基于ROS/NF-κB信号通路的天南星凝集素致炎机制及炮制对蛋白的影响
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摘要
目的:研究天南星科有毒中药天南星凝集素蛋白(AEL)的致炎机制及炮制对蛋白的影响。方法:通过体外培养RAW264.7巨噬细胞,经不同浓度AEL刺激1h后,ELISA法检测炎性因子肿瘤坏死因子α(TNF-α)、白细胞介素-1β(IL-1β)的释放,Western blot法检测p-IκB、IκB、p-p65、p65蛋白的表达,倒置荧光显微镜及荧光酶标仪检测细胞内活性氧(ROS)、线粒体膜电位及胞内游离钙离子水平;加入ROS的阻断剂N-乙酰半胱氨酸(NAC)后,采用ELISA和Western blot法检测TNF-α、IL-1β及p-IκB、IκB、p-p65、p65蛋白表达的变化。采用8%白矾溶液炮制AEL,ELISA法检测炮制前后炎性因子TNF-α、IL-1β释放的变化。结果:AEL刺激巨噬细胞可导致炎性因子TNF-α、IL-1β大量释放,促进p-IκB、p-p65水平升高,导致巨噬细胞中ROS水平升高,线粒体膜电位降低以及胞内游离钙离子水平升高。当加入ROS的阻断剂NAC后,可明显降低ROS的生成,同时抑制了IκB、p65的磷酸化以及炎性因子TNF-α、IL-1β的大量释放。AEL经8%白矾溶液炮制后,能显著降低巨噬细胞炎性因子TNF-α、IL-1β的大量释放。结论:AEL刺激巨噬细胞后可诱导氧化应激,生成过量ROS,进而激活NF-κB炎性信号通路并导致炎性因子大量释放。AEL经白矾炮制后毒性降低,推测凝集素蛋白在白矾溶液中发生变性或降解,这可能是矾制天南星减毒的机制之一。
Objective: To investigate the pro-inflammatory mechanism of Arisaema erubescens lectin(AEL) and its processing effect on protein. Methods: RAW 264.7 macrophage cells were cultured in vitro, and they were stimulated by different concentrations of AEL for one hour. ELISA assay was used for the detection of TNF-α, IL-1β. Western blot assay was used to detect the levels of p-IκB, IκB, p-p65 and p65, and fluorescent inverted microscope and microplate reader were applied for ROS, mitochondrial membrane potential(MMP) and cytosolic free Ca2+. N-acetylcysteine(NAC), an ROS blocking agent, was added to reduce ROS production, and the expression of TNF-α, IL-1β and p-IκB, IκB, p-p65, p65 were detected by ELISA and Western blot. AEL was processed by 8%alum solution, and ELISA assay was used for the detection of TNF-α, IL-1β before and after processing. Results: AEL could stimulate macrophage and cause the release of inflammation TNF-α, IL-1β and increase the level of p-IκB, p-p65, and up-regulation of ROS and cytosolic free Ca2+, but down-regulation of MMP. When NAC was added to the ROS, the production of ROS could be significantly reduced, and the phosphorylation of IκB and p65 and the release of inflammatory cytokines were inhibited. AEL which processed by 8%alum solution could inhibit the production of TNF-α and IL-1β. Conclusion: AEL could cause oxidative stress and generate excess ROS and stimulate the NF-κB signaling pathway, and the overproduction of pro-inflammatory factors. 8%alum solution could inhibit the toxicity of AEL, so it is speculated that the lectin hydrolyzed in alum solution denatured or dissolved under the action of ions, which may be one of the mechanisms of processing by alum.
