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目的研究桑枝不同提取部位及其配比对脂多糖(LPS)协同γ-干扰素(IFN-γ)刺激的巨噬细胞RAW264.7中炎症介质的影响。方法采用有机溶剂萃取和大孔树脂富集的方法制备桑枝乙醇提取物不同萃取部位Ⅰ～Ⅳ。各提取部位及不同部位组合分别作用于细胞后,Griess法测定RAW264.7细胞亚硝酸盐的量;MTT法检测细胞活力;三价铁还原抗氧化能力测试(FRAP)法测定细胞抗氧化能力;半定量PCR法测定炎症介质基因的表达;Western blotting法测定炎症介质蛋白的表达。结果桑枝4个提取部位中,Ⅰ和Ⅱ以剂量相关方式抑制细胞悬液中亚硝酸盐的量,IC50分别为145.23、152.14 mg/L。在Ⅰ与Ⅱ 3种配比(1∶1、1∶4、4∶1)组合中,1∶1配比时抑制细胞上清液中亚硝酸盐量的IC50值最低,为110.31 mg/L,且对细胞活力具有保护作用。与模型组相比,Ⅰ和Ⅱ在200 mg/L时显著下调白细胞介素-1β(IL-1β)、IL-6、诱生型NO合酶(iNOS)、环氧合酶-2(COX-2)、核因子-κB(NF-κB)基因表达水平(P<0.05、0.01),同时上调血红素加氧酶(HO-1)和过氧化物酶增殖体受体(PPAR-γ)基因表达水平(P<0.05),提高细胞抗氧化能力(P<0.05),且抑制ERK蛋白的磷酸化(P<0.05)。Ⅰ和Ⅱ(1∶1)组合对COX-2、IL-1β、HO-1、NF-κB、PPAR-γ的表达有一定的协同调控效果。结论提取部位Ⅰ和Ⅱ是桑枝抗炎的活性部位,其部分通过NF-κB和ERK/MAPK信号转导通路调控炎症介质的表达,其1∶1配比组合对炎症中某些靶点均有较好的协同调控作用,抗炎效果更佳。
Objective To study the effects of different extraction sites and their ratios on the inflammatory mediators in macrophages RAW264.7 stimulated by lipopolysaccharide (LPS) and interferon γ (IFN-γ). Methods The organic extracts and macroporous resin were used to prepare Ⅰ ~ Ⅳ of different extraction sites of ethanol extract from mulberry branches. The amount of nitrite in RAW264.7 cells was determined by Griess method after the combination of different parts and different parts of the cells were applied to the cells. The cell viability was measured by MTT assay. The antioxidant capacity of cells was measured by the ferric reduction-antioxidant capacity test (FRAP) Semi-quantitative PCR was used to detect the expression of inflammatory mediators. Western blotting was used to detect the expression of inflammatory mediators. Results In four extracts of mulberry branches, Ⅰ and Ⅱ dose-dependently inhibited the amount of nitrite in the cell suspension with IC50 of 145.23 and 152.14 mg / L, respectively. In the combination of Ⅰ and Ⅱ (1: 1, 1: 4, 4:1), IC50 value of inhibiting nitrite in the cell supernatant was the lowest at the ratio of 1: 1, which was 110.31 mg / L , And has a protective effect on cell viability. Compared with the model group, Ⅰ and Ⅱ significantly reduced the levels of interleukin-1β (IL-1β), IL-6, iNOS, COX (P <0.05, 0.01), upregulated the expression of HO-1 and PPAR-γ, and increased the expression of nuclear factor-κB (NF- (P <0.05), increased the antioxidant capacity of cells (P <0.05), and inhibited the phosphorylation of ERK protein (P <0.05). The combination of Ⅰ and Ⅱ (1: 1) had a synergistic effect on the expression of COX-2, IL-1β, HO-1, NF-κB and PPAR-γ. Conclusion The extracted fractions Ⅰ and Ⅱ are the active site of mulberry branch anti-inflammatory, which partly regulate the expression of inflammatory mediators through NF-κB and ERK / MAPK signal transduction pathways. Better coordination and control, anti-inflammatory effect is better.