补中益气丸对IEC-6细胞损伤模型NLRP3炎性体及相关细胞因子的影响
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摘要
目的:探讨补中益气丸含药血清对炎症刺激引起的大鼠小肠隐窝上皮细胞(ICE-6)损伤模型中NOD样受体-3(NLRP3)炎性体复合物及相关细胞因子的影响。方法:根据中药复方血清药理学实验方法,以IEC-6细胞为研究对象,设立胎牛血清组、肿瘤坏死因子-α(TNF-α)组、空白组、模型组,补中益气丸组、柳氮磺吡啶组。先用10%2,4,6-三硝基苯磺酸(TNBS)大鼠模型血清或者TNF-α(质量浓度100μg·L~(-1))刺激IEC-6细胞24 h,然后加入10%药物血清继续培养24 h后,采用细胞增殖与活性检测(CCK-8)方法检测IEC-6细胞活率;采用蛋白免疫印迹法(Western blot)测定NLRP3炎性体的相关蛋白表达量,采用酶联免疫吸附法(ELISA)检测细胞培养上清液中白细胞介素-1β(IL-1β)和白细胞介素-18(IL-18)的含量。结果:与空白组比较,TNF-α组及模型组均可引起IEC-6细胞增殖率显著下降(P<0.01),细胞内NLRP3炎性体的相关蛋白表达显著增高(P<0.01),细胞因子IL-1β,IL-18含量显著增高(P<0.01);与模型组比较,各给药组细胞增殖率显著增加(P<0.01),NLRP3炎性体复合物蛋白表达明显降低(P<0.05,P<0.01),IL-1β,IL-18分泌显著降低(P<0.01)。结论:补中益气丸含药血清可通过调节肠上皮细胞NLRP3炎性体复合物蛋白及细胞因子分泌,减轻炎症刺激引起的肠上皮细胞损伤。
Objective: To investigate the effect of Buzhong Yiqi Wan medicated serum on NOD like receptor-3( NLRP3) inflammasome and related cytokines in intestinal epithelial cell( IEC-6) injury model.Method: According to the methods of serum pharmacology of Chinese medicine,IEC-6 cells were divided into fetal bovine serum( FBS) group,tumor necrosis factor-α( TNF-α) group,blank group,model group,Buzhong Yiqi Wan group( BZYQ),and sulfasalazine group( SASP). First 10% 2,4,6-nitrobenzene sulfonic acid( TNBS)model serum or TNF-α( mass concentration of 100 g·L~(-1)) was used to stimulate IEC-6 cells for 24 h,and then10% drug containing serum was added for further culture for 24 h. Then cell proliferation was detected by cell counting kit-8( CCK-8) method and the protein expression level of NLRP3 inflammasome was detected by Western blot; the interleukin-1β( IL-1β) and IL-18 levels in cell culture supernatant were tested by enzyme-linked immunosorbent assay( ELISA). Result: As compared with the blank group, IEC-6 cell proliferation was significantly decreased in both TNF-α group and model group( P < 0. 01); the protein expression levels of NLRP3 inflammasome and cytokine( IL-1β,IL-18) levels were all increased( P < 0. 01). After administration with BZYQ medicated serum,cell proliferation rate was increased significantly,and the NLRP3 inflammasome and IL-1β,IL-18 levels were decreased in IEC-6 cells( P < 0. 05,P < 0. 01) as compared with model group. Conclusion:BZYQ medicated serum has protective effect on intestinal epithelial cell injury by down-regulating the NLRP3 inflammasome protein expression and cytokine level in injury epithelial cell.
Objective: To investigate the effect of Buzhong Yiqi Wan medicated serum on NOD like receptor-3( NLRP3) inflammasome and related cytokines in intestinal epithelial cell( IEC-6) injury model.Method: According to the methods of serum pharmacology of Chinese medicine,IEC-6 cells were divided into fetal bovine serum( FBS) group,tumor necrosis factor-α( TNF-α) group,blank group,model group,Buzhong Yiqi Wan group( BZYQ),and sulfasalazine group( SASP). First 10% 2,4,6-nitrobenzene sulfonic acid( TNBS)model serum or TNF-α( mass concentration of 100 g·L~(-1)) was used to stimulate IEC-6 cells for 24 h,and then10% drug containing serum was added for further culture for 24 h. Then cell proliferation was detected by cell counting kit-8( CCK-8) method and the protein expression level of NLRP3 inflammasome was detected by Western blot; the interleukin-1β( IL-1β) and IL-18 levels in cell culture supernatant were tested by enzyme-linked immunosorbent assay( ELISA). Result: As compared with the blank group, IEC-6 cell proliferation was significantly decreased in both TNF-α group and model group( P < 0. 01); the protein expression levels of NLRP3 inflammasome and cytokine( IL-1β,IL-18) levels were all increased( P < 0. 01). After administration with BZYQ medicated serum,cell proliferation rate was increased significantly,and the NLRP3 inflammasome and IL-1β,IL-18 levels were decreased in IEC-6 cells( P < 0. 05,P < 0. 01) as compared with model group. Conclusion:BZYQ medicated serum has protective effect on intestinal epithelial cell injury by down-regulating the NLRP3 inflammasome protein expression and cytokine level in injury epithelial cell.
