小檗碱通过抑制经典的NLRP3炎症小体活化通路来下调LPS诱导的HUVEC炎症反应
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摘要
目的:探讨小檗碱(Berberine, Ber)对脂多糖(Lipopolysaccharide, LPS)诱导的人脐静脉血管内皮细胞(Human Umbilical VeinEndothelial Cells, HUVEC)炎症反应的抑制作用及相关机制。方法:MTT法检测不同浓度的Ber对HUVEC存活率的影响。以0.05μg/mL LPS刺激HUVEC建立炎症反应模型,并分别给予1.25、2.5、5μM Ber干预,Elisa检测Ber对炎症因子TNF-α、IL-1β分泌的影响,免疫印迹法检测Ber对NLRP3炎症小体信号通路中NLRP3、My D88、IL-1β、TLR4、ASC和Caspase-1蛋白表达的影响。结果:不同浓度的Ber对HUVEC增殖均具有抑制作用,且基本呈现浓度梯度,IC50值接近5μM;与LPS组相比,不同浓度Ber(1.25、2.5、5μM)均可以显著下调HUVEC中TNF-α、IL-1β炎症因子的分泌(P<0.005及P<0.01),并可以显著抑制细胞中NLRP3、My D88、IL-1β、TLR4、ASC和Caspase-1蛋白的表达(P<0.05),其抑制作用基本呈剂量关系,以5μM Ber的抑制效果最佳。结论:Ber可以通过抑制经典的NLRP3炎症小体活化通路来下调LPS诱导的HUVEC炎症反应。
Objective: To investigate the inhibition and related molecular mechanisms of berberine on inflammation induced by LPS in HUVEC. Methods: The viability of different concentrations of berberine on HUVEC cells were determined by MTT.An inflammatory model of HUVEC was induced by 0.05 μg/mL LPS, then 1.25, 2.5, 5 μM berberine were used for intervention. The expressions of TNF-α, IL-1β in cell supernatants were evaluated by ELISA. The expressions of NLRP3, My D88, IL-1β, TLR4, ASC and Caspase-1 proteins associating with Classical NLRP3 signaling pathway were evaluated by Western blot. Results: The proliferation of HUVEC were restrained by different concentrations of berberine, and there existed a concentration gradient. The IC50 value of berberine on HUVEC was close to 5 μM. Compared with LPS group, different concentrations of berberine could significantly reduce the secretions of TNF-α, IL-1β and the expressions of NLRP3, My D88, IL-1β, TLR4, ASC and Caspase-1 proteins in HUVEC. The inhibition had a dose dependent manner, and 5 μM berberine showed the greatest effect. Conclusion: Berberine could reduce inflammation induced by LPS in HUVEC through suppressing Classical NLRP3 inflammasome activiation pathway.
Objective: To investigate the inhibition and related molecular mechanisms of berberine on inflammation induced by LPS in HUVEC. Methods: The viability of different concentrations of berberine on HUVEC cells were determined by MTT.An inflammatory model of HUVEC was induced by 0.05 μg/mL LPS, then 1.25, 2.5, 5 μM berberine were used for intervention. The expressions of TNF-α, IL-1β in cell supernatants were evaluated by ELISA. The expressions of NLRP3, My D88, IL-1β, TLR4, ASC and Caspase-1 proteins associating with Classical NLRP3 signaling pathway were evaluated by Western blot. Results: The proliferation of HUVEC were restrained by different concentrations of berberine, and there existed a concentration gradient. The IC50 value of berberine on HUVEC was close to 5 μM. Compared with LPS group, different concentrations of berberine could significantly reduce the secretions of TNF-α, IL-1β and the expressions of NLRP3, My D88, IL-1β, TLR4, ASC and Caspase-1 proteins in HUVEC. The inhibition had a dose dependent manner, and 5 μM berberine showed the greatest effect. Conclusion: Berberine could reduce inflammation induced by LPS in HUVEC through suppressing Classical NLRP3 inflammasome activiation pathway.
