蛋白激酶C在糖基化终末产物介导结肠平滑肌细胞内钙离子浓度中的作用

摘要

目的: 探讨糖基化终末产物(advanced glycation end products, AGEs)对结肠平滑肌细胞内钙离子浓度的影响及可能机制.

方法: 酶解法分离培养SD大鼠结肠平滑肌细胞(smooth muscle cell, SMC), α-actin免疫荧光鉴定; 激光共聚焦显微镜检测(SMC)钙闪烁; 蛋白激酶C(protein kinase C, PKC)活性检测试剂盒检测细胞PKC活性.

结果: 细胞免疫荧光鉴定大鼠结肠SMC. 不同浓度AGEs(50、100 μg/mL)刺激结肠SMC后, 与对照组比较, 钙离子浓度显著降低(56.7%±3.6%、78.6%±5% vs 99.6%±3.1%, P<0.05, P<0.01), 50 μg/mL为体外最大有效浓度. 与对照组相比, AGEs(50、100 μg/mL)升高细胞内PKC活性. PKC抑制剂chelerythrine可阻断AGEs介导的钙离子浓度降低(70.7%±3.7% vs 87.1%±2.5%, P<0.05).

结论: AGEs可激活PKC通路、从而降低胞内钙离子的浓度, 最终抑制大鼠结肠平滑肌收缩.

关键词: 糖基化终末产物; 结肠平滑肌细胞; 蛋白激酶C; 钙离子

核心提示: 本研究提示糖基化终末产物(advanced glycation end products, AGEs)可激活结肠平滑肌细胞内的蛋白激酶C(protein kinase C, PKC), PKC磷酸化下游底物丝/苏氨酸残基, 调节结肠平滑肌细胞内钙离子浓度, 参与细胞收缩等功能.

Advanced glycation end products inhibit intracellular calcium concentration in colon smooth muscle cells in a protein kinase C-dependent manner

Ying Zhu, Qing-E Wang, Yun Wang, Yao-Yao Gong, Xiao-Meng Sun, Lin Lin

Ying Zhu, Qing-E Wang, Yun Wang, Yao-Yao Gong, Xiao-Meng Sun, Lin Lin, Department of Gastroenterology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China

Supported by: National Natural Science Foundation of China, No. 81270462; the International Science and Technology Cooperation Program of Jiangsu Province, No. BZ2011044

Correspondence to: Lin Lin, Professor, Department of Gastroenterology, the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, Jiangsu Province, China. lin9100@aliyun.com

Received: December 6, 2013
Revised: December 30, 2013
Accepted: January 8, 2014
Published online: February 28, 2014

Abstract

AIM: To investigate the effect of advanced glycation end products (AGEs) on intracellular calcium concentration in isolated colonic smooth muscle cells and the possible mechanisms involved.

METHODS: Colonic smooth muscle cells were isolated from normal adult rats, and immumofluorescence staining for α-actin was used to identify smooth muscle cells. The responsiveness of colonic smooth muscle cells to AGEs was measured by confocal laser scanning microscopy. Intracellular Ca²⁺ concentration ([Ca²⁺]i) was determined by Fluo3/AM based digital microfluorimetric measurement. Protein kinase C (PKC) activity was detected by PKC activity assay. PKC inhibitor chelerythrine was used to examine the role of PKC in AGEs-mediated inhibition of [Ca²⁺]i in colonic smooth muscle cells.

RESULTS: Colonic smooth muscle cells were successfully isolated from normal rats and identified by immunofluorescence staining. AGEs inhibited [Ca²⁺]i in a concentration-dependent manner. AGEs at a concentration of 50 or 100 µg/mL significantly inhibited the mean [Ca²⁺]i compared with the control group (56.7% ± 3.6%, 78.6% ± 5% vs 99.6% ± 3.1%, P < 0.05, P < 0.01). PKC activity increased in SMCs treated with 50 µg/mL or 100 µg/mL of AGEs compared with the control group. Pretreatment with chelerythrine (1 µmol/L) reduced AGEs-mediated inhibition of [Ca²⁺]i (70.7% ± 3.7% vs 87.1% ± 2.5%, P < 0.05).

