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1 2016 7, 23(4): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J ZF4 ROS MAPK 许琼琼, 韩兵社, 罗军涛, 李艳, 侯艳雯, 胡鹏, 张俊芳,, 摘要 : (reactive oxygen species, ROS), (Danio rerio) ZF4 (18 10 ), (1 d 3 d 5 d) ROS (mitogen-activated protein kinase, MAPK) : (1) DCFH-DA, ROS ROS 3 d, 18 10, ROS (28 ) (1.23±0.04) (P<0.05) (2.31±0.08) (P<0.05) (2) Western Blot p38 (p-p38) JNK (p-jnk p54 p-jnk p46), p38 JNK, 10 3 d (3) DNA γh2a.x,, 18 10, 3 ROS ;, p-jnk p-p38 γh2a.x ROS, 3 d : ROS; ZF4 ; ; MAPKs; γh2a.x 中图分类号 : S917 文献标志码 : A 文章编号 : (2016) , [1 2],, [3 5],, [6],,,, (reactive oxygen species, ROS) (mitogen-activated protein kinase, MAPK) [7] ROS, ( O 2 ) (H 2 O 2 ) (-OH) [8], ROS, ROS,, [9] (mitogen-activated protein kinase, MAPK),, [10] MAPK 4 : ERK p38 收稿日期 : ; 修订日期 : 基金项目 : ( ); (13ZR ); ; (201457). 作者简介 : (1992 ),,,, @qq.com 通信作者 :,. jfzhang@shou.edu.cn

2 JNK ERK5, JNK, 46 kd 54 kd, p46 p54 MAPK, ROS MAPK ( : p38 JNK ASK1 ) [11 12] ROS [13 14], ROS MAPK (Danio rerio), [15] ZF4 1 d [16], [17] (1 d 3 d 5 d) (18 10 ) ZF4 ROS MAPK, ZF4 American Type Culture Collection (ATCC), 28 5% CO 2 DMEM: F12 (10% 100 U/mL 100 mg/ml ) 2 d, 90% 0.25% EDTA, ZF4 4~6 1.2 Gibco ; DMEM.. F12 (Hyclone, SH B) L-Glutamine(Hyclone, SH ) DPBS Thermo ; 2, 7 DCFH-DA DMSO Sigma ; p-p38(thr202/tyr204) p-jnk(thr183/tyr185) Cell Signaling Technology ; β-actin Sigma ; γh2a.x (phosphorylate S139) Abcam ; Western 5 SDS-PAGE TEMED ; Bradford ; ECL Bio-Rad; : (Galaxy170R, eppendorf) Accuri C6 (BD) (Amersham Imager 600); (Bio-Rad); (Zeiss) 1.3, mm, h, 70%~90% (18 10 ) 1 d 3 d 5 d, 1.4 ROS 35 mm (28 ) (1 d 3 d 5 d) (18 10 ), DCFH-DA ( 20 μmol/l) 30 min, DPBS 3, 1000 g 5 min, 500 μl DPBS 1.5 ml, FL1 ( 488 nm), ROS 1.5 Western Blot 35 mm, DPBS 3, 500 μl Western ( cocktail ), 1.5 ml, g 15 min,, Bradford 5 loading buffer, 95 5 min, 30 μg, 12% SDS-PAGE PVDF,, 5% 2 h, 4, 2 h, ECL,

