3.1. MP ameliorated the severity of EAE.
To evaluate the therapeutic efficacy of MP on EAE, we examined the clinical score of mice from day 0 to day 19 after immunization. Neurological deficits became apparent on the 9th day after immunization and progressively worsened thereafter. EAE+MP group had reduced clinical scores significantly compared with EAE group (p < 0.05, Figure 1). The EAE+FMT group demonstrated significantly reduced cumulative, maximum, and mean EAE scores in comparison to the EAE group. However, there was no significant difference between the onset days of the EAE and EAE+MP groups (Figure 1).
Figure 1. MP reduced the clinical score of EAE.
(A) Daily fluctuations in clinical scores between the two cohorts throughout the course of the disease. (mean ± SEM; n = 8 each; * P < 0.05, ** P < 0.01, and *** P < 0.001).
(B) Analysis was conducted on the maximal scores, cumulative scores, and mean scores as well as onset day for both the EAE group and MP group. (n = 8 each; * P < 0.05, ** P < 0.01, and *** P < 0.001).
3.2. MP attenuated inflammatory cell infiltration and demyelination in the spinal cord of EAE mice
To evaluate the extent of inflammatory infiltration and remyelination in the lumbar spinal cord of EAE mice, H&E and LFB staining were performed (p < 0.05, Figure 2 (A)). We observed decreased perivascular and parenchymal mononuclear cell infiltrates within white matter as well as reduced perivascular cuffs in the EAE+MP group compared to the EAE group (p < 0.05, Figure 2(A)). Additionally, LFB staining revealed less extensive demyelination in the spinal cords of mice treated with MP compared to those without treatment (p < 0.05, Figure 2(B)). These findings indicate that MP effectively alleviates inflammatory infiltration and demyelination in a murine model of EAE.
Figure 2. MP reduced the CNS inflammation and demyelination of EAE Mice.
(A) Inflammatory infiltration observed through H&E staining with corresponding inflammation scores.
Demyelination was detected by LFB staining with corresponding demyelination scores.
3.3 MP ameliorated EAE by suppressing the microglia and astrocytes activation
Microglia and astrocyte activation promotes the production of pro-inflammatory cytokines and inflammatory infiltration in the CNS of EAE animals[34]. We evaluated the response of glial fibrillary acidic protein (GFAP), a marker for astrocytes, and ionized-calcium binding adaptor protein (Iba-1), a marker for microglia, through immunofluorescence analysis on lumbar spinal cord sections. The mice in the EAE+MP group exhibited significantly fewer Iba-1-positive microglia compared to those in the P-EAE group ( p<0.05, Figure 3(A)). Moreover, there was a decrease in GFAP-positive astrocytes observed in the spinal cord of MP-treated EAE group when compared with P-EAE mice( p<0.05, Figure 3(B)). The Western blot analysis revealed a significant upregulation of GFAP expression in the EAE group compared to both the control and Pre-EAE groups. However, MP treatment reversed the upregulation within EAE group ( p<0.05, Figure 3(C)). Additionally, iNOS protein levels showed a consistent trend with Iba-1 among different groups indicating an anti-inflammatory effect but without statistical significance (Figure3(C)). Furthermore, Arg-1 protein levels decreased significantly within the EAE group as compared to control and Pre-EAE groups; however, MP treatment reversed this decline without statistical significance. These findings suggest that MP can ameliorate EAE by suppressing microglial and astrocytic activation in vivo while reducing inflammatory responses and promoting anti-inflammatory responses through inhibiting M1 microglial activity as well as promoting polarization towards M2 phenotype.
Figure 3:MP inhibited microglia and astrocyte activation in EAE mice.
(A)-(B) The expression of the anti-GFAP and anti-Iba-1 protein was detected by immunofluorescence staining in lumbar spinal cords of P-EAE, EAE+MP groups.
(C) The protein expression levels of GFAP, Iba-1, iNOS, and Arg-1 were investigated in lysates from lumbar spinal cords using Western blot analysis. Statistical significance was determined as * P <0.05, ** P < 0.01, and *** P < 0.001
3.4. MP inhibits Tfh cell differentiation, promotes Tfr cell differentiation, and restores the balance of Tfr/Tfh ratio in EAE mice
The Tfr cells were positive for Foxp3 expression while Tfh cells were negative[35, 36]. We determined the location of CD4+CXCR5+Foxp3+ Tfr cells and CD4+CXCR5+Foxp3- Tfh cells in the spleen (Figure 4(A)) using immunofluorescence staining. To further investigate the systemic immunoregulatory effects of MP, we performed flow cytometry analysis on individual spleen cells. Tfh cells were defined as CD4+CXCR5+Foxp3-, and their percentage expanded from an average of 1.22% in Pre-EAE mice to 2.07% in P-EAE mice. However, MP treatment significantly reduced the percentage of Tfh cells to anaverage of 0.88% (Figure 4(B)), indicating its inhibitory effect on Tfh cell differentiation in EAE mice.
