目的 研究桑枝不同提取部位及其配比對脂多糖（LPS）協(xié)同γ-干擾素（IFN-γ）刺激的巨噬細胞RAW264.7中炎癥介質(zhì)的影響。方法 采用有機溶劑萃取和大孔樹脂富集的方法制備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組合分別作用于細胞后，Griess法測定RAW264.7細胞亞硝酸鹽的量；MTT法檢測細胞活力；三價鐵還原抗氧化能力測試（FRAP）法測定細胞抗氧化能力；半定量PCR法測定炎癥介質(zhì)基因的表達；Western blotting法測定炎癥介質(zhì)蛋白的表達。結(jié)果 桑枝4個提取部位中，I和II以劑量相關(guān)方式抑制細胞懸液中亞硝酸鹽的量，IC₅₀分別為145.23、152.14 mg/L。在I與II 3種配比（1∶1、1∶4、4∶1）組合中，1∶1配比時抑制細胞上清液中亞硝酸鹽量的IC₅₀值最低，為110.31 mg/L，且對細胞活力具有保護作用。與模型組相比，I和II在200 mg/L時顯著下調(diào)白細胞介素-1β（IL-1β）、IL-6、誘生型NO合酶（iNOS）、環(huán)氧合酶-2（COX-2）、核因子-κB（NF-κB）基因表達水平（P＜0.05、0.01），同時上調(diào)血紅素加氧酶（HO-1）和過氧化物酶增殖體受體（PPAR-γ）基因表達水平（P＜0.05），提高細胞抗氧化能力（P＜0.05），且抑制ERK蛋白的磷酸化（P＜0.05）。I和II（1∶1）組合對COX-2、IL-1β、HO-1、NF-κB、PPAR-γ的表達有一定的協(xié)同調(diào)控效果。結(jié)論 提取部位I和II是桑枝抗炎的活性部位，其部分通過NF-κB和ERK/MAPK信號轉(zhuǎn)導通路調(diào)控炎癥介質(zhì)的表達，其1∶1配比組合對炎癥中某些靶點均有較好的協(xié)同調(diào)控作用，抗炎效果更佳。

Abstract: 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 IC₅₀ (110.31 mg/L) for the 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 of IL-1β, 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-γ. Conclusion Fractions I and II of Mori Ramulus show the major anti-inflammatory activity which is partly through NF-κB 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.

國家科技重大專項重大新藥創(chuàng)制（2009ZX09311-003）；國家自然科學青年基金資助項目（81001666）；上海市教育委員會科研創(chuàng)新項目（13YZ048）；上海高校選拔培養(yǎng)優(yōu)秀青年教師基金資助項目（SZY07029）