Ginsenoside Rd inhibits apoptosis following spinal cord ischemia/reperfusion injury

Abstract

Ginsenoside Rd has a clear neuroprotective effect against ischemic stroke. We aimed to verify the neuroprotective effect of ginsenoside Rd in spinal cord ischemia/reperfusion injury and explore its anti-apoptotic mechanisms. We established a spinal cord ischemia/reperfusion injury model in rats through the occlusion of the abdominal aorta below the level of the renal artery for 1 hour. Successfully established models were injected intraperitoneally with 6.25, 12.5, 25 or 50 mg/kg per day ginsenoside Rd. Spinal cord morphology was observed at 1, 3, 5 and 7 days after spinal cord ischemia/reperfusion injury. Intraperitoneal injection of ginsenoside Rd in ischemia/reperfusion injury rats not only improved hindlimb motor function and the morphology of motor neurons in the anterior horn of the spinal cord, but it also reduced neuronal apoptosis. The optimal dose of ginsenoside Rd was 25 mg/kg per day and the optimal time point was 5 days after ischemia/reperfusion. Immunohistochemistry and western blot analysis showed ginsenoside Rd dose-dependently inhibited expression of pro-apoptotic Caspase 3 and down-regulated the expression of the apoptotic proteins ASK1 and JNK in the spinal cord of rats with spinal cord ischemia/reperfusion injury. These findings indicate that ginsenoside Rd exerts neuroprotective effects against spinal cord ischemia/reperfusion injury and the underlying mechanisms are achieved through the inhibition of ASK1-JNK pathway and the down-regulation of Caspase 3 expression.

Keywords: ASK1; Caspase 3; JNK; apoptosis; ginsenoside Rd; ischemia/reperfusion injury; nerve regeneration; neural regeneration; spinal cord injury.

Figure 1

Effect of intraperitoneal injection of ginsenoside Rd on the morphology of L₂ spinal cord cross-sections in rats 5 days after spinal cord ischemia/reperfusion injury (hematoxylin-eosin staining). (a, A) Sham group; (b, B) ischemia/reperfusion in-jury group (I/R group); (c, C) 6.25 mg/kg ginseno-side Rd group; (d, D) 12.5 mg/kg Rd group; (e, E) 25 mg/kg ginsenoside Rd group; (f, F) 50 mg/kg ginsenoside Rd group. (A) Large neurons, slightly stained nuclei, clearly visible nucleolus, and patchy Nissl bodies (arrows) were found in the cytoplasm. (B) Large neurons and slightly stained nuclei were found, the nucleolus was visible. Nissl body content (arrows) was significantly lower than that in the sham group. (C) The neurons were large; Nissl body content (arrows) was higher than that in the I/R group. (D) The neurons were large, with a large amount of cytoplasmic Nissl bodies (arrows). (E) The neurons were large and round, with slightly stained nuclei and thick nucleolus, there were a large number of Nissl bodies (arrows) in the cytoplasm. (F) The neurons were large and round, with slightly stained nuclei and thick nucleolus, and there were a large number of Nissl bodies (arrows) in the cytoplasm. Original magnification, a–f: × 40, A–F: × 400; A–F are the magnified images of the boxes in a–f, respectively.

Figure 2

Hematoxylin-eosin staining score in rats with spinal cord ischemia/reperfusion injury to determine the optimal dose of ginsenoside Rd. *P < 0.05, vs. sham group; #P < 0.05, vs. ischemia/reperfusion group; †P < 0.05, vs. 12.5 mg/kg Rd group; P < 0.05, 12.5, 25 and 50 mg/kg ginsenoside Rd group vs. 6.25 mg/kg ginsenoside Rd group. Measure-ment data are expressed as the mean ± SD of five rats in each group, one-way analysis of variance and Student-Newman-Keuls test were used. I–VI are sham, ischemic/reperfusion, 6.25, 12.5, 25, and 50 mg/kg ginsenoside Rd groups, respectively.

Figure 3

Morphology of spinal cord in Sprague-Dawley rats for determination of the optimal treatment time of ginsenoside Rd (hematoxylin-eosin staining). (a, A) 1 day after modeling; (b, B) 3 days after mod-eling; (c, C) 5 days after modeling; (d, D) 7 days after modeling. (a, A) There were large and round neurons, slightly stained nuclei, large nucleolus, and many Nissl bodies (arrows) in the cytoplasm. (b, B) The nuclei were slightly stained, nucleoli were clearly visible, and many Nissl bodies (arrows) were found in the cytoplasm. (c, C) The nuclei were slightly stained and many Nissl bodies (arrows) were found, there were more large neurons than A and B. (d, D) The nuclei were slightly stained and many Nissl bodies (arrows) were found, there were more large neurons than A and B. Original magnifi-cation, a–d: × 40, A–D: × 400, A–D are the magni-fied images of the boxes in a–d, respectively.

