紫堇灵的分离制备及抗炎活性
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摘要
旨在从苦地丁药材中分离出紫堇灵,从TLRs/NF-κB信号传导通路角度探讨其抗炎作用机制。以80%乙醇提取、硅胶柱色谱等方法制备紫堇灵,UPLC、MS、1H NMR、~(13)C NMR等方法进行纯度及结构鉴定;利用MTT实验评价紫堇灵的细胞毒性;采用脂多糖(LPS)诱导的RAW264.7巨噬细胞建立炎症模型,用紫堇灵进行干预,以Western blot技术检测RAW264.7巨噬细胞中TLR4、TLR2、IκBα、p-IκBα、p65和p-p65蛋白的表达水平;进而通过实时荧光定量PCR、Western blot技术分别检测p65 mRNA及细胞核p65表达情况。实验发现,在LPS诱导的RAW264.7炎症细胞模型中,5~40μmol/L紫堇灵呈正相关下调RAW264.7细胞中TLR4、TLR2蛋白的表达水平,抑制IκBα磷酸化并从基因水平和蛋白水平抑制p65转位入核。结果表明,紫堇灵可能是通过TLRs/NF-κB信号通路发挥对LPS致RAW264.7细胞炎症的抑制作用。
To isolate corynoline from Corydalis bungeana Turcz.and study its anti-inflammatory mechanism via TLRs/NF-κB signal pathway.Corynoline was extracted by 80% ethanol and purified by silicagel column chromatography.The structure and purity of corynoline was determined by UPLC,MS,~1H NMR and ~(13)C NMR.In the course of experiment,the cytotoxicity of corynoline was evaluated by MTT assay.And the inflammation model was established by RAW264.7 macrophages induced by lipopolysaccharide(LPS),which was intervened by corynoline.The expression levels of TLR4,TLR2 and nuclear transcription factor-κB(NF-κB) signaling pathways related proteinsin RAW264.7 macrophages were detected by Western blot.Furthermore,the expressionof NF-κB p65 mRNA and nuclear p65 were determined by the real-time fluorescence quantitative PCR(RT-q PCR) and Western blot.Results showed that 5-40 μmol/L corynoline reduced the expression level of TLR4 and TLR2,and inhibited the phosphorylation level of IκBα and the phosphorylation and nuclear translocation of p65 at gene and protein levelin a dose-dependent manner in LPS-induced RAW264.7 cells.This study indicated the protective effect of corynoline on LPS-induced RAW264.7 macrophages may be related with the inhibition of TLRs/NF-κB inflammatory signaling pathway.
To isolate corynoline from Corydalis bungeana Turcz.and study its anti-inflammatory mechanism via TLRs/NF-κB signal pathway.Corynoline was extracted by 80% ethanol and purified by silicagel column chromatography.The structure and purity of corynoline was determined by UPLC,MS,~1H NMR and ~(13)C NMR.In the course of experiment,the cytotoxicity of corynoline was evaluated by MTT assay.And the inflammation model was established by RAW264.7 macrophages induced by lipopolysaccharide(LPS),which was intervened by corynoline.The expression levels of TLR4,TLR2 and nuclear transcription factor-κB(NF-κB) signaling pathways related proteinsin RAW264.7 macrophages were detected by Western blot.Furthermore,the expressionof NF-κB p65 mRNA and nuclear p65 were determined by the real-time fluorescence quantitative PCR(RT-q PCR) and Western blot.Results showed that 5-40 μmol/L corynoline reduced the expression level of TLR4 and TLR2,and inhibited the phosphorylation level of IκBα and the phosphorylation and nuclear translocation of p65 at gene and protein levelin a dose-dependent manner in LPS-induced RAW264.7 cells.This study indicated the protective effect of corynoline on LPS-induced RAW264.7 macrophages may be related with the inhibition of TLRs/NF-κB inflammatory signaling pathway.
引文
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[2]Akira S,Takeda K,Kaisho T.Toll-like receptors:critical proteins linking innate and acquired immunity[J].Nat Immunol,2001,2(8):675-680.
[3]Zhao L,Su SQ,Xia L,et al.Regulation of total glucosides of paeony TLR2,4/NF-κB signal pathway in the kidney from diabetic rats[J].Acta Univ Med Anhui(安徽医科大学学报),2012,47(5):518-521.
[4]Chinese Pharmacopoeia Commission.Chinese Pharmacopoeia:Part1(中华人民共和国药典:一部)[S].Beijing:China Medical Science Press,2015:201.
[5]Zheng JF,Qin MJ,Zheng Y,et al.Alkaloids from Corydalis bungeana[J].J China Pharm Univ(中国药科大学学报),2007,38(2):112-114.
[6]Zhang XT,Chen JQ,Jiang CH,et al.Corydalis bungeana attenuates LPS-induced inflammatory responses via the suppression of NF-κB signaling pathway in vitro and in vivo[J].J Ethnopharmacol,2016,194(24):153-161.
[7]He ZB,Chen P,Peng ZY,et al.Effect of Corynoline isolated from Corydalis bungeana Turcz on lipopolysaccharides-induced sepsis in vivo and in vitro[J].Tropical J Pharm Res January,2014,13(1):81-86.
[8]Yang CJ,Zhang CY,Wang ZB,et al.Corynoline isolated from Corydalis bungeana Turcz.exhibits anti-inflammatory effects via modulation of Nfr2 and MAPKs[J].Molecules,2016,21(975):2-15.
[9]Du YX,Zhai XT,Zhu FX,et al.Study on UPLC fingerprint of Corydalis bungeana[J].J Chin Med Mater(中药材),2015,38(8):78-81.
[10]Ma WG,Fukushi Y,Tahara S.Fungitoxic alkaloids from Hokkaido Corydalis species[J].Fitoterapia,1999,70:258-265.
[11]Matsuguchi T,Musikachroen T,Ogawa T,et al.Gene expressions of Toll-like receptors 2,but not Toll-like receptor 4,is induced by LPS and inflammatory cytokines in mouse macrophages[J].J Immunol,2000,165:5767-5772.
[12]Medvedev AE,Kopydlowski KM,Vogel SN,et al.Inhibition of lipopolysaccharide-induced signal transduction in endotoxin-tolerized mouse macrophages:dysregulation of cytokine,chemokine,and Toll-like receptor 2 and 4 gene expression[J].J Immunol,2000,164:5564-5574.
[13]Wang AF,Guan SH,Yang K,et al.Enhancement of LPS on the antiviral effect of IFN-αvia increasing the expression of TLR2 and TLR4[J].Acta Univ Med Anhui(安徽医科大学学报),2014,49(1):18-21.
[14]Hayden MS,Ghosh S.Shared principles in NF-κB signaling[J].Cell,2008,132(3):344-362.
[15]Ardizzone S,Porro GB.Biologic therapy for inflammatory bowel disease[J].Drugs,2005,65(16):2253-2286.