引文
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[9]Wang ZH,Mong MC,Yang YC,et al.Asiatic acid and maslinic acid attenuated kainic acid-induced seizure through decreasing hippocampal inflammatory and oxidative stress[J].Epilepsy Res,139:28-34
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[11]Yadav N,Chandra H.Suppression of inflammatory and infection responses in lung macrophages by eucalyptus oil and its constituent1,8-cineole:Role of pattern recognition receptors TREM-1 and NLRP3,the MAP kinase regulator MKP-1,and NFκB[J].PLo S One2017,12(11):e0188232
[12]毛开睿,孙兵.NLRP3炎症小体研究进展[J].现代免疫学,2011,31(1):1-3Mao Kai-rui,Sun Bing.The progress of NLRP3 inflammasome[J].Current Immunology,2011,31(1):1-3
[13]Zhu Y,Zhu H,Wang Z,et al.Wogonoside alleviates inflammation induced by traumatic spinal cord injury by suppressing NF-κB and NLRP3 inflammasome activation[J].Exp Ther Med,2017,14(4):3304-3308
[14]Song N,Liu ZS,Xue W,et al.NLRP3 Phosphorylation Is an Essential Priming Event for Inflammasome Activation[J].Mol Cell,2017,68(1):185-197
[15]Evavold C,Kagan JC.How inflammasomes inform adaptive immunity[J].J Mol Biol,2017,36(17):30470-30479
[16]Subramanian N,Natarajan K,Clatworthy MR,et al.The adaptor MAVS promotes NLRP3 mitochondrial localization and inflammasome activation[J].Cell,2013,153(2):348-361
[17]Freeman L,Guo H,David CN,et al.NLR members NLRC4 and NLRP3 mediate sterile inflammasome activation in microglia and astrocytes[J].J Exp Med,2017,214(5):1351-1370
[18]Liston A,Masters SL.Homeostasis-altering molecular processes as mechanisms of inflammasome activation[J].Nat Rev Immunol,2017,17(3):208-214
[19]Negash AA,Ramos HJ,Crochet N,et al.IL-1βproduction through the NLRP3 inflammasome by hepatic macrophages links hepatitis Cvirus infection with liver inflammation and disease[J].PLo S Pathog,2013,9(4):e1003330
[20]Broz P,Dixit VM.Inflammasomes:mechanism of assembly,regulation and signaling[J].Nat Rev Immunol,2016,16(7):407-420
[21]Liston A,Masters SL.Homeostasis-altering molecular processes as mechanisms of inflammasome activation[J].Nat Rev Immunol,2017,17(3):208-214
[22]Wang X,Gong P,Zhang X,et al.NLRP3 inflammasome activation in murine macrophages caused by Neospora caninum infection[J].Parasit Vectors,2017,10(1):266
[23]Kayagaki N,Wong MT,Stowe IB,et al.Noncanonical inflammasome activation by intracellular LPS independent of TLR4[J].Science,2013,341(6151):1246-1249
[24]Man SM,Karki R,Sasai M,et al.IRGB10 Liberates Bacterial Ligands for Sensing by the AIM2 and Caspase-11-NLRP3Inflammasomes[J].Cell,2016,167(2):382-396
[25]Zhang H,Shan Y,Wu Y,et al.Berberine suppresses LPS-induced inflammation through modulating Sirt1/NF-κB signaling pathway in RAW264.7 cells[J].Int Immunopharmacol,2017,52:93-100
