticoids (GCs), including dexamethasone (DEX), have been widely used as effective anti-inflammatory and immunosuppressant drugs. In this study, seven GCs had no obvious effect on cell viability of angiotensin converting enzyme 2 (ACE2) high expressed HEK293T cells when concentrations were under 10 µM. Molecular docking results revealed that DEX occupied with active binding site of ACE2 of SARS-CoV-2 spike protein. Surface plasmon resonance (SPR) results showed that KD value between DEX and ACE2 was (9.03 ± 0.78) e-6 M. Cell membrane chromatography (CMC) results uncovered that DEX had a chromatographic retention. DEX was found out to inhibiting the viropexis into ACE2h cells using SARS-CoV-2 spike pseudotyped virus. Therefore, DEX inhibits the entrance of SARS-CoV-2 spike pseudotyped virus into cell by binding to ACE2.