Effects of Alhagi pseudalhagi(M.B.)Desv.extract(APE)exerts on nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome and related cytokines in lipopolysaccharide induced intestinal epithelial cell(IEC-6)injury model
Effects of Alhagi pseudalhagi(M.B.)Desv.extract(APE)exerts on nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome and related cytokines in lipopolysaccharide induced intestinal epithelial cell(IEC-6)injury model
徐晓琴 1卿德刚 1陈良 2张娟 1孙宇 1夏提古丽·阿不利孜1
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作者信息
1. 新疆维吾尔自治区中药民族药研究所,乌鲁木齐 830002
2. 新疆医科大学第四附属医院,乌鲁木齐 830000
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摘要
目的 研究骆驼刺提取物(Alhagi pseudalhagi(M.B.)Desv.Extract,APE)对脂多糖诱导的大鼠小肠隐窝上皮细胞(Intestinal epithelial cell,IEC-6)损伤模型NLRP3炎症小体及相关细胞因子的影响.方法 培养IEC-6细胞,将其分为空白组、模型组、APE低、中、高浓度组,用1.0 μg/mL的脂多糖(Lipopolysaccharide,LPS)诱导建立细胞炎症损伤模型,APE(低、中、高浓度:15、25、35 μg/mL)干预后采用CCK-8法检测细胞的存活率,通过ELISA试剂盒检测炎症因子IL-1β、IL-18、TNF-α的分泌水平.蛋白质印迹法(WB)检测核苷酸结合寡聚化结构域样受体蛋白3(Nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)炎症小体信号通路 5个关键蛋白:NLRP3、半胱氨酸天冬氨酸蛋白酶1(Cystein-asparate protease-1,Caspase-l)、凋亡相关斑点样蛋白(Apoptosis-associated speck-like protein containing a CARD,ASC)及抗凋亡蛋白Bcl-2(Anti-apoptosis Protein Bcl-2)和 Bcl-xl(Anti-apoptosis Protein Bcl-xl)表达.结果 与空白组比较,模型组IEC-6细胞的存活率降低,NLRP3、Caspase-1、ASC蛋白表达水平升高,抗凋亡蛋白Bcl-2、Bcl-xl的表达水平降低,促炎因子IL-1β、IL-18和TNF-α的分泌水平升高,差异有统计学意义(P＜0.05).与模型组比较,APE低、中、高浓度组细胞存活率升高,35 μg/mL APE组IEC-6细胞的NLRP3、Caspase-1、ASC蛋白相对表达水平降低,抗凋亡蛋白Bcl-2、Bcl-xl的表达水平升高,差异有统计学意义(P＜0.05).中、高浓度的APE能够抑制炎症因子分泌,25 μg/mL APE对IL-1β、IL-18、TNF-α炎症因子分泌水平抑制率分别为31.60％、31.19％和31.09％(P＜0.05).结论 骆驼刺提取物通过提高抗凋亡蛋白Bcl-2、Bcl-xl的表达水平,下调NLRP3炎症小体组成成分以及促炎因子IL-1β、IL-18和TNF-α分泌,从而抑制NLRP3炎症小体组装和激活,实现缓解LPS对IEC-6细胞的损伤.
Abstract
Objective To study the effects of Alhagi pseudalhagi(M.B.)Desv.extract(APE)exerts on nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome and related cy-tokines in lipopolysaccharide induced intestinal epithelial cell(IEC-6)injury model.Methods The inflam-matory injury model of IEC-6 cells induced by LPS was established in vitro.The survival rate of IEC-6 cells was detected by CCK-8 method,the levels of IL-1β,IL-18 and TNF-a were measured by enzyme-linked immunosorbent assay(ELISA),and the expressions of NLRP3,Caspase-1,ASC,Bcl-2 and Bcl-xl were detected by Western blot.Results Compared with the blank group,the survival rate of IEC-6 cells in the model group decreased,and the expression levels of NLRP3,Caspase-1 and ASC proteins was in-creased.The expression levels of anti apoptotic proteins Bcl-2 and Bcl-xl weredecreased,and the pro-in-flammatory factor IL-1β,IL-18 and TNF-asecretion level of wasincreased,and the difference was statisti-cally significant(P＜0.05).Compared with model group,the cell survival rate of APE low,mediumand high concentration groups wereincreased by 35μg/mL.The relative expression levels of NLRP3,Caspase-1,and ASC proteins in IEC-6 cells in theAPE group weredecreased,while the expression levels of anti apoptotic proteins Bcl-2 and Bcl-xl were increased,with statistical significance(P＜0.05).Medium to high concentrations of APE can inhibit the secretion of inflammatory factors,25 μg/mL APE for IL-1 β,IL-18,TNF-α The inhibition rates of inflammatory cytokine secretion levels were 31.60％,31.19％and 31.09％,respectively(P＜0.05).Conclusion APE may inhibit the assembly and activation of NLRP3 inflammasome by increasing the expression levels of anti-apoptotic protein Bcl-2 and Bcl-xl,and down-regulate the secretion of pro-inflammatory factors IL-1β,IL-18 and TNF-α,thereby alleviating the damage of LPS to IEC 6 cells.
关键词
骆驼刺提取物/脂多糖/小肠隐窝上皮细胞/NLRP3炎症小体
Key words
Alhiagi pseudalhagi(M.B.)Desv.extract(APE)/lipopolysaccharide/intestinal crypt epi-thelial cells(IEC-6)/nucleotide-binding oligomerization domain-like receptor family pyrrolidine domain-like protein 3(NLRP3)
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