Topical moringin-cream relieves neuropathic pain by suppression of inflammatory pathway and voltage-gated ion channels in murine model of multiple sclerosis

Abstract

Background: Neuropathic pain represents the major public health burden with a strong impact on quality life in multiple sclerosis patients. Although some advances have been obtained in the last years, the conventional therapies remain poorly effective. Thus, the discovery of innovative approaches to improve the outcomes for multiple sclerosis patients is a goal of primary importance. With this aim, we investigated the efficacy of the 4-(α−L-rhamnopyranosyloxy)benzyl isothiocyanate (moringin), purified from Moringa oleifera seeds and ready-to-use as topical treatment in experimental autoimmune encephalomyelitis, murine model of multiple sclerosis. Female C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein (MOG35–55) were topically treated with 2% moringin cream twice daily from the onset of the symptoms until the sacrifice occurred about 21 days after experimental autoimmune encephalomyelitis induction.

Results: Our observations showed the efficacy of 2% moringin cream treatment in reducing clinical and histological disease score, as well as in alleviating neuropathic pain with consequent recovering of the hind limbs and response to mechanical stimuli. In particular, Western blot analysis and immunohistochemical evaluations revealed that 2% moringin cream was able to counteract the inflammatory cascade by reducing the production of pro-inflammatory cytokines (interleukin-17 and interferon-γ) and in parallel by increasing the expression of anti-inflammatory cytokine (interleukin-10). Interestingly, 2% moringin cream treatment was found to modulate the expression of voltage-gated ion channels (results focused on P2X7, Nav 1.7, Nav 1.8 KV4.2, and α2δ-1) as well as metabotropic glutamate receptors (mGluR5 and xCT) involved in neuropathic pain initiation and maintenance.

Conclusions: Finally, our evidences suggest 2% moringin cream as a new pharmacological trend in the management of multiple sclerosis-induced neuropathic pain.

Figure 1.

Moringin was produced by myrosinase-catalyzed hydrolysis of the glucosinolate glucomoringin (GMG). This glucosinolate was purified from Moringa oleifera (Moringaceae) seeds.

Figure 2.

(a) Clinical disease score. Naive vs. EAE ****p < 0.0001; EAE vs. EAE + moringin cream ****p < 0.0001; naive vs. EAE + moringin cream ****p < 0.0001. (b) Body weigh evaluations. Naive vs. EAE ****p < 0.0001; EAE vs. EAE + moringin cream °°°°p < 0.0001. The measure of clinical disease score and body weight variations are expressed as mean ± SEM of all measurements of each experimental group. Naive group (N = 5), EAE group (N = 15), and EAE + moringin cream mice (N = 10). Results were analyzed by one-way ANOVA followed by a Bonferroni test for multiple comparisons. EAE: experimental autoimmune encephalomyelitis.

Figure 3.

Eosin and hematoxylin (E/H) staining. Naive mice (a) did not show histological alterations in the spinal cord tissues, whereas EAE mice (b) displayed a wide area of infiltrating cells (see arrows). Topical treatment with 2% moringin cream led to a complete resolution of inflammatory cells infiltration (c). All images were acquired with 10× objective (size bar 200 µm). A positive staining for MBP was observed in naive (d), whereas a negative tissue localization was found in EAE mice (e) with evident reduction in myelin and axonal structures in spinal cord sections (as shown by arrows). A positive staining for MBP was noticed in EAE + moringin cream with a marked remyelination (f) Densitometric analysis for MBP (g). Naive vs. EAE ****p < 0.0001; EAE vs. EAE + moringin cream ****p < 0.0001. Immunohistochemical pictures (N = 5 photos from each samples collected from all mice in each experimental group) were acquired using light microscopy (LEICA DM 2000 combined with LEICA ICC50 HD camera) and assessed by densitometric analysis by Leica Application Suite V4.2.0 software. All images were acquired with 20× objective (size bar 100 µm).Values shown are mean ± SEM expressed of all mice for each group. All results were analyzed by one-way ANOVA followed by a Bonferroni test for multiple comparisons. EAE: experimental autoimmune encephalomyelitis.

Figure 4.

