6-溴-靛玉红-3'-肟抑制脂多糖诱导的小鼠乳腺上皮细胞凋亡
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摘要
研究6-溴-靛玉红-3'-肟(BIO)对脂多糖(lipopolysaccharide,LPS)诱导小鼠乳腺上皮细胞(mouse mammary epithelial cell,MMEC)凋亡的作用及机制。收集空白对照组、DMSO组、BIO单独处理组及不同浓度BIO(5、25、50nmol·L~(-1))+LPS共同处理组细胞,分别用流式细胞术检测凋亡率,利用qRT-PCR检测Bak、Bax、Bcl-2、Bcl-xl、Caspase-3和Caspase-8 mRNA的变化,Western blot检测Bcl-2、Caspase-3和Caspase-8蛋白水平的变化。结果显示:BIO能够降低LPS诱导的MMECs的凋亡率,抑制凋亡基因Bak和Bax及凋亡通路蛋白Caspase-3和Caspase-8活性(P <0. 001),促进抑凋亡基因Bcl-2及Bcl-xl的活性。BIO通过调节Bax、Bak、Bcl-2、Bcl-xl、Caspase-3和Caspase-8活性抑制LPS诱导的鼠乳腺上皮细胞凋亡。
The aim of this study was to investigate the effects of 6-bromo-indirubin-3'-oxime( BIO) on apoptosis of mouse mammary epithelial cell induced by lipopolysaccharide( LPS) and its regulatory mechanism. The cells from the blank control group,DMSO group,BIO alone treatment group and different concentrations of BIO( 5,25,50 nmol·L~(-1) treatment) + LPS co-treatment group were collected,respectively. Apoptosis rate was analyzed by flow cytometry. Bak,Bax,Bcl-2,Bcl-xl,Caspase-3 and Caspase-8 mRNA expression were detected by qRT-PCR. Bcl-2,Caspase-3 and Caspase-8 protein expression were tested by Western blot. Results were as follows: BIO reduced the apoptosis rate of MMECs induced by LPS. BIO decreased LPS-induced activities of apoptosis genes Bak and Bax,apoptotic pathway proteins Caspase-3 and Caspase-8( P < 0. 001) were also down-regulated. BIO increased the activities of anti-apoptosis genes Bcl-2 and Bcl-xl. BIO inhibited apoptosis of MMECs induced by LPS through regulating Bax,Bak,Bcl-2,Bcl-xl,Caspase-3 and Caspase-8 activity.
The aim of this study was to investigate the effects of 6-bromo-indirubin-3'-oxime( BIO) on apoptosis of mouse mammary epithelial cell induced by lipopolysaccharide( LPS) and its regulatory mechanism. The cells from the blank control group,DMSO group,BIO alone treatment group and different concentrations of BIO( 5,25,50 nmol·L~(-1) treatment) + LPS co-treatment group were collected,respectively. Apoptosis rate was analyzed by flow cytometry. Bak,Bax,Bcl-2,Bcl-xl,Caspase-3 and Caspase-8 mRNA expression were detected by qRT-PCR. Bcl-2,Caspase-3 and Caspase-8 protein expression were tested by Western blot. Results were as follows: BIO reduced the apoptosis rate of MMECs induced by LPS. BIO decreased LPS-induced activities of apoptosis genes Bak and Bax,apoptotic pathway proteins Caspase-3 and Caspase-8( P < 0. 001) were also down-regulated. BIO increased the activities of anti-apoptosis genes Bcl-2 and Bcl-xl. BIO inhibited apoptosis of MMECs induced by LPS through regulating Bax,Bak,Bcl-2,Bcl-xl,Caspase-3 and Caspase-8 activity.
引文
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[11]MOEN S H,WESTHRIN M,ZAHOOR M,et al.Caspase-8 regulates the expression of pro-and anti-inflammatory cytokines in human bone marrow-derived mesenchymal stromal cells[J]. Immun Inflamm Dis,2016,4(3):327-337.
[12]王正引,郭明章,陈燕.四物汤对血虚证小鼠肝细胞凋亡相关分子Caspase3及Caspase8表达的影响[J].陕西中医药大学学报,2017,40(6):98-102.WANG Z Y,GUO M Z,CHEN Y. Effects of Siwu decoction on expression of Caspase3 and Caspase8 in hepatocytes of mice with blood deficiency syndrome[J]. Journal of Shaanxi University of Chinese Medicine,2017,40(6):98-102.(in Chinese)
[2]赖金伦.中药单体靛玉红抗LPS诱导的小鼠乳腺炎作用及机制研究[D].武汉:华中农业大学,2017.LAI J L. Anti-inflammatory effect and mechanisms of indirubin in LPS-induced mouse mastitis[D]. Wuhan:Huazhong Agricultural University,2017.(in Chinese)
[3]SKLIROU A D,GABORIAUD-KOLAR N,PAPASSIDERI I,et al. 6-bromo-indirubin-3'-oxime(6BIO),a Glycogen synthase kinase-3βinhibitor,activates cytoprotective cellular modules and suppresses cellular senescence-mediated biomolecular damage in human fibroblasts[J]. Sci Rep,2017,7(1):11713.
[4]TRAN F H,ZHENG J J. Modulating the wnt signaling pathway with small molecules[J]. Protein Sci,2017,26(4):650-661.
[5]LU Y C,YEH W C,OHASHI P S. LPS/TLR4 signal transduction pathway[J]. Cytokine,2008,42(2):145-151.
[6]寇明明,刘玉辉,刘婧,等.奶牛乳腺炎治疗研究进展[J].动物医学进展,2015,36(6):126-129.KOU M M,LIU Y H,LIU J,et al. Progress on treatment of dairy cow mastitis[J]. Progress in Veterinary Medicine,2015,36(6):126-129.(in Chinese)
[7]赵彦超,顾耘.细胞凋亡通路研究进展[J].现代医学,2013,41(4):285-288.ZHAO Y C,GU Y. Progress in apoptosis pathway[J].Modern Medical Journal,2013,41(4):285-288.(in Chinese)
[8]史海敏,丁库克,张新.中药有效成分影响宫颈癌Hela细胞凋亡机制的研究进展[J].环球中医药,2015,8(1):124-127.SHI H M,DING K K,ZHANG X. Progress on the study of effective constituent of Chinese medicinals inducing cervical cancer Hela cell apoptosis[J]. Global Traditional Chinese Medicine,2015,8(1):124-127.(in Chinese)
[9]ARNOULT D. Apoptosis-associated mitochondrial outer membrane permeabilization assays[J]. Methods,2008,44(3):229-234.
[10]O'NEILL K L,HUANG K,ZHANG J J,et al. Inactivation of prosurvival Bcl-2 proteins activates Bax/Bak through the outer mitochondrial membrane[J]. Genes Dev,2016,30(8):973-988.
[11]MOEN S H,WESTHRIN M,ZAHOOR M,et al.Caspase-8 regulates the expression of pro-and anti-inflammatory cytokines in human bone marrow-derived mesenchymal stromal cells[J]. Immun Inflamm Dis,2016,4(3):327-337.
[12]王正引,郭明章,陈燕.四物汤对血虚证小鼠肝细胞凋亡相关分子Caspase3及Caspase8表达的影响[J].陕西中医药大学学报,2017,40(6):98-102.WANG Z Y,GUO M Z,CHEN Y. Effects of Siwu decoction on expression of Caspase3 and Caspase8 in hepatocytes of mice with blood deficiency syndrome[J]. Journal of Shaanxi University of Chinese Medicine,2017,40(6):98-102.(in Chinese)