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<p>Neonatal white matter injury (WMI), a leading cause of cerebral palsy, results from microglial-driven neuroinflammation that affects oligodendrocyte precursor cell (OPC) survival and differentiation. Our prior in vivo research indicated that inosine may protect against maternal inflammation-induced WMI by modulating microglial polarization and inhibiting TLR4/MyD88/NF-κB signaling, but its direct effects on microglia are unknown. This in vitro study used a microglia-conditioned medium (MCM)-OPC approach to explore this question. Primary microglia were stimulated with lipopolysaccharide (LPS) and treated with inosine, followed by the measurement of inflammatory cytokines (TNF-α, IL-1β, IL-6) and TLR4 pathway proteins via ELISA and Western blot. MCM derived from differentially treated microglia was then applied to OPC cultures, where OPC viability, death, proliferation, and differentiation were assessed using CCK-8 assay, propidium iodide (PI) staining, immunofluorescence, and Western blot. Inosine co-treatment significantly decreased LPS-induced secretion of TNF-α, IL-1β, and IL-6 from microglia (<i>P</i> &lt; 0.05) and downregulated TLR4, MyD88, and p-NF-κB p65 expression (<i>P</i> &lt; 0.001, <i>P</i> &lt; 0.01). MCM from inosine-treated microglia mitigated OPC damage caused by activated microglia, as demonstrated by enhanced OPC viability (<i>P</i> &lt; 0.01), reduced apoptosis (evidenced by decreased PI positivity and Cleaved Caspase-3 expression, <i>P</i> &lt; 0.01, <i>P</i> &lt; 0.05), increased proliferation (indicated by elevated Ki67 positivity and NG2 expression, <i>P</i>&lt;0.001, <i>P</i> &lt; 0.01), and improved differentiation (reflected by increased expression of CNPase, Olig2, and MBP, <i>P</i> &lt; 0.001, <i>P</i> &lt; 0.01). These findings suggest that inosine can directly inhibit the overactivation and inflammatory response of microglia in vitro, an effect associated with TLR4/MyD88/NF-κB downregulation. Furthermore, it can indirectly ameliorate the injurious microenvironment for OPC, thereby providing cellular-level mechanistic clues for explaining its neuroprotective role in vivo.</p>

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Inosine Ameliorates the Injurious Microenvironment for Oligodendrocyte Precursor Cells by Suppressing Microglial Activation and Neuroinflammation In Vitro

  • 期刊论文

  • 发表日期：

    2026年5月20日

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  • Yong Han,
  • Jin Chen,
  • Peilian Wang,
  • Jinping Sun,
  • Jianguo Niu,
  • Quanrui Ma

摘要

Neonatal white matter injury (WMI), a leading cause of cerebral palsy, results from microglial-driven neuroinflammation that affects oligodendrocyte precursor cell (OPC) survival and differentiation. Our prior in vivo research indicated that inosine may protect against maternal inflammation-induced WMI by modulating microglial polarization and inhibiting TLR4/MyD88/NF-κB signaling, but its direct effects on microglia are unknown. This in vitro study used a microglia-conditioned medium (MCM)-OPC approach to explore this question. Primary microglia were stimulated with lipopolysaccharide (LPS) and treated with inosine, followed by the measurement of inflammatory cytokines (TNF-α, IL-1β, IL-6) and TLR4 pathway proteins via ELISA and Western blot. MCM derived from differentially treated microglia was then applied to OPC cultures, where OPC viability, death, proliferation, and differentiation were assessed using CCK-8 assay, propidium iodide (PI) staining, immunofluorescence, and Western blot. Inosine co-treatment significantly decreased LPS-induced secretion of TNF-α, IL-1β, and IL-6 from microglia (P < 0.05) and downregulated TLR4, MyD88, and p-NF-κB p65 expression (P < 0.001, P < 0.01). MCM from inosine-treated microglia mitigated OPC damage caused by activated microglia, as demonstrated by enhanced OPC viability (P < 0.01), reduced apoptosis (evidenced by decreased PI positivity and Cleaved Caspase-3 expression, P < 0.01, P < 0.05), increased proliferation (indicated by elevated Ki67 positivity and NG2 expression, P<0.001, P < 0.01), and improved differentiation (reflected by increased expression of CNPase, Olig2, and MBP, P < 0.001, P < 0.01). These findings suggest that inosine can directly inhibit the overactivation and inflammatory response of microglia in vitro, an effect associated with TLR4/MyD88/NF-κB downregulation. Furthermore, it can indirectly ameliorate the injurious microenvironment for OPC, thereby providing cellular-level mechanistic clues for explaining its neuroprotective role in vivo.