天津医药

天津医药 ›› 2020, Vol. 48 ›› Issue (4): 248-252.doi: 10.11958/20191827

• 细胞与分子生物学 • 上一篇    下一篇

高迁移率族蛋白1在醛固酮诱导肾小管上皮细胞自噬中的作用 #br#

毛楠 1，林定彪 2，马欣 1，陈鸿禧 1，周琬秋 1，王少清 1△
  

  1. 1成都医学院第一附属医院肾脏内科（邮编610500）；2成都医学院生物科学与技术学院生物医学系
  • 收稿日期:2019-06-18 修回日期:2019-12-23 出版日期:2020-04-15 发布日期:2020-06-23
  • 通讯作者: 王少清 E-mail:wowosasa2003@163.com
  • 基金资助:
    2016年四川省医学科研青年创新项目（Q16016）

The role of high mobility group protein 1 in aldosterone-induced autophagy of renal tubular epithelial cells #br#

MAO Nan1, LIN Ding-biao2, MA Xin1, CHEN Hong-xi1, ZHOU Wan-qiu1, WANG Shao-qing1△ #br#   

  1. 1 Department of Nephrology, the First Affiliated Hospital of Chengdu Medical College, Chengdu 610500, China;
    2 Department of Biomedicine, College of Biological Science and Technology, Chengdu Medical College
  • Received:2019-06-18 Revised:2019-12-23 Published:2020-04-15 Online:2020-06-23
  • Contact: WANG Shao-qing E-mail:wowosasa2003@163.com
  • About author:毛楠（1985），女，博士，主治医师，主要从事慢性肾脏病发病机制的研究

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摘要： 目的 探讨高迁移率族蛋白 1（HMGB1）在醛固酮（ALDO）诱导大鼠肾小管上皮细胞自噬中的作用。方
法 取对数生长期的大鼠肾小管上皮细胞（NRK-52E），分为 Control组、ALDO组（10 nmol/L）、HMGB1抗体组（1 mg/
L）、IgG组（1 mg/L）和 ALDO+HMGB1抗体组（1 mg/L HMGB1抗体预处理 1 h，再加入 10 nmol/L ALDO刺激 24 h）和阳
性对照组（Rosup，100 µmol/L），处理24 h后利用荧光探针DCFH-DA标记后使用流式细胞仪检测NRK-52E细胞内活
性氧（ROS）水平的变化。另外，将培养的 NRK-52E 细胞分为 Control 组、ALDO 组（10 nmol/L）、N-乙酰半胱氨酸组
（NAC，50 µmol/L）和 NAC+ALDO组（50 µmol/L NAC预处理 1 h，再加入 10 nmol/L ALDO），处理 24 h后 Western blot法
检测白细胞介素（IL）-1β蛋白的表达水平。观察10 nmol/L ALDO刺激24 h后，HMGB1蛋白表达的变化。最后，予以
1 mg/L HMGB1抗体预处理（1 mg/L HMGB1抗体处理 1 h后，再加入 10 nmol/L ALDO刺激 24 h），Western blot法检测
LC3-Ⅱ、Beclin-1和p62蛋白的表达水平。结果 流式细胞仪检测结果显示，与Control组相比，ALDO组NRK-52E细
胞内 ROS 的产生明显增加（P＜0.05）；与 ALDO 组相比，ALDO+HMGB1 抗体组细胞内 ROS 的水平显著下降（P＜
0.05）。Western blot结果显示，与Control组相比，ALDO组IL-1β、HMGB1、LC3-Ⅱ和Beclin-1蛋白的表达上调，p62蛋
白的表达下调（P＜0.05）；与 ALDO组相比，ALDO+NAC组 IL-1β蛋白的表达下调（P＜0.05），ALDO+HMGB1抗体组
LC3-Ⅱ蛋白的表达降低、p62蛋白的表达增加（P＜0.05），Beclin-1蛋白的表达差异无统计学意义。结论 ALDO通
过促进NRK-52E细胞内ROS的产生，增加HMGB1的分泌，刺激IL-1β的释放，进而诱导自噬的发生；抑制HMGB1的
释放可逆转这一现象，本研究为慢性肾脏病的防治提供了新靶点和新思路。

关键词: 高迁移率族蛋白质类, 醛固酮, 自噬, 慢性肾脏病, 肾小管上皮细胞

Abstract: Objective To investigate the role of high mobility group protein 1 (HMGB1) in aldosterone (ALDO) -
induced autophagy of rat renal tubular epithelial cells. Methods Rat renal tubular epithelial cells (NRK-52E) in
logarithmic growth phase were divided into control group, ALDO group (10 nmol/L), HMGB1 antibody group (1 mg/L), IgG
group (1 mg/L), ALDO + HMGB1 antibody group (1 mg/L HMGB1 antibody pretreatment for 1 h, then 10 nmol/L ALDO
stimulation for 24 h) and positive control group (Rosup, 100 µmol/L). After 24 h treatment, the changes of reactive oxygen
species (ROS) levels in NRK-52E cells were detected by flow cytometry after labeling with fluorescent probe DCFH-DA. In
addition, the cultured NRK-52E cells were divided into control group, ALDO group (10 nmol/L), N-acetylcysteine group
(NAC, 50 µmol/L) and NAC+ALDO group (50 µmol/L NAC pretreatment for 1 hour, followed by 10 nmol/L ALDO). The
expression of IL-1β protein was detected by Western blot assay 24 hours after treatment. The expression of HMGB1 protein was observed 24 hours after 10 nmol/L ALDO stimulation.Finally, the expressions of LC3-Ⅱ, Beclin-1 and p62 proteins were detected by Western blot assay after pretreatment with 1 mg/L HMGB1 antibody for 1 hour and stimulation with 10 nmol/L ALDO for 24 hours. Results Flow cytometry showed that ROS production in NRK-52E cells increased significantly
in ALDO group compared with those of control group (P＜0.05). Compared with ALDO group, the intracellular ROS level
decreased significantly in ALDO+HMGB1 antibody group (P＜0.05). Western blot results showed that compared with control
group, the expressions of IL-1β, HMGB1, LC3- Ⅱ and Beclin-1 proteins were up-regulated in ALDO group, and the
expression of p62 protein was down-regulated (P＜ 0.05). Compared with ALDO group, the expression of IL-1β protein was
down-regulated in NAC+ALDO group (P＜0.05), the expression of LC3-Ⅱ protein was down-regulated and the expression
of p62 protein was increased in ALDO+HMGB1 antibody group (P＜0.05). There was no significant difference in the
expression of Beclin-1 protein (P＞0.05). Conclusion ALDO can induce autophagy by promoting ROS production,
increasing the secretion of HMGB1 and stimulating the release of IL-1β in NRK-52E cells. Inhibiting the release of HMGB1
can reverse this phenomenon and provide new targets and ideas for the prevention and treatment of chronic kidney diseases.

Key words: high mobility group proteins, aldosterone, autophagy, chronic kidney disease, renal tubular epithelial cells

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