引文
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[16]吴健宇,穆静,李仪奎.血清药理实验中异种动物血清对细胞的毒性作用和灭活后的减毒作用[J].中国药理学通报,2000,16(1):118-119.
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[18]Esper L,Utsch L,Soriani F M,et al.Regulatory effects of IL-18 on cytokine profiles and development of myocarditis during Trypanosoma cruzi infection[J].Microbes Infect,2014,16(6):481-490.
[19]唐月华,王小平.溃疡性结肠炎患者血清IL-1β和IL-18的表达及临床意义[J].河北医药,2015,37(10):1534-1535.
[2]王敬元,刘刚.补中益气汤加四神丸治疗溃疡性结肠炎临床观察[J].中医临床研究,2013,5(18):103.
[3]Zambetti L P,Mortellaro A.NLRPs,microbiota,and gut homeostasis:unravelling the connection[J].J Pathol,2014,233(4):321-330.
[4]Zaki M H,Boyd K L,Vogel P,et al.The NLRP3inflammasome protects against loss of epithelial integrity and mortality during experimental colitis[J].Immunity,2010,32(3):379-391.
[5]陈宁,宋冬雪,凌娜,等.中药血清药理学方法的研究进展[J].北京联合大学学报,2014,28(1):40-43.
[6]Akira S,Uematsu S,Takeuchi O.Pathogen recognition and innate immunity[J].Cell,2006,124(4):783-801.
[7]Perera A P,Kunde D,Eri R.NLRP3 inhibitors as potential therapeutic agents for treatment of inflammatory bowel disease[J].Curr Pharm Des,2017,23(46):1873.
[8]Kim J J,Jo E K.NLRP3 inflammasome and host protection against bacterial infection[J].J Korean Med Sci,2013,28(10):1415-1423.
[9]Martinon F,Burns K,Tschopp J.The inflammasome:a molecular platform triggering activation of inflammatory caspases and processing of pro-IL-beta[J].Mol Cell,2002,10(2):417-426.
[10]Sutterwala F S,Ogura Y,Zamboni D S,et al.NALP3:a key player in caspase-1 activation[J].J Endotoxin Res,2006,12(4):251-256.
[11]Bauer C,Duewell P,Lehr H A,et al.Protective and aggravating effects of Nlrp3 inflammasome activation in IBD models:influence of genetic and environmental factors[J].Dig Dis,2012,30(S1):82-90.
[12]Bauer C,Duewell P,Mayer C,et al.Colitis induced in mice with dextran sulfate sodium(DSS)is mediated by the NLRP3 inflammasome[J].Gut,2010,59(9):1192-1199.
[13]Zaki M H,Lamkanfi M,Kanneganti T D.The Nlrp3inflammasome:contributions to intestinal homeostasis[J].Trends Immunol,2011,32(4):171-179.
[14]刘凯,王本军,马恒,等.补中益气汤对胃癌术后气虚血瘀证胃肠功能恢复和营养状况的影响[J].中国实验方剂学杂志,2015,21(24):152-156.
[15]李燕舞,王汝俊,王建华,等.补脾方药对脾虚大鼠壁细胞胃泌素受体及细胞内钙离子浓度的影响[J].中医杂志,2006,47(8):616-618.
[16]吴健宇,穆静,李仪奎.血清药理实验中异种动物血清对细胞的毒性作用和灭活后的减毒作用[J].中国药理学通报,2000,16(1):118-119.
[17]郑海涵,吴正祥.TNF-α在IBD发病机制中的调节[J].胃肠病学和肝病学杂志,2011,20(2):191-193.
[18]Esper L,Utsch L,Soriani F M,et al.Regulatory effects of IL-18 on cytokine profiles and development of myocarditis during Trypanosoma cruzi infection[J].Microbes Infect,2014,16(6):481-490.
[19]唐月华,王小平.溃疡性结肠炎患者血清IL-1β和IL-18的表达及临床意义[J].河北医药,2015,37(10):1534-1535.