降低Rho激酶抑制剂促神经元突起生长

doi:10.3969/j.issn.1673-5374.2012.34.008

中国神经再生研究(英文版) ›› 2012, Vol. 7 ›› Issue (34): 2705-2712.doi: 10.3969/j.issn.1673-5374.2012.34.008

• 原著:颅神经损伤修复保护与再生 • 上一篇下一篇

降低Rho激酶抑制剂促神经元突起生长

收稿日期:2012-02-06 修回日期:2012-07-24 出版日期:2012-12-05 发布日期:2012-07-24
基金资助:
云南省科技发展项目2009CD079 ，国家自然基金项目81060109.

Tolerance of neurite outgrowth to Rho kinase inhibitors decreased by cyclooxygenase-2 inhibitor

Weigang Duan¹, Ling Que¹, Xiaoman Lv¹, Qifeng Li¹, Hua Yin¹, Luyong Zhang²

1 Key Laboratory of Molecular Biology for Sinomedicine, Yunnan University of Traditional Chinese Medicine, Kunming 650500, Yunnan Province, China
2 New Drug Screening Center, China Pharmaceutical University, Nanjing 210009, Jiangsu Province, China

Received:2012-02-06 Revised:2012-07-24 Online:2012-12-05 Published:2012-07-24
Contact: Weigang Duan, Key Laboratory of Molecular Biology for Sinomedicine, Yunnan University of Traditional Chinese Medicine, Kunming 650500, Yunnan Province, China deardwg@126.com
About author:Weigang Duan☆, M.D., Associate professor, Key Laboratory of Molecular Biology for Sinomedicine, Yunnan University of Traditional Chinese Medicine, Kunming 650500, Yunnan Province, China
Supported by:
This study was financially supported by Yunnan Provincial Science and Technology Department, No. 2009CD079 and by the National Natural Science Foundation of China, No. 81060109.

摘要/Abstract

摘要：

以Rho相关激酶抑制剂Y27632，法舒地尔、环氧合酶1选择性抑制剂SC560、环氧合酶2抑制剂NS398干预PC12 Adh细胞和Neuro-2a 细胞。发现Rho相关激酶抑制剂干预3d以上的细胞出现明显的耐受现象，即突起生长减慢，环氧合酶2通路因子环氧合酶2和前列腺素E合成酶表达增加；NS398能减轻神经元突起生长的耐受现象，但SC560作用很弱。说明长时间接触Rho相关激酶抑制剂对神经元突起能产生耐受现象，其机制可说明至少部分与环氧合酶2通路上调有关，而环氧合酶2抑制剂能减轻这种耐受作用。

关键词: Rho激酶抑制剂, Y 27632, 神经元突起, 环氧合酶2抑制剂, 耐受

Abstract:

In this study, PC12 Adh cells and Neuro-2a cells were treated with Rho-associated kinase inhibitors (Y27632 and Fasudil), a cyclooxygenase-1 selective inhibitor (SC560), and a cyclooxygenase-2 inhibitor (NS398). We found that these cells became tolerant to Rho-associated kinase inhibitors, as neurite outgrowth induced by these inhibitors diminished following more than 3 days of exposure in either cell line. The proteins cyclooxygenase-2 and cytosolic prostaglandin E synthetase were upregulated at day 3. NS398 decreased the tolerance to neurite outgrowth induction in both cell lines, whereas SC560 had almost no effect. These findings indicate that cells become tolerant to neurite outgrowth induced by Rho-associated kinase inhibitors, this is at least partly associated with upregulation of proteins involved in the cyclooxygenase-2 pathway, and cyclooxygenases-2 inhibition prevents this tolerance.

Key words: Rho-associated kinase inhibitors, Y27632, fasudil, neurite, cyclooxygenase 2 inhibitors, drug tolerance

. 降低Rho激酶抑制剂促神经元突起生长[J]. 中国神经再生研究(英文版), 2012, 7(34): 2705-2712.