CONCLUSION: AGEs inhibit [Ca²⁺]i in colonic smooth muscle cells in a PKC-dependent manner.

Key Words: Advanced glycation end products; Smooth muscle cell; Protein kinase C; Ca2+

0 引言

糖尿病胃肠动力障碍是糖尿病(diabetes mellitus, DM)的慢性并发症之一, 发生于75%的DM患者[1,2]. 糖基化终末产物(advanced glycation end products, AGEs)在DM患者血清及各种组织中均显著升高, 他与DM肾病、DM血管病变的研究颇多, 与DM胃肠动力障碍的研究很少[3,4]. 细胞外钙内流和内钙释放是平滑肌细胞(smooth muscle cell, SMC)收缩的决定因素; DM结肠SMC内钙离子信号通路异常, 导致钙离子浓度异常[5,6]. 本文探讨AGEs是否影响结肠SMC内钙离子浓度及可能机制, 为DM胃肠道动力障碍的研究提供依据.

1 材料和方法

1.1 材料

清洁级SD大鼠, 体质量150-200 g, 由南京医科大学实验动物中心提供; DMEM培养基、胎牛血清、青链霉素混悬液、大豆胰蛋白酶抑制剂(Gibco, USA); α-actin抗体(Abgent, USA); AGEs(Merck Millipore, Germany); FITC标记山羊抗大鼠IgG二抗(Bioworld, USA); 蛋白激酶C(protein kinase C, PKC)检测试剂盒(Enzo, USA); PKC抑制剂chelerythrine、Ⅱ型胶原酶(Sigma, USA); Fluo-3/AM(Invitrogen, USA).

1.2 方法

1.2.1 结肠SMCs的分离及原代培养: SD大鼠断颈处死, 快速自肛门上2 cm取结肠10 cm左右, 用Hepes-Ringer缓冲液反复冲洗, 去除黏膜和浆膜层、剪碎平滑肌组织、加入消化液(0.1%的II型胶原酶和0.01%的大豆胰蛋白酶抑制剂)消化、离心, 含10%胎牛血清及DMEM培养液重悬细胞、过筛; 锥虫蓝染色确认细胞活力>90%, 于37 ℃、95%O₂和5%CO₂条件下培养, SMCs长至致密单层时, 传代培养, 采用2代SMCs进行实验.

1.2.2 结肠SMCs的鉴定: 取对数生长期的SMCs, 胰蛋白酶消化、制成单细胞悬液、接种到放有载玻片的培养皿中、CO₂培养箱培养1-3 d, 待SMCs长至单层时, 吸去培养液, PBS冲洗、冰丙酮固定、PBS冲洗、BSA封闭30 min后吸去、加α-actin一抗(1:100), 阴性对照不加一抗, 4 ℃过夜, PBS冲洗; 加羊抗兔IgG二抗(1:500), 室温避光湿盒中孵育1 h, 冲洗、hoechest33258染核3 min、PBS冲洗、封片、镜下观察特异性荧光.

1.2.3 细胞内钙离子浓度的检测: 用DMSO溶解的Fluo-3/AM为钙荧光探针, SMCs接种于玻底皿中, PBS冲洗后, 加5 μmol/L的Fluo-3/AM, 置于37 ℃、95%O₂和5%CO₂孵育箱40 min、PBS冲洗; 激光共聚焦显微镜激发光波488 nm, 发射光波为515 nm, 采样间歇为2 s, 记录单个SMC在给予AGEs前后荧光强度变化(荧光强度表示为F/F0, F0为初始荧光强度).

1.2.4 PKC活性检测: 蛋白裂解液提取各组细胞蛋白, 4 ℃、12000 r/min、5 min、取上清, BCA法测定蛋白总量, 加待检样品30 µL于包被的反应孔中, 37 ℃孵育2 h, 洗涤、加酶标抗体30 µL, 室温孵育2 h, 洗涤、加显色液30 µL、显色30-45 min、终止反应, 在450 nm波长读板.

统计学处理 采用SPSS17.0软件包分析, 非参数检验之K-S检验进行数据正态性分析, 各组数据均呈正态分布, 以mean±SD表示, 各组间比较采用单因素方差分析和成组t检验, P<0.05为差异有统计学意义.

