NFκB-Orai1参与NEFA诱导的内质网应激
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摘要
为探究NFκB-Orai1是否参与游离脂肪酸(NEFA)诱导的内质网应激,首先通过体外添加一定浓度的非酯化脂肪酸(nonestesterifiedfatty acid,NEFAs)(1.2 mmol/L)处理大鼠肝细胞不同时间,运用Western blot和实时荧光定量PCR(qRT-PCR)方法,测定NEFAs对内质网应激(ER stress)标志分子GRP78表达量以及钙通道相关蛋白Orai1表达水平的影响。结果显示NEFAs处理12 h时GRP78、Orai1表达量极显著高于对照组(P<0.01),证明12 h NEFAs刺激诱导产生内质网应激效果最佳。而后又通过分别添加NFκB抑制剂wogonin与Orai1抑制剂2APB,结果显示抑制NFκB和Orai1后NEFAs刺激可明显降低GRP78、NFκB p65、Orai1的表达水平。结果表明NFκB-Orai1参与NEFA诱导的内质网应激。
The purpose of this study was to explore whether NFκB p65-Orai1 is involved in NEFA induced endoplasmic reticulum stress.Firstly,BRL-3 A cell lines were treated with a certain concentration of non-esterified fatty acid(NEFAs)(1.2 mmol/L) at different time.The expression of ER stress marker GRP78 and Orai1 was examined by Western blot and real-time quantitative PCR(qRT-PCR).The results showed that the expression of GRP78 and Orai1 was significantly increased at 12 hours after NEFAs treatment(P<0.01),demonstrating that the cells treated with NEFAs for 12 h produces the strongest endoplasmic reticulum stress.and the cells were treated with the NFκB inhibitor wogonin and Orai1 inhibitor 2 APB,respectively.The results showed that inhibition of NFκB and Orai1 were significantly reduced the expression of GRP78,NFκB p65,and Orai1.NFκB-Orai1 participates in NEFA induced endoplasmic reticulum stress.
The purpose of this study was to explore whether NFκB p65-Orai1 is involved in NEFA induced endoplasmic reticulum stress.Firstly,BRL-3 A cell lines were treated with a certain concentration of non-esterified fatty acid(NEFAs)(1.2 mmol/L) at different time.The expression of ER stress marker GRP78 and Orai1 was examined by Western blot and real-time quantitative PCR(qRT-PCR).The results showed that the expression of GRP78 and Orai1 was significantly increased at 12 hours after NEFAs treatment(P<0.01),demonstrating that the cells treated with NEFAs for 12 h produces the strongest endoplasmic reticulum stress.and the cells were treated with the NFκB inhibitor wogonin and Orai1 inhibitor 2 APB,respectively.The results showed that inhibition of NFκB and Orai1 were significantly reduced the expression of GRP78,NFκB p65,and Orai1.NFκB-Orai1 participates in NEFA induced endoplasmic reticulum stress.
引文
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[8] 麦晓仪.抑制Orai1通道介导的Ca～(2+)内流减轻肾脏纤维化和肾功能损害及其机制研究[D].广东广州:中山大学,2016.
[9] LANG F,SHUMILINA E.Regulation of ion channels by the serum-and glucocorticoid-inducible kinase SGK1[J].Faseb J Nlm,2013,27(1):3-12.
[10] ZHANG B,YAN J,UMBACH A T,et al.NFκB-sensitive Orai1 expression in the regulation of FGF23 release[J].J Mol Med,2016,94(5):557-566.
[11] ASSELAH T,BIèCHE I,MANSOURI A,et al.In vivo hepatic endoplasmic reticulum stress in patients with chronic hepatitis C[J].J Pathol,2010,221(3):264-274.
[2] UMEMURA M,BALJINNYAM E,FESKE S,et al.Store-operated Ca2+ entry (SOCE) regulates melanoma proliferation and cell migration[J].PLoS One,2014,9(2):e89292.
[3] FUJIMOTO Y,ONODUKA J,HOMMA K J,et al.Long-chain fatty acids induce lipid droplet formation in a cultured human hepatocyte in a manner dependent of Acyl-CoA synthetase[J].Biol Pharm Bull,2006,29(11):2174-2180.
[4] 黄啸,周植星,段为钢,江振洲,孙丽新,张陆勇.内质网应激与肝脏损伤的治疗[J].现代药物与临床,2011,26(3):168-173.
[5] MU Y,OGAWA T,KAWADA N.Reversibility of fibrosis,inflammation,and endoplasmic reticulum stress in the liver of rats fed a methionine-choline-deficient diet[J].Lab Inves T,2010,90(2):245.
[6] 闵敏,陈东风,王军,等.葡萄糖调节蛋白78在大鼠非酒精性脂肪性肝炎中的表达及意义[J].第三军医大学学报,2009,31(1):79-81.
[7] WU P,WANG Y,DAVIS M E,et al.Store-operated Ca2+ channels in mesangial cells inhibit matrix protein expression[J].J Am Soc Nephrol,2015,26(11):2691-2702.
[8] 麦晓仪.抑制Orai1通道介导的Ca～(2+)内流减轻肾脏纤维化和肾功能损害及其机制研究[D].广东广州:中山大学,2016.
[9] LANG F,SHUMILINA E.Regulation of ion channels by the serum-and glucocorticoid-inducible kinase SGK1[J].Faseb J Nlm,2013,27(1):3-12.
[10] ZHANG B,YAN J,UMBACH A T,et al.NFκB-sensitive Orai1 expression in the regulation of FGF23 release[J].J Mol Med,2016,94(5):557-566.
[11] ASSELAH T,BIèCHE I,MANSOURI A,et al.In vivo hepatic endoplasmic reticulum stress in patients with chronic hepatitis C[J].J Pathol,2010,221(3):264-274.