LOX-1 mediates inflammatory activation of microglial cells through the p38-MAPK/NF-κB pathways under hypoxic-ischemic conditions

Abstract

Background: Microglial cells play an important role in the immune system in the brain. Activated microglial cells are not only injurious but also neuroprotective. We confirmed marked lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) expression in microglial cells in pathological lesions in the neonatal hypoxic-ischemic encephalopathy (nHIE) model brain. LOX-1 is known to be an activator of cytokines and chemokines through intracellular pathways. Here, we investigated a novel role of LOX-1 and the molecular mechanism of LOX-1 gene transcription microglial cells under hypoxic and ischemic conditions.

Methods: We isolated primary rat microglial cells from 3-day-old rat brains and confirmed that the isolated cells showed more than 98% Iba-1 positivity with immunocytochemistry. We treated primary rat microglial cells with oxygen glucose deprivation (OGD) as an in vitro model of nHIE. Then, we evaluated the expression levels of LOX-1, cytokines and chemokines in cells treated with or without siRNA and inhibitors compared with those of cells that did not receive OGD-treatment. To confirm transcription factor binding to the OLR-1 gene promoter under the OGD conditions, we performed a luciferase reporter assay and chromatin immunoprecipitation assay. In addition, we analyzed reactive oxygen species and cell viability.

Results: We found that defects in oxygen and nutrition induced LOX-1 expression and led to the production of inflammatory mediators, such as the cytokines IL-1β, IL-6 and TNF-α; the chemokines CCL2, CCL5 and CCL3; and reactive oxygen/nitrogen species. Then, the LOX-1 signal transduction pathway was blocked by inhibitors, LOX-1 siRNA, the p38-MAPK inhibitor SB203580 and the NF-κB inhibitor BAY11-7082 suppressed the production of inflammatory mediators. We found that NF-κB and HIF-1α bind to the promoter region of the OLR-1 gene. Based on the results of the luciferase reporter assay, NF-κB has strong transcriptional activity. Moreover, we demonstrated that LOX-1 in microglial cells was autonomously overexpressed by positive feedback of the intracellular LOX-1 pathway.

Conclusion: The hypoxic/ischemic conditions of microglial cells induced LOX-1 expression and activated the immune system. LOX-1 and its related molecules or chemicals may be major therapeutic candidates. Video abstract.

Keywords: Hypoxia; Ischemia; LOX-1; Microglia; NF‐kappa B (NF‐κB); OLR-1; p38-MAPK.

Fig. 1

OGD-treated microglial cells express LOX-1. Untreated microglial cells (control; CTL) did not exhibit LOX-1 expression (A). OGD-treated microglial cells (OGD) exhibited LOX-1 expression in the cytoplasm (B). Immunoblot analysis shows LOX-1 overexpression in OGD-treated microglial cells (C, D). Immunocytochemistry, blue: Hoechst 33342 (A, B), red; LOX-1 (B). *P < 0.05. Scale bar = 10 µm

Fig. 2

LOX-1 siRNA inhibits OGD-induced cytokine and chemokine production. The cytokines IL-1β, IL-6 and TNF-α (A) and the chemokines CCL2, CCL5 and CCL3 (B) were significantly increased. LOX-1 siRNA downregulates the expression of these molecules. **P < 0.01

Fig. 3

LOX-1 inhibition alters the distribution of NF-κB p65 from the cytoplasm to the nucleus. OGD-treated microglial cells show NF-κB p65 in the nucleus, but LOX-1 siRNA induces cytoplasmic expression of NF-κB p65 (A). Western blots exhibit significantly increased NF-κB p65 expression levels in the nuclear fraction of OGD-treated microglial cells but no significant difference in NF-κB p65 expression in the cytoplasmic fraction (B). Hoechst; Hoechst 33342, *P < 0.05. Scale bar = 10 µm

Fig. 4

LOX-1 suppression induces different expression patterns of p38-MAPK and ERK phosphorylation in OGD-treated microglial cells. The expression of phospho-p38-MAPK and the ratio of phospho/total p38-MAPK were significantly increased in OGD-treated microglial cells but were reduced to the control level by LOX-1 siRNA (A). However, the expression of phospho-ERK and the ratio of phospho/total ERK were relatively increased in OGD-treated microglial cells but were not significantly reduced to the control level by LOX-1 siRNA (B). *P < 0.05

Fig. 5

p38-MAPK and NF-κB inhibitors do not change LOX-1 expression levels in OGD-treated microglial cells. OGD-treated microglial cells expressed LOX-1 protein (red) in the cytoplasm (A). The same photographs of untreated microglial cells and OGD-treated microglial cells are shown in Fig. 1A, B. LOX-1 expression is not influenced by the p38-MAPK or NF-κB inhibitors SB or BAY, respectively. The relative LOX-1 expression level was the same as that shown by immunocytochemistry (B). SB; SB203580, BAY; BAY11-7082, Scale bar = 10 µm. *P < 0.05

Fig. 6

The production of cytokines and chemokines in OGD-treated microglial cells is downregulated by p38-MAPK and NF-κB inhibitors. The production of the cytokines IL-1β, IL-6 and TNF-α (A) and the chemokines CCL2, CCL5 and CCL3 (B), which are increased by OGD treatment, are suppressed by SB and BAY to the same levels as those of the control. SB; SB203580, BAY; BAY11-7082, **P < 0.01

Fig. 7

p38-MAPK and NF-κB inhibitors alter the distribution pattern of NF-κB p65 from the cytoplasm to the nucleus. NF-κB p65 is located in the nucleus in OGD-treated microglial cells but in the cytoplasm in other microglial cells (A). The same photographs of untreated microglial cells and OGD-treated microglial cells are shown in Fig. 3A. Western blots exhibit significantly increased NF-κB p65 expression levels in the nuclear fraction of OGD-treated microglial cells but no significant differences in NF-κB p65 expression levels in the cytoplasmic fraction (B). Hoechst; Hoechst 33,342, Scale bar = 10 µm. *P < 0.05

Fig. 8

A luciferase reporter assay revealed the NF-κB binding site of − 1628 to − 1621 of the OLR-1 promoter region. The OLR-1 transcription activity is less affected by the binding sites of OCT-1 and HIF-1α. #P < 0.05 compared with the mock; , not significant compared with the SV40 promoter. , TATA box; , NF-κB binding site; , OCT-1 binding site; , HIF-1α binding site; , mutation position