亚甲蓝对大鼠椎间盘纤维环细胞的毒性作用。

Toxicity Effects of Methylene Blue on Rat Intervertebral Disc Annulus Fibrosus Cells.

机构信息

Clinical College of Yangzhou University, China.

Dalian Medical University, China.

出版信息

Pain Physician. 2019 Mar;22(2):155-164.

PMID:30921981

Abstract

BACKGROUND

There is an increasing local application of methylene blue (MB) in the treatment of discogenic low back pain (LBP) and percutaneous transforaminal endoscopic discectomy (PTED) procedures. MB could generate DNA damage and induce apoptosis in different cell types; however, the effects of MB on intervertebral disc (IVD) annulus fibrosus (AF) cells are not clearly understood.

OBJECTIVE

The objective of this study was to investigate the effects of different concentrations of MB on rat AF cells in vitro.

STUDY DESIGN

This study used an experimental design.

SETTING

This research was conducted at the Orthopaedic Institute of the Clinical Medical College of Yangzhou University.

METHODS

AF cells were isolated and cultured with different concentrations of MB (0, 2, 20, and 200 mu-g/mL) and assessed to determine the possible cytotoxic effects of MB. The cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. The inverted phase-contrast microscopy was used to perform morphological observation of apoptotic cells, and flow cytometry was used to measure the incidence of cell apoptosis. The mRNA and protein expression levels of apoptosis-associated genes (caspase-3, Bcl-2, and Bax) and other related genes (collagen type I, transforming growth factor beta1 [TGF-beta1], fibroblast growth factor [bFGF], and tissue inhibitor of metalloproteinase-1 [TIMP-1]) were analyzed by quantitative real-time PCR (RT-PCR) and Western blotting.

RESULTS

Our results indicated that MB reduced cell viability in a concentration- and time-dependent manner. MB also induced marked AF cell apoptosis in a concentration-dependent manner observed by inverted phase-contrast microscopy, flow cytometry, and indicated by the increased expression of caspase-3. Both RT-PCR and Western blotting revealed significant up-regulation of Bax and caspase-3 expression levels accompanied by decreased expression of Bcl-2 in a concentration-dependent manner. Moreover, collagen type I, TGF-beta1, bFGF, and TIMP-1 mRNA and protein levels were also found to be decreased by MB in a concentration-dependent manner.

LIMITATIONS

Limitations of this study were the in vitro study design and lack of in vivo validation of the observed effects of MB on human IVD cells.

CONCLUSIONS

Our results indicate that a high concentration of MB can not only inhibit proliferation and paracrine function of AF cells, but can also induce cell apoptosis in a concentration-dependent manner, suggesting that it is necessary to choose low concentrations of MB in practical application and limit the use of MB in the treatment of discogenic LBP to research protocols.

KEY WORDS

Methylene blue, annulus fibrosus cell, proliferation, apoptosis, paracrine.

摘要

背景

甲脒蓝（MB）在治疗椎间盘源性腰痛（LBP）和经皮椎间孔内窥镜椎间盘切除术（PTED）方面的局部应用越来越多。MB 可在不同细胞类型中产生 DNA 损伤并诱导细胞凋亡，但 MB 对椎间盘（IVD）纤维环（AF）细胞的影响尚不清楚。

目的

本研究旨在探讨不同浓度 MB 对大鼠 AF 细胞的体外影响。

研究设计

本研究采用实验设计。

设置

本研究在扬州大学临床医学院骨科研究所进行。

方法

分离培养不同浓度 MB（0、2、20、200 μ-g/mL）的 AF 细胞，并评估 MB 可能产生的细胞毒性作用。用细胞计数试剂盒-8（CCK-8）检测细胞增殖。倒置相差显微镜观察细胞凋亡形态，流式细胞术检测细胞凋亡率。通过实时定量 PCR（RT-PCR）和 Western blot 分析凋亡相关基因（caspase-3、Bcl-2 和 Bax）和其他相关基因（胶原 I、转化生长因子β1[TGF-β1]、成纤维细胞生长因子[bFGF]和金属蛋白酶组织抑制剂-1[TIMP-1]）的 mRNA 和蛋白表达水平。

结果

结果表明，MB 呈浓度和时间依赖性降低细胞活力。倒置相差显微镜、流式细胞术观察到 MB 呈浓度依赖性诱导 AF 细胞明显凋亡，并伴有 caspase-3 表达增加。RT-PCR 和 Western blot 均显示 Bax 和 caspase-3 表达水平显著上调，Bcl-2 表达水平呈浓度依赖性下调。此外，MB 还呈浓度依赖性降低胶原 I、TGF-β1、bFGF 和 TIMP-1 的 mRNA 和蛋白水平。