金钗石斛有效成分对脂多糖激活星形胶质细胞产生炎症因子的影响
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摘要
目的：观察金钗石斛生物总碱、石斛碱、石斛多糖对外源性内毒素脂多糖(lipopolysaecharide, LPS)激活大鼠大脑皮层星形胶质细胞(astrocyte, Ac)及诱导其产生和释放炎性介质的影响,重点探讨石斛生物总碱对星形胶质细胞的保护作用及机制。方法：分离、制备新生大鼠大脑皮层星形胶质细胞,传代至第三代时用LPS刺激细胞,观察不同浓度的石斛生物总碱(0.025、0.25、2.5mg/L)、石斛碱(0.025、2.5mg/L)、石斛多糖(0.025、0.25、2.5mg/L)对细胞激活的影响。通过MTS检测细胞存活率,试剂盒检测NO释放量,ELISA法检测NF-α、IL-6炎性因子蛋白的表达,实时定量多聚酶链反应(RT-PCR)检测炎症相关基因TNF-α、IL-6 mRNA的表达。结果：1.LPS刺激星形胶质细胞后MTS检测吸光度明显升高,NO含量、TNF-a和IL-6 mRNA及其蛋白表达均明显升高,与正常组比较有显著性差异(P<0.01)；2.石斛生物总碱3个剂量组的吸光度值均比模型组低,并减少LPS诱导的NO释放,均降低LPS所致的TNF-α、IL-6基因及蛋白的高表达(P<0.05)；3.石斛多糖3个剂量组对LPS诱导的NO释放具有改善作用(P<0.05)；石斛碱只有在高剂量时才能减少LPS诱导的NO释放。结论：1.石斛生物总碱能明显抑制LPS对星形胶质细胞的激活,减少炎性因子的释放,提示其具有神经保护作用；2.石斛多糖和石斛碱减少LPS诱导的NO释放,提示两者均可能是金钗石斛抗炎作用的主要有效成分。
Objective To investigate the impact of total alkaloids, dendrobine and polysaccharides extracted from Dendrobium nobile on inflammatory factors, which are produced and released by lipopolysaecharide (LPS)-activated rat cerebral astrocytes (Ac), and to explore the protective mechanisms of Dendrobium total alkaloids against LPS-induced astrocytes damage. Methods:The cerebral cortex of newborn rat was dissected and astrocytes were isolated and cultured. The 3rd generation of cultural astrocytes were randomly divided into control, model (LPS 10 ng/ml), total alkaloids (0.025,0.25 and 2.5 mg/L), dendrobine (0.025,2.5 mg/L), polysaccharide (0.025,0.25 and 2.5 mg/L). LPS stimulated astrocytes to produce and release a large number of inflammatory cytokines, on which the effects of different concentrations of total alkaloids, dendrobine and polysaccharide were examined. The cell viability was tested by MTS; the expressions of TNF-a, IL-6 mRNA in astrocytes were detected through real-time RT-PCR; the generation and release of TNF-a, IL-6 from astrocytes were evaluated via enzyme link immunosorbent assay (ELISA) assay. Results:1. LPS activated astrocytes resulting in significantly elevated absorbance tested by MTS, increased the level of NO, up-regulated the expressions of TNF-a and IL-6 mRNA and protein (compared with control group P<0.01); 2. The different concentrations of total alkaloids can decrease LPS-induced high absorbance, depress the high-level of NO, ameliorate the up-expression of TNF-a, IL-6 mRNA and protein by LPS (P<0.05); 3. The different concentrations of polysaccharides can significantly decrease the high-level of NO induced by LPS, whereas Dendrobine only at the high dose can depress the level of NO. Conclusion:1. Dendrobium total alkaloids can significantly inhibit the activation of astrocytes induced by LPS and reduce the release of inflammatory factors. These results indicated that Dendrobium total alkaloids exert neuroprotection through depressing the actived astrocytes.2. Dendrobine and polysaccharides can reduce the LPS-induced release of NO, this suggested the two components may play a role in anti-inflammatory effects of Dendrobium nobile.
Objective To investigate the impact of total alkaloids, dendrobine and polysaccharides extracted from Dendrobium nobile on inflammatory factors, which are produced and released by lipopolysaecharide (LPS)-activated rat cerebral astrocytes (Ac), and to explore the protective mechanisms of Dendrobium total alkaloids against LPS-induced astrocytes damage. Methods:The cerebral cortex of newborn rat was dissected and astrocytes were isolated and cultured. The 3rd generation of cultural astrocytes were randomly divided into control, model (LPS 10 ng/ml), total alkaloids (0.025,0.25 and 2.5 mg/L), dendrobine (0.025,2.5 mg/L), polysaccharide (0.025,0.25 and 2.5 mg/L). LPS stimulated astrocytes to produce and release a large number of inflammatory cytokines, on which the effects of different concentrations of total alkaloids, dendrobine and polysaccharide were examined. The cell viability was tested by MTS; the expressions of TNF-a, IL-6 mRNA in astrocytes were detected through real-time RT-PCR; the generation and release of TNF-a, IL-6 from astrocytes were evaluated via enzyme link immunosorbent assay (ELISA) assay. Results:1. LPS activated astrocytes resulting in significantly elevated absorbance tested by MTS, increased the level of NO, up-regulated the expressions of TNF-a and IL-6 mRNA and protein (compared with control group P<0.01); 2. The different concentrations of total alkaloids can decrease LPS-induced high absorbance, depress the high-level of NO, ameliorate the up-expression of TNF-a, IL-6 mRNA and protein by LPS (P<0.05); 3. The different concentrations of polysaccharides can significantly decrease the high-level of NO induced by LPS, whereas Dendrobine only at the high dose can depress the level of NO. Conclusion:1. Dendrobium total alkaloids can significantly inhibit the activation of astrocytes induced by LPS and reduce the release of inflammatory factors. These results indicated that Dendrobium total alkaloids exert neuroprotection through depressing the actived astrocytes.2. Dendrobine and polysaccharides can reduce the LPS-induced release of NO, this suggested the two components may play a role in anti-inflammatory effects of Dendrobium nobile.
引文
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8.陈云龙等,细叶石斛有效成分分析及其水溶性提取物的血管舒张活性.植物资源与环境学报,2003,12(1)：6-9
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11.施红等,石斛复方制剂的抗氧化功能和降低血糖作用.福建中医学院学报1997,7(3)：26-28
12. Lee Y H, Park J D, Baek N I, Kim S I, Ahn BZ. In vitro and in vivo antit urmoral phenanthrenes from the aerial parts of Dendrobium nobile Planta Med.1 995 Apr; 6(2):178-80
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