Abstract: BACKGROUND: In the occurrence and development of demyelinating diseases of the central nervous system, neuroinflammation caused by microglia is the main pathological feature, so inhibiting the inflammatory response is very important to alleviate demyelination. Hydroxysafflor yellow A can protect the blood-brain barrier, inhibit neuronal apoptosis, and improve neurological function.
OBJECTIVE: To explore the mechanism of hydroxysafflor yellow A inhibiting bicyclohexanone oxalyl dihydrazone-induced demyelination in mice.
METHODS: (1) In vivo: Thirty healthy male C57BL/6 mice were randomly divided into three groups: normal group, cuprizone group, and hydroxysafflor yellow A group. The mice in the cuprizone group and the hydroxysafflor yellow A group were fed with 0.2 % cuprizone diet for 6 weeks to establish mouse models of demyelination. The mice in the normal group were fed with normal diet. At the end of the 4th week, the mice in the hydroxysafflor yellow A group were intraperitoneally injected with hydroxysafflor yellow A 20 mg/kg per day. The mice in the normal and cuprizone groups were intraperitoneally injected with normal saline for 2 weeks. The behavioral changes of mice were evaluated by open field test and elevated plus maze test. The loss of myelin sheath in corpus callosum was detected by black gold staining, myelin basic protein and degraded myelin basic protein immunofluorescence staining. The activation of microglia and the expression of inflammatory factors were detected by Iba-1 immunofluorescence staining and ELISA, respectively. The protein expression levels of Toll-like receptor 4, myeloid differentiation factor 88, and nuclear factor κB p65 in the brain of mice in each group were detected by western blot assay. (2) In vitro experiment: The inflammation model of BV2 microglia was established by lipopolysaccharide induction. BV2 cells were divided into normal group, lipopolysaccharide group (1 μg/mL), and lipopolysaccharide (1 μg/mL) + hydroxysafflor yellow A (25 μmol/L) group. The expression levels of tumor necrosis factor α and interleukin 6 in the supernatant were detected by ELISA. 
RESULTS AND CONCLUSION: (1) Compared with the normal group, the mice in the cuprizone group had severe anxiety, abnormal autonomic movement ability, and a large amount of myelin sheath loss in the corpus callosum. The average fluorescence intensity of myelin basic protein was significantly reduced, and the average fluorescence intensity of degraded myelin basic protein was significantly increased. The number of Iba1+ microglia increased, the contents of interleukin 1β, tumor necrosis factor α, and interleukin 6 in the brain increased, and the protein expression levels of Toll-like receptor 4, myeloid differentiation factor 88, and nuclear factor κB p65 increased significantly. The above symptoms and indexes of mice were reversed after hydroxysafflor yellow A treatment. (2) Hydroxysafflor yellow A significantly inhibited the expression of inflammatory factors such as tumor necrosis factor α, and interleukin 6 induced by lipopolysaccharide in BV2 microglia. (3) The above results demonstrate that hydroxysafflor yellow A can significantly improve cuprizone-induced demyelination in mice. The mechanism of action is related to the inhibition of microglial activation-mediated inflammatory response through the Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor κB p65 signaling pathway.

Key words: hydroxysafflor yellow A, cuprizone, inflammation, demyelination, microglia, mouse