NMDARs antagonist MK801 suppresses LPS-induced apoptosis and mitochondrial dysfunction by regulating subunits of NMDARs via the CaM/CaMKII/ERK pathway

Abstract

Lipopolysaccharide (LPS) displays a robust immunostimulatory ability upon Toll-like receptor 4 (TLR4) recognition. N-methyl-D-aspartate receptors (NMDARs) are highly compartmentalized in most cells and implicated in various inflammatory disorders. However, the relationship between TLR4 and NMDARs has not been explored deeply. This study aimed to examine the role of NMDARs and its specific inhibitor MK801 in LPS-treated endothelial cell dysfunction and the related mechanism in vivo and in vitro. The results showed that pre-treatment with MK801 significantly decreased LPS-induced cell death, cellular Ca²⁺, cellular reactive oxygen species, and glutamate efflux. Moreover, MK801 restrained LPS-induced mitochondrial dysfunction by regulating mitochondrial membrane potential and mitochondrial Ca²⁺ uptake. The oxygen consumption, basal and maximal respiration rate, and ATP production in LPS-treated HUVECs were reversed by MK801 via regulating ATP synthesis-related protein SDHB2, MTCO1, and ATP5A. The molecular pathway involved in MK801-regulated LPS injury was mediated by phosphorylation of CaMKII and ERK and the expression of MCU, MCUR1, and TLR4. LPS-decreased permeability in HUVECs was improved by MK801 via the Erk/ZO-1/occluding/Cx43 axis. Co-immunoprecipitation assay and western blotting showed three subtypes of NMDARs, NMDAζ1, NMDAε2, and NMDAε4 were bound explicitly to TLR4, suppressed by LPS, and promoted by MK801. Deficiency of NMDAζ1, NMDAε2, or NMDAε4 induced cell apoptosis, Ca²⁺ uptake, ROS production, and decreased basal and maximal respiration rate, and ATP production, suggesting that NMDARs integrity is vital for cell and mitochondrial function. In vivo investigation showed MK801 improved impairment of vascular permeability, especially in the lung and mesentery in LPS-injured mice. Our study displayed a novel mechanism and utilization of MK801 in LPS-induced ECs injury and permeability.

Fig. 1. MK801 protects against LPS-treated HUVECs.

A MTT assay showed MK801 (1, 5, 10 μM) did not affect cell viability in HUVECs. B MTT assay showed MK801 (1, 5, 10 μM) effectively countered viability reduction induced by LPS (20 μg/ml). C Intracellular glutamate and D extracellular glutamate concentrations in HUVECs after treatment with LPS (20 μg/ml) for 24 h in the absence or presence of MK801(1, 5, 10 μM). E Flow cytometry graphs and F analysis of Ca²⁺ influx in HUVECs with the fluorescent probe Fluo-4-AM in response to LPS (20 μg/ml) and MK801 (1, 5, 10 μM). G Flow cytometry graphs and (H) analysis of ROS production in HUVECs treated with LPS (20 μg/mL) in the absence or presence of MK801 (1, 5, 10 μM) for 24 h. (Data are presented as mean ± SEM, ***P < 0.001, **P < 0.01, *P < 0.05 vs. Control; ###P < 0.001, ##P < 0.01, #P < 0.05 vs. LPS).

Fig. 2. Protective effects of MK801 against LPS-induced mitochondrial dysfunction in HUVECs.

A Flow cytometry graphs and B analysis of mitochondrial transmembrane potential in HUVECs treated with LPS in the absence or presence of indicated doses of MK801. Representative of traces (C) and quantification at 10 min (D) of mitochondria Ca²⁺ influx labeled with Rhod-2 AM in HUVECs treated with LPS in the presence or absence of Ru360 (mitochondrial calcium uptake inhibitor), DS16570511 (MCU inhibitor), or MK801 for 2 h. E–H O₂ consumption rates (D), basal (E) and maximal respiration rate (F), and ATP production (G) in HUVECs treated with LPS in the presence or absence of MK801. Representative of western blotting images (I) and quantification (J) of NDUFB8, SDHB2, UQCRC2, MTCO1, and ATP5a in HUVECs treated with LPS in the presence or absence of MK-801 at indicated doses. (Data are presented as mean ± SEM, ***P < 0.001, **P < 0.01, *P < 0.05 vs. Control; ###P < 0.001, ##P < 0.01, #P < 0.05 vs. LPS).

Fig. 3. Molecular signal pathway in MK801 regulated dysfunction of HUVEC induced by LPS.

