苦地丁的抗炎药效物质基础组分及作用机制研究

苦地丁常用于治疗多种炎症,为确定其抗炎药效物质基础,该文利用自动纯化系统对苦地丁提取物进行分离制备,制备了黄酮组分和生物碱组分,并对所制备的组分进行了质谱鉴定;进一步检测组分对LPS诱导RAW264.7细胞炎症模型的影响。首先,用LPS处理小鼠巨噬细胞(RAW264.7),观察其细胞存活率与LPS浓度的关系;然后检测不同给药浓度的黄酮组分和生物碱组分对细胞存活率的影响,确定最大给药浓度。其次,分别加入质量浓度为2.5,5,10,20μg·mL^-1生物碱组分和黄酮组分,观察不同浓度生物碱组分和黄酮组分对LPS导致的RAW264.7小鼠巨噬细胞炎症的影响及其作用机制。Griess试剂法检测细胞上清液NO含量;ELISA法检测细胞上清液炎症因子(TNF-α,IL-1β,IL-6)的表达;Western blot法检测细胞内核因子κB(NF-κB)IκBα磷酸化(p-IκBα)、p65磷酸化(p-p65)以及TLR4和TLR2的蛋白表达。结果显示,生物碱组分在2.5~20μg·mL^-1呈剂量依赖地对NO,TNF-α,IL-1β,IL-6有显著抑制作用。在炎症上游通路,生物碱组分对TLR4表达水平的抑制作用强于TLR2,在炎症下游NF-κB通路,2.5~20μg·mL-1生物碱组分呈剂量依赖方式显著抑制IκBα及p65磷酸化。由此可知,生物碱组分是苦地丁抗炎药效组分,其抗炎作用机制可能为,通过抑制炎症信号在以TLR4为主的TLRs/NF-κB信号通路的传导,干扰炎性基因的激活抑制其过量表达,下调炎性因子的分泌水平达到炎症抑制作用。

Study on anti-inflammatory active components and mechanism of Corydalis Bungeanae Herba

Corydalis Bungeanae Herba is often used to treat a variety of inflammatory diseases in traditional Chinese medicine.In order to determine its chemical material basis,the components of Corydalis Bungeanae Herba were isolated by automated purification system.Flavonoids and alkaloids were prepared,and all such components were identified by mass spectrometry.The effects of the components on the production of inflammatory mediators and pharmacological mechanisms in the lipopolysaccharide(LPS)-induced RAW264.7 cell inflammation model were examined.Mouse macrophages(RAW264.7)were first treated with LPS.The relationship between cell viability and LPS concentration was observed.Then,the effects of flavonoids components and alkaloid components with different administration concentrations on cell viability were detected to determine the maximum administration concentration.Secondly,2.5,5,10 and 20μg·mL^-1 flavonoids components and alkaloid components were added respectively to observe the effects and mechanism of different concentrations of flavonoids components and alkaloid components on LPS-induced inflammation of RAW264.7 macrophages.Griess reagent assay was used to detect NO content in cell supernatant.The inflammatory cytokines(TNF-α,IL-1βand IL-6)in cell supernatant were determined by ELISA method.Western blot method was used to detect the intracellular nuclear factor(NF-κB)IκBαphosphorylation(p-IκBα),p65 phosphorylation(p-p65)and protein expression of TLR4,TLR2.The results showed that the alkaloid components inhibited the production of NO,TNF-α,IL-1βand IL-6 in a dose-dependent mannerin the concentration range of 2.5-20μg·mL^-1.In inflammation upstream pathways,the inhibitory effect of the alkaloid components on the TLR2 expression level was weaker than that of TLR4.In inflammation downstream,alkaloid components significantly inhibited phosphorylation of IκBαand p65 in a dose-dependent manner.These data suggested that the alkaloid components were the material basis components of Corydalis Bungeanae Herba,and its anti-inflammatory mechanism might be related to inhibiting the transmission of inflammatory signals in TLRs/NF-κB signaling pathways dominated by TLR4,interfering with the activation of inflammatory genes and inhibiting their over expression,and down-regulating the secretion level of inflammatory factors.