TLR4对H_2O_2诱导的PC12细胞凋亡及β-catenin、c-myc蛋白表达的影响
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摘要
目的:探讨Toll样受体4(TLR4)对H_2O_2诱导的PC12细胞凋亡及Wnt/β-catenin信号通路的影响。方法:分别用200μmol/L H_2O_2干预感染TLR4 shRNA慢病毒(shRNA+H_2O_2组)或空病毒(空病毒+H_2O_2组)的PC12细胞6 h,分别用0和200μmol/L H_2O_2干预PC12细胞(空白对照组和H_2O_2组) 6 h。用qRT-PCR和Western blot法检测细胞中TLR4 mRNA和蛋白表达水平,MTT法测定细胞存活率,Annexin V-FITC和PI双染法测定细胞凋亡率,Western blot法检测细胞中β-连环蛋白(β-catenin)、c-myc表达水平。结果:与空白对照组比较,H_2O_2组和空病毒+H_2O_2组PC12细胞中TLR4 mRNA和蛋白表达水平均升高;细胞存活率降低,凋亡率升高;细胞中β-catenin、c-myc蛋白水平降低(P <0. 05)。与H_2O_2组和空病毒+H_2O_2组比较,shRNA+H_2O_2组PC12细胞中TLR4 mRNA和蛋白表达水平降低;细胞存活率提高,凋亡率降低;细胞中β-catenin、c-myc蛋白水平提高(P <0. 05)。结论:敲减TLR4可以通过提高Wnt/β-catenin信号通路激活水平,促进PC12细胞抵抗氧化应激损伤的能力。
Aim: To investigate the effects of TLR4 on apoptosis and Wnt/β-catenin signaling pathway of PC12 cells induced by H_2O_2. Methods: PC12 cells infected with lentiviruses loaded TLR4 shRNA and empty viruses were cultured in200 mmol/L H_2O_2 for 6 hours,named by shRNA + H_2O_2 group and empty virus + H_2O_2 group. PC12 cells without lentivirusinfection were cultured with 0 and 200 mmol/L H_2O_2 for 6 hours,respectively,named by blank control group and H_2O_2 group. The expressions of TLR4 mRNA and protein in PC12 cells were detected by qRT-PCR and Western blot. Cell proliferation was measured by MTT,apoptosis was detected by Annexin V-FITC/PI staining,and Western blot was used to detect the expression levels of β-catenin and c-myc proteins. Results: Compared with the blank control group,the expressions of TLR4 mRNA and protein in PC12 cells of H_2O_2 group and empty virus + H_2O_2 group were higher,the proliferation rate decreased and apoptosis rate increased,and the expression levels of β-catenin and c-myc proteins decreased( P < 0. 05).Compared with the empty virus + H_2O_2 group and H_2O_2 group,the expressions of TLR4 mRNA and protein in PC12 cells of shRNA + H_2O_2 group were lower,the proliferation rate increased and apoptosis rate decreased,and the expression levels ofβ-catenin and c-myc proteins increased( P < 0. 05). Conclusion: Knockdown of TLR4 could promote the ability of PC12 cells to resist oxidative stress by increasing the activation level of Wnt/β-catenin signaling pathway.
Aim: To investigate the effects of TLR4 on apoptosis and Wnt/β-catenin signaling pathway of PC12 cells induced by H_2O_2. Methods: PC12 cells infected with lentiviruses loaded TLR4 shRNA and empty viruses were cultured in200 mmol/L H_2O_2 for 6 hours,named by shRNA + H_2O_2 group and empty virus + H_2O_2 group. PC12 cells without lentivirusinfection were cultured with 0 and 200 mmol/L H_2O_2 for 6 hours,respectively,named by blank control group and H_2O_2 group. The expressions of TLR4 mRNA and protein in PC12 cells were detected by qRT-PCR and Western blot. Cell proliferation was measured by MTT,apoptosis was detected by Annexin V-FITC/PI staining,and Western blot was used to detect the expression levels of β-catenin and c-myc proteins. Results: Compared with the blank control group,the expressions of TLR4 mRNA and protein in PC12 cells of H_2O_2 group and empty virus + H_2O_2 group were higher,the proliferation rate decreased and apoptosis rate increased,and the expression levels of β-catenin and c-myc proteins decreased( P < 0. 05).Compared with the empty virus + H_2O_2 group and H_2O_2 group,the expressions of TLR4 mRNA and protein in PC12 cells of shRNA + H_2O_2 group were lower,the proliferation rate increased and apoptosis rate decreased,and the expression levels ofβ-catenin and c-myc proteins increased( P < 0. 05). Conclusion: Knockdown of TLR4 could promote the ability of PC12 cells to resist oxidative stress by increasing the activation level of Wnt/β-catenin signaling pathway.
