过敏是人和养殖动物常见的疾病,而且发生的频率呈现逐渐增加的趋势。为了深入了解过敏反应的机制,本研究利用CRISPR/Cas9基因编辑技术,构建钙调磷酸酶A beta(calcineurin A beta,CnAβ)基因敲除的大鼠RBL-2H3细胞株,并探讨CnAβ基因对RB...过敏是人和养殖动物常见的疾病,而且发生的频率呈现逐渐增加的趋势。为了深入了解过敏反应的机制,本研究利用CRISPR/Cas9基因编辑技术,构建钙调磷酸酶A beta(calcineurin A beta,CnAβ)基因敲除的大鼠RBL-2H3细胞株,并探讨CnAβ基因对RBL-2H3细胞生长及脱颗粒的影响。选取大鼠CnAβ基因第一外显子为敲除靶点,设计并合成3个单导向RNA(single guide RNA,sgRNA),构建pX459-CnAβ-sgRNA质粒,并用脂质体3000将构建好的质粒转染到RBL-2H3细胞内;利用嘌呤霉素对转染细胞进行筛选,通过DNA测序验证获得CnAβ基因敲除的RBL-2H3细胞株;并检测CnAβ基因缺失对细胞增殖和脱颗粒的影响。结果表明,成功构建了CnAβ单基因敲除的大鼠RBL-2H3细胞株;CnAβ基因缺失对RBL-2H3细胞增殖、细胞的颗粒形成以及颗粒含量无显著影响,但显著抑制由细胞表面受体介导的RBL-2H3细胞脱颗粒作用。该研究结果有助于深入了解动物过敏性疾病的发生机制,为动物过敏性疾病的预防提供了理论基础。展开更多
Food allergy has become a global concern.Spleen tyrosine kinase(SYK)inhibitors are promising therapeutics against allergic disorders.In this study,a total of 300 natural phenolic compounds were firstly subjected to vi...Food allergy has become a global concern.Spleen tyrosine kinase(SYK)inhibitors are promising therapeutics against allergic disorders.In this study,a total of 300 natural phenolic compounds were firstly subjected to virtual screening.Sesamin and its metabolites,sesamin monocatechol(SC-1)and sesamin dicatechol(SC-2),were identified as potential SYK inhibitors,showing high binding affinity and inhibition efficiency towards SYK.Compared with R406(a traditional SYK inhibitor),sesamin,SC-1,and SC-2 had lower binding energy and inhibition constant(Ki)during molecular docking,exhibited higher bioavailability,safety,metabolism/clearance rate,and distribution uniformity ADMET predictions,and showed high stability in occupying the ATP-binding pocket of SYK during molecular dynamics simulations.In anti-dinitrophenyl-immunoglobulin E(Anti-DNP-Ig E)/dinitrophenyl-human serum albumin(DNP-HSA)-stimulated rat basophilic leukemia(RBL-2H3)cells,sesamin in the concentration range of 5-80μmol/L influenced significantly the degranulation and cytokine release,with 54.00%inhibition againstβ-hexosaminidase release and 58.45%decrease in histamine.In BALB/c mice,sesamin could ameliorate Anti-DNP-Ig E/DNP-HSA-induced passive cutaneous anaphylaxis(PCA)and ovalbumin(OVA)-induced active systemic anaphylaxis(ASA)reactions,reduce the levels of allergic mediators(immunoglobulins and pro-inflammatory cytokines),partially correct the imbalance of T helper(Th)cells differentiation in the spleen,and inhibit the phosphorylation of SYK and its downstream signaling proteins,including p38 mitogen-activated protein kinases(p38 MAPK),extracellular signalregulated kinases(ERK),and p65 nuclear factor-κB(p65 NF-κB)in the spleen.Thus,sesamin may be a safe and versatile SYK inhibitor that can alleviate Ig E-mediated food allergies.展开更多
基金Incubation Program of Youth Innovation in Shandong ProvinceKey Research and Development Program of Shandong Province(2021TZXD007)。
文摘Food allergy has become a global concern.Spleen tyrosine kinase(SYK)inhibitors are promising therapeutics against allergic disorders.In this study,a total of 300 natural phenolic compounds were firstly subjected to virtual screening.Sesamin and its metabolites,sesamin monocatechol(SC-1)and sesamin dicatechol(SC-2),were identified as potential SYK inhibitors,showing high binding affinity and inhibition efficiency towards SYK.Compared with R406(a traditional SYK inhibitor),sesamin,SC-1,and SC-2 had lower binding energy and inhibition constant(Ki)during molecular docking,exhibited higher bioavailability,safety,metabolism/clearance rate,and distribution uniformity ADMET predictions,and showed high stability in occupying the ATP-binding pocket of SYK during molecular dynamics simulations.In anti-dinitrophenyl-immunoglobulin E(Anti-DNP-Ig E)/dinitrophenyl-human serum albumin(DNP-HSA)-stimulated rat basophilic leukemia(RBL-2H3)cells,sesamin in the concentration range of 5-80μmol/L influenced significantly the degranulation and cytokine release,with 54.00%inhibition againstβ-hexosaminidase release and 58.45%decrease in histamine.In BALB/c mice,sesamin could ameliorate Anti-DNP-Ig E/DNP-HSA-induced passive cutaneous anaphylaxis(PCA)and ovalbumin(OVA)-induced active systemic anaphylaxis(ASA)reactions,reduce the levels of allergic mediators(immunoglobulins and pro-inflammatory cytokines),partially correct the imbalance of T helper(Th)cells differentiation in the spleen,and inhibit the phosphorylation of SYK and its downstream signaling proteins,including p38 mitogen-activated protein kinases(p38 MAPK),extracellular signalregulated kinases(ERK),and p65 nuclear factor-κB(p65 NF-κB)in the spleen.Thus,sesamin may be a safe and versatile SYK inhibitor that can alleviate Ig E-mediated food allergies.
