Efficacy and mechanism of Wuzi Yanzong pill on the prevention and treatment of EAE

Abstract

Objective: Studies have shown that Wuzi Yanzong Pill (WYP) can be used to treat neurological diseases, but its mechanisms for multiple sclerosis (MS) remain unclear. This study aims to determine the effect of WYP on MS in an animal model of experimental autoimmune encephalomyelitis (EAE), and explore its mechanism. To provide theoretical basis for the clinical treatment of MS with WYP.

Methods: C57BL/6 female mice were randomly divided into Blank control, EAE control, low dose WYP, medium dose WYP, and high dose WYP groups. One week before model generation, the mice were gavaged with saline (50 mL/kg/d) in Blank control and EAE control groups. The treatment groups was gavaged with different doses of WYP solution (4, 8, or 16 g/kg/d respectively) Clinical scores were recorded daily. Sample collection was conducted on the 14th and 28th days, respectively The expressions of IL-10, IL-17, IL-12, TNF-α and IFN-γ in spleen were detected by ELISA. The expressions of ROCKII, P-MYPT1, TLR4, NF-κB/p65, MCP-1, CCR2 in spleen, brain and spinal cord were detected by Western Blot. The types of macrophages and the contents of intracellular IL-10 and IL-12 were detected by Flow Cytometry. The contents of TNF-α and TLR4 mRNA in the spleen were detected by RT-PCR.

Results: WYP treatment improved the clinical score of EAE mice in a significant dose-dependent manner, with the WYP high-dose group showed the most significant improvement in clinical score. Compared with the EAE control group, WYP high dose group had significantly lower levels of IL-17, IFN-γ, ROCKII, P-MYPT1, TLR4, NF-κB/p65, MCP-1, and CCR2 as well as TNF-α and TLR4 mRNA, but increased the number of M₂ macrophages and IL-10.

Conclusion: WYP treatment relieves clinical symptoms in EAE mice, which may be related to regulate inflammatory pathway and inhibiting expressions of inflammatory cytokines.

Keywords: Experimental autoimmune encephalomyelitis; Immunomodulation; Wuzi Yanzong Pill; anti-inflammation.

Fig. 1

Treatment with WYP delayed EAE onset time and relieved the neurological symptoms. A-B. There were progressive increases in mean neurological scores at day 9 p.i., suggesting that the EAE model induced significant neurological deficits. Treatment with three different doses of WYP for 3 weeks caused significant decreases in neurological function scores in all the three groups (A). In contrast, there were only significant decreases in neurological function scores of the high dose WYP group than the EAE control group after 5 weeks of treatment (B). ^△P < 0.05, ^△△P < 0.01, ^△△△P < 0.001 (compared with Blank control); *P < 0.05, **P < 0.01, ***P < 0.001 (compared with EAE control); ^☆P < 0.05, ^☆☆P < 0.01, ^☆☆☆P < 0.001 (compared with WYP high dose).

Fig. 2

WYP treatment improved spinal cord inflammation and demyelination in the EAE model. HE staining and Luxol Fast Blue staining were used to evaluate inflammation and demyelination of the spinal cord tissues. Among the EAE control, Blank control, and WYP high dose groups, treatment with high dose WYP for either 3 weeks (day 14 p.i.) or 5 weeks (day 28 p.i.) resulted in significant decreases in inflammatory foci (Fig. 2A) and myelin loss (Fig. 2B). The results were analyzed with Image Pro software. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3

M₁ phenotype macrophage is marked with CD16/32 and excretes pro-inflammatory factor IL-12; M₂ phenotype macrophage is marked with CD206 and excretes an anti-inflammation factor IL-10. The WYP group had significantly higher levels of CD206 and IL-10, suggesting that WYP improved the expression of M₂ phenotype macrophage. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3

M₁ phenotype macrophage is marked with CD16/32 and excretes pro-inflammatory factor IL-12; M₂ phenotype macrophage is marked with CD206 and excretes an anti-inflammation factor IL-10. The WYP group had significantly higher levels of CD206 and IL-10, suggesting that WYP improved the expression of M₂ phenotype macrophage. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4

Treatment with WYP for 3 weeks inhibited inflammatory pathway activity and down-regulated inflammatory chemokines and chemotactic factors and their receptors. At day 14 p.i., the contents of ROCKII, P-MYPT1, TLR-4, NF-κB, CCR2, and MCP-1 in brain (A), spinal cord (B), and the macrophages of spleen (C) were detected by Western blot. The results were analyzed from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 (Compared with EAE control).

Fig. 4

Treatment with WYP for 3 weeks inhibited inflammatory pathway activity and down-regulated inflammatory chemokines and chemotactic factors and their receptors. At day 14 p.i., the contents of ROCKII, P-MYPT1, TLR-4, NF-κB, CCR2, and MCP-1 in brain (A), spinal cord (B), and the macrophages of spleen (C) were detected by Western blot. The results were analyzed from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 (Compared with EAE control).

Fig. 5

Treatment with WYP for 5 weeks inhibited inflammatory pathway activity and down-regulateed inflammatory chemokines and chemotactic factors and their receptors. At day 28 p.i., the contents of ROCKII, P-MYPT1, TLR-4, NF-κB, CCR2, and MCP-1 in brain (A), spinal cord (B) and macrophages of spleen (C) were detected respectively by Western blot. The results were obtained from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 (Compared with EAE control).

Fig. 5

Treatment with WYP for 5 weeks inhibited inflammatory pathway activity and down-regulateed inflammatory chemokines and chemotactic factors and their receptors. At day 28 p.i., the contents of ROCKII, P-MYPT1, TLR-4, NF-κB, CCR2, and MCP-1 in brain (A), spinal cord (B) and macrophages of spleen (C) were detected respectively by Western blot. The results were obtained from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 (Compared with EAE control).

Fig. 6

Expressions of inflammatory factors in splenocytes supematant. IL-10, IL-12, IL-17, TNF-α and IFN-γ were detected by ELISA kits. The results were expressed as pg/mL. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 7

Expressions of TLR-4 and TNF-α genes in spleen cells were detected by RT-PCR technique. The results were obtained from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.