[26]张红霞,赵娟,于希忠,等.小檗碱和人参皂苷Rb1合用对脂肪细胞炎症因子表达和炎症信号通路的影响[J].食品与生物技术学报,2014,33(6):597-603Zhang Hong-xia,Zhao Juan,Yu Xi-zhong,et al.Effect of Combination of Berberin and Ginsenosides Rb1 on Inflammatory Adipocytokines and Inflammatory Signaling Pathways in Adipocytes[J].Journal of Food Science and Biotechnology,2014,33(6):597-603
[27]刘司漩.小檗碱对脂多糖诱导的THP-I细胞相关炎症因子的影响[D].太原:山西医科大学,2014Liu Si-xuan.Effect of Berberine on Inflammatory Cytokines Induced by Lipopolysaccharide in THP-1 Cells[D].Tai Yuan:Shanxi medical university,2014
[2]吴佳蕾,许惠琴,谭真,等.生地黄-山茱萸对对AGEs致HUVEC细胞损伤的保护作用[J].南京中医药大学学报,2013,29(3):243-246Wu Jia-lei,Xu Hui-qin,Tan Zhen,et al.The protective effect of Radix Rehmanniae and Cornus of ficinalis on HUVEC Stimulated by AGEs[J].Journal of Nanjing University of Traditional Chinese Medicine,2013,29(3):243-246
[3]郝钰,赵斌,郭钰琪,等.脂多糖诱导小鼠内皮损伤早期T细胞亚群的变化[J].中国免疫学杂志,2011,27(6):488-452Hao Yu,Zhao Bin,Guo Yu-qi,et al.Alteration of early T cell subsets in lipopolysaccharide-induced vascular endothelial cells injury in C57BL/6 mice[J].Chinese Journal of Immunology,2011,27(6):488-452
[4]毛秋娴,龙启才.盐酸小檗碱对L P S诱导的小鼠R A W 2 6 4.7细胞PGE2、COX-2表达的影响[J].现代医院,2008,8(12):7-9Mao Qiu-xian,Long Qi-cai.The Effect of berberine on PEG2 and COX2 expression in LPS-induced RAW264.7 cells[J].Modern Hospital,2008,8(12):7-9
[5]吴阳阳,董燕,易浪,等.小檗碱对LPS、I L-4诱导的小鼠RAW 264.7细胞分泌TNF-α、I L-10的影响[J].中药新药与临床药理,2014,25(3):268-272Wu Yang-yang,Dong Yan,Yi Lang,et al.Effect of berberine on TNF-α,I L-10 secretion induced by LPS and IL-4 in RAW 264.7[J].Traditional Chinese Drug Research and Clinical Pharmacology,2014,25(3):268-272
[6]万强,周凤华,贾钰华,等.小檗碱抑制p38 MAPK通路降低visfatin诱导HUVEC损伤的研究[J].中药新药与临床药理,2015,26(5):586-590Wan Qiang,Zhou Feng-hua,Jia Yu-hua,et al.Study of Berberine in Reducing Visfatin-induced HUVVEC Injury through p38 MAPKSignal Pathway[J].Traditional Chinese Drug Research&Clinical Pharmacology,2015,26(5):586-590
[7]牟娜娜,娄晓盈,王仁庆,等.LPS激活巨噬细胞NLRP3炎性小体的作用研究[J].热带医学杂志,2015,15(4):441-466Mu Na-na,Lou Xiao-ying,Wang Ren-qing,et al.Study of LPS-induced activaction of NLRP3 inflammasome in macrophages[J].J Trop Med,2015,15(4):441-466
[8]Xiao HB,Wang CR,Liu ZK,et al.LPS induces pro-inflammatory response in mastitis mice and mammary epithelial cells:Possible involvement of NF-κB signaling and OPN[J].Pathol Biol,2015,63(1):11-16
[9]Wang ZH,Mong MC,Yang YC,et al.Asiatic acid and maslinic acid attenuated kainic acid-induced seizure through decreasing hippocampal inflammatory and oxidative stress[J].Epilepsy Res,139:28-34
[10]Strowig T,Henao-Mejia J,Elinav E,et al.Inflammasomes in health and disease[J].Nature,2012,481(7381):278-286
[11]Yadav N,Chandra H.Suppression of inflammatory and infection responses in lung macrophages by eucalyptus oil and its constituent1,8-cineole:Role of pattern recognition receptors TREM-1 and NLRP3,the MAP kinase regulator MKP-1,and NFκB[J].PLo S One2017,12(11):e0188232