Objective: To investigate the pro-inflammatory mechanism of Arisaema erubescens lectin(AEL) and its processing effect on protein. Methods: RAW 264.7 macrophage cells were cultured in vitro, and they were stimulated by different concentrations of AEL for one hour. ELISA assay was used for the detection of TNF-α, IL-1β. Western blot assay was used to detect the levels of p-IκB, IκB, p-p65 and p65, and fluorescent inverted microscope and microplate reader were applied for ROS, mitochondrial membrane potential(MMP) and cytosolic free Ca2+. N-acetylcysteine(NAC), an ROS blocking agent, was added to reduce ROS production, and the expression of TNF-α, IL-1β and p-IκB, IκB, p-p65, p65 were detected by ELISA and Western blot. AEL was processed by 8%alum solution, and ELISA assay was used for the detection of TNF-α, IL-1β before and after processing. Results: AEL could stimulate macrophage and cause the release of inflammation TNF-α, IL-1β and increase the level of p-IκB, p-p65, and up-regulation of ROS and cytosolic free Ca2+, but down-regulation of MMP. When NAC was added to the ROS, the production of ROS could be significantly reduced, and the phosphorylation of IκB and p65 and the release of inflammatory cytokines were inhibited. AEL which processed by 8%alum solution could inhibit the production of TNF-α and IL-1β. Conclusion: AEL could cause oxidative stress and generate excess ROS and stimulate the NF-κB signaling pathway, and the overproduction of pro-inflammatory factors. 8%alum solution could inhibit the toxicity of AEL, so it is speculated that the lectin hydrolyzed in alum solution denatured or dissolved under the action of ions, which may be one of the mechanisms of processing by alum.
引文
[1]国家中医药管理局《中华本草》编委会.中华本草精选本(下册).上海:上海科学技术出版社,1998:2212
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[7]Jiang Q L,Zhang S,Tian M,et al.Plant lectins,from ancient sugarbinding proteins to emerging anti-cancer drugs in apoptosis and autophagy.Cell Proliferation,2015,48(1):17-28
[8]Panda P K,Mukhopadhyay S,Behera B,et al.Antitumor effect of soybean lectin mediated through reactive oxygen speciesdependent pathway.Life Sciences,2014,111(1-2):27-35
[9]Tao L,Lei W,Di W,et al.Role of reactive oxygen species mediated MAPK and NF-?B activation in Polygonatum cyrtonema lectin induced apoptosis and autophagy in human lung adenocarcinoma A549 cells.Journal of Biochemistry,2016,160(6):315-324
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[13]Vajravijayan S,Pletnev S,Pletnev V Z,et al.Structural analysis ofβ-prism lectin from Colocasia esculenta(L.)Schott.International Journal of Biological Macromolecules,2016,91:518-523
[14]Heim C,Hertzberg H,Butschi A,et al.Inhibition of Haemonchus contortus,larval development by fungal lectins.Parasites&Vectors,2015,8(1):425-435
[15]段晓亮.三种植物凝集素基因在转基因小麦中的表达及其抗蚜效果分析.北京:中国农业大学,2013
[16]Pilar J,Patricia C,Basterrechea J E,et al.Isolation and Molecular Characterization of Two Lectins from Dwarf Elder(Sambucus ebulus L).Blossoms Related to the Samn1 Allergen.Toxins,2013,5(10):1767-1779
[17]Kabir S R,Nabi M M,Nurujjaman M,et al.Momordica charantia,Seed Lectin:Toxicity,Bacterial Agglutination and Antitumor Properties.Applied Biochemistry and Biotechnology,2015,175(5):2616-2628
[18]潘耀宗.天南星科有毒中药毒性作用机制.南京:南京中医药大学,2016
[19]Yu H,Pan Y,Wu H,et al.The alum-processing mechanism attenuating toxicity of Araceae Pinellia ternata and Pinellia pedatisecta.Archives of Pharmacal Research,2015,38(10):1810-1821