The percentage of CD4+CXCR5+Foxp3+ Tfr cells was lower in both Pre-EAE and P-EAE groups compared to the control group (p<0.05, Figure 4(B)). Nevertheless, MP treatment partially restored the percentage of Tfr cells. The ratio of Tfr/Tfh cells was significantly reduced in both Pre-EAE and P-EAE groups compared to the control group but was reversed after treatment with MP (p<0.05, figure 4(B)). We observed an increase in the percentage of Tfh cell subtype, identified as CD4+CXCR5+Foxp3-ICOS+, increased in Pre-EAE, reached a maximum at the P-EAE, and decreased after MP treatment(p<0.05, Figure5(B)). Similar results were obtained when using CD4+CXCR5+Foxp3-PD-1 as another sybtype of Tfh cells (p<0.05, Figure5(B)). Here, our findings suggest that MP treatment can correct the imbalance between Tfr and Tfh cells by inhibiting the differentiation of Tfh cells and promotes the differentiation of Tfr cells, thereby exerting its systemic immuno-inflammatory regulation effect in EAE mice.
Figure 4: Confocal photomicrographs of Tfr cell localization in spleen characterized by the distinct density of CD4 T cells (red (A)), CXCR5 (blue (B)), Foxp3 (green (C)), and Merge (D) and Tfh cell localization in spinal cord characterized by the distinct density of CD4 T cells (red (A)), CXCR5 (blue (B)), and Merge (D).
Figure 5: Flow cytometry analysis of different subsets of spleen Tfr and Tfh cells in the control, Pre-EAE, P-EAE, and MP groups.
The percentage of CD4+CXCR5+Foxp3+Tfr and CD4+CXCR5+Foxp3-Tfh cells in the spleen of EAE mice in these four groups.
The percentages of CD4+CXCR5+Foxp3-ICOS+, CD4+CXCR5+Foxp3-PD-1+ Tfh cells in the spleen of EAE mice in these four groups.
3.5. MP treatment resulted in the downregulation of Tfh-related molecules and pro-inflammatory factors while promoting the expression of Tfr-related molecules and anti-inflammatory factors.
We detected various cell surface and intracellular markers associated with Tfh in spleen tissues from control, Pre-EAE, P-EAE, and EAE+MP groups using Western Blot, including CXCR5, ICOS, PD-1, as well as IL-21. Compared to the Control and Pre-EAE group, the expression of CXCR5, ICOS, PD-1, and IL-21 were elevated in the EAE group. However, administration of MP effectively prevented the upregulation of CXCR5, ICOS, PD-1, and IL-21 protein expression observed in the EAE group ( p<0.05, Figure 6). Furthermore, in this study, the level of TGF-β1 protein expression significantly decreased in the EAE group compared to that in Control and Pre-EAE groups, however, treatment with MP reversed this reduction specifically within P-EAE Group (p<0.05, Figure 6). Moreover, the expression levels of Foxp3 protein exhibited a consistent correlation with TGF-β1, notably, only the EAE+MP group demonstrated a significantly elevated level compared to the P-EAE group (p<0.05, Figure 6).
Figure 6: The EAE mice were classified into the control group, the Pre-EAE group, the P-EAE group and the EAE+MP group. Representative blots band of CXCR5, PD-1, iCOS, TGF-β1, FoxP3, IL-21 in spinal cord of EAE mice were analyzed.
3.6. MP modulated Tfr/Tfh proliferation in EAE via the PI3K/AKT/FoxO1 and PI3K/AKT/ mTOR pathway.
To gain further insight into the molecular mechanism of MP in the treatment of EAE, we quantitatively analyzed the proteins associated with PI3K/AKT/FoxO1 and PI3K/AKT/ mTOR pathway. Western blot analysis revealed significantly increased levels of PI3K, phosphorylated AKT (p-AKT), phosphorylated FoxO1 (p-FoxO1), and phosphorylated mTOR (p-mTOR) in the EAE group compared to the control and Pre-EAE groups. However, total AKT protein, total Foxo1 protein, and total mTOR protein did not show significant changes among the three groups. Interestingly, MP treatment could significantly reduce the expression of PI3K, p-AKT, p-FoxO1, and p-mTOR compared to P-EAE group (p<0.05, Figure 7). This result further supports involvement of the PI3K/AKT/FoxO1 and PI3K/AKT/mTOR pathways in EAE occurrence and development while highlighting that MP treatment can inhibit activation of these signaling pathways. Our findings suggest that MP may modulate Tfr/Tfh cell ratio through the PI3K/AKT signaling pathway.
Figure 7. MP relieved EAE through the PI3K/AKT/mTOR and PI3K/AKT/FOXO1 signaling pathways.
Western blotting analysis of total PI3K, p-AKT, total AKT, p-FoxO1, total FoxO1, p-mTOR, and total mTOR.
(B) Quantitative analysis revealed decreased levels of expression for PI3K, p-AKT, p-FoxO1 and p-mTOR in the spinal cord following MP treatment.