2 结果

2.1 结肠SMCs鉴定(免疫荧光)

细胞核Hoechst染色呈蓝色, 大部分细胞α-actin荧光反应阳性, 细胞呈梭形(胞质见红色荧光)(图1).

图1 大鼠结肠SMC鉴定(细胞免疫荧光×200). A: Hoechest33258染色细胞核(蓝色); B: α-actin染色(绿色); C: 融合.

2.2 不同浓度AGEs对结肠SMCs中钙离子浓度的影响

与对照组相比, AGEs在50和100 µg/mL时, 降低Fluo-3/AM负载的钙离子荧光强度(56.7%±3.6%、78.6%±5% vs 99.6%±3.1%, P<0.05, P<0.01), 说明AGEs可降低SMCs内钙离子浓度; AGEs在10 µg/mL时, 与对照组的差异无统计学意义; 实验结束后加入50 mmol/L氯化钾, 钙离子显著升高表示细胞活性良好(图2).

图2 Fluo-3/AM负载大鼠结肠SMC钙离子, 给予不同浓度AGEs荧光强度的变化. A: 对照组; B: 10 µg/mL AGEs; C: 50 µg/mL AGEs; D: 100 µg/mL AGEs; E: 不同浓度AGEs介导结肠SMC荧光强度, 用F/F0表示, F: 实时荧光强度; F0: 基础荧光强度. ^aP<0.05, ^bP<0.01 vs 对照组.

2.3 不同浓度AGEs对结肠SMCs中PKC活性的影响

与对照组相比, AGEs在50 µg/mL和100 µg /mL时, 升高细胞内PKC活性(2.3600±0.0723、2.1060±0.0625 vs 0.5123±0.0614, P<0.05, P<0.01), 而AGEs在10 mg/mL时, 与对照组差异无统计学意义(图3).

图3 不同浓度AGEs对结肠SMC中PKC活性的影响. AGEs在50 µg/mL和100 µg/mL时, 升高细胞内PKC活性. ^aP<0.05, ^bP<0.01 vs 对照组.

2.4 PKC抑制剂chelerythrine阻断AGEs介导的钙离子浓度降低

预先给予PKC抑制剂chelerythrine(1 µmol/L)可显著升高Fluo-3/AM负载的钙离子荧光强度, 说明细胞内钙离子浓度明显增加; 再加入AGEs后荧光强度下降, 但下降较溶剂对照组(DMSO, 1 µL)明显减少. 提示PKC抑制剂chelerythrine阻断AGEs介导的钙离子浓度降低(70.7%±3.7% vs 87.1%±2.5%, P<0.05)(图4).

图4 PKC抑制剂chelerythrine阻断AGEs介导的钙离子浓度降低. A: 溶剂对照组; B: PKC抑制剂chelerythrine组; C: PKC抑制剂chelerythrine阻断AGEs介导的钙离子浓度降低, 用F/F0%表示, F: 实时荧光强度, F0: 基础荧光强度. ^aP<0.05 vs 溶剂对照组. AGEs: 糖基化终末产物.

3 讨论

本实验证实: AGEs可以影响细胞内钙离子浓度, 其通过活化PKC, 来降低细胞内钙离子浓度; 而PKC抑制剂chelerythrine则可阻断AGEs降低细胞内钙离子浓度的作用. 提示PKC参与了AGEs抑制大鼠结肠SMCs钙信号的作用, 减少钙离子释放[7,8].

细胞外钙内流和内钙释放是SMCs收缩的决定因素, 经典的钙致敏平滑肌收缩机制是: 钙离子与钙调蛋白结合, 再结合并激活肌球蛋白轻链激酶(myosin light chain kinase, MLCK), 激活的MLCK磷酸化肌球蛋白轻链(myosin light chain, MLC), 促进肌动蛋白和肌球蛋白之间的横桥周期、导致平滑肌收缩. 而肌球蛋白轻链磷酸酶(myosin light chain phosphase, MLCP)可使肌球蛋白轻链去磷酸化、导致平滑肌松弛[9,10].