引文
[1]赖丽娟,刘松,谢佳丽,等.三氟淫羊藿素通过抑制氧化应激对脑缺血再灌注损伤的保护作用[J].赣南医学院学报,2017,37(3):343
[2] SAHASRABUDHE NM,DOKTER-FOKKENS J,DE VOS P. Particulateβ-glucans synergistically activate TLR4 and Dectin-1 in human dendritic cells[J]. Mol Nutr Food Res,2016,60(11):2514
[3] ZHANG T,LI CY,JIA JJ,et al. Combination therapy with LXW7 and ceria nanoparticles protects against acute cerebral ischemia/reperfusion injury in rats[J]. Curr Med Sci,2018,38(1):144
[4] SHI CX,DING YB,JIN FYJ,et al. Effects of sevoflurane post-conditioning in cerebral ischemia-reperfusion injury via TLR4/NF-κB pathway in rats[J]. Eur Rev Med Pharmacol Sci,2018,22(6):1770
[5]陈雪,沈楠,安英,等.丹酚酸B对氧化应激损伤乳鼠心肌细胞的保护作用及其机制[J].吉林大学学报(医学版),2018,44(5):974
[6]赵忆宁,何颖,沈先荣,等.西咪替丁对低剂量电离辐射致大鼠氧化应激的保护作用[J].解放军医学杂志,2017,42(2):128
[7] WANG M,LI YJ,DING Y,et al. Silibinin prevents autophagic cell death upon oxidative stress in cortical neurons and cerebral ischemia-reperfusion injury[J]. Mol Neurobiol,2016,53(2):932
[8] WANG JE,CAO B,HAN D,et al. Long non-coding RNA H19 induces cerebral ischemia reperfusion injury via activation of autophagy[J]. Aging Dis,2017,8(1):71
[9] LOU J,CAO GX,LI RR,et al.β-caryophyllene attenuates focal cerebral ischemia-reperfusion injury by Nrf2/HO-1pathway in rats[J]. Neurochem Res,2016,41(6):1291
[10]ZHANG JF,SHI LL,ZHANG L,et al. MicroRNA-25 negatively regulates cerebral ischemia/reperfusion injury-induced cell apoptosis through Fas/Fas L pathway[J]. J Mol Neurosci,2016,58(4):507
[11]GUO J,CHENG C,CHEN CS,et al. Overexpression of Fibulin-5 attenuates ischemia/reperfusion injury after middle cerebral artery occlusion in rats[J]. Mol Neurobiol,2016,53(5):3154
[12]WU BW,NI HC,LI J,et al. The impact of circulating mitochondrial DNA on cardiomyocyte apoptosis and myocardial injury after TLR4 activation in experimental autoimmune myocarditis[J]. Cell Physiol Biochem,2017,42(2):713
[13]WANG X,ZHOU Y,ZHU N,et al. Effects of hepatitis B virus X gene on apoptosis and expression of immune molecules of human proximal tubular epithelial cells[J]. Arch Virol,2013,158(12):2479
[14]WANG CP,SHI YW,TANG M,et al. Isoquercetin ameliorates cerebral impairment in focal Ischemia through anti-oxidative,anti-inflammatory, and anti-apoptotic effects in primary culture of rat hippocampal neurons and hippocampal CA1 region of rats[J]. Mol Neurobiol,2017,54(3):2126
[15]李会哲,周晓红,张颖,等.黄芪甲苷对缺氧缺糖/复氧复糖PC12细胞TLR4通路的影响[J].中国药理学通报,2016,32(12):1772
[16]白庆霞. TLR4在破骨细胞中的生物学作用及其与经典Wnt信号相互作用机制的初步研究[D].西安:第四军医大学,2013.
[17]尹晶. TLR4活化Wnt/β-catenin信号通路促进乳腺癌转移[D].天津:南开大学,2013.
