Apoptotic action of botulinum toxin on masseter muscle in rats: early and late changes in the expression of molecular markers.

Young-Min Moon,Min-Keun Kim,Seong-Gon Kim,Tae-Woo Kim

doi:10.1186/s40064-016-2680-9

Young-Min Moon, Min-Keun Kim + Show 2 more

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https://doi.org/10.1186/s40064-016-2680-9

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Abstract

The purpose of this study was to compare the early or late expression levels of p65, Bcl-2, and type II myosin and the frequency of TUNEL-positive nuclei in the rat masseter muscle after injection of different concentrations of botulinum toxin-A (BTX-A). We injected either 5 U or 10 U of BTX-A into both masseter muscles of the rat. As a control group, the same volume of saline was injected. After 2 or 12 weeks, the animals were sacrificed. Subsequently, a biopsy and immunohistochemical staining of the samples were performed using a p65, Bcl-2, or type II myosin antibody. Additionally, a TUNEL assay and transmission electron microscopic analysis were performed. The expression of p65, Bcl-2, and type II myosin increased significantly with increasing concentrations of BTX-A at 2 weeks after BTX-A injection (P < 0.05). The number of TUNEL-positive nuclei was also significantly increased in the BTX-A-treated groups in comparison to the saline-treated control at 2 weeks after BTX-A injection (P < 0.05). Elevated expression of Bcl-2 was also observed in 10-unit BTX-A-treated group at 12 weeks after injection (P < 0.05). At 12 weeks after injection, the number of enlarged mitochondria was increased, and many mitochondria displayed aberrations in cristae morphology after BTX-A injection. In conclusion, BTX-A injection into the masseter muscle increased the expression level of p65, Bcl-2, and type II myosin at an early stage. The morphological changes of mitochondria were more evident at 12 weeks after injection.

Highlights

Botulinum toxin-A (BTX-A) is a protein produced by bacterium Clostridium botulinum (Schiavo et al 1992)
The transferase biotin dUTP nick-end labelling (TUNEL) assay demonstrated that the number of TUNEL-positive nuclei was significantly higher in the 10 U BTX-Atreated group than in the saline-treated group (Fig. 1b; P < 0.001)
The post hoc test revealed differences between the groups treated with 10 U BTX-A and the other groups, with significantly higher values in the 10 U BTX-A group than in the saline-treated control and 5 U BTX-A-treated group (P < 0.001 and 0.004 for the saline-treated group and the 5 U BTX-A-treated group, respectively)

Summary

Introduction

Botulinum toxin-A (BTX-A) is a protein produced by bacterium Clostridium botulinum (Schiavo et al 1992). Synaptosomal-associated protein of 25 kDa (SNAP-25) has been regarded as the only target for BTX-A until recently (Rossetto et al 2014). BTX-A mediated SNAP25 proteolysis inhibits acetylcholine release from nerve endings (Rossetto et al 2014). New findings suggest that BTX-A may have antimitotic and antitumor properties (Matak and Lackovic 2015). BTX-A induces apoptosis mediated by capase-3 and capase-7 in breast cancer cells (Bandala et al 2013). These fibroblasts and breast cancer cells do not express SNAP-25 (Matak and Lackovic 2015). BTX-A activates apoptotic pathways in the prostate via sympathetic nerve impairment (Gorgal et al 2012).

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