研究发现PKC有多种亚型, 其中α、β、γ等亚型主要分布于胃肠SMC上, PKC可被DAG、Ca2+或磷脂激活, 参与调节细胞收缩、细胞增殖、代谢和凋亡等多种过程[11-13]. 生理条件下, 磷脂酶C(phospholipase C, PLC)激活, 水解4, 5-二磷酸磷脂酰肌醇(4, 5-phosphatidylinositol bisphosphate, PIP2)产生三磷酸肌醇(inositol triphosphate, InsP3)和甘油二酯(diaglycerides, DAG), InsP3与内质网上InsP3R结合、引起内质网钙离子释放, 细胞内钙离子浓度升高; 同时PKC活化负反馈调节钙离子的释放[14-16], PKC这种保护性负反馈机制一方面源于细胞内钙过度释放、导致内质网自身应急信号的激活, 另一方面可防止细胞内钙过度释放引起能量浪费和细胞损伤[17,18]; 该生理过程决定着多种生物学效应, 如钙依赖性酶和通道的激活, 细胞收缩等[19-21]. 另有文献报道, 其他一些蛋白激酶如PKG、PKA也能负反馈调节细胞内钙离子浓度, 发挥相应的生物学功能[22-24].

文献报道AGEs在DM患者血清及组织中比正常人明显升高, 他可通过氧化应激、糖基化修饰某些蛋白在DM并发症中起重要作用[25,26]. 本实验的新颖点: 从DM结肠SMCs病变入手, 探讨AGEs是否通过SMCs内钙离子途径、最终影响SMCs收缩的. 遗憾的是: AGEs作用受体及信号通路尚不明确(尤其是胃肠平滑肌AGEs的作用受体不明), 因此没有特异性受体拮抗剂[27-29], 不能直接证明AGEs降低SMCs内钙离子浓度的信号通路. 另外, 调节SMCs内钙离子的机制很多, AGEs是否影响细胞外钙离子内流? 是作用于胞膜上T型钙通道或者L型钙通道还是抑制内质网上InsP3R3或ryanodine受体释放钙离子[30]? 仍有待研究证实. 最后, SMCs内钙离子浓度的检测还可以结合流式细胞术及膜片钳技术多重证实.

总之, AGEs可以降低结肠SMC内钙离子浓度从而抑制收缩. AGEs激活PKC降低细胞内钙离子浓度, 是细胞内钙离子降低的重要因素. DM患者结肠动力障碍与患者血清及组织中AGEs增多相关, AGEs对结肠平滑肌是否有直接抑制作用, 以及AGEs降低结肠SMCs内钙离子的其他机制, 仍待研究.

评论

背景资料

糖尿病(diabetes mellitus, DM)胃肠动力障碍与胃肠自主神经、Cajal间质细胞及平滑肌细胞病变相关, 平滑肌病变在DM胃肠动力障碍中的研究已成为重点, 其中DM结肠平滑肌细胞内钙离子信号通路异常, 导致钙离子浓度异常, 是本病的病理基础之一.

同行评议者

刘长征, 副教授, 中国医学科学院基础医学研究所

研发前沿

糖基化终末产物(advanced glycation end products, AGEs)在DM患者血清及各种组织中均显著升高, 他与DM肾病、DM血管病变的研究颇多. 但AGEs与 DM结肠平滑肌病变相关迄今为止尚未报道.

相关报道

国内外研究报道DM胃肠动力障碍存在胃肠道平滑肌病变, 且DM结肠平滑肌细胞内存在钙离子信号通路异常.

创新盘点

文本首次采用激光共聚焦的方法观测AGEs对结肠平滑肌细胞内钙离子浓度的影响及可能机制, 为DM胃肠道动力障碍研究提供依据.

应用要点

本研究提示AGEs抑制结肠平滑肌细胞内钙离子浓度, 可能是DM胃肠动力障碍的相关机制, 为临床治疗DM胃肠动力障碍提供治疗靶点.

同行评价

本文有一定的学术价值, 研究论点新颖, 为探索DM胃肠动力障碍、平滑肌病变提供了新思路.

备注

编辑:郭鹏 电编:鲁亚静

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