[Abstract]
Background: Neuroinflammation is the major pathogenesis of cerebral ischemia. Microglia are activated and polarized to either the pro-inflammatory M1 phenotype or anti-inflammatory M2 phenotype, which act as a critical mediator of neuroinflammation. Sestrin2 has pro-survival properties against ischemic brain injury. However, whether sestrin2 has an anti-inflammatory function by shifting microglia polarization and its underlying mechanism is unknown.
Methods: Adult male C57BL/6 mice (N=108) underwent transient middle cerebral artery occlusion (tMCAO) and were treated with exogenous sestrin2. Neurological deficit scores and infarct volume were determined. Cell apoptosis was examined by TUNEL staining and western blotting. The expression of inflammatory mediators, M1/M2 specific markers, and signaling pathways were detected by reverse transcription-polymerase chain reaction, immunostaining, and western blotting. To explore the underlying mechanism, primary neurons were subjected to oxygen-glucose deprivation (OGD) and then treated with oxygenated condition medium of BV2 cells incubated with different doses of sestrin2.
Results: Sestrin2 attenuated the neurological deficits, infarction volume, and cell apoptosis after tMCAO compared to those in the control (p<0.05). Sestrin2 had an anti-inflammatory effect and could suppress M1 microglia polarization and promote M2 microglia polarization. Condition medium from BV2 cells cultured with sestrin2 reduced neuronal apoptosis after OGD in vitro. Furthermore, we demonstrated that sestrin2 drives microglia to the M2 phenotype by inhibiting the mammalian target of rapamycin (mTOR) signaling pathway and restoring autophagic flux.
Conclusions: Sestrin2 exhibited neuroprotection by shifting microglia polarization from the M1 to M2 phenotype in ischemic mouse brain, which may be due to suppression of the mTOR signaling pathway and the restoration of autophagic flux.
【中文摘要】
背景:神經炎症是腦缺血後組織損傷的重要病理生理機制，腦缺血後小膠質細胞被激活並極化為促炎的M1表型或抗炎的M2表型，對神經炎症起關鍵的調節作用。Sestrin2對腦缺血存在保護作用，然而其是否通過改變小膠質細胞分型進行抗炎和免疫調節功能尚不清楚。
方法:應用成年雄性C57BL/6小鼠建立短暫性大腦中動脈栓塞模型，並於側腦室給予外源性的sestrin2重組蛋白。模型成功後評估神經功能缺損和梗死體積，應用TUNEL染色和western blotting方法檢測細胞凋亡。同時，通過逆轉錄-聚合酶鏈反應、免疫染色和western blotting的方法檢測炎症指標、M1/M2分型標記物及相關信號通路的表達。為探討其作用機制，對原代神經元進行糖氧剝奪處理，然後用梯度濃度sestrin2培養的BV2細胞糖氧剝奪後再灌的條件培養液處理原代神經元。
結果:與對照組相比，Sestrin2可減輕腦缺血小鼠的神經功能缺損、梗死體積和細胞凋亡。Sestrin2具有減輕炎症的作用，可抑制M1型小膠質細胞極化並促進M2型小膠質細胞極化。用sestrin2培養的BV2細胞條件培養液能夠減少糖氧剝奪後的原代神經元凋亡。此外，我們證明sestrin2是通過抑制 mTOR信號通路並恢復自噬流的機制促進小膠質細胞向M2表型轉化。
結論:Sestrin2通過改變腦缺血小鼠的小膠質細胞表型轉變產生保護作用，其機制可能與抑制mTOR信號通路並恢復自噬流有關。