Weigang Duan, Ling Que, Xiaoman Lv, Qifeng Li, Hua Yin, Luyong Zhang. Tolerance of neurite outgrowth to Rho kinase inhibitors decreased by cyclooxygenase-2 inhibitor[J]. Neural Regeneration Research, 2012, 7(34): 2705-2712.

参考文献

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[2] Mueller BK, Mack H, Teusch N. Rho kinase, a promising drug target for neurological disorders. Nat Rev Drug Discov. 2005;4(5):387-398.

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[4] Katoh H, Aoki J, Ichikawa A, et al. p160 RhoA-binding kinase ROKalpha induces neurite retraction. J Biol Chem. 1998;273(5):2489-2492.

[5] Sakurada S, Takuwa N, Sugimoto N, et al. Ca2+- dependent activation of Rho and Rho kinase in membrane depolarization-induced and receptor stimulation-induced vascular smooth muscle contraction. Circ Res. 2003;93(6): 548-556.

[6] Que L, Duan W, Zhang L, et al. Differences of neurite outgrowth induced by rho kinase inhibitors between in PC12 cell line and PC12 Adh cell line. Yunnan Daxue Xuebao: Ziran Kexue Ban. 2011;33(3):370-372.

[7] Duan WG, Shang J, Jiang ZZ, et al. Rho kinase inhibitor Y-27632 down-regulates norepinephrine synthesis and release in PC12 cells. Basic Clin Pharmacol Toxicol. 2009;104(6):434-440.

[8] Wylie SR, Chantler PD. Myosin IIA drives neurite retraction. Mol Biol Cell. 2003;14(11):4654-4666.

[9] Yamazaki M, Miyazaki H, Watanabe H, et al. Phosphatidylinositol 4-phosphate 5-kinase is essential for ROCK-mediated neurite remodeling. J Biol Chem. 2002; 277(19):17226-17230.

[10] Chan CC, Roberts CR, Steeves JD, et al. Aggrecan components differentially modulate nerve growth factor- responsive and neurotrophin-3-responsive dorsal root ganglion neurite growth. J Neurosci Res. 2008;86(3): 581-592.

[11] Shiotani S, Shimada M, Suehiro T, et al. Involvement of Rho-kinase in cold ischemia-reperfusion injury after liver transplantation in rats. Transplantation. 2004;78(3): 375-382.

[12] Hiraga A, Kuwabara S, Doya H, et al. Rho-kinase inhibition enhances axonal regeneration after peripheral nerve injury. J Peripher Nerv Syst. 2006;11(3):217-224.

[13] Madura T, Kubo T, Tanag M, et al. The Rho-associated kinase inhibitor fasudil hydrochloride enhances neural regeneration after axotomy in the peripheral nervous system. Plast Reconstr Surg. 2007;119(2):526-535.

[14] Limongelli V, Bonomi M, Marinelli L, et al. Molecular basis of cyclooxygenase enzymes (COXs) selective inhibition. Proc Natl Acad Sci U S A. 2010;107(12): 5411-5416.

[15] Menter DG, Schilsky RL, DuBois RN. Cyclooxygenase-2 and cancer treatment: understanding the risk should be worth the reward. Clin Cancer Res. 2010;16(5): 1384-1390.

[16] Dehmelt L, Nalbant P, Steffen W, et al. A microtubule- based, dynein-dependent force induces local cell protrusions: Implications for neurite initiation. Brain Cell Biol. 2006;35(1):39-56.

[17] Gopalakrishnan SM, Teusch N, Imhof C, et al. Role of Rho kinase pathway in chondroitin sulfate proteoglycan- mediated inhibition of neurite outgrowth in PC12 cells. J Neurosci Res. 2008;86(10):2214-2226.

[18] Evangelopoulos ME, Weis J, Kruttgen A. Neurotrophin effects on neuroblastoma cells: correlation with trk and p75NTR expression and influence of Trk receptor bodies. J Neurooncol. 2004;66(1-2):101-110.

[19] Tamura M, Nakao H, Yoshizaki H, et al. Development of specific Rho-kinase inhibitors and their clinical application. Biochim Biophys Acta. 2005;1754(1-2):245-252.

