Total flavonoids of hawthorn leaves promote motor function recovery via inhibition of apoptosis after spinal cord injury

Abstract

Flavonoids have been reported to have therapeutic potential for spinal cord injury. Hawthorn leaves have abundant content and species of total flavonoids, and studies of the effects of the total flavonoids of hawthorn leaves on spinal cord injury have not been published in or outside China. Therefore, Sprague-Dawley rats were used to establish a spinal cord injury model by Allen's method. Rats were intraperitoneally injected with 0.2 mL of different concentrations of total flavonoids of hawthorn leaves (5, 10, and 20 mg/kg) after spinal cord injury. Injections were administered once every 6 hours, three times a day, for 14 days. After treatment with various concentrations of total flavonoids of hawthorn leaves, the Basso, Beattie, and Bresnahan scores and histological staining indicated decreases in the lesion cavity and number of apoptotic cells of the injured spinal cord tissue; the morphological arrangement of the myelin sheath and nerve cells tended to be regular; and the Nissl bodies in neurons increased. The Basso, Beattie, and Bresnahan scores of treated spinal cord injury rats were increased. Western blot assays showed that the expression levels of pro-apoptotic Bax and cleaved caspase-3 were decreased, but the expression level of the anti-apoptotic Bcl-2 protein was increased. The improvement of the above physiological indicators showed a dose-dependent relationship with the concentration of total flavonoids of hawthorn leaves. The above findings confirm that total flavonoids of hawthorn leaves can reduce apoptosis and exert neuroprotective effects to promote the recovery of the motor function of rats with spinal cord injury. This study was approved by the Ethics Committee of the Guangxi Medical University of China (approval No. 201810042) in October 2018.

Keywords: Bax protein; Bcl-2 protein; Nissl bodies; apoptosis; cleaved caspase-3; inflammation; motor function recovery; neuroprotection; spinal cord injury; total flavonoids of hawthorn leaves.

Figure 1

Effect of TFHL on motor function recovery of SCI rats. BBB scores of different groups were recorded at 0, 1, 3, 7, and 14 days after SCI. All rats in the sham operation group exhibited a BBB score of 21 for the entire measurement period. The SCI group had the lowest BBB score. After 14 days of continuous treatment with TFHL (5, 10, and 20 mg/kg), the scores of the SCI + TFHL groups gradually increased with increasing drug concentration. However, the BBB scores were considered statistically significant among groups at 7 and especially 14 days (*P < 0.05, **P < 0.001). Data are expressed as the mean ± SD (n = 6; one-way analysis of variance followed by a least-significant difference post hoc test). BBB: Basso, Beattie, and Bresnahan; SCI: spinal cord injury; TFHL: total flavonoids of hawthorn leaves.

Figure 2

Effect of TFHL on histomorphological changes of the injured spinal cord. (A) Assessment of injury in spinal cord sections of different groups using hematoxylin-eosin staining (n = 3 per group, cross-section of the T10 segment of the spinal cord, scale bars: 100 μm). Inflammatory cells and the lesion cavity are clearly visible in each group. The arrows indicate severely injured cavities caused by inflammation. It is worth noting that because of injury or technical reasons, the spinal cord morphology data of the spinal cord injury (SCI) group are incomplete, but this did not affect the observation. (B) The proportion of the lesion cavity area of the total area was determined. The SCI group had the largest proportion of lesion cavity. After 14 days of continuous treatment with TFHL (5, 10, and 20 mg/kg), the proportions of lesion cavity areas of the SCI + TFHL groups decreased with increasing drug concentration. *P < 0.05, vs. sham operation group; #P < 0.05, vs. SCI group; †P < 0.05, vs. SCI + 5 mg/kg TFHL group. Data are expressed as the mean ± SD (one-way analysis of variance followed by a least-significant difference post hoc test). SCI: Spinal cord injury; TFHL: total flavonoids of hawthorn leaves.

