内质网应激关键因子PREK在应力加载下成肌细胞凋亡中的作用及机制
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摘要
目的:研究周期性张应力对L6大鼠成肌细胞凋亡的影响,探讨蛋白激酶R样内质网激酶(protein kinase Rlike ER kinase,PERK)在这一过程中的作用及机制。方法:构建L6细胞力学刺激模型,加载参数为细胞拉伸形变率15%,频率10个循环/min,每循环包括3 s拉伸及3 s舒张,加力时间为2、6、12、24 h,以不加力组为对照组。应用DAPI染色观察各组细胞凋亡情况;应用实时荧光定量PCR检测各组PERK、CHOP mRNA的表达;应用Western免疫印迹检测各组PERK、p-PERK的表达变化。加入PERK抑制剂后重复实验,检测各组细胞凋亡情况,PERK、CHOP mRNA的表达变化及p-PERK的蛋白表达变化。采用SPSS17.0软件包进行统计学分析。结果:DAPI染色显示,L6大鼠成肌细胞在周期性应力诱导下发生凋亡,24 h时达到凋亡最大值。PERK被抑制后,加力组的细胞凋亡程度与对照组无显著差别。RT-PCR结果显示,随着加力时间的延长,CHOP mRNA的表达逐渐增加;PERK mRNA表达无差异。Western免疫印迹结果显示,p-PERK蛋白表达水平随加力时间延长而逐渐升高,总蛋白PERK的表达无明显变化;加入PERK抑制剂后,CHOP的mRNA表达与对照组无显著差异,p-PERK蛋白表达与对照组无显著差异。结论:内质网应激关键因子PERK参与了周期性应力条件下的成肌细胞凋亡,其作用机制可能与PERK磷酸化有关。
PURPOSE:This study was aimed to figure out the way that cyclic-stretch influenced the apoptosis of myoblasts and evaluate the importance of PERK and its possible mechanism involved.METHODS:L6 rat myoblasts were cultured in vitro and mechanical stimulation model was constructed successfully.The myoblasts were imposed tension for0,2,6,12 and 24 hours respectively by multi-channel cell stress loading system.The force value was 15% cell deformation and the frequency was 10 cycles/min.Each cycle was consisted of stretch for 3 seconds and relaxation for 3 seconds,and the group without tension was used as the control group.The apoptotic myoblasts were dyed by DAPI and observed through fluorescence microscopy to detect the apoptosis rate;the mRNA levels of PERK and CHOP in different groups were detected by real-time PCR and protein levels of PERK and p-PERK in different groups were detected by Western blot.PERK inhibitor was used to clear the role of PERK in apoptosis induced by cyclic-stretch and clarify the relationship between the endoplasmic reticulum stress and apoptosis induced by cyclic-stretch.SPSS 17.0 software package was used to analyze the data statistically.RESULTS:DAPI nuclear stain showed that cyclical tensile stress can induce apoptosis in vitro cultured myoblast.Apoptosis rate showed a trend of rising gradually over time,peaked at 24 h.After dealt with the inhibitor of PERK,the apoptosis rate of the 24 h group under the cyclic stretch showed no difference compared with the control.The results of real-time PCR showed that the m RNA of CHOP was increased with the extension loading time,while the mRNA of PERK showed no difference compared with the control.Western blot results showed that the protein level of p-PERK was increased with the extension of loading time,while the expression of PERK showed no difference compared with the control group.When PERK inhibitor added,the m RNA level of CHOP along with the protein expression level of p-PERK showed no significant difference compared to the control.CONCLUSIONS:PERK signaling pathway is involved in the apoptosis of myoblasts induced by cyclic stretch,and the possible mechanism may be closely related to the phosphorylation of PERK.
