Anti-cerebral ischemic neuronal injury mechanism of Zhenlong Xingnao capsules: role of the Notch/NF-κB signaling pathway

Abstract

Objective: To investigate the anti-cerebral ischemia-reperfusion injury (CIRI) effect and mechanism of Zhenlong Xingnao capsules based on Notch/NF-κB signaling pathway.

Methods: The rat model of middle cerebral artery occlusion (MCAO) was established using the Longa suture occlusion method, and 70 rats were divided into sham-operated, model, low dose Zhenlong Xingnao capsule group (125 mg/kg Zhenlong Xingnao capsule solution) and high dose Zhenlong Xingnao capsule group (250 mg/kg Zhenlong Xingnao capsule solution), low dose Zhenlong Xingnao capsule + neurogenic site notch homologous protein 1 (Notch1) antibody (Jagged1 group, 125 mg/kg capsule solution + 25 mg/kg Jagged1 solution), high dose Zhenlong Xingnao capsule + Jagged1 group (250 mg/kg capsule solution + 25 mg/kg Jagged1 solution), and Jagged1 group (25 mg/kg Jagged1 solution). The learning and memory abilities (behavioral score, spontaneous movement, and rotarod test), neurological function score, inflammatory factors and oxidative stress levels [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD)] in hippocampal tissue, and Bcl-2, Bax, and Caspase-3 mRNA levels were measured by reverse transcription quantitative polymerase chain reaction, and Notch1/NF-κB signaling pathway-related protein expression was assessed by Western blot.

Results: The low and high dose interventions of Zhenlong Xingnao capsules significantly improved the learning and memory abilities of MCAO rats, reduced the neurological impairment scores, improved the levels of IL-6, TNF-α, MDA, GSH-Px, SOD, and inhibited the expression levels of Notch1, p-NF-κB p65, and Hes-1 proteins. However, the protective effect of Zhenlong Xingnao capsules on neurons in rat brain tissue could be reduced after treatment with Jagged1.

Conclusions: Zhenlong Xingnao capsules can promote neuronal repair during ischemia-reperfusion, and its mechanism may be related to inhibiting the activation of Notch/NF-κB signaling pathway and reducing inflammation and oxidative stress response.

Keywords: Notch; Zhenlong Xingnao capsules; inflammation; ischemia-reperfusion injury; oxidative stress.

Figure 1

Effect of Jenlong Awakening capsules on the learning motor ability of rats. It shows that the (A) behavioral scores and (B) spontaneous movement levels of rats in the low and high dose groups of Zhenlong Xingnao capsules were significantly reduced and (C) the level of the motor ability on the rotarod was significantly increased, but the treatment with Jagged1 could weaken the improvement effect of Zhenlong Xingnao capsules. Note: ^aindicates comparison with the sham-operated group, P<0.05; ^bindicates comparison with the model group, P<0.05; ^cindicates comparison with the low-dose group of Zhenlong Xingnao capsules, P<0.05; ^dindicates comparison with the high-dose group of Zhenlong Xingnao capsules, P<0.05.

Figure 2

Comparison of cerebral infarction volume in each group. Note: (A) Sham-operated group; (B) Model group; (C) High-dose group; (D) High-dose + Jagged1 group; (E) Jagged1 group.

Figure 3

Hippocampal neuronal injury in rats (× 400). It shows that in the model group, the structure of hippocampal neurons was damaged, the nuclei were deformed, shrunken and broken, the staining was deepened, the cells were loosely arranged, and cavities were formed. The damage of hippocampal neurons was significantly improved in the low and high dose groups. Note: (A) Sham-operated group; (B) Model group; (C) Low-dose group; (D) High-dose group; (E) Low-dose + Jagged1 group; (F) High-dose + Jagged1 group; (G) Jagged1 group.

Figure 4

Effect of Zenglong Xingnao capsules on the level of inflammation and oxidative stress in hippocampal tissue of rats. It shows that the levels of (A) IL-6, (B) TNF-α, (C) MDA, (D) GSH-Px, and (E) SOD activity in hippocampal tissue were significantly decreased and increased in the low and high dose groups, but the treatment with Jagged1 could weaken the ameliorative effect of Zhenlong Xingnao capsules. Note: ^aindicates comparison with the sham-operated group, P<0.05; ^bindicates comparison with the model group, P<0.05; ^cindicates comparison with the low-dose group, P<0.05; ^dindicates comparison with the high-dose group, P<0.05.

Figure 5

Effect of Zenglong Xingnao capsules on the expression of apoptosis-related genes in hippocampal tissues of rats. It shows that the levels of (A) Bcl-2, (B) Bax, and (C) Caspase-3 in hippocampal tissues were significantly decreased and increased in the low and high dose groups, but the treatment with Jagged1 weakened the ameliorative effect of the capsules. Note: ^aindicates comparison with the sham-operated group, P<0.05; ^bindicates comparison with the model group, P<0.05; ^cindicates comparison with the low-dose group, P<0.05; ^dindicates comparison with the high-dose group, P<0.05.

Figure 6

Effect of Zenglong Xingnao capsules on the expression of Notch1/NF-κB signaling pathway-related proteins in hippocampal tissue of rats. (A) Western Blot images of expression of Notch1/NF-κB signaling pathway-related proteins. It shows that (B) Notch1, (D) p-NF-κB p65, and (E) Hes-1 protein expression levels in hippocampal tissues were significantly reduced in the low and high dose groups of Zhenlong Xingnao capsules, but the treatment with Jagged1 could weaken the improvement effect of Zhenlong Xingnao capsules; the difference of NF-κB p65 protein expression level in each group (C) was not statistically significant. Note: ^aindicates comparison with the sham-operated group, P<0.05; ^bindicates comparison with the model group, P<0.05; ^cindicates comparison with the low-dose group, P<0.05; ^dindicates comparison with the high-dose group, P<0.05.