ABSTRACT
Objective:To investigate the role and regulatory mechanism of triggering receptor expressed on myeloid cell 2 (TREM2) in mice lung ischemia/reperfusion injury (LIRI).Methods:Thirty-six healthy male C57BL/6 mice were divided into six groups according to the random number method ( n = 6): normal control group, and LIRI 2, 6, 12, 24, 48 hours group. Mice LIRI models were established by clamping the left hilum. The wet/dry weight ratio (W/D) of left lung tissue was measured. Lung injury was observed and evaluated by hematoxylin-eosin (HE) staining and electron microscopy. The levels of interleukins (IL-1β, IL-18) in lung tissue were detected by enzyme linked immunosorbent assay (ELISA). The mRNA expressions of TREM2 and caspase-1 were determined by polymerase chain reaction (PCR). The protein expressions of TREM2, caspase-1, Gasdermin-D (GSDMD) were determined by Western blotting. Results:At 2 hours after LIRI, lung injury began to appear, the lung ultrastructure changed, and the lung injury score increased; at 6 hours, the degree of lung injury was the most serious; after 12 hours, the lung injury gradually reduced and the lung injury score gradually decreased. Compared with the normal control group, lung W/D ratio and lung injury score of LIRI 2, 6, 12, 24, 48 hours groups were significantly higher, the differences were statistically significant (lung W/D ratio: 7.06±0.52, 8.34±0.17, 6.42±0.35, 5.34±0.25, 5.59±0.45 vs. 4.69±0.23; lung injury score: 5.50±0.54, 9.75±0.89, 5.88±0.84, 3.63±0.74, 4.13±0.64 vs. 1.13±0.35, all P < 0.05). Compared with the normal control group, the levels of IL-1β and IL-18 in lung tissue were significantly increased at 2 hours after LIRI, reached a peak at 6 hours [IL-1β (ng/L): 502.76±12.25 vs. 56.50±8.07, IL-18 (ng/L): 414.02±10.75 vs. 81.63±5.29, both P < 0.05], then decreased gradually, and were still significantly higher than the normal control group at 48 hours. The PCR and Western blotting showed that the expression of TREM2 was significantly lower than that in the normal control group at 2 hours after LIRI, and reached a valley at 6 hours [TREM2 mRNA (2 -ΔΔCt): 0.47±0.05 vs. 1.02±0.05, TREM2/GAPDH: 0.23±0.13 vs. 0.48±0.17, both P < 0.05], then gradually increased, and reached the peak at 24 hours [TREM2 mRNA (2 -ΔΔCt): 3.98±0.15 vs. 1.02±0.05, TREM2/GAPDH: 0.71±0.17 vs. 0.48±0.17, both P < 0.05]. The trend of expression of caspase-1 and GSDMD were opposite to that of TREM2, which increased at first and then decreased, and reached a peak at 6 hours after reperfusion [caspase-1 mRNA (2 -ΔΔCt): 2.20±0.13 vs. 1.01±0.02, caspase-1/GAPDH: 0.64±0.02 vs. 0.20±0.06, GSDMD/GAPDH: 1.23±0.01 vs. 0.87±0.02, all P < 0.05]. Conclusions:TREM2 might be involved in LIRI in mice. The mechanism may be related to the effect of TREM2 on caspase-1-mediated pyroptosis.
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.