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. 2023 Feb 3;25(3):123.
doi: 10.3892/etm.2023.11822. eCollection 2023 Mar.

Cyclooxygenase‑2 contributes to the hypoxia‑induced aggravation of the neuroinflammation response stimulated by lipopolysaccharide in microglia

Yifan Yang  1   2 , Yanan Geng  2 , Xiang Cheng  2 , Jiayue Gao  2 , Zibi Shi  2 , Ming Zhao  2
Affiliations

Cyclooxygenase‑2 contributes to the hypoxia‑induced aggravation of the neuroinflammation response stimulated by lipopolysaccharide in microglia

Yifan Yang et al. Exp Ther Med. .

Abstract

Hypoxia and neuroinflammation are key risk factors involved in various pathophysiological neural disorders. Hypoxia can aggravate neuroinflammation in vitro and in vivo; however, the underlying mechanisms remain unknown. In the present study, hypoxia [either 3 or 1% oxygen (O2)] increased lipopolysaccharide (LPS)-induced expression of the IL-6, IL-1β and TNF-α proinflammatory cytokines in BV2 cells. At the molecular level, both hypoxia and FG-4592, an hypoxia inducible factor 1 pathway activator, effectively induced cyclooxygenase-2 (COX-2) expression. The COX-2 inhibitor celecoxib significantly reduced the expression of cytokines induced by LPS under hypoxic conditions. Additionally, the administration of celecoxib inhibited the activation of microglia as well as cytokine expression in mice administered with hypoxia exposure and LPS injection. The present data demonstrated that COX-2 is involved in the hypoxia-induced aggravation of neuroinflammation stimulated by LPS.

Keywords: cyclooxygenase-2; hypoxia; lipopolysaccharide; microglia; neuroinflammation.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Hypoxia aggravates the LPS-induced neuroinflammatory response in BV2 cells. (A-C) BV2 cells were treated with 1% or 3% O2 hypoxia and/or LPS (100 ng/ml) for 24 h. Total RNA was isolated and used to perform reverse transcription-quantitative PCR assays to measure the mRNA levels of (A) IL-6, (B) IL-1β and (C) TNF-α. The results are expressed as the mean ± SEM (n=3). *P<0.05 and **P<0.01. LPS, lipopolysaccharide; SEM, standard error of the mean; Nor, Normoxia; Hy, hypoxia; ns, not significant.
Figure 2
Figure 2
Hypoxia increases COX-2 expression at both the mRNA and protein levels in BV2 cells. (A and B) BV2 cells were exposed to 1% O2 hypoxia for 6, 12 and 24 h. (A) Protein levels of HIF-1α were measured by western blotting and β-actin served as the internal control. (B) Quantification of the protein band densities of HIF-1α are presented as a bar graph. (C) BV2 cells were exposed to 1% O2 hypoxia for 6, 12 and 24 h. Total RNA was isolated and used to perform reverse transcription-quantitative PCR assays to measure the mRNA levels of COX-2. (D) BV2 cells were treated with hypoxia (1% O2) and/or LPS (100 ng/ml) for 6, 12 and 24 h. The protein levels of COX-2 were measured by western blotting and β-actin served as the internal control. (E) Quantification of the protein band densities of COX-2. The results are expressed as the mean ± SEM (n=3). *P<0.05 and **P<0.01. SEM, standard error of the mean; COX-2, cyclooxygenase-2; HIF-1α, hypoxia inducible factor 1α; Nor, normoxia; Hy, hypoxia; LPS, lipopolysaccharide.
Figure 3
Figure 3
FG-4592 induces COX-2 expression. (A) BV2 cells were treated with FG-4592 (10 µM) for 6, 12 and 24 h. The protein levels of HIF-1α, COX-2 and β-actin in BV-2 cells in different groups were determined by western blotting. Quantification of the protein band densities of (B) HIF-1α and (C) COX-2. The results are expressed as the mean ± standard error of the mean (n=3). **P<0.01. COX-2, cyclooxygenase-2; HIF-1α, hypoxia inducible factor 1α; ns, not significant.
Figure 4
Figure 4
Celecoxib inhibits the neuroinflammatory response induced by the combination treatment of LPS and hypoxia in BV2 cells. (A-C) BV2 cells were pretreated with different doses of celecoxib (10 and 20 µM) for 1 h and then treated with 100 ng/mg LPS combined with 1% O2 hypoxia for 12 and 24 h. Total RNA was isolated and used to perform RT-qPCR assays to measure the mRNA levels of (A) IL-6, (B) IL-1β and (C) TNF-α. The results are expressed as the mean ± standard error of the mean (n=3). *P<0.05 and **P<0.01. LPS, lipopolysaccharide; Hy, hypoxia; Cel, celecoxib; ns, not significant.
Figure 5
Figure 5
Celecoxib inhibits microglial activation and decreases the mRNA levels of proinflammatory cytokines in the mouse model. (A) Mice were pretreated with celecoxib (20 mg/kg) and then treated with lipopolysaccharide injection (0.5 mg/kg) and hypoxia exposure (mimicking 6,000 m of high altitude) for 24 h. The brain slices were subjected to immunofluorescence staining. Representative images of ionized calcium-binding adapter molecule 1 staining in the hippocampus. The nucleus was stained with DAPI. Scale bar, 100 µm. (B) Statistical analysis of microglial activation, as presented in A. The mRNA levels of (C) IL-6, (D) IL-1β and (E) TNF-α were measured via reverse transcription-quantitative PCR assay. The results are expressed as the mean ± SEM (n=4). *P<0.05 and **P<0.01. SEM, standard error of the mean; IBA1, ionized calcium-binding adapter molecule 1; LPS, lipopolysaccharide; Nor, normoxia; Hy, hypoxia; Cel, celecoxib; ns, not significant.
Figure 6
Figure 6
Schematic representation depicting the role of COX-2 in mediating hypoxia-induced aggravation of neuroinflammation stimulated by LPS. Hypoxia aggravated LPS-induced neuroinflammation responding in vitro and in vivo. Under hypoxic conditions, the expression of COX-2 was induced in microglia. Celecoxib, a specific inhibitor of COX-2, inhibited neuroinflammation response under hypoxia conditions. LPS, lipopolysaccharide; COX-2, cyclooxygenase-2.
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Grants and funding

Funding: This work was supported by the National Natural Science Foundation of China (grant no. 81930054).
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