[20] Wu J, Zhang L, Sun ZW, et al. Mutated recombinant human glucagon-like peptide-1 induces differentiation of PC12 cells. Neural Regen Res. 2011;6(6):457-461.

[21] Rojo AI, Innamorato NG, Martín-Moreno AM, et al. Nrf2 regulates microglial dynamics and neuroinflammation in experimental Parkinson's disease. Glia. 2010;58(5): 588-598.

[22] Stolp HB, Ek CJ, Johansson PA, et al. Factors involved in inflammation-induced developmental white matter damage. Neurosci Lett. 2009;451(3):232-236.

[23] Duan W, Zhang L. Cyclooxygenase inhibitors not inhibit resting lung cancer A549 cell proliferation. Prostaglandins Leukot Essent Fatty Acids. 2006;74(5):317- 321.

[24] Acquatella-Tran Van Ba I, Marchal S, François F, et al. Regenerating islet-derived 1α (Reg-1α) protein is new neuronal secreted factor that stimulates neurite outgrowth via exostosin Tumor-like 3 (EXTL3) receptor. J Biol Chem. 2012;287(7):4726-4739.

[25] Qin Z, Sun Z, Huang J, et al. Mutated recombinant human glucagon-like peptide-1 protects SH-SY5Y cells from apoptosis induced by amyloid-beta peptide (1-42). Neurosci Lett. 2008;444(3):217-221.

[26] Wang TC, Chiu H, Chang YJ, et al. The adaptor protein SH2B3 (Lnk) negatively regulates neurite outgrowth of PC12 cells and cortical neurons. PLoS One. 2011;6(10): e26433.

[27] Yamazaki M, Chiba K, Mohri T, et al. Cyclic GMP-dependent neurite outgrowth by genipin and nerve growth factor in PC12h cells. Eur J Pharmacol. 2004; 488(1-3):35-43.

[28] Fukuda T, Takekoshi K, Nanmoku T, et al. Inhibition of the RhoA/Rho kinase system attenuates catecholamine biosynthesis in PC 12 rat pheochromocytoma cells. Biochim Biophys Acta. 2005;1726(1):28-33.

引言

材料方法

Design

An observational cytology experiment.

Time and setting

This study was performed at the Key Laboratory of Molecular Biology for Sinomedicine, Yunnan University of Traditional Chinese Medicine, China from March 2009 to May 2011.

Materials
Cells

PC12 Adh cells were obtained from the American Type Culture Collection via the Kunming Institute of Zoology, Chinese Academy of Sciences, China. Neuro-2a cells were purchased from the Beijing Dingguo Biotechnology Company Ltd., China. Both cell lines were identified by comparing their morphologies with those described online (http://www.atcc.org/ ATCCAdvancedCatalogSearch/ProductDetails/tabid/ 452/Default.aspx?ATCCNum=CRL-1721.1&Template=cellBiology, http://www.atcc.org/ATCCAdvanced CatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CCL-131&Template= cellBiology).

Drugs

The ROCK inhibitors Y-27632 ((R)-(+)-trans-N-(4- pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide·2HCl, purity ≥ 98%) and fasudil (5-(1,4-diazepan-1- ylsulfonyl) isoquinoline, purity ≥ 98%) were purchased from Alexis Biochemicals (San Diego, USA). The COX-1 selective inhibitor SC560 (5-(4-chlorophenyl)-1- (4-methoxyphenyl)-3-trifluoromethylpyrazole, purity ≥ 98%, IC₅₀ = 9 nM)^[23]

and the COX-2 selective inhibitor NS398 (N-[2-(cyclohexoxy)-4-nitro-phenyl]- methanesulfonamide, purity ≥ 98%, IC₅₀ = 1.77 μM)^{[23 were purchased from Cayman Chemical Company (San Diego, USA). NGF-7S (130 kDa) was purchased from Sigma-Aldrich (St. Louis, MO, USA). ]}