[12]毛开睿,孙兵.NLRP3炎症小体研究进展[J].现代免疫学,2011,31(1):1-3Mao Kai-rui,Sun Bing.The progress of NLRP3 inflammasome[J].Current Immunology,2011,31(1):1-3
[13]Zhu Y,Zhu H,Wang Z,et al.Wogonoside alleviates inflammation induced by traumatic spinal cord injury by suppressing NF-κB and NLRP3 inflammasome activation[J].Exp Ther Med,2017,14(4):3304-3308
[14]Song N,Liu ZS,Xue W,et al.NLRP3 Phosphorylation Is an Essential Priming Event for Inflammasome Activation[J].Mol Cell,2017,68(1):185-197
[15]Evavold C,Kagan JC.How inflammasomes inform adaptive immunity[J].J Mol Biol,2017,36(17):30470-30479
[16]Subramanian N,Natarajan K,Clatworthy MR,et al.The adaptor MAVS promotes NLRP3 mitochondrial localization and inflammasome activation[J].Cell,2013,153(2):348-361
[17]Freeman L,Guo H,David CN,et al.NLR members NLRC4 and NLRP3 mediate sterile inflammasome activation in microglia and astrocytes[J].J Exp Med,2017,214(5):1351-1370
[18]Liston A,Masters SL.Homeostasis-altering molecular processes as mechanisms of inflammasome activation[J].Nat Rev Immunol,2017,17(3):208-214
[19]Negash AA,Ramos HJ,Crochet N,et al.IL-1βproduction through the NLRP3 inflammasome by hepatic macrophages links hepatitis Cvirus infection with liver inflammation and disease[J].PLo S Pathog,2013,9(4):e1003330
[20]Broz P,Dixit VM.Inflammasomes:mechanism of assembly,regulation and signaling[J].Nat Rev Immunol,2016,16(7):407-420
[21]Liston A,Masters SL.Homeostasis-altering molecular processes as mechanisms of inflammasome activation[J].Nat Rev Immunol,2017,17(3):208-214
[22]Wang X,Gong P,Zhang X,et al.NLRP3 inflammasome activation in murine macrophages caused by Neospora caninum infection[J].Parasit Vectors,2017,10(1):266
[23]Kayagaki N,Wong MT,Stowe IB,et al.Noncanonical inflammasome activation by intracellular LPS independent of TLR4[J].Science,2013,341(6151):1246-1249
[24]Man SM,Karki R,Sasai M,et al.IRGB10 Liberates Bacterial Ligands for Sensing by the AIM2 and Caspase-11-NLRP3Inflammasomes[J].Cell,2016,167(2):382-396
[25]Zhang H,Shan Y,Wu Y,et al.Berberine suppresses LPS-induced inflammation through modulating Sirt1/NF-κB signaling pathway in RAW264.7 cells[J].Int Immunopharmacol,2017,52:93-100
[26]张红霞,赵娟,于希忠,等.小檗碱和人参皂苷Rb1合用对脂肪细胞炎症因子表达和炎症信号通路的影响[J].食品与生物技术学报,2014,33(6):597-603Zhang Hong-xia,Zhao Juan,Yu Xi-zhong,et al.Effect of Combination of Berberin and Ginsenosides Rb1 on Inflammatory Adipocytokines and Inflammatory Signaling Pathways in Adipocytes[J].Journal of Food Science and Biotechnology,2014,33(6):597-603
[27]刘司漩.小檗碱对脂多糖诱导的THP-I细胞相关炎症因子的影响[D].太原:山西医科大学,2014Liu Si-xuan.Effect of Berberine on Inflammatory Cytokines Induced by Lipopolysaccharide in THP-1 Cells[D].Tai Yuan:Shanxi medical university,2014
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