Needle test to evaluate mechanical allodynia. The measure of sensibility score is expressed as mean ± SEM of all measurements of each experimental group. Naive group (N = 5), EAE group (N = 15), EAE + moringin cream mice (N = 10). Naive vs. EAE ****p < 0.0001; EAE vs. EAE + moringin cream °°°°p < 0.0001. Results were analyzed by one-way ANOVA followed by a Bonferroni test for multiple comparisons. EAE: experimental autoimmune encephalomyelitis.

Figure 5.

Immunohistochemical localization for IL-17. A negative staining for IL-17 was noticed in naive mice (a). An intense positive staining in the vascular endothelium of EAE mice was observed (b; see arrows), compared to EAE mice topically treated with 2% moringin cream (c) Densitometric analysis for IL-17 (d) Naive vs. EAE ****p < 0.0001; EAE vs. EAE + moringin cream ****p < 0.0001. Immunohistochemical pictures (N = 5 photos from each samples collected from all mice in each experimental group) were acquired using light microscopy (LEICA DM 2000 combined with LEICA ICC50 HD camera) and assessed by densitometric analysis by Leica Application Suite V4.2.0 software. All images were acquired with 20× objective (size bar 100 µm). Values shown are mean ± SEM expressed of all mice for each group. ELISA assay for TNF-α (E). Naive vs. EAE ****p < 0.0001; EAE vs. EAE + moringin cream ****p < 0.0001. The data are representative of three independent experiments. All results were analyzed by one-way ANOVA followed by a Bonferroni test for multiple comparisons. EAE: experimental autoimmune encephalomyelitis.

Figure 6.

Western blot analysis for IFN-γ (a). Naive vs. EAE ***p = 0.0004; EAE vs. EAE + moringin cream **p = 0.0054. Western blot analysis for IL-10 (b). Naive vs. EAE **p = 0.0024; EAE vs. EAE + moringin cream **p = 0.0080. GAPDH was used as the internal control. The figure is representative of three separate experiments. In detail, a representative blot of samples obtained from two naive, two EAE, and three EAE + moringin cream mice is shown and densitometry analysis of all animals is reported. Data are expressed as mean ± SEM. All results were analyzed by one-way ANOVA followed by a Bonferroni test for multiple comparisons. EAE: experimental autoimmune encephalomyelitis.

Figure 7.

Western blot analysis for P2X7 (a). Naive vs. EAE **p = 0.0039; EAE vs. EAE + moringin cream *p = 0.0293. Western blot analysis for Nav 1.7 (b). Naive vs. EAE ****p < 0.0001; EAE vs. EAE + moringin cream ****p < 0.0001. Western blot analysis for Nav 1.8 (c). Naive vs. EAE ****p < 0.0001; EAE vs. EAE + moringin cream ****p < 0.0001. Western blot analysis for KV4.2 (d). Naive vs. EAE **p = 0.0032; EAE vs. EAE + moringin cream **p = 0.0015. GAPDH was used as the internal control. The figure is representative of three separate experiments. In detail, a representative blot of samples obtained from two naive, two EAE, and three EAE + moringin cream mice is shown and densitometry analysis of all animals is reported. Data are expressed as mean ± SEM. All results were analyzed by one-way ANOVA followed by a Bonferroni test for multiple comparisons. EAE: experimental autoimmune encephalomyelitis; ND: not detectable.

Figure 8.

Western blot analysis for α2δ-1 (a). Naive vs. EAE **p = 0.0029. Western blot analysis for xCT (b). Naive vs. EAE ****p < 0.0001; EAE vs. EAE + moringin cream ****p < 0.0001. Western blot analysis for mGluR5 (c). Naive vs. EAE ***p = 0.0001; EAE vs. EAE + moringin cream ***p = 0.0001. GAPDH was used as the internal control. The figure is representative of three separate experiments. In detail, a representative blot of samples obtained from two naive, two EAE, and three EAE + moringin cream mice is shown and densitometry analysis of all animals is reported. Data are expressed as mean ± SEM. All results were analyzed by one-way ANOVA followed by a Bonferroni test for multiple comparisons. EAE: experimental autoimmune encephalomyelitis.