目的 通过研究解毒祛瘀滋阴方含药血清对脂多糖(lipopolysaccharide, LPS)刺激后小鼠单核巨噬细胞白细胞介素-1受体相关激酶(interleukin-1 receptor-associated kinase 1, IRAK1)信号通路的影响,探讨解毒祛瘀滋阴方对IRAK1、NF-κB炎症信号通路的影响,为其抗炎临床用药提供良好的理论支持。方法 该研究拟采用体外培养小鼠单核巨噬细胞,随机分为空白组、LPS刺激组、中药血清组、空白血清组、LPS加中药血清组、LPS加空白血清组、IRAK1抑制剂组、抑制剂加LPS组、抑制剂加中药血清组及抑制剂加空白血清组。干预24 h后,采用CCK8法检测解毒祛瘀滋阴方对细胞的活力,采用ELISA法检测细胞上清中肿瘤坏死因-α(tumor necrosis factor-α, TNF-α)含量;采用免疫荧光化学染色法检测解毒祛瘀滋阴方对小鼠单核巨噬细胞中IRAK1表达的影响;采用RT-PCR法检测IRAK1、核转录因子κB(nuclear factor κB, NF-κB) 、TNF-α及白介素6(interleukin-6,IL-6)mRNA的表达;采用Western-blot法检测IRAK1、p-IRAK1、NF-κB蛋白水平表达;采用LC-MS检测中药组血清中的有效成分。结果 中药含药血清组以2.5%为最佳给药浓度,解毒祛瘀滋阴方能下调TNF-α和IL-6的表达及抑制IRAK1表达和NF-κB的活化(P<0.05),中药组含药血清中检测到芍药苷和阿魏酸。结论 解毒祛瘀滋阴方能抑制LPS刺激后小鼠单核巨噬细胞IRAK1、NF-κB的表达,为其抗炎临床用药提供良好的理论支持。
Abstract
OBJECTIVE To study the effect of serum containing Jieduquyuziyin-prescription (JP) on signal pathway of interleukin-1 receptor-associated kinase 1 (IRAK1) in mononuclear macrophages of mice stimulated by lipopolysaccharide (LPS), and to explore the effect of Jieduquyuziyin-prescription on IRAK1 and NF-κB inflammatory signaling pathways, which providing a good theoretical support for its anti-inflammatory clinical medication. METHODS In this study, mice mononuclear macrophages cultured in vitro were randomly divided into blank group, LPS group, JP serum group, blank serum group, LPS plus JP serum group, LPS plus blank serum group, IRAK1 inhibitor group, inhibitor plus LPS group, inhibitor plus JP serum group and inhibitor plus blank serum group. After intervention for 24 h, the activity of JP on macrophages was tested by CCK8 method. The IRAK1 expression in macrophages was tested by immunofluorescence chemical staining. The content of TNF-α in the supernatant of the cells was detected by ELISA. The mRNA expressions of IRAK1, NF-κB, TNF-α and IL-6 were detected by RT-PCR. The protein expressions of IRAK1, p-IRAK1 and NF-κB were detected by Western-blot. The LC-MS was used to detect the active ingredients in JP serum. RESULTS The results show that 2.5% of JP serum is the optimal concentration. Jieduquyuziyin-prescription could down-regulate the expression of TNF-α and IL-6 and inhibit the expression of IRAK1 and activate NF-κB(P<0.05). Paeoniflorin and ferulic acid were detected in the JP serum. CONCLUSION Jieduquyuziyin-prescription can inhibit the expression of IRAK1 and NF-κB in mouse monocyte-macrophage cells after LPS stimulation and provide a good theoretical support for its anti-inflammatory clinical medication.
关键词
解毒祛瘀滋阴方 /
含药血清 /
RAW264.7巨噬细胞 /
单核巨噬细胞白细胞介素-1受体相关激酶 /
核转录因子-κB
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Key words
Jieduquyuziyin-prescription /
drug-containing serum /
RAW264.7 macrophage /
interleukin-1 receptor-associated kinase 1 /
nuclear factor κB
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中图分类号：
R285.5
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参考文献
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脚注
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基金
国家自然科学基金面上项目资助(81673863);浙江省自然科学基金一般项目资助(LY19H290006)
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