3 第4期 1.6 许琼琼等: 低温胁迫诱导斑马鱼 ZF4 细胞 ROS 及 MAPK 相关蛋白表达的影响数据统计分析收缩成长条状, 细胞之间的空隙变大, 而在 10 所有实验结果均是来自至少 3 次独立实验, 处理 5 d 的时候, 能够在培养基中发现明显可见图中所示结果均为均值±标准差( x ±SD) 单因素的细胞脱落悬浮在培养基中方差分析(one-way ANOVA)用于检测低温处理的 2.2 显著性, 两两之间的显著差异 (P<0.05) 利用 Turkey s 多重比较来检测低温诱导 ROS 产生与常温下细胞相比, 无论是在 18 还是在 10, 低温均能诱导细胞内 ROS 水平上升 (图 2 A) 与对照组(28 )相比, 在低温处理 5 d 后, 18 处理的细结果与分析胞其 ROS 含量显著升高到(1.42±0.06)倍(P<0.05); ZF4 低温下形态学检测而 10 处理的细胞其 ROS 含量显著升高到倒置相差显微镜下观察可见, 在常温下(28 ), (2.46±0.08)倍(P<0.05)(图 2B) 从 ROS 上升的时 ZF4 细胞呈现多边形, 细胞核呈椭圆形, 细胞呈间梯度点上来看, 18 处理 5 d 后, 细胞的 ROS 含量现铺路石排列(图 1) 在 18 低温处理下, 细胞形有显著的升高; 而在 10 处理 1 d 后, ROS 含量就显态变化不大, 处理 5 d 的时候, 有少量贴壁细胞脱著上升[达到(1.36±0.16)倍, P<0.05], 在处理 3 d 后, 落而在 10 低温处理 3 d 的时候, 可以看见细胞 ROS 升高更加显著[达到(2.31±0.05)倍, P<0.05] 图1 Fig ZF4 细胞在常温 28 以及低温(18 和 10 )处理 1 d, 3 d 和 5 d 的细胞形态图 The cellular morphology at normal (28 ) and low (18 and 10 ) temperatures at indicated time points (1 d, 3 d and 5 d) 低温诱导 MAPK 通路中关键蛋白 p-p38 p-jnk 以及 γh2a.x 的表达 MAPK 蛋白激酶家族蛋白 p38 和 JNK 的活化现了类似的结果(图 3) γh2a.x 是组蛋白 H2A.X 的磷酸化形式, DNA 发生损伤时, H2A.X 可被磷酸化为 γh2a.x 需要在其内部特定位点进行磷酸化修饰, 所以检为了查看是否低温引起了 DNA 断裂, 我们检测测其磷酸化水平(p-p38 和 p-jnk)可以反映其活性了 DNA 断裂标记蛋白 γh2a.x 的表达, 我们同样状态 Western Blot 结果显示, 无论在 18 还是发现 10 处理 3 d 后, γh2a.x 的表达有明显的升 10, 低温都诱导了 p-jnk p54 的表达值得注高; 而在 18 处理 3 d 后, 我们同样也观察到意的是在 10 处理 3 d 后, p-jnk p54 和 p-jnk γh2a.x 的表达被强烈的诱导, 说明 18 的低温 p46 的表达量大幅度上升, 同样 p-p38 的蛋白也发胁迫也引起了细胞内的 DNA 断裂(图 3)

774 23 2 ROS A. ROS ; B. ROS. ±, 4.. Fig. 2 Determination of ROS level in cells at different temperatures by flow cytometry A: The detection of ROS under various temperatures and times points.

05) between samples are indicated by different lower cases. 3 Western Blot p-jnk (Thr183/Tyr185) p-p38 (Thr202/Tyr204) γh2a.x (phosphorylate S139) ACTB (β-actin). Fig.

4 ROS A. ROS ; B. ROS. ±, 4.. Fig. 2 Determination of ROS level in cells at different temperatures by flow cytometry A: The detection of ROS under various temperatures and times points. B: The fold change of ROS level of each cell population was quantified. Data shown are the means ± standard deviation of at least four independent experiments. Significances (P<0.05) between samples are indicated by different lower cases. 3 Western Blot p-jnk (Thr183/Tyr185) p-p38 (Thr202/Tyr204) γh2a.x (phosphorylate S139) ACTB (β-actin). Fig. 3 Western Blot showing the protein levels of phosphorylated-jnk(thr183/tyr185), phosphorylated-p38 (Thr202/Tyr204) and γh2a.x (phosphorylate S139) at low temperatures ACTB served as a control. 3, (Dissostichus mawsoni) 5 (Danio rerio) (Salmo salar) (Gasterosteus aculeatus) 鳉 (Fundulus heteroclitus) 鳉 (Oryzias latipes) 4, Chen [7] ROS, PHGPx Prdx5 SOD ROS,, ZF4 ROS, (18 10 ) (1~5 d)( 2) ROS, 10 3~5 d, ROS ( 2B), ROS,, SOD ROS, SOD [18], 18 [18] 16 [19] 12 [20],, [17], 2 : : 18 ; 10, ROS, : 18 ; 10 ( 1) MAPK 3 : ERK JNK p38, ( ),

