Triggering receptor expressed on myeloid cell-2 modulating the polarization of mouse M2 microglia with oxygen-glucose deprivation/re-oxygenation model / 解剖学报

ABSTRACT

Objective To investigate the mechanism of TREM2 modulating the polarization of M2 microglia treated by oxygen-glucose deprivation/reoxygenation (OGD/R). Methods Mouse N9 microglial cells were cultured in vitro. N9 cells were transfected with lenti virus for TREM-2-overexpression (LV-TREM2), and LV-scramble acted as control group. OGD/R model was established. The OGD/R cells were randomly divided into OGD/R, OGD/R+LV-scramble and OGD/R+LV-TREM2 groups. Real-time PCR was used to detect the expression of TREM2 mRNA in OGD/R N9 cells within 72 hours after re-oxygenation. Immunofluorescence was applied to observe transfection of lentivirus LV-scramble and LV-TREM2 for normal N9 microglia, and Real-time PCR and Western blotting were used to verify the efficiency of lentivirus transfection. The mRNA and protein contents of Ml microglial markers tumor necrosis factor-a (TNF-α), interleukin-lβ (IL-lβ) and inducible nitric oxide synthase (iNOS), M2 microglial markers arginase-1 (Arg-1) and interleukin-10 (IL-10) were detected by Real-time PCR and ELISA. The expressions of phosphorylated-phosphatidylinositol 3-kinases (p-PI3K), PI3K, phosphorylated protein kinase B (p-Akt), Akt, phosphorylated inhibitor of nuclear factor-κB ( NF-κB)α (p-lκBα) and IκBα protein were detected by Western blotting. The distribution of NF-κB P65 (NF-κB P65) protein in N9 cells was analyzed by immunofluorescence method. Results TREM2 mRNA content in the OGD/R group cells increased significantly within 72 hours after re-oxygenation, and peaked at hour 24 and hour 48. Lenti virus LV- TREM2 effectively promoted the expression of TREM2 mRNA and protein of N9 cells in OGD/R model (P<0.001, P<0.01). Compared with the OGD/R group, the mRNA and protein content of TNF-α, IL-lβ and iNOS decreased significantly, while Arg-1 and IL-10 in OGD/R+LV-TREM2 group increased significantly(P<0.05). Besides, the ratios of P-PI3K/PI3K and p-Akt/Akt increased obviously (P<0.05), the ratio of p-IκBα/IκBα decreased significantly in OGD/ R+ LV-TREM2 group (P<0.001), and the nuclear translocation of NF-κB P65 protein was obviously weakened. Conclusion TREM-2 overexpression exerts anti-inflammatory effect by modulating the polarization of microglia from Ml to M2 type, which is associated with PI3K/Akt and NF-κB signaling pathways regulated by TREM2 in N9 microglia with OGD/R model.