Honokiol exerts protective effects on neural myelin sheaths after compressed spinal cord injury by inhibiting oligodendrocyte apoptosis through regulation of ER-mitochondrial interactions

Abstract

Objective: To investigate the effect of honokiol on demyelination after compressed spinal cord injury (CSCI) and it's possible mechanism.

Design: Animal experiment study.

Setting: Institute of Neuroscience of Chongqing Medical University.

Interventions: Total of 69 Sprague-Dawley (SD) rats were randomly divided into 3 groups: sham group (n=15), honokiol group (n=27) and vehicle group (n=27). After established CSCI model by a custom-made compressor successfully, the rats of sham group were subjected to the limited laminectomy without compression; the rats of honokiol group were subjected to CSCI surgery and intraperitoneal injection of 20 mg/kg honokiol; the rats of vehicle group were subjected to CSCI surgery and intraperitoneal injection of an equivalent volume of saline.Outcome measures: The locomotor function of each group was assessed using the Basso, Beattie and Bresnahan (BBB) rating scale. The pathological changes of myelinated nerve fibers of spinal cord in 3 groups were detected by osmic acid staining and transmission electron microcopy (TME). Immunofluorescence and Western blot were used to research the experessions of active caspase-3, caspase-12, cytochrome C and myelin basic protein (MBP) respectively.

Results: In the vehicle group, the rats became paralyzed and spastic after injury, and the myelin sheath became swollen and broken down along with decreased number of myelinated nerve fibers. Western blot analysis manifested that active caspase-3, caspase-12 and cytochrome C began to increase 1 d after injury while the expression of MBP decreased gradually. After intervened with honokiol for 6 days, compared with the vehicle group, the locomotor function and the pathomorphological changes of myelin sheath of the CSCD rats were improved with obviously decreased expression of active caspase-3, caspase-12 and cytochrome C.

Conclusions: Honokiol may improve locomotor function and protect neural myelin sheat from demyelination via prevention oligodendrocytes (OLs) apoptosis through mediate endoplasmic reticulum (ER)-mitochondria pathway after CSCI.

Keywords: Compressed spinal cord injury; Demyelination; Honokiol; Oligodendrocyte; Remyelination.

Figure 1

Model of injury.

Figure 2

Assessment of neurological function. Locomotor scores were assessed based on the BBB rating scale. Data represent mean ± SEM. *P < 0.05, compared with the sham group; ^#P < 0.05, compared with the vehicle group. n = 5 per group.

Figure 3

Morphological changes and MBP expression. (A) Ultrastructural features of myelinated nerve fibers in white matter on day 7 after CSCI. In the vehicle group, the myelin sheaths were swollen or fragmented (arrow point). The degree of swelling in the myelin sheaths in the honokiol group was milder than that in the vehicle group, and layers of the myelin sheaths tended to be more compact (arrow point). Scale bar = 1 µm. (B) Representative images (×400) showing osmic acid staining of myelinated nerve fibers in white matter from the first day to the seventh day in different groups after CSCI. Scale bar = 40 µm. (arrow point: myelinated nerve fiber). (C) The number of myelinated nerve fibers in different groups on day 1, 3, and 7 after CSCI. (D 1) Western blot showing MBP expression in different groups on day 1, 3, and 7 after CSCI. (D 2) Expression of MBP was normalized to β-actin as estimated by optical density and expressed as the percentage of the value recorded for the sham group. Data represent mean ± SEM. *P < 0.05, compared with the sham group; ^#P < 0.05, compared with the vehicle group. n = 5 per group.

Figure 4

Expression of apoptosis-related factors in different groups after CSCI. (A) Immunofluorescence of caspase-12- and active caspase-3-positive CNPase cells in white matter of the spinal cord in different groups on day 7 after CSCI. Scale bar = 40 µm. Caspase-12- and active caspase-3-positive CNPase cells in the white matter (arrow point). (C) Immunofluorescence of cytochrome C- and active caspase-3-positive CNPase cells in white matter of the spinal cord in different groups on day 7 after CSCI. Scale bar = 40 µm. Cytochrome C- and active caspase-3-positive CNPase cells (arrow point). (E) Immunofluorescence of cytochrome C- and caspase-12-positive CNPase cells in white matter of the spinal cord in different groups on day 7 after CSCI. Scale bar = 40 µm. Cytochrome C- and caspase-12-positive CNPase cells (arrow point). (B1-2; D1-2; F1-2) Active caspase-3, caspase-12, and cytochrome C protein expression in different groups on day 1, 3, and 7 after CSCI were examined by western blot. Protein expression was normalized to β-actin or GAPDH, as estimated by optical density and expressed as percentage of the sham group. Data represent mean ± SEM. Expression levels of active caspase-3, caspase-12, and cytochrome C were lower in the honokiol group when compared with the vehicle group. *P < 0.05, compared with the sham group; ^#P < 0.05, compared with the vehicle group. n = 5 per group.