A Representative of western blotting images and B analysis of phosphorylation of Erk1/2 (p-Erk1/2) and CaMKII (p-CaMKII), and expression of CaM, MCU, MCUR, and TLR4 in HUVECs exposed to LPS in the presence or absence of different concentrations of MK801. (Data are presented as mean ± SEM, ***P < 0.001, **P < 0.01, *P < 0.05 vs. Control; ###P < 0.001, ##P < 0.01, #P < 0.05 vs. LPS).

Fig. 4. MK801 rescued LPS-increased permeability in HUVECs.

A Immunostaining images of F-actin labeled with TRITC Phalloidin and B analysis in HUVECs induced by LPS and treated with MK801; the peripheral bands of F-actin and intercellular gaps appeared between adjacent cells. C Vascular Permeability Assay showed the relative intensity of FITC-Dextran in HUVECs induced by LPS and treated with MK801. Representative of western blotting images (D) and analysis (E) of expression of ZO-1, Occludin, and Cx43 in LPS-exposed HUVECs in the presence or absence of different concentrations of MK801. (Data are presented as mean ± SEM, ***P < 0.001, **P < 0.01, *P < 0.05 vs. Control; ###P < 0.001, ##P < 0.01, #P < 0.05 vs. LPS).

Fig. 5. The relationship between TLR4 and NMDARs.

A Flow cytometry images and B analysis of FITC-conjugated LPS-treated HUVECs in the presence or absence of indicated doses of MK-801. C MTT assay showed relative cell viability in NMDA- or LPS-treated HUVECs in the presence or absence of MK801 or TAK242. D MTT assay showed relative cell viability in control siRNA or TLR4 siRNA transfected HUVECs treated with LPS or MK801. E Co-IP detection showed the interaction of NMDARs subunits, NMDAζ1, NMDAε1, NMDAε2, NMDAε3, and NMDAε4, with TLR4 induced by LPS in HUVECs. F The western blotting image showed the expression of NMDAζ1, NMDAε1, NMDAε2, and NMDAε4 treated with LPS in the presence or absence of different concentrations of MK801 in HUVECs. (Data are presented as mean ± SEM, ***P < 0.001, **P < 0.01 vs. Control or control siRNA; ##P < 0.01 vs. LPS or control siRNA + LPS; &P < 0.05 vs. NMDA; $P < 0.05 vs. TLR4 siRNA).

Fig. 6. Knockdown of NMDAR subunits NMDAζ1, NMDAε2, and NMDAε4 promoted apoptosis, intracellular Ca²⁺ influx, ROS production, and restrained mitochondrial O₂ consumption rate.

A Representative flow cytometry images and analysis of cell apoptosis with live cells (B), death (C), early apoptosis (D), and late apoptosis in HUVECs transfected with control siRNA, NMDAζ1 siRNA, NMDAε1 siRNA, NMDAε2 siRNA, NMDAε3 siRNA, or NMDAε4 siRNA. F Flow cytometry graphs and G analysis of Ca²⁺ influx in HUVECs transfected with control siRNA, NMDAζ1 siRNA, NMDAε1 siRNA, NMDAε2 siRNA, NMDAε3 siRNA, or NMDAε4 siRNA. H Flow cytometry graphs and I analysis of ROS in HUVECs transfected with control siRNA, NMDAζ1, NMDAε1 siRNA, NMDAε2 siRNA, NMDAε3 siRNA, or NMDAε4 siRNA. J Seahorse XF24 Extracellular Flux Analyzer detected mitochondrial O₂ consumption rate, K basal respiration rate, L maximal respiration rate, and M ATP production in HUVECs after transfection with different siRNA. (Data are presented as mean ± SEM, ***P < 0.001, **P < 0.01, *P < 0.05 vs. control siRNA).

Fig. 7. MK801 ameliorated LPS-induced vascular permeability in mice.

A EB extravasation in C57/BL6 mice treated with vehicle, LPS (5 mg/kg, i/p), or LPS plus MK-801 for 12 h. Evans blue dye (30 ml/kg, i/v) was injected 2 h before the termination of the experiment. B Wet/dry ratio of lungs from the control group, LPS-induced mice group, and LPS-induced mice treated with MK-801. C The schematic image of the present finding. (Data are presented as mean ± SEM, ***P < 0.001, **P < 0.01, *P < 0.05 vs. Control; ##P < 0.01, #P < 0.05 vs. LPS).