Baihe Dihuang decoction activates cannabinoid type 2 receptor to regulate microglial polarization and alleviate anxiety-like behavior in chronic restraint stress mice.

OBJECTIVE: To investigate the anxiolytic effects of Baihe Dihuang decoction (, BDD) and its underlying molecular mechanisms, focusing on cannabinoid type 2 receptor (CB2R)-mediated regulation of microglial polarization and the toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-kappa B (NF-κB) signaling pathway. METHODS: A total of 60 male C57BL/6J mice were randomly divided into six groups (n= 10 per group): control group, model group, diazepam (2 mg/kg) group, high-dose BDD (16 g/kg) group, low-dose BDD (8 g/kg) group, and high-dose BDD (16 g/kg) combined with CB2R antagonist AM630 (1 mg/kg) group. Except for the control group, all other groups were subjected to chronic restraint stress (CRS) to establish an anxiety model. Anxiety-like behaviors were assessed using the open field test, light-dark box test, and elevated plus maze. Serum levels of interleukin (IL)-1β, IL-6, IL-10, and IL-4 and amygdala concentrations of 2-arachidonoylglycerol (2-AG) and anandamide (AEA) were quantified by enzyme-linked immunosorbent assay. Hematoxylin and eosin, Nissl, and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining were used to evaluate neuronal damage in the amygdala. Immunofluorescence staining was employed to assess microglial polarization and CB2R expression. The expression levels of CB2R and downstream signaling molecules TLR4/MyD88/NF-κB in the amygdala were determined by quantitative polymerase chain reaction and Western blot. RESULTS: BDD significantly alleviated CRS-induced anxiety-like behaviors and reduced neuronal damage in the amygdala. It downregulated pro-inflammatory cytokines IL-1β, IL-6 and upregulated anti-inflammatory cytokines IL-10, IL-4 in serum while increasing 2-AG and AEA levels in the amygdala. Furthermore, BDD promoted microglial polarization toward the M2 phenotype and enhanced CB2R expression, while suppressing the TLR4/MyD88/NF-κB signaling pathway. The administration of the CB2R antagonist AM630 reversed these effects, confirming the essential role of CB2R activation in the anxiolytic and neuroprotective effects of BDD. CONCLUSION: BDD treatment activated CB2R, which in turn modulated microglial polarization in the amygdala viathe TLR4/MyD88/NF-κB signaling pathway, thereby alleviating anxiety-like behaviors and exerting neuro-protective effects.