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Published on: November 9, 2020
Inosine Ameliorates the Injurious Microenvironment for Oligodendrocyte Precursor Cells by Suppressing Microglial
Yong Han1, Jin Chen1, Peilian Wang1
1Department of Human Anatomy and Histoembryology, Basic Medical College of Ningxia Medical University, Yinchuan, 750004, Ningxia, P.R. China.
Abstract:
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.
Insights
Inosine directly reduces microglial inflammation by inhibiting the TLR4/MyD88/NF-κB pathway. This protects oligodendrocyte precursor cells (OPCs) from damage, offering neuroprotection against neonatal white matter injury (WMI).
Area of Science:
- Neuroscience
- Cell Biology
- Developmental Biology
Background:
- Neonatal white matter injury (WMI) is a primary cause of cerebral palsy, linked to microglial-driven neuroinflammation.
- Microglial activation impairs oligodendrocyte precursor cell (OPC) survival and differentiation.
- Inosine's in vivo neuroprotective effects against WMI are known, but its direct impact on microglia requires elucidation.
Purpose of the Study:
- To investigate the direct effects of inosine on microglial activation and inflammatory responses in vitro.
- To determine if inosine modulates the TLR4/MyD88/NF-κB signaling pathway in microglia.
- To assess the impact of inosine-modulated microglia on OPC viability, proliferation, and differentiation.
Main Methods:
- Primary microglia were stimulated with lipopolysaccharide (LPS) and treated with inosine.
- Cytokine secretion (TNF-α, IL-1β, IL-6) and TLR4 pathway protein expression were measured.
- Microglia-conditioned medium (MCM) was applied to OPC cultures to evaluate OPC viability, apoptosis, proliferation, and differentiation.
Main Results:
- Inosine significantly reduced LPS-induced secretion of TNF-α, IL-1β, and IL-6 from microglia.
- Inosine downregulated the expression of TLR4, MyD88, and p-NF-κB p65 in microglia.
- MCM from inosine-treated microglia enhanced OPC viability, reduced apoptosis, increased proliferation, and promoted differentiation.
Conclusions:
- Inosine directly inhibits microglial overactivation and inflammation in vitro via TLR4/MyD88/NF-κB pathway downregulation.
- Inosine indirectly protects OPCs by mitigating the detrimental effects of activated microglia.
- These findings provide cellular mechanisms for inosine's neuroprotective role in neonatal WMI.
