目的 探讨草苁蓉多糖(Boschniakia rossica polysaccharides,BRPS)对脂多糖(lipopolysaccharide,LPS)诱导的RAW264.7巨噬细胞炎症反应的影响。方法 体外利用LPS刺激RAW264.7细胞,建立巨噬细胞炎症模型。将巨噬细胞随机分为正常组、模型组、BRPS低、中、高剂量组(BRPS质量浓度分别为25、50、100 mg·L-1)。采用细胞增殖检测试剂盒(cell counting kit-8,CCK-8)检测细胞活力;酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测白介素-1β(interleukin-1β,IL-1β)、白介素-6(interleukin-6,IL-6)以及肿瘤坏死因子-α(tumor necrosis factor alpha,TNF-α)的分泌;采用Western blot法检测环氧合酶-2(cyclooxygenase-2,COX-2)、髓样分化因子88(myeloid differentiation factor 88,MyD88)、白介素-1受体相关激酶1(interleukin-1 receptor-associated kinase1,IRAK1)蛋白的表达以及核转录因子-κB(nuclear factor-кB,NF-кB)、丝裂原活化蛋白激酶(mitogen activated protein kinases,MAPK)、信号传导与转录激活因子-3(signal transducers and activators of transcription-3,STAT3)蛋白的活化水平。结果 LPS在100~2 000 μg·L-1内对RAW264.7细胞活力无影响;但能够显著上调RAW264.7细胞COX-2蛋白的表达,提示巨噬细胞炎症模型构建成功。BRPS在0～100 mg·L-1内对RAW264.7细胞活力无影响,不显示细胞毒性;但能够下调LPS诱导的RAW264.7细胞IL-1β、IL-6、TNF-α的分泌。与模型组比较,BRPS低、中、高剂量组细胞对IL-1β分泌的抑制率分别为14.2%、47.6%、72.4%,对IL-6分泌的抑制率分别为5.5%、22.2%、45.1%,对TNF-α分泌的抑制率分别为9.5%、29.1%、55.3%。同时,BRPS能够抑制LPS诱导的COX-2蛋白表达,抑制MyD88、IRAK1蛋白表达,抑制NF-кB p65的磷酸化和核内转移,抑制ERK、JNK、p38磷酸化以及STAT3磷酸化。结论 BRPS对LPS诱导的RAW264.7巨噬细胞炎症反应具有抑制作用,其作用可能与其抑制NF-кB、MAPK、STAT3活化有关。
Abstract
OBJECTIVE To investigate the effect of Boschniakia rossica polysaccharides (BRPS) on the inflammatory response of RAW264.7 macrophages induced by lipopolysaccharide (LPS). METHODS The cellular model was established by treating RAW264.7 macrophages with LPS. The macrophages were randomly assigned into normal group, model group, BRPS low-, medium- and high-dose groups (the final concentrations of BRPS were 25, 50 and 100 mg·L-1, respectively). Cell viability was detected by the cell counting kit-8 (CCK-8); the levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured with enzyme-linked immunosorbent assay (ELISA); the protein expressions of cyclooxygenase-2 (COX-2), myeloid differentiation factor 88 (MyD88) and interleukin-1 receptor-associated kinase1 (IRAK1), and the activation of nuclear factor-кB (NF-кB), mitogen activated protein kinases (MAPK) and signal transducers and activators of transcription-3 (STAT3) were determined with Western blot method. RESULTS LPS had no significant effect on the viabilities of RAW264.7 macrophages in the concentration range of 100-2 000 μg·L-1, but could significantly up-regulate the expression of COX-2 protein in RAW264.7 cells, indicating the establishment of a macrophage model of inflammation. BRPS had no significant effect on the viabilities of RAW264.7 cells in the concentration range of 0-100 mg·L-1, however, it could down-regulate the LPS-induced secretions of IL-1β, IL-6 and TNF-α into the culture media of RAW264.7 cells. Compared with the model group, the inhibition ratios of IL-1β secretion were 14.2%, 47.6% and 72.4%; inhibition ratios of IL-6 secretion were 5.5%, 22.2% and 45.1%; and inhibition ratios of TNF-α secretion were 9.5%, 29.1% and 55.3% in BRPS low-, medium- and high-dose groups, respectively. At the same time, BRPS could reduce the LPS-induced COX-2 protein expression, down-regulate the expressions of MyD88 and IRAK1 proteins, inhibit the phosphorylation and nuclear translocation of NF-кB p65, and inhibit the phosphorylation of ERK, JNK, p38 and STAT3. CONCLUSION BRPS has an inhibitory effect on LPS-induced RAW264.7 macrophage inflammatory response, maybe through the suppression of NF-кB, MAPK, and STAT3 activation.
关键词
草苁蓉 /
多糖 /
脂多糖 /
RAW264.7 /
炎症因子
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Key words
Boschniakia rossica /
polysaccharide /
LPS /
RAW264.7 /
inflammatory factor
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中图分类号：
R285.5
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脚注
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基金
国家自然科学地区科学基金项目资助(82060113)
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