桑枝提取部位及其组合对巨噬细胞炎症介质的影响  

Effects of different extracting fractions from Mori Ramulus and their combinations on inflammatory mediators in macrophage

作　　者：章丹丹[1] 唐宁[1] 华晓东[1] 卞卡[1,2] 

机构地区：[1]上海中医药大学穆拉德中药现代化研究中心,上海201203 [2]美国乔治华盛顿大学生物化学与分子生物学系,华盛顿特区20052

出　　处：《中草药》2013年第2期186-192,共7页Chinese Traditional and Herbal Drugs

基　　金：国家科技重大专项重大新药创制(2009ZX09311-003);国家自然科学青年基金资助项目(81001666);上海市教育委员会科研创新项目(13YZ048);上海高校选拔培养优秀青年教师基金资助项目(SZY07029)

摘　　要：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 investigate the effects of different extracting fractions from Mori Ramulus and their combinations on the inflammatory mediators in RAW264.7 macrophages stimulated with interferon-γ （IFN-γ） plus lipopolysaccharide （LPS）. Methods Organic solvent extraction, separation, and macroporous resin purification were performed to obtain the fractions I--IV from the ethanol extract of Mori Ramulus. After cells were treated with the different extraccting fractions from Mori Ramulus and their combinations, Griess reaction for nitric oxide production, MTT assay for cell viability, and FRAP assay for anti-oxidant activity with trolox as control. RT-PCR for mRNA expression and Western blotting for protein expression examination were performed. Results The fractions I and II inhibited nitrite/nitrate of stimulated macrophage in a dose-dependent manner with IC50 145.23 and 152.14 mg/L, respectively. Among the three combinations of fractions I and II （1 ： 4, 1 ： 1, and 4 ： 1）, the lowest ICs0 （110.31 mg/L） forthe nitrite/nitrate inhibition was demonstrated and protective effect against cell viability was shown in 1 ： 1 group. Fractions I and II （200 mg/L） suppressed the gene expression ofIL-1γ, IL-6, iNOS, COX-2, and NF-κB （P 〈 0.05, 0.01）, while up-regulated HO-1 （P 〈 0.05）, PPAR-γ （P 〈 0.05） expression and improved anti-oxidant activity （P 〈 0.05）. The fractions also inhibited ERK protein phosphorylation （P 〈 0.05）. The combination of fractions I and II （1 ： 1） exhibited the coordinated expression on COX-2, IL-1β, HO-1, NF-κB, and PPAR-7. Conclusion Fractions I and II ofMori Ramulus show the major anti-inflammatory activity which is partly through NF-κ3 and ERK/MAPK signaling pathway to regulate the expression of inflammatory mediators. The combination of fractions I and II （1 ： 1） shows the coordinated regulation towards some targets in the inflammation with better anti-inflammatory activity.

关 键 词：桑枝提取物 巨噬细胞 炎症介质 ERK MAPK信号转导通路 核因子-ΚB 

分 类 号：R282.710.5[医药卫生—中药学]