天津医药

天津医药 ›› 2016, Vol. 44 ›› Issue (7): 834-838.doi: 10.11958/20160025

• 细胞与分子生物学 • 上一篇    下一篇

Rho激酶在氢气保护LPS诱导的Caco-2细胞上皮屏障功能障碍中的机制研究

马小叶1,于洋2,张红涛3,谢克亮4,于泳浩1   

  1. 1 天津医科大学总医院麻醉科（邮编 300052）；2天津市麻醉学研究所
  • 收稿日期:2016-01-22 修回日期:2016-03-03 出版日期:2016-07-15 发布日期:2016-07-15
  • 通讯作者: 于泳浩 E-mail:yuyonghao@126.com
  • 作者简介:马小叶（1989）， 女， 硕士在读， 主要从事氢气对脓毒症器官功能保护方面的研究
  • 基金资助:
    国家自然科学基金资助项目（81071533， 81372033）；天津市应用基础及前沿技术研究计划（13JCQNJC11400）

The role of Rho kinase in the protection effects of hydrogen on Caco-2 epithelial barrier against LPS in vitro

MA Xiaoye, YU Yang, ZHANG Hongtao, XIE Keliang, YU Yonghao   

  1. 1 Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin 300052, China; 2 Tianjin Institute of Anesthesiology
  • Received:2016-01-22 Revised:2016-03-03 Published:2016-07-15 Online:2016-07-15
  • Contact: YU Yonghao E-mail:yuyonghao@126.com

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摘要： 目的 探讨Rho激酶（ROCK）在氢气改善离体脓毒症肠屏障功能中的作用。方法 常规培养人结肠上皮细胞Caco-2，随机分为6组（n=3）：对照组、富氢培养基组、LPS处理组、富氢培养基+LPS组、Rho激酶抑制剂组、Rho激酶抑制剂+LPS组。富氢培养基组给予0.6mmol/L富氢培养基；LPS 和Y-27632处理浓度分别为100μg/ml、25μmol/L。建立Transwell小室模型，定期检测跨上皮电阻值（TEER），当TEER达到800Ω?cm2后给予处理，于6h、12h、24h检测TEER值，24h时检测FITC-右旋糖苷通过率。细胞接种于6孔板，融合达80%~90%后给予处理，实时聚合酶链式反应（RT-PCR）技术检测ZO-1 mRNA和ROCK mRNA表达情况；蛋白免疫印迹技术（Western Blot）检测ZO-1蛋白和ROCK蛋白表达水平。结果 与对照组比较，富氢培养基组12h、24h TEER值升高（P＜0.05），FITC-右旋糖苷通过率、ZO-1和ROCK mRNA及蛋白表达水平无显著变化（P＞0.05）；ROCK抑制剂组各时间点TEER值升高（P＜0.05），FITC-右旋糖苷通过率无显著变化（P＞0.05），ZO-1 mRNA表达增加，ROCK mRNA表达减少（均P＜0.05）；LPS处理组各时间点TEER值降低，FITC-右旋糖苷通过率增高，ZO-1 mRNA和蛋白表达均下降，ROCK mRNA和蛋白表达均增加（P＜0.05）。与LPS处理组比较ROCK抑制剂+LPS组TEER值增高，FITC-右旋糖苷率降低，ZO-1 mRNA表达增加（P＜0.05）。与LPS处理组比较，富氢培养基+LPS组各时间点TEER值增高，FITC-右旋糖苷通过率降低，各时间点ZO-1蛋白表达上升，ROCK蛋白表达下降（均P＜0.05）。结论 氢气可保护脓毒症肠屏障功能，改善肠上皮屏障完整性，减少肠壁通透性，增加肠细胞间紧密连接蛋白表达，这些保护机制可能与氢气抑制LPS诱导的Rho激酶过度表达有关。

关键词: rho 相关激酶类, 氢, 脂多糖类, 脓毒症, Caco-2 细胞, 肠上皮屏障功能

Abstract: [Abstract] Objective To investigate the role of Rho kinase in the protection effects of hydrogen to intestinal epithelial barrier function against sepsis. Methods Caco-2 cells were cultured routinely, and divided into 6 groups randomly: control group, hydrogen-rich medium group, LPS-treatment group, hydrogen+ LPS-treament group, Y-27632 (ROCK inhibitor) treatment group and Y-27632+ LPS-treatment group. Hydrogen-rich medium group were treated with the 0.6mmol/L hydrogen-rich media. The concentration of LPS and Y-27632 were 100μg/ml and 25μmol/L separately. After the Caco-2 monolayer models were established, the transepithelial electrical resistance (TEER) values were measured regularly. When the TEER values reached 800Ω?cm2, the treatments were administered. Then TEER values were measured at 6h, 12h and 24h, and FITC-dextran permeability were detected at 24h. Cells were seeded on 6-well cell culture clusters. After cell density reached 80%~90%, treatments were given randomly. The real time-polymerase chain reaction (RT-PCR) was conducted to assess mRNA levels of ZO-1 and Rho kinase; ZO-1 and Rho kinase protein levels were detected with Western Blot. Results Compared with control group, TEER values of hydrogen-rich medium group elevated in 12h and 24h (P<0.05), no statistical significance in FITC-dextran permeability, neither in mRNA or protein levels of ZO-1 and ROCK (P>0.05); TEER values of Y-27632 treatment group at 6h, 12h and 24h elevated (P<0.05), FITC-dextran permeability did not change obviously (P>0.05), mRNA of ZO-1 increased and mRNA of ROCK decreased (P<0.05); the TEER values of LPS-treatment group reduced at 6h, 12h and 24h, FITC-dextran permeability increased markedly, mRNA and protein s of ZO-1 decreased, mRNA and protein s of ROCK increased (all P<0.05); compared with LPS-treatment group, TEER values in Y-27632+ LPS-treatment group rose significantly, FITC-dextran permeability decreased, mRNA s of ZO-1 increased, mRNA s of ROCK decreased (P<0.05); TEER values in hydrogen+ LPS-treament group increased, FITC-dextran permeability reduced markedly, protein s of ZO-1 increased, protein s of ROCK decreased (P<0.05). Conclusion Hydrogen can protect intestinal barrier function against sepsis, ameliorating the integrity of intestinal epithelium, reducing its permeability and increasing the s of tight junction proteins. The suppression of Rho kinase over reduced by LPS maybe involved in these protective effects of hydrogen.

Key words: Rho kinase, Hydrogen, LPS, epithelial barrier