[18]陈文明,龙仙萍,赵然尊,等. Toll样受体4激活Wnt/β-catenin信号通路促进骨髓间充质干细胞增殖[J].中国老年学杂志,2018,38(3):666
[19]SHANG YC,WANG SH,XIONG F,et al. Wnt3 a signaling promotes proliferation,myogenic differentiation,and migration of rat bone marrow mesenchymal stem cells[J]. Acta Pharmacol Sin,2007,28(11):1761
[20]严梦玲,王新金,赵利娜,等. Wnt信号通路在百草枯诱导PC12细胞损伤中的作用[J].中华劳动卫生职业病杂志,2015,33(11):806
[2] SAHASRABUDHE NM,DOKTER-FOKKENS J,DE VOS P. Particulateβ-glucans synergistically activate TLR4 and Dectin-1 in human dendritic cells[J]. Mol Nutr Food Res,2016,60(11):2514
[3] ZHANG T,LI CY,JIA JJ,et al. Combination therapy with LXW7 and ceria nanoparticles protects against acute cerebral ischemia/reperfusion injury in rats[J]. Curr Med Sci,2018,38(1):144
[4] SHI CX,DING YB,JIN FYJ,et al. Effects of sevoflurane post-conditioning in cerebral ischemia-reperfusion injury via TLR4/NF-κB pathway in rats[J]. Eur Rev Med Pharmacol Sci,2018,22(6):1770
[5]陈雪,沈楠,安英,等.丹酚酸B对氧化应激损伤乳鼠心肌细胞的保护作用及其机制[J].吉林大学学报(医学版),2018,44(5):974
[6]赵忆宁,何颖,沈先荣,等.西咪替丁对低剂量电离辐射致大鼠氧化应激的保护作用[J].解放军医学杂志,2017,42(2):128
[7] WANG M,LI YJ,DING Y,et al. Silibinin prevents autophagic cell death upon oxidative stress in cortical neurons and cerebral ischemia-reperfusion injury[J]. Mol Neurobiol,2016,53(2):932
[8] WANG JE,CAO B,HAN D,et al. Long non-coding RNA H19 induces cerebral ischemia reperfusion injury via activation of autophagy[J]. Aging Dis,2017,8(1):71
[9] LOU J,CAO GX,LI RR,et al.β-caryophyllene attenuates focal cerebral ischemia-reperfusion injury by Nrf2/HO-1pathway in rats[J]. Neurochem Res,2016,41(6):1291
[10]ZHANG JF,SHI LL,ZHANG L,et al. MicroRNA-25 negatively regulates cerebral ischemia/reperfusion injury-induced cell apoptosis through Fas/Fas L pathway[J]. J Mol Neurosci,2016,58(4):507
[11]GUO J,CHENG C,CHEN CS,et al. Overexpression of Fibulin-5 attenuates ischemia/reperfusion injury after middle cerebral artery occlusion in rats[J]. Mol Neurobiol,2016,53(5):3154
[12]WU BW,NI HC,LI J,et al. The impact of circulating mitochondrial DNA on cardiomyocyte apoptosis and myocardial injury after TLR4 activation in experimental autoimmune myocarditis[J]. Cell Physiol Biochem,2017,42(2):713
[13]WANG X,ZHOU Y,ZHU N,et al. Effects of hepatitis B virus X gene on apoptosis and expression of immune molecules of human proximal tubular epithelial cells[J]. Arch Virol,2013,158(12):2479
[14]WANG CP,SHI YW,TANG M,et al. Isoquercetin ameliorates cerebral impairment in focal Ischemia through anti-oxidative,anti-inflammatory, and anti-apoptotic effects in primary culture of rat hippocampal neurons and hippocampal CA1 region of rats[J]. Mol Neurobiol,2017,54(3):2126
[15]李会哲,周晓红,张颖,等.黄芪甲苷对缺氧缺糖/复氧复糖PC12细胞TLR4通路的影响[J].中国药理学通报,2016,32(12):1772
[16]白庆霞. TLR4在破骨细胞中的生物学作用及其与经典Wnt信号相互作用机制的初步研究[D].西安:第四军医大学,2013.
[17]尹晶. TLR4活化Wnt/β-catenin信号通路促进乳腺癌转移[D].天津:南开大学,2013.
[18]陈文明,龙仙萍,赵然尊,等. Toll样受体4激活Wnt/β-catenin信号通路促进骨髓间充质干细胞增殖[J].中国老年学杂志,2018,38(3):666
[19]SHANG YC,WANG SH,XIONG F,et al. Wnt3 a signaling promotes proliferation,myogenic differentiation,and migration of rat bone marrow mesenchymal stem cells[J]. Acta Pharmacol Sin,2007,28(11):1761
[20]严梦玲,王新金,赵利娜,等. Wnt信号通路在百草枯诱导PC12细胞损伤中的作用[J].中华劳动卫生职业病杂志,2015,33(11):806