OBJECTIVE To investigate the mechanism by which Modified Baishile Decoction(BSL)-medicated serum alleviates glutamatergic neuron synaptic damage in the anterior cingulate cortex(ACC) of post-stroke depression(PSD) via regulation of the cytidine/uridine monophosphate kinase 2(CMPK2)/N-methyl-D-aspartate receptor(NR) signaling pathway.

METHODS  Microglia and ACC neurons were primarily isolated and cultured from Sprague-Dawley(SD) rats. An in vitro cellular model simulating the PSD pathological microenvironment was established by oxygen-glucose deprivation(OGD) combined with lipopolysaccharide and corticosterone intervention for 6 h. The cultured cells were randomly divided into 8 groups: normal, model, lipopolysaccharide endotoxin (LOS, CMPK2 activator) group (LOS group), nordihydroguaiaretic acid (NDGA, CMPK2 inhibitor) group (NDGA group), positive control group, BSL group, BSL+LOS group, and BSL+NDGA group. Cell morphology and structure of microglia and ACC neurons were observed using a cell imaging analysis system. ACC neuron viability was assessed by the CCK-8 assay. Calcein-AM/PI staining was employed to observe the dead/alive cell ratio of ACC neurons. Nissl staining was used to evaluate synaptic damage in ACC neurons. The levels of serotonin(5-HT) and dopamine(DA) in the cell supernatant were measured by enzyme-linked immunosorbent assay(ELISA). Immunofluorescence staining was performed to detect the protein expression of CMPK2 and ionized calcium-binding adaptor molecule 1(IBA1) in microglia, as well as glutamate receptor 2A(GluN2A), glutamate receptor 2B(GluN2B), synaptophysin 1(SYN1), and postsynaptic density protein 95(PSD95) in ACC glutamatergic neurons. Western blotting was used to quantify the protein expression of CMPK2 in microglia and GluN2A, GluN2B, SYN1, and PSD95 in ACC neurons.

RESULTS  BSL-medicated serum effectively ameliorated morphological and structural damage to microglia and ACC neurons, inhibited aberrant microglial activation, increased ACC neuronal viability, and elevated the levels of the monoamine neurotransmitters 5-HT and DA, thereby alleviating ACC neuronal synaptic damage in PSD. Further mechanistic investigation revealed that the CMPK2 activator LOS exacerbated abnormal microglial activation and ACC synaptic damage, whereas the CMPK2 inhibitor NDGA attenuated these pathological changes. Additionally, BSL-medicated serum suppressed the expression of synaptic-related proteins GluN2A and GluN2B in ACC glutamatergic neurons while promoting the expression of the presynaptic membrane protein SYN1 and the postsynaptic membrane protein PSD95, an effect mediated by downregulation of CMPK2 signaling in microglia. Notably, BSL-medicated serum significantly reversed the abnormal upregulation or downregulation of the aforementioned proteins induced by the CMPK2 activator LOS, ultimately repairing glutamatergic synaptic damage in ACC neurons of PSD.

CONCLUSION BSL-medicated serum effectively promotes neuronal survival, elevates monoamine neurotransmitter(5-HT and DA) levels, and ameliorates glutamatergic synaptic damage in the ACC. The underlying molecular mechanism may involve the regulation of aberrant CMPK2/NR signaling.