Morroniside ameliorates experimental autoimmune encephalomyelitis by regulating gut microenvironment, immune balance, and NF-κB neuroinflammatory pathway.

Multiple sclerosis (MS) is a chronic disease characterized by demyelination and neuroinflammation in the central nervous system. Experimental autoimmune encephalomyelitis (EAE) is a classic animal model for MS. Existing therapeutic drugs for MS have limitations such as high cost, significant side effects, and the inability to reverse nerve damage. Morroniside, as a natural iridoid glycoside compound, has anti-inflammatory and antioxidant activities, but its therapeutic mechanism in MS remains unclear. Morroniside significantly delayed the onset time of EAE mice, reduced the clinical symptom score, and improved the weight loss. DTI showed that it repaired the microstructure of the corpus callosum and cerebellum; decreased the values of ADC, MD, and RD; and increased the FA value. Histopathology confirmed that it reduced inflammatory infiltration and demyelination. Mechanistically, morroniside had the effects of improving peripheral immune balance and neuroinflammation. Flow cytometry showed that morroniside downregulated the proportions of Th1 and Th17 cells in the spleen and upregulated the proportion of Treg cells. RT-qPCR showed that morroniside inhibited the expressions of IL-1β, IL-6, and TNF-α in the brain and spinal cord tissue. Transcriptome analysis revealed that there were 30 overlapping differentially expressed genes among the differentially expressed genes in the EAE group versus the control group and the treatment group versus the EAE group. GO and KEGG pathway enrichment analyses found that the differentially expressed genes were significantly enriched in biological processes such as the inflammatory response and immune response, as well as signaling pathways such as NF-κB, PI3K-AKT, and NOD-like receptor. WB, RT-qPCR, and ELISA verified that morroniside could alleviate neuroinflammation by inhibiting the gene and protein expressions of Tnfsf8 and the NF-κB pathway. Molecular docking showed that morroniside had a good binding ability with Tnfsf8, with a binding energy of - 6.3 kcal/mol. In addition, morroniside also improved the gut microbiota dysbiosis in EAE mice. Although it failed to completely restore the level of the genus Lactobacillus, its regulatory effect on the gut microenvironment is worthy of further study. Morroniside improves the pathological process of EAE by multi-target regulation of the peripheral immune balance, inhibition of NF-κB-mediated neuroinflammation, and regulation of the gut microenvironment.