中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (34): 5503-5507.doi: 10.3969/j.issn.2095-4344.1484

• 生物材料基础实验 basic experiments of biomaterials • 上一篇    下一篇

萝卜硫素干预纳米细菌诱导肾小管上皮细胞凋亡及线粒体自噬相关蛋白的表达

蒲道静1,郑  府2,徐先顺2,丁文娟2,陈  艳2,邹照银2 
  

  1. 恩施土家族苗族自治州中心医院,1感染管理科,2泌尿外科,湖北省恩施市  445000
  • 收稿日期:2019-04-26 出版日期:2019-12-08 发布日期:2019-12-08
  • 通讯作者: 邹照银,主治医师,恩施土家族苗族自治州中心医院泌尿外科,湖北省恩施市 445000
  • 作者简介:蒲道静,女,1974年生,湖北省恩施市人,1987年湖北民族大学毕业,副主任护师,主要从事肾脏疾病研究。

Effects of sulforaphane on mitophagy-related proteins in nanobacteria-induced renal tubular epithelial cell apoptosis

Pu Daojing1, Zheng Fu2, Xu Xianshun2, Ding Wenjuan2, Chen Yan2, Zou Zhaoyin2 
  

  1. 1Department of Infection Management, 2Department of Urology, the Central Hospital of Enshi Tujia and Miao Autonomous Prefecture, Enshi 445000, Hubei Province, China
  • Received:2019-04-26 Online:2019-12-08 Published:2019-12-08
  • Contact: Zou Zhaoyin, Attending physician, Department of Urology, the Central Hospital of Enshi Tujia and Miao Autonomous Prefecture, Enshi 445000, Hubei Province, China
  • About author:Pu Daojing, Associate chief nurse, Department of Infection Management, the Central Hospital of Enshi Tujia and Miao Autonomous Prefecture, Enshi 445000, Hubei Province, China

摘要:

文章快速阅读:

 

文题释义:
线粒体自噬:指细胞选择性清除受损、衰老的线粒体,从而维持细胞内环境稳定;是一种进化上保守的细胞过程,以清除功能失调或多余的线粒体,从而微调线粒体数和保持能量代谢;还一种特异性的过程,线粒体通透性孔道通透性的改变在这个过程中起着重要的作用。
纳米细菌:胞内存在一种原核微生物,能通过100 nm的滤菌器,1990年Kajander等将此种微生物命名为纳米细菌。纳米细菌是革兰阴性菌,呈球状或球杆状,细胞壁厚,无荚膜与鞭毛结构,20-200 nm,可通过0.1-0.4 μm的滤菌膜,体积极小,通过电子显微镜和其他的高分辨率显微镜(如原子显微镜)可发现。
 
 
背景:萝卜硫素能改善肾小管上皮细胞氧化应激损伤,抑制细胞凋亡,对肾小管上皮细胞损伤作用具有保护作用,但萝卜硫素对肾小管上皮细胞线粒体自噬的影响还有待研究。
目的:验证萝卜硫素对纳米细菌诱导人肾小管上皮细胞(HK-2)凋亡及线粒体自噬相关蛋白表达的影响。
方法:研究方案的实施符合恩施土家族苗族自治州中心医院对研究的相关伦理要求。纳米细菌取自恩施土家族苗族自治州中心医院泌尿外科临床确诊肾结石且未服药或手术患者的尿液,患者对实验方案完全知情同意。将人肾小管上皮细胞HK-2分为3组:对照组、纳米细菌组及萝卜硫素干预组(给予纳米细菌刺激同时加30 μmol/L萝卜硫素)。采用CCK-8法检测HK-2细胞相对存活率,流式细胞仪检测HK-2细胞凋亡率,Western Blot方法检测凋亡相关蛋白(Bax和Bcl-2)及同源性磷酸酶张力蛋白诱导的激酶1(PINK1)/帕金蛋白(Parkin)介导的线粒体自噬相关蛋白PINK1、Parkin、LC3-Ⅱ、LC3-Ⅰ的表达。
结果与结论:①萝卜硫素能有效改善纳米细菌对HK-2细胞增殖的抑制作用(P < 0.05);②纳米细菌可上调促凋亡蛋白Bax表达,下调抗凋亡蛋白Bcl-2表达,诱导线粒体自噬相关蛋白PINK1、Parkin的表达及LC3-Ⅱ/LC3-Ⅰ比值上升(P < 0.05);而萝卜硫素处理可明显下调促凋亡蛋白Bax表达,上调抗凋亡蛋白Bcl-2表达,抑制PINK1、Parkin蛋白表达及LC3-Ⅱ/ LC3-Ⅰ比值(P < 0.05);③结果说明,萝卜硫素可抑制纳米细菌诱导的肾小管上皮细胞HK-2的凋亡,缓解其由PINK1/Parkin介导的线粒体自噬。

关键词: 萝卜硫素, 肾小管上皮细胞, 纳米细菌, 线粒体自噬, 细胞凋亡, Bax, Bcl-2, LC3-Ⅱ, LC3-Ⅰ

Abstract:

BACKGROUND: Sulforaohane has been shown to effectively improve the oxidative stress injury of renal tubular epithelial cells and inhibit the cell apoptosis to protect against renal tubular epithelial cell damage. However, the effect of sulforaohane on the mitophagy in renal tubular epithelial cells needs to be further studied.
OBJECTIVE: To explore the effects of sulforaphane on apoptosis- and mitophagy-related proteins in nanobacteria-induced renal tubular epithelial cell (HK-2) apoptosis.
METHODS: The study was in accordance with the ethics requirements of the Central Hospital of Enshi Tujia and Miao Autonomous Prefecture. Nanobacteria were from the urine of patients with kidney stone receiving no administration or undergoing surgery at Department of Urology, the Central Hospital of Enshi Tujia and Miao Autonomous Prefecture, and the patients signed the informed consents. HK-2 cells were divided into three groups, control group, nanobacteria-infected group, and sulforaphane intervention group (treated with nanobacteria and 30 μmol/L sulforaphane). The relative survival rates of HK-2 cells were determined using cell counting kit-8. Flow cytometry was used to detect the apoptotic rate of HK-2 cells. The expression levels of apoptosis-related proteins (Bax and Bcl-2) and proteins including PINK1, Parkin, LC3-II, LC3-I associated with mitophagy mediated by phosphatase and tensin homolog deleted on chromosome ten induced kinase 1 (PINK1)/Parkin were detected by western blot assay.
RESULTS AND CONCLUSION: (1) The inhibition of nanobacteria-induced cell proliferation was effectively lightened by sulforaphane (P < 0.05). (2) The expression levels of pro-apoptotic protein Bax and mitophagy-associated proteins PINK1 and Parkin and the ratio of LC3-II/LC3-I in HK-2 cells were up-regulated by nanobacteria (P < 0.05), and then were significantly down-regulated by sulforaphane (P < 0.05). The expression levels of anti-apoptotic protein Bcl-2 in HK-2 cells were down-regulated by nanobacteria (P < 0.05), and then was significantly up-regulated by sulforaphane (P < 0.05). (3) In summary, sulforaphane can inhibit the apoptosis of nanobacteria-induced renal tubular epithelial cells HK-2, and alleviate PINK1/Parkin-mediated mitophagy.

Key words: sulforaphane, renal tubular epithelial cells, nanobacteria, mitophagy, apoptosis, Bax, Bcl-2, LC3-II, LC3-I

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