PURPOSE:This study was aimed to figure out the way that cyclic-stretch influenced the apoptosis of myoblasts and evaluate the importance of PERK and its possible mechanism involved.METHODS:L6 rat myoblasts were cultured in vitro and mechanical stimulation model was constructed successfully.The myoblasts were imposed tension for0,2,6,12 and 24 hours respectively by multi-channel cell stress loading system.The force value was 15% cell deformation and the frequency was 10 cycles/min.Each cycle was consisted of stretch for 3 seconds and relaxation for 3 seconds,and the group without tension was used as the control group.The apoptotic myoblasts were dyed by DAPI and observed through fluorescence microscopy to detect the apoptosis rate;the mRNA levels of PERK and CHOP in different groups were detected by real-time PCR and protein levels of PERK and p-PERK in different groups were detected by Western blot.PERK inhibitor was used to clear the role of PERK in apoptosis induced by cyclic-stretch and clarify the relationship between the endoplasmic reticulum stress and apoptosis induced by cyclic-stretch.SPSS 17.0 software package was used to analyze the data statistically.RESULTS:DAPI nuclear stain showed that cyclical tensile stress can induce apoptosis in vitro cultured myoblast.Apoptosis rate showed a trend of rising gradually over time,peaked at 24 h.After dealt with the inhibitor of PERK,the apoptosis rate of the 24 h group under the cyclic stretch showed no difference compared with the control.The results of real-time PCR showed that the m RNA of CHOP was increased with the extension loading time,while the mRNA of PERK showed no difference compared with the control.Western blot results showed that the protein level of p-PERK was increased with the extension of loading time,while the expression of PERK showed no difference compared with the control group.When PERK inhibitor added,the m RNA level of CHOP along with the protein expression level of p-PERK showed no significant difference compared to the control.CONCLUSIONS:PERK signaling pathway is involved in the apoptosis of myoblasts induced by cyclic stretch,and the possible mechanism may be closely related to the phosphorylation of PERK.
引文
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[14]Jiang Q,Li F,Shi K,et al.Involvement of p38 in signal switching from autophagy to apoptosis via the PERK/eIF2alpha/ATF4 axis in selenite-treated NB4 cells[J].Cell Death Dis,2014,5:e1270.
[15]Feng Y,Yang Y,Fan C.Pterostilbene inhibits the growth of human esophageal cancer cells by regulating endoplasmic reticulum stress[J].Cell Physiol Biochem,2016,38(3):1226-1244.
[16]Vattem KM,Wek RC.Reinitiation involving upstream ORFs regulates ATF4 m RNA translation in mammalian cells[J].Proc Natl Acad Sci USA,2004,101(31):11269-11274.
[17]方芳,龚平生,宋祥福,等.低剂量电离辐射诱导小鼠睾丸细胞内质网应激及PERK-CHOP信号通路的激活[J].中华男科学杂志,2012,18(9):777-782.
[18]Wei J,Rahman S,Ayaub EA,et al.Protein misfolding and endoplasmic reticulum stress in chronic lung disease[J].Chest,2013,143(4):1098-1105.
[19]Matsumoto H,Miyazaki S,Matsuyama S,et al.Selection of autophagy or apoptosis in cells exposed to ER-stress depends on ATF4 expression pattern with or without CHOP expression[J].Biol Open,2013,2(10):1084-1090.
[20]Wang Q,He Z,Zhang J,et al.Overexpression of endoplasmic reticulum molecular chaperone GRP94 and GRP78 in human lung cancer tissues and its significance[J].Cancer Detect Prev,2005,29(6):544-551.
[21]Yoshida H,Okada T,Haze K,et al.ATF6 activated by proteolysis binds in the presence of NF-Y(CBF)directly to the cis-acting element responsible for the mammalian unfolded protein response[J].Mol Cell Biol,2000,20(18):6755-6767.
[22]Safra M,Ben-Hamo S,Kenyon C,et al.The ire-1 ER stressresponse pathway is required for normal secretory-protein metabolism in C.elegans[J].J Cell Sci,2013,126(Pt 18):4136-4146.
[2]Barnouti ZP,Owtad P,Shen G,et al.The biological mechanisms of PCNA and BMP in TMJ adaptive remodeling[J].Angle Orthod,2011,81(1):91-99.
[3]王昕,罗颂椒,周秀坤,等.功能矫形前伸下颌后幼年大鼠下颌前伸肌的组织化学研究[J].华西口腔医学杂志,1992,10(3):220-223.
[4]Yagci A,Uysal T,Kara S,et al.The effects of myofunctional appliance treatment on the perioral and masticatory muscles in Class II,Division 1 patients[J].World J Orthod,2010,11(2):117-122.
