1. 2019;22;155-164Toxicity Effects of Methylene Blue on Rat Intervertebral Disc Annulus Fibrosus Cells
   Basic Science
   Liang Zhang, MD, PhD, Yang Liu, MD, Zenan Huang, MD, Liping Nan, MD, Feng Wang, MD, Shifeng Zhou, MD, Jingcheng Wang, MD, PhD, and Xinmin Feng, MD.

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