没食子酸对脂多糖诱导的人THP1巨噬细胞炎症反应的抑制作用
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延边大学医学院

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国家自然科学地区科学(82060113)


Inhibition of gallic acid on lipopolysaccharide-induced inflammatory response in human THP1 macrophages
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Medical College of Yanbian University

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    摘要:

    目的 探讨没食子酸(gallic acid,GA)对脂多糖(lipopolysaccharide,LPS)诱导的人THP1巨噬细胞炎症反应的抑制作用。方法 将人THP1单核细胞用佛波酯(phorbol myristate acetate,PMA)分化为巨噬细胞,用LPS诱导建立巨噬细胞炎症模型,给予不同浓度GA进行保护。CCK-8法筛选 LPS 和GA对THP1细胞的安全浓度;设正常组、模型组(100 μg/L LPS)、GA组(100 μg/L LPS +不同浓度GA)。ELISA法检测各组细胞培养液白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)水平,微板法检测各组细胞培养液乳酸脱氢酶(lactate dehydrogenase,LDH)活性和一氧化氮(nitric oxide,NO)含量,荧光染色检测各组细胞活性氧(ROS)水平、细胞损伤情况和线粒体跨膜电位的变化,Western blotting法检测各组细胞环氧合酶-2(COX-2)、高迁移率族蛋白B1(HMGB1)、c-Jun氨基末端激酶(JNK)、细胞外调节蛋白激酶(ERK)、P38丝裂原活化蛋白激酶(P38)、核转录因子-κB(NF-κB)、NOD样受体3(NOD-like receptor protein 3,NLRP3)、半胱氨酸天冬氨酸蛋白酶-1(Caspase-1)、IL-1β、消皮素D(Gasdermin D,GSDMD)蛋白表达的影响。结果 与正常组比较,模型组细胞培养液IL-6、TNF-α、IL-1β和NO的分泌增多(P<0.05),COX-2、HMGB1、p-ERK、p-JNK和p-P38和p-NF-κB、NLRP3、Caspase-1、IL-1β蛋白表达升高(P<0.05),GSDMD蛋白表达水平降低(P<0.05), ROS生成和细胞损伤明显增多,线粒体跨膜电位较正常组显著降低,LDH活性升高(P<0.05);与模型组比较,GA组细胞培养液IL-6、TNF-α、IL-1β和NO的分泌减少(P<0.05),COX-2、HMGB1、p-ERK、p-JNK和p-P38和p-NF-κB、NLRP3、Caspase-1、IL-1β蛋白表达降低(P<0.05),GSDMD蛋白表达水平升高(P<0.05), ROS生成和细胞损伤减少,线粒体跨膜电位逐渐升高,LDH活性降低(P<0.05)。结论 GA对LPS诱导的人THP1巨噬细胞炎症反应具有抑制作用,其抗炎机制可能涉及MAPK和NF-κB信号通路。

    Abstract:

    Objective To investigate the inhibitory effect of gallic acid (GA) on lipopolysaccharide (LPS)-induced inflammatory responses in human THP1 macrophages. Methods THP1 monocytes were differentiated into macrophages with phorbol myristate acetate (PMA) and then the macrophage inflammation model was established with LPS induction,and GA was given in different concentrations. The safe concentrations of LPS and GA on THP1 cells were screened by CCK-8 method,and the normal group, model group (100 μg/L LPS ) and GA group(100 μg/L LPS + different concentrations of GA)were set up. ELISA was used to detect the levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin-1β(IL-1β) in the cell culture fluid of each group. Microplate method was used to detect LDH activity and NO content in cell cultures of each group, and fluorescence staining was used to detect ROS levels, cell damage and changes in mitochondrial transmembrane potential in each group.Western blotting assay was performed to detect the levels of cyclooxygenase-2 (COX-2), HMGB1,c-Jun amino-terminal kinase (JNK), extracellular-regulated protein kinase (ERK), P38 mitogen-activated protein kinase (P38), nuclear transcription factor-κB (NF-κB), NOD-like receptor protein 3(NLRP3), Caspase-1,IL-1β、Gasdermin D(GSDMD). Results Compared with the normal group, the secretion of IL-6, TNF-α, IL-1β and NO in cell cultures was increased in the model group (P<0.05), and the secretion of COX-2, HMGB1, p-ERK, p-JNK, and p-P38 and p-NF-κB, NLRP3, Caspase-1, IL-1? protein expression was elevated (P<0.05), the expression level of GSDMD protein activation fragment was reduced (P<0.05), ROS generation and cellular damage were significantly increased, mitochondrial transmembrane potential was significantly lower than that of the normal group, and the activity of LDH was elevated (P<0.05); in comparison with the model group, the cell culture fluid of the GA group IL-6, TNF-α, IL-1β and NO secretion were decreased (P<0.05), COX-2, HMGB1, p-ERK, p-JNK and p-P38 and p-NF-κB, NLRP3, Caspase-1, IL-1β protein expression was decreased (P<0.05), GSDMD protein activation fragment expression level was increased (P<0.05), ROS generation and cellular damage were decreased, mitochondrial transmembrane potential was gradually increased, and LDH activity was decreased (P<0.05).Conclusion GAhas an inhibitory effect on LPS-induced inflammatory response in THP1 macrophages, and its anti-inflammatory mechanism may involve MAPK and NF-κB signalingpathways.

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  • 收稿日期:2024-01-18
  • 最后修改日期:2024-03-14
  • 录用日期:2024-04-18
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