5 4 : ZF4 ROS MAPK 775, [21] MAPKs ZF4 MAPK, p-jnk p54 p-jnk p46 p-p d, DNA γh2a.x ( 3),, ROS ( 2B), ZF4 ROS MAPK, ROS MAPK, ROS MAPK [22 23] ROS, ASK1 ASK1, JNK p38 [24] MAPK [25], ZF4, p-jnk p54 p-jnk p46 p-p38 10 ( 3),, ROS ASK1 JNK p38,, 18 3 d, p-p38, ROS p38,,, ROS MAPKs (p-jnk p54 p-jnk p46 p-p38), 10 3 d, ROS MAPK, (3 d) ZF4 参考文献 : [1] Brett J R. Temperature and fish[j]. Chesapeake Sci, 1969, 10(3): [2] Perry A L, Low P J, Ellis J R, et al. Climate change and distribution shifts in marine fishes[j]. Science, 2005, 308(5730): [3] Donaldson M R, Cooke S J, Patterson D A, et al. Cold shock and fish[j]. J Fish Biol, 2008, 73(7): [4] Bonga S W. The stress response in fish[j]. Physiol Rev, 1997, 77(3): [5] Long H. The effect of temperature to fish survival[j]. Fishery Modernization, 2005(2): [. [J]., 2005(2): ] [6] Gracey A Y, Fraser E J, Li W, et al. Coping with cold: An integrative, multitissue analysis of the transcriptome of a poikilothermic vertebrate[j]. Proc Natl Acad Sci USA, 2004, 101(48): [7] Chen Z, Cheng C H, Zhang J, et al. Transcriptomic and genomic evolution under constant cold in Antarctic notothenioid fish[j]. Proc Natl Acad Sci USA, 2008, 105(35): [8] Nordberg J, Arner E S. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system[j]. Free Radic Biol Med, 2001, 31(11): [9] Chinnusamy V, Zhu J, Zhu J K. Cold stress regulation of gene expression in plants[j]. Trends Plant Sci, 2007, 12(10): [10] Chen J Y, Wang C, Wang J, et al. Advances in MAPK signaling pathway[j]. China Medicine and Pharmacy, 2011, 1(8): [,,,. MAPK [J]., 2011, 1(8): ] [11] Ray P D, Huang B W, Tsuji Y. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling[j]. Cell Signal, 2012, 24(5): [12] Son Y, Cheong Y K, Kim N H, et al. Mitogen-Activated Protein Kinases and Reactive Oxygen Species: How Can ROS Activate MAPK Pathways?[J]. J Signal Transduct, 2011: Article ID [13] Lesser M P. Oxidative stress in marine environments: biochemistry and physiological ecology[j]. Annu Rev Physiol, 2006, 68: [14] Heise K, Estevez M, Puntarulo S, et al. Effects of seasonal and latitudinal cold on oxidative stress parameters and activation of hypoxia inducible factor (HIF-1) in zoarcid fish[j]. J Comp Physiol B, 2007, 177(7): [15] Howe K, Clark M D, Torroja C F, et al. The zebrafish reference genome sequence and its relationship to the human genome[j]. Nature, 2013, 496(7446): [16] Driever W, Rangini Z. Characterization of a cell line derived from zebrafish (Brachydanio rerio) embryos[j]. In Vitro Cell Dev Biol-Anim, 1993, 29(9): [17] Hu P, Liu M, Zhang D, et al. Global identification of the genetic networks and cis-regulatory elements of the cold re-

