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目的:考察芪麝丸体外对小鼠RAW264.7巨噬细胞炎症模型的抗炎机制。方法:干扰素γ(1×104U.mL-1)和脂多糖(100μg.L-1)协同诱导小鼠RAW264.7巨噬细胞(1×105/mL)24 h造成炎症模型;Griess反应测定细胞上清液中一氧化氮(NO)产量;Western blot检测诱导型一氧化氮合酶(iNOS)、环氧合酶(COX-2)的蛋白表达及丝裂原活化蛋白激酶(MAPK)信号转导通路的活化情况。结果:芪麝丸(250,500,1 000 mg.L-1)呈剂量依赖性抑制细胞上清液中NO含量,且无细胞毒性。较模型组,经芪麝丸500,1 000 mg.L-1剂量处理后,iNOS蛋白表达从(1.00±0.06)下调至(0.61±0.07)和(0.02±0.15),(P<0.01),COX-2蛋白表达从(0.56±0.03)下调至(0.42±0.02),(0.30±0.03),(P<0.01)。胞外信号调节激酶(ERK)磷酸化水平从(1.04±0.04)下调至(0.79±0.06),(0.73±0.10),(P<0.01)。p38磷酸化水平从(0.51±0.06)下调至(0.39±0.07),(0.29±0.15),(P<0.05),c-Jun氨基末端激酶(JNK)磷酸化水平从(1.05±0.03)下调至(0.65±0.02),(0.66±0.033),(P<0.01)。结论:芪麝丸部分通过抑制MAPK信号转导通路活化过程中关键蛋白胞外信号调节激酶(ERK)、JNK和p38丝裂原活化蛋白激酶(p38)磷酸化,下调iNOS基因和蛋白的表达从而减少NO的产量,同时下调COX-2蛋白表达,而发挥其抗炎效果。
Objective: To investigate the anti-inflammatory mechanism of Qishewan in mouse RAW264.7 macrophage inflammatory model in vitro. METHODS: Collagen γ (1 × 104U.mL-1) and lipopolysaccharide (100μg.L-1) synergistically induced mouse RAW264.7 macrophages (1 × 105 / mL) for 24 h to induce inflammation model. Griess reaction assay The production of nitric oxide (NO) in the cell supernatant was measured. The protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) and mitogen-activated protein kinase (MAPK) Activation of transduction pathway. Results: QSK (250, 500, 1000 mg.L-1) inhibited the NO content in the cell supernatant in a dose-dependent manner and showed no cytotoxicity. Compared with the model group, the iNOS protein expression decreased from (1.00 ± 0.06) to (0.61 ± 0.07) and (0.02 ± 0.15), (P <0.01) after treated with Qijishenwan 500,1 000 mg.L-1, The expression of COX-2 protein decreased from (0.56 ± 0.03) to (0.42 ± 0.02) and (0.30 ± 0.03), respectively (P <0.01). The phosphorylation of extracellular signal-regulated kinase (ERK) was decreased from (1.04 ± 0.04) to (0.79 ± 0.06), (0.73 ± 0.10), (P <0.01). The level of phosphorylation of c-Jun N-terminal kinase (JNK) was downregulated from (1.05 ± 0.03) to (0.39 ± 0.07), (0.29 ± 0.15), (0.65 ± 0.02), (0.66 ± 0.033), (P <0.01). Conclusion: QSK can down-regulate the expression of iNOS gene and protein by inhibiting phosphorylation of key proteins such as extracellular signal-regulated kinase (ERK), JNK and p38 mitogen-activated protein kinase (p38) during the activation of MAPK signal transduction pathway Reduce NO production while down-regulating COX-2 protein expression while exerting its anti-inflammatory effect.