[5]Erdem A,Kilic N,Erz B.Changes in soft tissue profile and electromyographic activity after activator treatment[J].Aust Orthod J,2009,25(2):116-122.
[6]倪琳,刘洪臣,徐娟,等.功能矫形前伸青春期大鼠下颌对翼外肌Na+/K+-ATPase功能活性的影响[J].口腔颌面修复学杂志,2009,10(6):324-326.
[7]Kerr JF,Wyllie AH,Currie AR.Apoptosis:a basic biological phenomenon with wide-anging implications in tissue kinetics[J].Br J Cancer,1972,26(4):239-257.
[8]黄宁,陈开云,罗颂椒.青春期大鼠前伸下颌后浅层嚼肌细胞膜乙酰胆碱受体研究[J].华西口腔医学杂志,2008,26(4):365-367.
[9]Cucina A,Fuso A,Coluccia P,et al.Nicotine inhibits apoptosis and stimulates proliferation in aortic smooth muscle cells through a functional nicotinic acetylcholine receptor[J].J Surg Res,2008,150(2):227-235.
[10]赵云罡,徐建兴.线粒体,活性氧和细胞凋亡[J].生物化学与生物物理进展,2001,28(2):168-171.
[11]Bollini R,Chrispeels MJ.The rough endoplasmic reticulum is the site of reserve-protein synthesis in developing Phaseolus vulgaris cotyledons[J].Planta,1979,146(4):487-501.
[12]Rizzolo LJ,Kornfeld R.Post-translational protein modification in the endoplasmic reticulum.Demonstration of fatty acylase and deoxymannojirimycin-sensitive alpha-mannosidase activities[J].J Biol Chem,1988,263(19):9520-9525.
[13]Karnik AB,Thakore KN,Nigam SK,et al.Studies onglucose-6-phosphatase,fructose-1,6-diphosphatase activity,glycogen distribution and endoplasmic reticulum changes during hexachlorocyclohexane induced hepatocarcinogenesis in pure inbred Swiss mice[J].Neoplasma,1981,28(5):575-584.
[14]Jiang Q,Li F,Shi K,et al.Involvement of p38 in signal switching from autophagy to apoptosis via the PERK/eIF2alpha/ATF4 axis in selenite-treated NB4 cells[J].Cell Death Dis,2014,5:e1270.
[15]Feng Y,Yang Y,Fan C.Pterostilbene inhibits the growth of human esophageal cancer cells by regulating endoplasmic reticulum stress[J].Cell Physiol Biochem,2016,38(3):1226-1244.
[16]Vattem KM,Wek RC.Reinitiation involving upstream ORFs regulates ATF4 m RNA translation in mammalian cells[J].Proc Natl Acad Sci USA,2004,101(31):11269-11274.
[17]方芳,龚平生,宋祥福,等.低剂量电离辐射诱导小鼠睾丸细胞内质网应激及PERK-CHOP信号通路的激活[J].中华男科学杂志,2012,18(9):777-782.
[18]Wei J,Rahman S,Ayaub EA,et al.Protein misfolding and endoplasmic reticulum stress in chronic lung disease[J].Chest,2013,143(4):1098-1105.
[19]Matsumoto H,Miyazaki S,Matsuyama S,et al.Selection of autophagy or apoptosis in cells exposed to ER-stress depends on ATF4 expression pattern with or without CHOP expression[J].Biol Open,2013,2(10):1084-1090.
[20]Wang Q,He Z,Zhang J,et al.Overexpression of endoplasmic reticulum molecular chaperone GRP94 and GRP78 in human lung cancer tissues and its significance[J].Cancer Detect Prev,2005,29(6):544-551.
[21]Yoshida H,Okada T,Haze K,et al.ATF6 activated by proteolysis binds in the presence of NF-Y(CBF)directly to the cis-acting element responsible for the mammalian unfolded protein response[J].Mol Cell Biol,2000,20(18):6755-6767.
[22]Safra M,Ben-Hamo S,Kenyon C,et al.The ire-1 ER stressresponse pathway is required for normal secretory-protein metabolism in C.elegans[J].J Cell Sci,2013,126(Pt 18):4136-4146.
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