Anti-inflammatory effect of Mongolian drug Naru-3 on traumatic spinal cord injury and its mechanism of action

Abstract

Objective This study was performed to confirm the anti-inflammatory effect of the Mongolian drug Naru-3 on traumatic spinal cord injury (TSCI) and its possible mechanism of action. Methods We prepared a TSCI model using Sprague-Dawley rats. The rats were divided into a Naru-3 group and a methylprednisolone group. Real-time polymerase chain reaction and western blotting were performed to measure the expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β. Enzyme-linked immunosorbent assay kits were employed to detect serum inflammatory cytokine levels. The localization and expression of monocyte chemotactic protein-1 (MCP-1) in spinal cord tissue was determined by immunohistochemical analysis. Flow cytometry was performed to analyze the ratio of M1- and M2-phenotype macrophages. The locomotor function recovery was evaluated by the Basso, Beattie, and Bresnahan score. Results Naru-3 significantly inhibited the inflammatory response and reduced the expression of TNF-α, IL-6, and IL-1β in both spinal cord and blood in a time- and concentration-dependent manner. Immunohistochemical analysis indicated that Naru-3 significantly reduced MCP-1 expression in spinal cord and promoted M2-phenotype macrophage differentiation. Conclusions Naru-3 is an effective treatment for impact-induced TSCI in rats. Naru-3 treatment affects inflammatory cytokine levels and macrophage differentiation, which play a role in TSCI remission.

Keywords: Naru-3; inflammatory cytokines; macrophages; methylprednisolone; monocyte chemotactic protein-1; traumatic spinal cord injury.

Figure 1.

Relative expression levels of IL-1β, IL-6, and TNF-α mRNA in (a) different dose groups and (b) different administration time groups. Data are presented as the mean ± standard deviation from three independent experiments. IL, interleukin; TNF, tumor necrosis factor.

Figure 2.

Western blot detected the expression of IL-1β, IL-6, TNF-α, and MCP-1 in each group. (a) Schematic image of western blotting. (b) Statistical results of relative gray values in each group. GAPDH was used as a loading control. Data are presented as the mean ± standard deviation from three independent experiments. ^#P < 0.05 compared with *; ^&P < 0.05 compared with *; ^&P > 0.05 compared with #. IL, interleukin; TNF, tumor necrosis factor; MCP-1, monocyte chemotactic protein-1; MP, methylprednisolone.

Figure 3.

Concentrations of serum proinflammatory cytokines in each group. Data are presented as the mean ± standard deviation from three independent experiments. ^#P < 0.05 compared with *; ^&P < 0.05 compared with *; ^&P > 0.05 compared with #. IL, interleukin; TNF, tumor necrosis factor; MP, methylprednisolone.

Figure 4.

Immunohistochemical analysis of MCP-1 in the spinal cord. (a) Schematic image of immunohistochemical staining. (1) Sham group. (2) Model group. (3) Naru-3 group. (4) MP group. (b) MCP-1-positive rates in each group. Data are presented as the mean ± standard deviation from three independent experiments. ^#P < 0.05 compared with *; ^&P < 0.05 compared with *; ^&P > 0.05 compared with #. MCP-1, monocyte chemotactic protein-1; MP, methylprednisolone;

Figure 5.

Flow cytometric analysis of M1- and M2-phenotypes. (a) M1-phenotype and M2-phenotype macrophages measured using flow cytometry. (1) M1-phenotype-positive macrophages of the TSCI model group. (2) M2-phenotype-positive macrophages of the TSCI model group. (3) M1-phenotype-positive macrophages of the Naru-3 group. (4) M2-phenotype-positive macrophages of the Naru-3 group. (5) M1-phenotype-positive macrophages of the MP group. (6) M2-phenotype-positive macrophages of the MP group. The red line in (1) to (6) represents the sham group, and the blue line represents the corresponding group. (b) The M1- to M2-phenotype macrophage ratio in each group. Values are presented as mean ± standard deviation; n = 3. ^*P < 0.05 compared with sham group; ^#P < 0.05 compared with *. TSCI, traumatic spinal cord injury; MP, methylprednisolone.

Figure 6.

Locomotor recovery of hind limb function after TSCI in each group during 48 days. Values are presented as mean ± standard deviation; n = 6. There were no significant differences in the BBB scores among the model group, Naru-3 group, and MP group 8 days after TSCI (^*P > 0.05). Rats in the Naru-3 and MP groups showed significantly (^#P < 0.05) greater locomotor recovery at 16, 32, and 48 days after TSCI when compared with the sham group. There was no significant difference between the Naru-3 and MP groups at 48 days (^@P < 0.05). TSCI, traumatic spinal cord injury; BBB, Basso, Beattie, and Bresnahan; MP, methylprednisolone.