The Extract of Roots of Sophora flavescens Enhances the Recovery of Motor Function by Axonal Growth in Mice with a Spinal Cord Injury

Abstract

Although axonal extension to reconstruct spinal tracts should be effective for restoring function after spinal cord injury (SCI), chondroitin sulfate proteoglycan (CSPG) levels increase at spinal cord lesion sites, and inhibit axonal regrowth. In this study, we found that the water extract of roots of Sophora flavescens extended the axons of mouse cortical neurons, even on a CSPG-coated surface. Consecutive oral administrations of S. flavescens extract to SCI mice for 31 days increased the density of 5-HT-positive axons at the lesion site and improved the motor function. Further, the active constituents in the S. flavescens extract were identified. The water and alkaloid fractions of the S. flavescens extract each exhibited axonal extension activity in vitro. LC/MS analysis revealed that these fractions mainly contain matrine and/or oxymatrine, which are well-known major compounds in S. flavescens. Matrine and oxymatrine promoted axonal extension on the CSPG-coated surface. This study is the first to demonstrate that S. flavescens extract, matrine, and oxymatrine enhance axonal growth in vitro, even on a CSPG-coated surface, and that S. flavescens extract improves motor function and increases axonal density in SCI mice.

Keywords: Sophora flavescens; axonal growth; chondroitin sulfate proteoglycan; matrine; oxymatrine; spinal cord injury.

Figure 1

S. flavescens extract promotes axonal extension on the CSPG coating. Cortical neurons were cultured for 1 day and then treated with S. flavescens extract (1 and 10 μg/ml) or vehicle solution. Four days after treatment, the cells were fixed and immunostained for pNF-H and MAP2. (A) Representative immunofluorescence images of pNF-H for each treatment. (B) The densities of pNF-H-positive axons per neuron were quantified for each treatment. The numbers in parentheses indicate the measured number of captured images. ^*p < 0.05 vs. CSPG coating/vehicle-treated group, One-way ANOVA post hoc Bonferroni test. Scale bar = 200 μm.

Figure 2

S. flavescens extract improves motor dysfunction in SCI mice. SCI mice were administered S. flavescens extract (black squares, nine mice, 18 hindlimbs, n = 18) or vehicle solution (white squares, eight mice, 16 hindlimbs, n = 16) once a day from 1 h after the injury for 31 days. The motor function of hindlimbs was assessed using BMS (A), BSS (B), and TMS (C). At 31 days after SCI, the ambulation of a representative SCI mouse treated with vehicle solution (D) or S. flavescens extract (E) was captured. Sequentially captured images obtained every 0.05 s are shown. ^*p < 0.0001, time × drug interaction analyzed by repeated measures Two-way ANOVA. #p < 0.05, post hoc Bonferroni test.

Figure 3

S. flavescens extract increases the axon density at lesion sites in SCI mice. Sagittal sections of spinal cords were obtained from S. flavescens extract—or vehicle-treated SCI mice at 31 days after the injury. The sections were immunostained for 5-HT, GFAP, and CSPG. (A) Representative immunofluorescence images of 5-HT. The yellow solid lines indicate outlines of the spinal cords. The yellow dotted line surrounds the lesion sites identified using the immunofluorescence images of GFAP. (B–D) The 5-HT-positive area at the lesion site (B), size of the lesion site (C), and expression of CSPG at the lesion site (D) were quantified. The numbers in parentheses indicate the numbers of mice. ^*p < 0.05 vs. vehicle group, unpaired two-tailed t-test. Scale bar = 500 μm.

Figure 4

The alkaloid fraction and the water fraction promote axonal extension on the CSPG coating. Cortical neurons were cultured for 1 day and then treated with the ethyl acetate fraction (1 and 10 μg/ml), the alkaloid fraction (1 and 10 μg/ml), the water fraction (1 and 10 μg/ml), S. flavescens extract (10 μg/ml), or vehicle solution. Four days after treatment, the cells were fixed and immunostained for pNF-H and MAP2. (A) Representative immunofluorescence images of pNF-H for each treatment. (B) The densities of pNF-H-positive axons per neuron were quantified for each treatment. The numbers in parentheses indicate the measured number of captured images. ^*p < 0.05 vs. CSPG coating/vehicle-treated group, One-way ANOVA post hoc Bonferroni test. Scale bar = 200 μm.

Figure 5

LC/MS analysis of the alkaloid and water fractions. Photodiode array (PDA) total scan chromatograms, total ion chromatograms, and extracted ion chromatograms at m/z 249.1967 ± 0.003 and 265.1916 ± 0.003 to detect matrine and oxymatrine, respectively, in the alkaloid fraction and the water fraction. ^*A specific peak of matrine; ^#a specific peak of oxymatrine.

Figure 6

Matrine and oxymatrine promote axonal extension on the CSPG coating. Cortical neurons were cultured for 1 day and then treated with matrine (1 and 10 μM), oxymatrine (1 and 10 μM), or vehicle solution. Four days after the treatment, the cells were fixed and immunostained for pNF-H and MAP2. (A) Representative immunofluorescence images of pNF-H for each treatment. (B) The density of pNF-H-positive axons per neuron were quantified for each treatment. The numbers in parentheses indicate the measured number of captured images. ^*p < 0.05 vs. CSPG coating/vehicle-treated group, One-way ANOVA post hoc Bonferroni test. Scale bar = 200 μm.

Figure 7

Matrine, oxymatrine, and S. flavescens extract have no effects on axonal extension on the PDL coating. Cortical neurons were cultured for 1 day and then treated with matrine (1 and 10 μM), oxymatrine (1 and 10 μM), S. flavescens extract (1 and 10 μg/ml), or vehicle solution. Four days after the treatment, the cells were fixed and immunostained for pNF-H and MAP2. The density of pNF-H-positive axons per neuron were quantified for each treatment. The numbers in parentheses indicate the measured number of captured images.