木香烃内酯通过抑制NLRP3缓解LPS诱导小鼠巨噬细胞的炎症反应
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摘要
目的:探讨木香烃内酯(Cos)对脂多糖(LPS)诱导的小鼠原代巨噬细胞炎性小体激活和炎症反应的影响。方法:提取并培养小鼠腹腔原代巨噬细胞,用不同浓度Cos预处理巨噬细胞1 h后,1 mg/L LPS处理,分为空白对照组、模型组(单用LPS)、不同浓度(5、10和20μmol/L) Cos+LPS组; ELISA法检测培养液上清中TNF-α/IL-6的含量,q PCR法检测TNF-α/IL-6 mRNA表达;为明确Cos对LPS诱发的炎症反应的保护机制,增加NLRP3抑制剂MCC950组(10μmol/L),LPS刺激4h、6 h,Western blot检测NLRP3、ERK及IκB的激活,细胞免疫荧光实验观察NLRP3的蛋白水平。结果:与LPS组比较,LPS+Cos组TNF-α/IL-6的mRNA表达及细胞培养液上清TNF-α/IL-6分泌均显著降低(P <0. 05); Western blot和细胞免疫荧光实验结果表明,与LPS组比较,LPS+Cos组的NLRP3蛋白水平显著降低,其抑制效果与NLRP3抑制剂MCC950相当(P <0. 05); Western blot结果表明,与LPS组比较,LPS+MCC950组和LPS+Cos组的ERK及IκB的激活均显著降低(P <0. 01)。结论:Cos能有效抑制LPS诱导的巨噬细胞炎性小体和NF-κB的激活而缓解炎症反应。
Objective: To investigate the effect of Costunolide on lipopolysaccharide(LPS)-induced activation of inflammatory corpuscles and inflammatory response in murine primary macrophages. Methods: Mouse peritoneal macrophages were extracted and cultured,and macrophages were divided into blank control group,model group(LPS),LPS + Costunolide group. After pretreatment with1 h,LPS was used to stimulate 6 h or 24 h,and total RNA or protein and culture medium were extracted. ELISA was used to detect the content of TNF-α/IL-6 in the culture medium,and q PCR was used to detect the expression of TNF-α/IL-6 mRNA. Western blot was used to detect the activation of NLRP3,ERK and IκB,and the level of NLRP3 protein was observed by cellular immunofluorescence assay. Results: Compared with group LPS,the mRNA expression and secretion of TNF-α/IL-6 significantly decreased in LPS + Costunolide group(P < 0. 001 or P < 0. 05). Experimental results of Western blot and immunofluorescence showed that,compared with LPS group,NLRP3 protein level in LPS + Costunolide group was significantly decreased(P < 0. 01). The results of Western blot showed that compared with LPS group,ERK and IκB activation in LPS + Costunolide group decreased significantly(P < 0. 01). Conclusion: Costunolide can effectively inhibit LPS induced macrophage inflammatory corpuscle and activation of NF-κB and alleviate inflammatory responses.
Objective: To investigate the effect of Costunolide on lipopolysaccharide(LPS)-induced activation of inflammatory corpuscles and inflammatory response in murine primary macrophages. Methods: Mouse peritoneal macrophages were extracted and cultured,and macrophages were divided into blank control group,model group(LPS),LPS + Costunolide group. After pretreatment with1 h,LPS was used to stimulate 6 h or 24 h,and total RNA or protein and culture medium were extracted. ELISA was used to detect the content of TNF-α/IL-6 in the culture medium,and q PCR was used to detect the expression of TNF-α/IL-6 mRNA. Western blot was used to detect the activation of NLRP3,ERK and IκB,and the level of NLRP3 protein was observed by cellular immunofluorescence assay. Results: Compared with group LPS,the mRNA expression and secretion of TNF-α/IL-6 significantly decreased in LPS + Costunolide group(P < 0. 001 or P < 0. 05). Experimental results of Western blot and immunofluorescence showed that,compared with LPS group,NLRP3 protein level in LPS + Costunolide group was significantly decreased(P < 0. 01). The results of Western blot showed that compared with LPS group,ERK and IκB activation in LPS + Costunolide group decreased significantly(P < 0. 01). Conclusion: Costunolide can effectively inhibit LPS induced macrophage inflammatory corpuscle and activation of NF-κB and alleviate inflammatory responses.
引文
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[2]Vanaja SK,Russo AJ,Behl B,et al.Bacterial outer membrane vesicles mediate cytosolic localization of LPS and caspase-11 activation[J].Cell,2016,165(5):1106-1119.
[3]Schroder K,TSchopp J.The inflammasomes[J].Cell,2010,140(6):821-832.
[4]Ehsan M,Singh KK,Lovren F,et al.Adiponectin limits monocytic microparticle-induced endothelial activation by modulation of the AMPK,Akt and NF-κB signaling pathways[J].Atherosclerosis,2015,245(1):1-11.
[5]Ko JH,Yoon SO,Lee HJ,et al.Rapamycin regulates macrophage activation by inhibiting NLRP3 inflammasome-p38 MAPK-NF-κBpathways in autophagy-and p62-dependent manners[J].Oncotarget,2017,8(25):40817-40831.
[6]Rayan NA,Baby N,Pitchai D,et al.Costunolide inhibits proinflammatory cytokines and i NOS in activated murine BV2 microglia[J].Front Biosci,2011,3:1079-1091.
[7]Wang Y,Zhang X,Zhao L,et al.Costunolide protects lipopolysaccharide/d-galactosamine-induced acute liver injury in mice by inhibiting NF-kappaB signaling pathway[J].J Surgical Res,2017,220(6):40-45.
[8]Pae HO,Jeong GS,Kim HS,et al.Costunolide inhibits production of tumor necrosis factor-alpha and interleukin-6 by inducing heme oxygenase-1 in RAW264.7 macrophages[J].Inflamm Res,2007,56(12):520-526.
[9]Wang L,Huang Z,Huang W,et al.Inhibition of epidermal growth factor receptor attenuates atherosclerosis via decreasing inflammation and oxidative stress[J].Sci Rep,2017,8:45917-45931.
[10]Xu F,Wang F,Wen T,et al.Inhibition of NLRP3 inflammasome:a new protective mechanism of cinnamaldehyde in endotoxin poisoning of mice[J].Immunopharmacol Immunotoxicol,2017,39(5):296-304.
[11]Vandanmagsar B,Youm YH,Ravussin A,et al.The NLRP3 inflammasome instigates obesity-induced inflammation and insulin resistance[J].Nat Med,2011,17(2):179-188.
[12]Lee S,Nakahira K,Dalli J,et al.NLRP3 inflammasome deficiency protects against microbial sepsis via increased lipoxin B4synthesis[J].Am J Resp Critical Care Med,2017,196(6):713-726.