[20]Zhou R,Yazdi A S,Menu P,et al.A role for mitochondria in NLRP3inflammasome activation.Nature,2011,469(7329):221-225
[21]Olsson M,Zhivotovsky B.Caspases and cancer.Cell Death&Differentiation,2011,18(18):1441-1449
[22]马文宇,陈晓昱,吴邓婷.沙棘水提物对短波紫外线诱导Ha Ca T细胞凋亡及线粒体膜电位的影响.中华中医药杂志,2017,32(12):5633-5636
[23]Upton,Jason W,Kaiser,et al.Virus Inhibition of RIP3-Dependent Necrosis.Cell Host&Microbe,2010,7(4):302-313
[2]倪朱谟.本草汇言.上海:上海科学技术出版社,2005
[3]葛秀允.天南星科有毒中药刺激性毒性成分及矾制解毒共性机制研究.南京:南京中医药大学,2009
[4]Liu X Q,Wu H,Yu H L,et al.Purification of a lectin from Arisaema erubescens(Wall.)Schott and its pro-inflammatory effects.Molecules,2011,16(11):9480-9494
[5]Yu H L,Zhao T F,Wu H,et al.Pinellia ternata lectin exerts a proinflammatory effect on macrophages by inducing the release of pro-inflammatory cytokines,the activation of the nuclear factor-κB signaling pathway and the overproduction of reactive oxygen species.International Journal of Molecular Medicine,2015,36(4):1127-1135
[6]潘耀宗,郁红礼,吴皓,等.掌叶半夏毒针晶及凝集素蛋白的致炎作用与巨噬细胞的相关性研究.中华中医药杂志,2014,29(5):1397-1401
[7]Jiang Q L,Zhang S,Tian M,et al.Plant lectins,from ancient sugarbinding proteins to emerging anti-cancer drugs in apoptosis and autophagy.Cell Proliferation,2015,48(1):17-28
[8]Panda P K,Mukhopadhyay S,Behera B,et al.Antitumor effect of soybean lectin mediated through reactive oxygen speciesdependent pathway.Life Sciences,2014,111(1-2):27-35
[9]Tao L,Lei W,Di W,et al.Role of reactive oxygen species mediated MAPK and NF-?B activation in Polygonatum cyrtonema lectin induced apoptosis and autophagy in human lung adenocarcinoma A549 cells.Journal of Biochemistry,2016,160(6):315-324
[10]Tajeddine N.How do reactive oxygen species and calcium trigger mitochondrial membrane permeabilisation?.Biochimica Et Biophysica Acta,2016,1860(6):1079-1088
[11]Ma S,Liu H,Jiao H,et al.Neuroprotective effect of ginkgolide K on glutamate-induced cytotoxicity in PC 12 cells via inhibition of ROS generation and Ca2+influx.Neurotoxicology,2012,33(1):59-69
[12]Liu X,Tian X,Liu T,et al.Disclosure of the Tuberous Lectin Composed of Homogeneous Tetramers in Pinellia pedatisecda,Schott.Applied Biochemistry and Biotechnology,2010,162(4):1214-1223
[13]Vajravijayan S,Pletnev S,Pletnev V Z,et al.Structural analysis ofβ-prism lectin from Colocasia esculenta(L.)Schott.International Journal of Biological Macromolecules,2016,91:518-523
[14]Heim C,Hertzberg H,Butschi A,et al.Inhibition of Haemonchus contortus,larval development by fungal lectins.Parasites&Vectors,2015,8(1):425-435
[15]段晓亮.三种植物凝集素基因在转基因小麦中的表达及其抗蚜效果分析.北京:中国农业大学,2013
[16]Pilar J,Patricia C,Basterrechea J E,et al.Isolation and Molecular Characterization of Two Lectins from Dwarf Elder(Sambucus ebulus L).Blossoms Related to the Samn1 Allergen.Toxins,2013,5(10):1767-1779
[17]Kabir S R,Nabi M M,Nurujjaman M,et al.Momordica charantia,Seed Lectin:Toxicity,Bacterial Agglutination and Antitumor Properties.Applied Biochemistry and Biotechnology,2015,175(5):2616-2628
[18]潘耀宗.天南星科有毒中药毒性作用机制.南京:南京中医药大学,2016
[19]Yu H,Pan Y,Wu H,et al.The alum-processing mechanism attenuating toxicity of Araceae Pinellia ternata and Pinellia pedatisecta.Archives of Pharmacal Research,2015,38(10):1810-1821
[20]Zhou R,Yazdi A S,Menu P,et al.A role for mitochondria in NLRP3inflammasome activation.Nature,2011,469(7329):221-225
[21]Olsson M,Zhivotovsky B.Caspases and cancer.Cell Death&Differentiation,2011,18(18):1441-1449
[22]马文宇,陈晓昱,吴邓婷.沙棘水提物对短波紫外线诱导Ha Ca T细胞凋亡及线粒体膜电位的影响.中华中医药杂志,2017,32(12):5633-5636
[23]Upton,Jason W,Kaiser,et al.Virus Inhibition of RIP3-Dependent Necrosis.Cell Host&Microbe,2010,7(4):302-313