关键词: 柚皮苷, 实验性自身免疫性脑脊髓炎, 小胶质细胞, STAT1, 自噬, 小鼠

Abstract: BACKGROUND: Microglia M1/M2 phenotypic imbalance plays an important role in the progression of many neurodegenerative and autoimmune diseases, and regulating microglia phenotypic transformation is a target of immunotherapy.
OBJECTIVE: To investigate the effect of naringin on microglial polarization in experimental autoimmune encephalomyelitis mice via STAT1 pathway and autophagy. 
METHODS: Fifty female C57BL/6 mice were randomly divided into five groups: blank control group, model group and low-, medium- and high-dose naringin groups (n=10 per group). In the latter four groups, animal models of experimental autoimmune encephalomyelitis were established. The naringin groups were intraperitoneally injected with 10, 20, 40 mg/(kg·d) naringin for 10 consecutive days. Simultaneously, the blank control and model groups were intraperitoneally injected with the same amount of saline. Disease conditions in each mouse were observed. Hematoxylin-eosin staining and Lux fast blue staining were used to observe the pathological changes of spinal cord tissue. Immunofluorescence method was used to observe the expression of Iba-1 and LC3 protein in spinal cord tissue. Western blot assay was used to detect the protein levels of STAT1, Beclin1, p62, LC3, CD16, CD206, inducible nitric oxide synthase, and Arg-1 in the spinal cord. Real-time fluorescence quantitative PCR method was used to detect the mRNA level of STAT1 in spinal cord tissue. 
RESULTS AND CONCLUSION: No mice in the blank control group had an attack, but mice in the other groups developed experimental autoimmune encephalomyelitis to different extents. Compared with the model group, the incubation period was prolonged (P < 0.01), the peak of onset was delayed (P < 0.01 or P < 0.05) and the neurological deficit scores (P < 0.01 or P < 0.05) were reduced in all naringin groups. The higher dose of naringin indicated milder symptoms in mice. Compared with the blank control group, the model group had a significant increase in inflammatory cell infiltration, demyelination, inflammation and demyelination scores in spinal cord tissue during the onset peak period (P < 0.01), while inflammatory cell infiltration and demyelination were alleviated and inflammation and demyelination scores were lower in all naringin groups (P < 0.01). The higher dose of naringin indicated the better effect (P < 0.01 or P < 0.05). Compared with the blank control group, the average absorbance value of positive Iba-1 and LC3 expression area in spinal cord tissue were increased in the model group (P < 0.01), while the average absorbance value of positive Iba-1 and LC3 expression area were decreased in all naringin groups (P < 0.01 or P < 0.05). The higher dose of naringin indicated the less reduction in the average absorbance value (P < 0.01 or P < 0.05). Compared with the blank control group, the LC3 II/LC3 I ratio and the levels of LC3, Beclin1, STAT1, STAT1 mRNA, CD16, and inducible nitric oxide synthase were increased (P < 0.01 or P < 0.05), while Arg-1, CD206, and P62 expressions were decreased (P < 0.01 or P < 0.05) in the model group. Compared with the model group, the LC3 II/LC3 I ratio and the levels of LC3, Beclin1, STAT1, STAT1 mRNA, CD16, and inducible nitric oxide synthase were decreased (P < 0.01 or P < 0.05), while 
Arg-1, CD206, and P62 expressions were increased (P < 0.01 or P < 0.05) in all naringin groups. The higher dose of naringin indicated the better effect (P < 0.01 or P < 0.05). To conclude, naringin can prevent and treat experimental autoimmune encephalomyelitis in mice in a dose-dependent manner. Its mechanism may be related to the regulation of microglial polarization and the correction of M1/M2 microglia imbalance in experimental autoimmune encephalomyelitis mice by down-regulating STAT1 pathway and inhibiting autophagy.

Key words: naringin, experimental autoimmune encephalomyelitis, microglia, STAT1, autophagy, mouse