Huangqin flavonoid extraction for spinal cord injury in a rat model

Abstract

Flavonoids from Huangqin (dried roots of Scutellaria baicalensis Georgi) have anti-inflammatory effects, and are considered useful for treatment of spinal cord injury. To verify this hypothesis, the T9-10 spinal cord segments of rats were damaged using Allen's method to establish a rat spinal cord injury model. Before model establishment, Huangqin flavonoid extraction (12.5 g/kg) was administered intragastrically for 1 week until 28 days after model establishment. Methylprednisolone (30 mg/kg) was injected into the tail vein at 30 minutes after model establishment as a positive control. Basso, Beattie, and Bresnahan locomotor scale scores were used to assess hind limb motor function. Hematoxylin-eosin staining was used to detect pathological changes in the injured spinal cord. Immunofluorescence and western blot assays were performed to measure immunoreactivity and expression levels of brain-derived neurotrophic factor, neuronal marker neurofilament protein, microglial marker CD11b and astrocyte marker glial fibrillary acidic protein in the injured spinal cord. Huangqin flavonoid extraction markedly reduced spinal cord hematoma, inflammatory cell infiltration and cavities and scars, and increased the Basso, Beattie, and Bresnahan locomotor scale scores; these effects were identical to those of methylprednisolone. Huangqin flavonoid extraction also increased immunoreactivity and expression levels of brain-derived neurotrophic factor and neurofilament protein, and reduced immunoreactivity and expression levels of CD11b and glial fibrillary acidic protein, in the injured spinal cord. Overall, these data suggest that Huangqin flavonoid extraction can promote recovery of spinal cord injury by inducing brain-derived neurotrophic factor and neurofilament protein expression, reducing microglia activation and regulating reactive astrocytes.

Keywords: Scutellaria baicalensis; astrocytes; brain-derived neurotrophic factor; flavonoids; microglia; nerve regeneration; neural regeneration; neurofilament; spinal cord injury.

Figure 1

Effect of Huangqin flavonoid extraction on motor function of rats after spinal cord injury (SCI). (A) The exposed spinal cord before and after the contusion injury induced with a weight-drop device. Hemorrhages and spinal cord edema are labeled with white arrows. (B) The stripped spinal cord tissue of different groups. (C) Effect of flavonoid extraction on motor function, as assessed using the Basso, Beattie, and Bresnahan (BBB) scoring system up to 4 weeks after SCI (n = 6 per group). (D) Effect of flavonoid extraction on cavities areas at 14 and 28 d after SCI. Data are presented as the mean ± SD, and analyzed by one-way analysis of variance followed by the Student-Newman-Keuls post hoc test. **P < 0.01, vs. model group. N: Normal group; M: model group; H: Huangqin group; d: days.

Figure 2

Effect of Huangqin flavonoid extraction on the histomorphological changes of the injured spinal cord. Assessment of injury in spinal cord sections using hematoxylin and eosin staining (original magnification, 4×). All cell nuclei were stained dark purple, and the cytoplasm was stained pink. The cavities (arrows) are labeled with a dotted line. Scale bars: 500 μm. N: Normal group; M: model group; H: Huangqin group; d: days.

Figure 3

Effect of Huangqin flavonoid extraction on the expression of neurofilament-H (NF-H) and brain-derived neurotrophic factor (BDNF) in neurons in the injured spinal cord of SCI rats. (A) NF-H (TRITC, red) and BDNF (FITC, green) expression with double-immunofluorescence labeling, and 4′,6-diamidino-2-phenylindole (DAPI) counterstaining, of the injured spinal cord at 3, 14 and 28 d of recovery in each group. (B) The integrated optical density of NF-H and BDNF double-immunofluorescence expression at 3, 14 and 28 d of recovery in each group (n = 8 per group). Western blot results (C) and bar graphs (D) showing NF-H and BDNF expression at 3, 14 and 28 d of recovery in each group (n = 6 per group). Data are presented as the mean ± SD, and analyzed by one-way analysis of variance followed by the Student-Newman-Keuls post hoc test. **P < 0.01, vs. normal group; #P < 0.05, ##P < 0.01, vs. model group. N: Normal group; M: model group; H: Huangqin group; BDNF: brain-derived neurotrophic factor; NF-H: neurofilament-H; DAPI: 4′,6-diamidino-2-phenylindole; d: days.

Figure 4

Effect of Huangqin flavonoid extraction on the expression of glial fibrillary acidic protein (GFAP) and CD11b in astrocytes and microglia in the injured spinal cord of SCI rats. (A) GFAP (DyLight 594, red) and CD11b (Dylight 488, green) expression with double-immunofluorescence labeling, and DAPI counterstaining, of the injured spinal cord at 3, 14 and 28 d of recovery in each group. (B) The integrated optical density of GFAP and CD11b double-immunofluorescence expression at 3, 14 and 28 d recovery in each group (n = 8 per group). Western blot results (C) and bar graphs (D) showing GFAP (50 kDa) and CD11b (127 kDa) expression at 3, 14 and 28 d of recovery in each group (n = 6 per group). Data are presented as the mean ± SD, and analyzed by one-way analysis of variance followed by the Student-Newman-Keuls post hoc test. **P < 0.01, vs. normal group; #P < 0.05, ##P < 0.01, vs. model group. N: Normal group; M: model group; H: Huangqin group; GFAP: glial fibrillary acidic protein; DAPI: 4′,6-diamidino-2-phenylindole; d: days.