Methods
Cell cultures

PC12 Adh cells were incubated in Ham’s F12K medium (Invitrogen, New York, NY, USA) with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate with 15% horse serum (Invitrogen) and 2.5% fetal bovine serum (Invitrogen) in an atmosphere containing 5% CO₂ and 95% air at 37°C. When the PC12 Adh cells occupied 80% of the flask, they were dispersed and seeded in 96-well plates wrapped with 0.01% polylysine for 4 hours. Then, we exposed the PC12 Adh cells to mixtures of ROCK inhibitors, COXs inhibitors, and NGF at different concentrations. We renewed the media every 2 days, and the concentration of the ROCK inhibitors, COXs inhibitors, and NGF in the media was maintained after each renewal^{[6, 24].}

Neuro-2a cells were incubated in Eagle’s Minimum Essential Medium (Invitrogen) with 10% fetal bovine serum in an atmosphere containing 5% CO₂ and 95% air at 37°C. When Neuro-2a cells occupied 80% of the flask, they were dispersed with a 0.25% trypsin and 0.53 mM ethylene diamine tetraacetic acid solution and seeded in 96-well plates for 4 hours. Then, we exposed the Neuro-2a cells to mixtures of ROCK inhibitors and COX inhibitors at different concentrations. We renewed each medium every 2 days, and the concentrations of the ROCK inhibitors and COX inhibitors in each medium were maintained after each renewal^[2.4]

The baseline period in the PC12 Adh cells and Neuro-2a cells before exposure to ROCK inhibitors, COX inhibitors, or NGF served as the negative control. NGF was used as the positive control for PC12 Adh cells.

Observations of neurite outgrowth

PC12 Adh cells^[25] and Neuro-2a cells grew neurites when exposed to ROCK inhibitors. The morphology of the treated PC12 Adh and Neuro-2a cells was categorized into neurite positive cells, round cells, and other cells was observed with a microscope (IX71-A21PH; Olympus, Tokyo, Japan). Cells with neurite(s) longer than their soma were defined as neurite positive cells. Other cells had various features including micro-spikes, ruffles, and a flattened appearance^[26-27] (supplementary Figures 1, 2 online).

The number of cells observed in each well was at least 100. If there were fewer than 100 cells in a well, all cells in that well were observed. The neurite positive cell rate was calculated based on the following formula: Positive cell rate (%) = (positive cells/total cells) × 100%. The EC₅₀ for each ROCK inhibitor and NGF was calculated with a sigmoidal dose-response equation^[26].

Western blot assays

PC12 Adh cells were exposed to Y-27632 or NGF for 3 days, and the cells were lysed with lyse buffer containing 20 mM Tris-HCl (pH 7.5), 0.1% Triton X-100, 1 mM ethylene diamine tetraacetic acid, 0.2 mM dithiothreitol, and 2 mM NaF. The lysate was spun (6 000 × g) for 10 minutes and the supernatant was collected as a protein sample for western blot assay. Based on previously reported methods^[28] with some modifications, protein (20 μg) from different samples was applied for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein in the electrophoresis was transferred to a nitrocellulose membrane by an electrical current of 10 V for 60 minutes in a semi-dry transfer system (Bio-Rad, Hercules, CA, USA). The nitrocellulose membrane was blocked with 3% bovine serum albumin at an ambient temperature (20°C) for 2 hours. It was then bathed in rabbit anti-COXs polyclonal antibodies (1:400; Boster, Wuhan, China), a rabbit anti-cytosolic prostaglandin E₂ synthase polyclonal antibody (1:500; Cell Signaling Technology, Beverly, MA, USA), and a rabbit anti-β-actin polyclonal antibody (1:600; Biovision, Milpitas, CA, USA) at 4°C overnight. The membrane was rinsed with TST solution that contained 20 mM Tris HCl (pH 7.5) and 0.05% Tween-20 for 10 minutes three times, and bathed in a solution of goat anti-rabbit antibody linked with horseradish peroxidase (1:1 000; Boster) for another 2 hours at the ambient temperature. The membrane was rinsed with the TST solution for 10 minutes three times and developed through chemiluminescence, and the sodium dodecyl sulfate-polyacrylamide gel electrophoresis bands in nitrocellulose membrane were recorded on X-ray film.

