草乌叶生物碱成分及其抗炎活性研究

Study on Anti-Inflammatory Active Components and Mechanism of Caowuye(Aconiti Kusnezoffii Folium)

目的探讨蒙古族习用药材草乌叶抗炎作用的物质基础及作用机制。方法制备草乌叶生物碱组分,并利用LC-MS技术进行质谱鉴定;进一步检测鉴定生物碱对LPS诱导的RAW264.7细胞炎症模型的影响。首先,采用CCK-8法检测生物碱对RAW264.7细胞的增殖-毒性作用,确定最大给药浓度;其次,通过PCR实验检测TNF-α、IL-10、IL-1β和iNOS的基因表达情况;采用Griess法测定NO浓度,ELISA法测定炎症因子TNF-α、IL-10、IL-1β含量;最后通过Western blotting实验检测COX-2蛋白表达情况。结果通过LC-MS技术同时鉴定了乌头碱、次乌头碱、新乌头碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、苯甲酰新乌头原碱等6种生物碱;上述6种生物碱对LPS诱导的炎症因子TNF-α、IL-10、IL-1β及NO具有不同程度的抑制作用,且同时可抑制iNOS基因表达及COX-2蛋白表达。结论草乌叶生物碱组分可含双酯型生物碱乌头碱、次乌头碱、新乌头碱及单酯型生物碱苯甲酰乌头原碱、苯甲酰次乌头原碱、苯甲酰新乌头原碱等6种生物碱;而这6种生物碱具有不同程度的体外抗炎效应,抗炎作用机制可能与抑制iNOS/COX-2炎症信号传导有关。

Objective To investigate the material basis and mechanism of anti-inflammatory effect of the traditional Mongolian medicine Caowuye(Aconiti Kusnezoffii Folium).Methods The alkaloid components of Caowuye(Aconiti Kusnezoffii Folium)were isolated and representative components in alkaloids were identified by liquid chromatograph mass spectrometer(LC-MS).Then,a cell counting kit-8 assay was used to determine the effects of alkaloid components on the viability of RAW264.7 macrophages.Secondly,anti-inflammation effects of alkaloid components in lipopolysaccharide(LPS)-stimulated RAW264.7 macrophages were measured based on the detection of tumor necrosis factor-α(TNF-α),interleukin-10(IL-10),interleukin-1β(IL-1β)and inducible nitric oxide synthase(iNOS)gene expressions.Griess reagent assay and ELISA assay were used to detect NO content and the inflammatory cytokines(TNF-α,IL-10 and IL-1β)in cell supernatant.Finally,the expression of COX-2 protein was detected by Western blotting method.Results Six alkaloids including aconitine,hypaconitine,neoaconitine,benzoyl aconitine,benzoyl hypaconitine and benzoyl neoaconitine were identified by LC-MS.These alkaloid components significantly reduced LPS-induced overproduction of NO and TNF-α,IL-10 and IL-1β.The alkaloid components also markedly decreased LPS-activated iNOS gene overexpression.Furthermore,the treatment with alkaloid components suppressed the COX-2 overexpression.Conclusion The data suggested that the alkaloid components were the material basis components of Caowuye(Aconiti Kusnezoffii Folium),and its anti-inflammatory mechanism might be related to inhibiting the iNOS/COX-2 signaling pathways.