Figure 3

Effect of TFHL on ultrastructural changes of the injured spinal cord. The changes in ultrastructure of spinal cord tissues were observed by transmission electron microscopy in each group (n = 3; cross-section of the T10 segment of the spinal cord; scale bars: 2 μm for the sham operation group and 1 μm for other four groups). In the sham operation group, the shape and structure of the myelin sheath were regular, the nerve cell nucleus was large and round, and the mitochondria were full. In the spinal cord injury (SCI) group, the shape and arrangement of the myelin sheath were irregular and disordered; the slab structure was loosely arranged, broken, and twisted; the mitochondria were swollen; and the nerve cell nucleus was irregular. In the SCI + TFHL groups, the conditions improved with drug concentration. In particular, the SCI + 20 mg/kg TFHL group demonstrated an obvious effect. Arrows indicate the myelin sheath and nerve cell nucleus of spinal cord tissue. SCI: Spinal cord injury; TFHL: total flavonoids of hawthorn leaves.

Figure 4

Effect of TFHL on the number of Nissl bodies in neurons in the injured spinal cord. (A) The number of Nissl bodies in neurons was observed by Nissl staining in each group (n = 3; cross-section of the T10 segment of the spinal cord; scale bars: 200 μm). Blue staining represents a Nissl body, as shown by the arrows. The spinal cord morphology of each group was observed. The Nissl bodies, nucleus, and morphology of nerve cells were clearly visible in each group. (B) The number of Nissl bodies/mm² was determined. The spinal cord injury (SCI) group had the lowest number of Nissl bodies/mm². After 14 days of continuous treatment with 5, 10, and 20 mg/kg TFHL, the number of Nissl bodies/mm² in the TFHL groups increased with increasing drug concentration. *P < 0.05, vs. sham operation group; #P < 0.05, vs. SCI group; †P < 0.05, vs. SCI + 5 mg/kg TFHL group. Data are expressed as the mean ± SD (one-way analysis of variance followed by a least-significant difference post hoc test). SCI: Spinal cord injury; TFHL: total flavonoids of hawthorn leaves.

Figure 5

Effect of TFHL on cell apoptosis in the injured spinal cord. (A) Apoptosis of cells was observed by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining in each group (n = 3; cross-section of the T10 segment of the spinal cord; scale bars: 200 μm). Apoptotic cells were stained by the TUNEL method, and nuclei were stained with 4′,6-diamidino-2-phenylindole. Green and blue fluorescence represent apoptotic cells and normal cells, respectively. (B) The spinal cord injury (SCI) group exhibited the largest percentage of TUNEL-positive cells. After 14 days of continuous treatment with TFHL (5, 10, and 20 mg/kg), the percentage of TUNEL-positive cells of the SCI + TFHL groups decreased with increasing drug concentration. *P < 0.05, vs. sham operation group; #P < 0.05, vs. SCI group; †P < 0.05, vs. SCI + 5 mg/kg TFHL group. Data are expressed as the mean ± SD (one-way analysis of variance followed by a least-significant difference post hoc test). DAPI: 4′,6-Diamidino-2-phenylindole; SCI: spinal cord injury; TFHL: total flavonoids of hawthorn leaves; TUNEL: terminal-deoxynucleotidyl transferase mediated nick end labeling.

Figure 6

Effect of TFHL on expression levels of Bcl-2, Bax, and cleaved caspase-3 in the injured spinal cord. (A) After 14 days of continuous treatment with TFHL (5, 10, and 20 mg/kg), the protein expression levels of Bcl-2, Bax, and cleaved caspase-3 were analyzed by western blot assays in each group (n = 6). (B–D) Gray value ratio of protein bands of Bcl-2, cleaved caspase-3, and Bax. After injury, protein expression levels of Bcl-2, Bax, and cleaved caspase-3 increased. After treatment with TFHL, the protein expression level of Bcl-2 increased, while the protein expression levels of Bax and cleaved caspase-3 decreased with increasing drug concentration in the spinal cord injury (SCI) + TFHL groups. *P < 0.05, vs. sham operation group; #P < 0.05, vs. SCI group; †P < 0.05, vs. SCI+5 mg/kg TFHL group. Data are expressed as the mean ± SD (one-way analysis of variance followed by a least-significant difference post hoc test). SCI: Spinal cord injury; TFHL: total flavonoids of hawthorn leaves.