Figure 4

Hematoxylin-eosin staining scorein rats with spinal cord ischemia/reperfusion injury for the determination of the optimal treatment time of ginsenoside Rd. *P < 0.05, vs. 1 day; #P < 0.05, vs. 3 days. Measurement data are ex-pressed as the mean ± SD of five rats in each group, one-way analysis of variance and Student-Newman-Keuls test were used. I–IV represent 1, 3, 5 and 7 days after treatment with ginsenoside Rd, respectively.

Figure 5

Morphology of spinal cord sections in rats 5 days after spinal cord ischemia/reperfusion injury for the determination of the optimal dose of ginsenoside Rd (Nissl staining, × 400). (A) Sham group; (B) ischemia/reperfusion injury group (I/R group); (C–F) 6.25, 12.5, 25, 50 mg/kg ginsenoside Rd groups. (A) In the sham group, spinal anterior horn moto-neurons were large; the nuclei were lightly stained, with a clearly visible nucleolus and a large amount of Nissl bodies in the cyto-plasm. (B) In the I/R group, there were large anterior horn motoneurons, lightly stained nuclei and clearly visible nucleoli, Nissl bod-ies that accumulated in the cytoplasm and were darkly stained. (C) 6.25 mg/kg ginse-noside Rd group: spinal anterior horn mo-toneurons were lightly stained and showed clear nucleoli, the content of Nissl bodies was high in the cytoplasm, which was lower than that in the I/R group, and the intensity of staining was lighter than that in the I/R group. (D–F) 12.5, 25, 50 mg/kg ginsenoside Rd groups: spinal anterior horn motor neu-rons showed light staining, clear nucleolus, and high content of Nissl bodies. Arrows indicate Nissl bodies.

Figure 6

Morphology of spinal cord sections in rats with spinal cord ischemia/reperfusion injury for the determination of the optimal treatment time of ginsenoside Rd (Nissl staining, × 400). (A–D) 1, 3, 5, 7 days after spinal cord ischemia/reperfusion injury. In all groups, spinal cord anterior horn motor neurons showed lightly stained cell nuclei, clearly visible nucleolus, and high content of Nissl bodies in the cytoplasm. Arrows indicate Nissl bodies.

Figure 7

Caspase 3 immunoreactivity in the spinal cord of rats at 5 days after spinal cord ischemia/reperfusion injuny for the determination of the optimal dose of ginsenoside Rd (immunohistochemistry, × 400). (A) Sham group; (B) ischemia/reperfusion injury group; (C–F) 6.25, 12.5, 25, 50 mg/kg ginsenoside Rd groups. (A) In the sham group, the neurons were large, the nuclei were round and lightly stained, the nucleolus was clearly visible, and no Caspase 3 posi-tive particles were found in the cytoplasm. (B) In the ischemia/reperfusion injury group, a large amount of Caspase 3 positive particles were found in the cytoplasm of some neurons. (C) In the 6.25 mg/kg ginsenoside Rd group, Caspase 3 positive particles were found in the cytoplasm of some neurons, but the number of particles and cells was significantly lower than that in the ischemia/reperfusion injury group. (D) In the 12.5 mg/kg ginsenoside Rd group, Caspase 3 positive particles were visible in a small number of neurons. (E, F) In the 25 and 50 mg/kg ginsenoside Rd groups, the neurons were large, a small amount of Caspase 3 positive particles were found in nerve cells. Arrows refer to apoptotic nerve cells.

Figure 8

Caspase 3 immunoreactivity of the spinal cord in rats for the determination of the optimal treatment time of ginsenoside Rd (immunohistochemistry, × 400). (A–D) 1, 3, 5, 7 days after spinal cord isch-emia/reperfusion injury. In all groups, the neurons were large and the nuclei were round, no Caspase 3 positive cells formed. Stars refer to normal nerve cells.

Figure 9

Effect of ginsenoside Rd treatment on ASK1, JNK, p38 MAPK protein expression in spinal cord of rats with ischemia/reperfusion injury (western blot analysis). Measurement data are expressed as the mean ± SD and the experiment was repeated three times, one-way analysis of variance and Student-Newman-Keuls test. *P < 0.01, vs. sham group; #P < 0.05, vs. ischemia/reperfusion group. I: Control group; II: sham group; III: ischemia/reperfusion group; IV: ginsenoside-treated group.