Neuroprotective Effects of Sinomenine on Experimental Autoimmune Encephalomyelitis via Anti-inflammatory and Nrf2-dependent Anti-oxidative Stress Activity

Research Background on Multiple Sclerosis and Central Nervous System Inflammation

Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS), often accompanied by chronic inflammation, leading to oligodendrocyte loss, activation of brain-resident immune cells, blood-brain barrier disruption, extensive demyelination, and axonal damage. Symptoms include dizziness, pain, fatigue, loss of coordination, numbness, depression, vision loss, and bowel and bladder dysfunction. MS primarily affects young and middle-aged populations, and there is currently no ideal treatment, urgently necessitating effective therapeutic approaches.

Experimental autoimmune encephalomyelitis (EAE) is a widely used mouse model for MS, employed to study disease pathways and evaluate potential treatments. Research has shown that reactive oxygen species (ROS) are closely associated with neuroinflammation in MS and EAE. Excessive accumulation of ROS leads to oxidative stress, damaging intracellular macromolecules (such as proteins, DNA, and lipids), thereby causing demyelination and neurodegeneration.

Considering that an “anti-oxidative inflammation” therapeutic strategy may be an ideal treatment for MS/EAE, increasing research has focused on compounds with anti-inflammatory and antioxidant properties. Among these, sinomenine (SIN) is an alkaloid monomer extracted from the Chinese medicinal plant Sinomenium acutum, known for its potent anti-inflammatory and immunosuppressive effects. Previous studies have shown that sinomenine enhances antioxidant stress capacity in PC12 neuronal cells through the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway.

Paper Source and Author Information

This paper, titled “Neuroprotective effects of sinomenine on experimental autoimmune encephalomyelitis via anti-inflammatory and Nrf2-dependent anti-oxidative stress activity,” was published in the journal “Neuromolecular Medicine.” It was jointly completed by Fan Hua, Yang Yang, Bai Qianqian, and others. The authors are primarily from the First Affiliated Hospital and School of Basic Medical Sciences of Henan University of Science and Technology. The paper was published by Springer Nature in September 2023.

Detailed Introduction of the Original Research Experiment

a. Experimental Procedure

Experimental Animals and Grouping: 6-8 week-old C57BL/6J mice were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. and kept in a specific pathogen-free environment. The experiment followed the animal use permit from the First Affiliated Hospital of Henan University of Science and Technology and used the EAE model. The study used 200µL of sinomenine at different concentrations (50mg/kg and 100mg/kg).

EAE Induction: Recombinant myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) and Complete Freund’s Adjuvant (CFA) were subcutaneously injected into mice, followed by intraperitoneal injections of pertussis toxin on days 0 and 2. The experiment was divided into multiple groups (control group, EAE group, sinomenine treatment groups, etc.) and lasted for 30 days.

Drug Treatment: Sinomenine was dissolved in a 2% DMSO solution and diluted to ensure a final DMSO concentration of 0.02%. The sinomenine solution was administered intraperitoneally daily at 10 AM.

Tissue Processing and Experimental Measurements: Spinal cord and serum samples from experimental mice were collected on day 19 for various analyses including HE staining, LFB staining, immunofluorescence staining, qPCR, Western Blot, etc., to detect key indicators such as inflammatory cells and ROS.

b. Experimental Results

Reduction in Disease Severity: Through EAE scoring and symptom monitoring of mice, sinomenine was found to significantly alleviate the disease, especially at the 100 mg/kg dose. Data showed that the sinomenine treatment groups significantly reduced the maximum and cumulative disease scores of EAE.

Decrease in Demyelination: LFB staining results showed that in the spinal cords of mice in the sinomenine treatment groups, the demyelinated areas were significantly reduced, with the 100 mg/kg dose showing better effects than 50 mg/kg. MBP (Myelin Basic Protein) immunofluorescence staining further verified this point.

Alleviation of Inflammatory Cell Infiltration and Axonal Damage: HE staining results showed reduced inflammatory cell infiltration in the sinomenine treatment groups, while neurofilament protein immunofluorescence results demonstrated the effectiveness of sinomenine in reducing axonal damage.

Regulation of Inflammatory Factors: ELISA detection showed that sinomenine significantly reduced levels of inflammatory factors such as TNF-α, IL-1β, MCP-1 in the spinal cord and serum of EAE mice, while increasing levels of the anti-inflammatory factor IL-10.

Inhibition of Microglia and Astrocyte Activation: Immunofluorescence results showed that after sinomenine treatment, the number of microglia (Iba1-positive cells) and astrocytes (GFAP-positive cells) in the spinal cords of EAE mice was significantly reduced, with changes in morphology, suggesting inhibition of neuroinflammation.

Reduction of Reactive Oxygen Species Generation and Activation of the Nrf2 Antioxidant System: Through detection of antioxidant proteins (such as HO-1, NQO1), it was found that sinomenine reduced ROS generation through the Nrf2 signaling pathway, reducing oxidative stress damage to proteins and lipids.

c. Research Conclusions

The study showed that sinomenine significantly alleviated the disease severity in EAE mice, reduced spinal cord demyelination and axonal damage by activating the Nrf2/HO-1 antioxidant system and inhibiting neuroinflammation. Overall, sinomenine demonstrated potent neuroprotective effects, suggesting its potential as a therapeutic drug for multiple sclerosis.

d. Research Highlights

The highlight of this study is that it systematically revealed the neuroprotective effects of sinomenine on the EAE model for the first time, and detailed its molecular mechanism of anti-inflammatory and anti-oxidative stress effects through the Nrf2/HO-1 signaling pathway. The research not only confirmed the anti-inflammatory and immunosuppressive efficacy of sinomenine but also demonstrated its inhibitory effect on ROS generation, proposing a new strategy for treating brain inflammation. Additionally, the series of experimental methods and design in this study provide valuable references for subsequent scientific research.