讨论

文章快阅

延伸阅读

1文章国内外研究状况的微观分析

经PubMed检索，Rho激酶抑制剂能促进神经突起生长，也能改善多种原因的神经损伤预后。COX-2抑制剂是一个经典的非甾体抗炎药，能够减轻炎症反应，也能改善某些神经损伤的预后，但COX-2抑制剂本身没有促神经生长作用。本研究是在筛选Rho激酶抑制剂的过程中无意中发现：Rho激酶抑制Y27632能促进PC12 Adh细胞突起生长，但其作用在第2天达到最大效应，其后突起不但不继续长长，还出现了萎缩。经检索，未发现此类报道。作者以此为契机，系统地阅读了Rho激酶抑制剂与神经突起生长方面的文献，发现Rho激酶抑制剂能对抗PGE2导致的神经生长锥坍塌，而PGE2是COX-2通路的主要产物。在此提示下，我们试图证明Rho激酶抑制剂长时间作用后会不会导致COX-2通路上调？这样，我们便有了该文的研究。

2文章创新性的分析

PubMed 检索参见下表（所有年）：

Search	Add to builder	Query	Items found	Time
#9	Add	Search #1 AND #5 AND #4	0	07:29:20
#8	Add	Search #5 AND #4	2	07:28:44
#7	Add	Search #1 AND #5	5	07:28:14
#6	Add	Search #1 AND #4	141	07:27:41
#5	Add	Search cyclooxygenase 2 inhibitors	9936	07:27:02
#4	Add	Search neurites	13530	07:26:47
#3	Add	Search fasudil	583	07:26:29
#2	Add	Search y27632	2004	07:26:07
#1	Add	Search rho associated kinase	4288	07:25:31

万方数据库检索结果参见下表（2009-2012）：

	检索词（题名/关键词）	检索结果
1	Rho激酶	期刊论文 (131) 学位论文 (39) 会议论文 (4)
2	环氧酶抑制剂	期刊论文 (19) 学位论文 (4) 会议论文 (0)
3	神经突起	期刊论文 (21) 学位论文 (9) 会议论文 (1)
4	1+3	期刊论文 (0) 学位论文 (0) 会议论文 (0)
5	2+3	刊论文 (0) 学位论文 (0) 会议论文 (0)

① 理论假设创新：Rho激酶抑制剂促神经生长已经被大量的研究证实，COX-2抑制剂对神经的保护作用也有报道，但COX-2抑制剂无促神经突起生长作用，COX-2产物导致神经突起生长抑制也有文献证实。单本研究首次发现Rho抑制剂在促神经突起生长方面存在一定的耐受现象，并找到其耐受的产生至少部分地与COX-2通路上调有关。因此本研究神经突起生长的大理论上无创新，但在此框架下有新发现，此新发现为以后Rho激酶抑制剂的使用提供依据。

② 技术方法创新：该研究所用方法均为细胞生物研究的常规方法（包括阳性细胞计数、Western Blot等），唯一不同的是该研究对神经突起生长作用进行了长时间观察。

③ 结果创新：得到了以前没有观测到的结果，即，长时间作用后，Rho激酶抑制剂在促神经突起生长方面存在耐受现象，此现象能被COX-2抑制剂部分减轻。

3 文章区别于他人他篇的特点。

① 干预措施的不同。

在Rho激酶抑制促神经突起生长的研究方面，我们进行了长期干预和观察，这一点与别人的文章不同，由此也观察到了新现象。

②结果结论的不同。

以前观察到的都是Rho激酶抑制剂促神经突起生长的作用，没有此方面的“反面”报道。该研究发现，Rho激酶抑制剂促神经生长是主要作用，但长时间作用其作用会减弱。此作用与beta受体的扩支气管（治疗哮喘）作用相似，因此称之为“耐受”。

降低Rho激酶抑制剂促神经元突起生长

Tolerance of neurite outgrowth to Rho kinase inhibitors decreased by cyclooxygenase-2 inhibitor

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