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

doi:10.19439/j.sjos.2019.03.007

上海口腔医学 ›› 2019, Vol. 28 ›› Issue (3): 259-263.doi: 10.19439/j.sjos.2019.03.007

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

嵇国平¹, 田一宏², 刘美希², 沈莹¹, 沈刚³

1.上海恒正口腔门诊部,上海200040;
2.青岛大学附属医院正畸科,青岛山东 266555;
3.上海拜博口腔医院,拜博口腔医疗集团正畸学科联合体,上海 200001

收稿日期:2019-03-08 修回日期:2019-05-25 出版日期:2019-06-25 发布日期:2019-08-09
通讯作者: 沈刚,E-mail：gangshen@orthosh.com
作者简介:嵇国平（1974-）,男,博士, E-mail：gpji741009@163.com

The role and mechanism of endoplasmic reticulum stress key factor PREK in myoblast apoptosis under stress loading

JI Guo-ping¹, TIAN Yi-hong², LIU Mei-xi², SHEN Ying¹, SHEN Gang³

1. Shanghai Hengzheng Dental Clinic. Shanghai 200040;
2. Department of Orthodontics, School and Hospital of Stomatology, Qingdao University. Qingdao 266555, Shandong Province;
3. Shanghai ByBo Dental Hospital,United Orthodontic Institutions, ByBo Dental Group. Shanghai 200001, China

Received:2019-03-08 Revised:2019-05-25 Online:2019-06-25 Published:2019-08-09

摘要/Abstract

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 R-like 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。

关键词: PERK, 周期性应力, 内质网应激, 成肌细胞凋亡

Abstract: 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 for 0, 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 mRNA 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 mRNA 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.

Key words: PERK, Cyclic stress, Endoplasmic reticulum stress, Myoblast apoptosis

中图分类号:

R783.5

嵇国平, 田一宏, 刘美希, 沈莹, 沈刚. 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[J]. 上海口腔医学, 2019, 28(3): 259-263.

JI Guo-ping, TIAN Yi-hong, LIU Mei-xi, SHEN Ying, SHEN Gang. The role and mechanism of endoplasmic reticulum stress key factor PREK in myoblast apoptosis under stress loading[J]. Shanghai Journal of Stomatology, 2019, 28(3): 259-263.

参考文献

[1] Owtad P, Potres Z, Shen G, et al.A histochemical study on condylar cartilage and glenoid fossa during mandibular advancement[J]. Angle Orthod, 2011, 81(2): 270-276.
[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, Eröz 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 mRNA 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 stress-response pathway is required for normal secretory-protein metabolism in C. elegans[J]. J Cell Sci, 2013, 126(Pt 18): 4136-4146.

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

The role and mechanism of endoplasmic reticulum stress key factor PREK in myoblast apoptosis under stress loading

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