Abstract
OBJECTIVE: Microglia can be polarized into a proinflammatory M1 phenotype or an anti-inflammatory M2 phenotype. An excess of the M1 phenotype and a deficiency of the M2 phenotype are crucial to the pathological process of ischemic stroke, but the molecular mechanism is still unclear. Although several studies have confirmed the therapeutic effects of PNS (Panax notoginseng saponins) on ischemic stroke, the precise molecular mechanisms of these effects remain poorly understood. The aim of this study was to investigate the molecular mechanism of PNS influencing microglia polarization via hematopoietic progenitor kinase 1 (HPK1) signaling pathway regulation. METHODS: BV2 cells were pretreated with PNS or GNE-1858 (HPK1 inhibitor) and then polarization into M1- and M2-like phenotypes via lipopolysaccharide + interferon-gamma or interleukin (IL)-4, respectively. Detection of M1- and M2-like phenotypes by flow cytometry. The mRNA levels of tumor necrosis factor-alpha, L-1β, Arg1, and IL-10 were measured by real-time PCR. The phosphorylation levels of HPK1, nuclear factor kappa-B (NF-κB), and c-Jun N-terminal kinase (JNK) were detected by western blot. RESULTS: The phosphorylation levels of HPK1, NF-κB, and JNK gradually increased under the M1 polarization condition. Under the M2 polarization condition, the phosphorylation levels of HPK1, NF-κB, and JNK gradually decreased. Inhibition of HPK1 activity effectively inhibited the activation of NF-κB and JNK during M1 polarization. PNS can inhibit the activation of JNK and NF-κB by inhibiting the activity of HPK1, thereby inhibiting the polarization of M1-like phenotype and promoting the polarization of M2-like phenotype. CONCLUSIONS: This research confirmed that PNS effectively inhibits M1 microglial polarization while stimulating M2 microglial polarization via HPK1, JNK, and NF-κB signaling pathway suppression.