6 sponse in zebrafish[j]. Nucleic Acids Res, 2015, 43(19): [18] Tseng Y C, Chen R D, Lucassen M, et al. Exploring uncoupling proteins and antioxidant mechanisms under acute cold exposure in brains of fish[j]. PLoS ONE, 2011, 6(3): e [19] Long Y, Li L, Li Q, et al. Transcriptomic characterization of temperature stress responses in larval zebrafish[j]. PLoS ONE, 2012, 7(5): e [20] Tang S J, Sun K H, Sun G H, et al. Cold-induced ependymin expression in zebrafish and carp brain: implications for cold acclimation[j]. FEBS Lett, 1999, 459(1): [21] Johnson G L, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases[j]. Science, 2002, 298(5600): [22] Torres M, Forman H J. Redox signaling and the MAP kinase pathways[j]. Biofactors, 2003, 17(1 4): [23] Mccubrey J A, Lahair M M, Franklin R A. Reactive oxygen species-induced activation of the MAP kinase signaling pathways[j]. ARS, 2006, 8(9 10): [24] Tobiume K, Matsuzawa A, Takahashi T, et al. ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis[j]. EMBO Rep, 2001, 2(3): [25] Widmann C, Gibson S, Jarpe M B, et al. Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human[j]. Physiol Rev, 1999, 79(1): Effects of cold stress on ROS production and expression of MAPK proteins in zebrafish ZF4 cells XU Qiongqiong, HAN Bingshe, LUO Juntao, LI Yan, HOU Yanwen, HU Peng, ZHANG Junfang Ministry of Education; College of Fisheries and Life Science, Shanghai Ocean University, Shanghai , China Abstract: In our previous study, we compared the transcriptome between Antarctic fish and temperate fish and found that genes were involved in reactive oxygen species (ROS) scavenge and Mitogen-activated protein kinase (MAPK) pathway associated with the cold adaptation in fish. To further study the regulatory network and the reactive oxygen species (ROS) homeostasis under cold stress in fish, we examined the level of ROS and the expression of stress-related proteins in zebrafish Danio rerio-derived ZF4 cells exposed to mildly and severely cold temperatures, 18 and 10, at various time points (1 d, 3 d, and 5 d). Results showed that (1) DCFH-DA probe method was used to determine the level of ROS in cells. We found that the level of ROS in cell positively correlated with the intensity of cold stress. After 3 days of cold treatment, the level of ROS in cells significantly increased to 1.23±0.04 (P<0.05) and 2.31±0.08 (P<0.05) times, respectively, at 18 and 10, compared with that in cells at 28. (2) Western blot showed that the expression of MAPKs (p-jnk p54, p-jnk p46 and p-p38) was induced under cold stress, and reached the highest at 3 d of 10. (3) In addition, the expression of γh2a.x reached peak after 3 days both at 18 and 10. These findings showed that cold temperature could induce the production of ROS in ZF4 cells. The level of ROS in ZF4 cells is dependent on the stress intensity and duration. We observed the markedly induction of ROS in the cells at the first 3 days of cold treatment, while the expression of p-jnk, p-p38, and γh2a.x was markedly induced at the 3 d, indicating that 3 d of cold treatment might be a key time point to determine the protein expression in ZF4 cells. Key words: ROS; zebrafish ZF4 cells; low temperature stress; MAPKs; γh2a.x Corresponding author: ZHANG Junfang. jfzhang@shou.edu.cn

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环球中医药 2016 年 5 月第 9 卷第 5 期摇 Global Traditional Chinese Medicine, May 2016,Vol 郾 9, No 郾 5 摇 557 两种电针对脊髓损伤 14 天后大鼠运动功能神经元及 MEK2 p 鄄 ERK1 表达的影响论著宋良玉摇吕威摇景泉凯摇莫雨