ABSTRACT
We investigated the ability of Dracocephalum moldavica (EPDM) flavonoids to protect human brain microvascular endothelial cells (HBMECs) from necroptosis induced by ischemia-reperfusion injury. To mimic the process of cerebral ischemia-reperfusion injury, a necroptosis model was established by treatment with the pan-cysteine aspartic acid protease (caspase) inhibitor Z-VAD-FMK combined with oxygen-glucose deprivation/re-oxygenation (OGD/R) injury using HBMECs. Cell proliferation and cytotoxicity (cell counting kit-8, CCK-8) was used to measure cell viability. A Hoechst33342/PI fluorescent double-staining method was exploited to determine the rate of cell necroptosis. A commercial kit was used to detect lactate dehydrogenase in the cell culture supernate. DCFH-DA probes, calcein AM and JC-1 probes were used to measure changes in ROS production, mitochondrial membrane permeability transformation pore (MPTP) opening and mitochondrial membrane potential (MMP), respectively. Enzyme-linked immunosorbent assay (ELISA) kits were chosen to detect the release of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Western blotting was used to detect necroptosis-related proteins. The results show that relative to control group, Z-VAD-FMK combined with OGD/R injury reduced cell viability, increased the necroptosis rate and the levels of LDH and ROS in HBMECs. The MPTP of the model group cells opened and the MMP reduced. TNF-α, IL-1β, and IL-6 levels were significantly elevated. Furthermore, the expression of receptor-interacting protein kinase 3 (RIP3) and mitochondrial phosphoglycerate mutase 5 (PGAM5) was significantly increased, accompanied by an increase of phosphorylated mixed-lineage kinase domain-like protein (p-MLKL)/MLKL. EPDM partially reversed the changes of the above-mentioned factors in HBMECs induced by Z-VAD-FMK plus OGD/R injury. These results indicate that EPDM may protect HBMECs from cerebral ischemia-reperfusion injury by inhibiting the RIP3/MLKL/PGAM5 pathway and MPTP opening to maintain mitochondrial function, thereby providing a scientific basis for the use of EPDM in the treatment of cerebral ischemia-related diseases.
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.