NRF1 knockdown alleviates lipopolysaccharide-induced pulmonary inflammatory injury by upregulating DKK3 and inhibiting the GSK-3β/β-catenin pathway

Abstract

Excessive inflammatory injury is the main cause of the incidence of severe neonatal pneumonia (NP) and associated deaths. Although dickkopf-3 (DKK3) exhibits anti-inflammatory activity in numerous pathological processes, its role in NP is still unknown. In this study, human embryonic lung WI-38 and MRC-5 cells were treated with lipopolysaccharide (LPS) to induce inflammatory injury of NP in vitro. The expression of DKK3 was downregulated in LPS-stimulated WI-38 and MRC-5 cells. DKK3 overexpression decreased LPS-induced inhibition of cell viability, and reduced LPS-induced apoptosis of WI-38 and MRC-5 cells. DKK3 overexpression also reduced LPS-induced production of pro-inflammatory factors such as ROS, IL-6, MCP-1, and TNF-α. Nuclear respiratory factors 1 (NRF1) knockdown was found to upregulate DKK3 and inactivate the GSK-3β/β-catenin pathway in LPS-injured WI-38 and MRC-5 cells. NRF1 knockdown also suppressed LPS-induced inhibition on cell viability, repressed LPS-induced apoptosis, and inhibited the accumulation of ROS, IL-6, MCP-1, and TNF-α in LPS-injured WI-38 and MRC-5 cells. DKK3 knockdown or re-activation of the GSK-3β/β-catenin pathway reversed the inhibitory effects of NRF1 knockdown on LPS-induced inflammatory injury. In conclusion, NRF1 knockdown can alleviate LPS-triggered inflammatory injury by regulating DKK3 and the GSK-3β/β-catenin pathway.

Keywords: DKK3; GSK-3β/β-catenin; NRF1; inflammatory injury; neonatal pneumonia.

Graphical Abstract

Figure 1.

The expression of DKK3 in LPS-induced pneumonia cell model. WI-38 and MRC-5 cells were exposed to 0, 5, 10, or 20 μg/ml of LPS for 24 h. (A) RT–qPCR showed the DKK3 mRNA level in WI-38 and MRC-5 cells (n = 6). ^*P < 0.05, compared with the 0 μg/ml group; ^#P < 0.05, compared with the 5 μg/ml group; ^&P < 0.05, compared with the 10 μg/ml group. (B) Representative western blotting images showed the DKK3 protein level in LPS-stimulated cells (n = 6). ^*P < 0.05, compared with the 0 μg/ml group; ^#P < 0.05, compared with the 5 μg/ml group; ^&P < 0.05, compared with the 10 μg/ml group. (C) RT–qPCR showed the DKK3 mRNA level in WI-38 and MRC-5 cells (n = 6).*P < 0.05, compared with the control group. Data were expressed as mean ± SD, and analyzed using one-way ANOVA with the Bonferroni test.

Figure 2.

DKK3 overexpression increases cell viability and inactivates GSK-3β/β-catenin pathway in LPS-stimulated cells. WI-38 and MRC-5 cells transfected with pGL3–DKK3 or its control pGL3 empty vectors were exposed to 0 or 10 μg/ml of LPS. (A) RT–qPCR showed the DKK3 mRNA level in LPS-stimulated WI-38 and MRC-5 cells (n = 6). (B) Representative western blotting images showed showed the protein levels of DKK3, p-GSK-3β, β-catenin, p-JNK, and c-Jun/AP-1 in LPS-stimulated WI-38 and MRC-5 cells (n = 6). (C) CCK-8 method was used to evaluate the cell viability of WI-38 and MRC-5 cells exposed to LPS for 24 h, 48 h, or 72 h (n = 6). (D) Commercial kits were used to detect ROS production in cells exposed to 10 μg/ml of LPS for 12 h (n = 6). Data were expressed as mean ± SD, and analyzed using one-way ANOVA with the Bonferroni test. ^*P < 0.05, compared with the control group; ^#P < 0.05, compared with the LPS group; ^&P < 0.05, compared with the LPS + pGL3 group.

Figure 3.

DKK3 overexpression ameliorates LPS-induced apoptosis and inflammatory response in WI-38 and MRC-5 cells. WI-38 and MRC-5 cells transfected with pGL3–DKK3 or its control pGL3 empty vectors were exposed to 0 or 10 μg/ml of LPS. (A) Representative flow cytometry images showed cell apoptosis in cells exposed to 10 μg/ml of LPS for 12 h (n = 6). (B) Representative western blotting images showed the protein level of Bax, Bcl-2, and c-Capase3 in cells exposed to 10 μg/ml of LPS for 24 h (n = 6). (C) ELISA assay displayed the levels of IL-6, MCP-1, and TNF-α in cells exposed to 10 μg/ml of LPS for 12 h (n = 6). Data were expressed as mean ± SD, and analyzed using one-way ANOVA with the Bonferroni test. ^*P < 0.05, compared with the control group; ^#P < 0.05, compared with the LPS group; ^&P < 0.05, compared with the LPS + pGL3 group.

Figure 4.

NRF1 downregulates DKK3 and activates the GSK-3β/β-catenin pathway in LPS-stimulated WI-38 and MRC-5 cells. (A) The putative binding sites of NRF1 on the promoter region of DKK3. (B) Enrichment of DKK3 promoter NRF1 with anti-NRF1 or anti-IgG was determined in WI-38 and MRC-5 cells by ChIP and RT–qPCR (n = 3). ^*P < 0.05, compared with the anti-IgG group. (C) Luciferase reporter assay showed the luciferase activity in WI-38 and MRC-5 cells co-transfected with the pGL3–DKK3 promoter or pGL3 and sh-NRF1 or sh-NC (n = 3). ^*P < 0.05, compared with the sh-NC group. (D) RT-qPCR showed the NRF1 mRNA level in sh-NC or sh-NRF1-transfected WI-38 cells treated with 0 or 10 μg/ml of LPS for 24 h (n = 6). ^*P < 0.05, compared with the control group; ^#P < 0.05, compared with the LPS group; ^&P < 0.05, compared with the LPS + sh-NC group. (E) Representative western blotting images showed the protein level of NRF1, DKK3, p-GSK-3β, and β-catenin in cells exposed to 10 μg/ml of LPS for 24 h (n = 6). ^*P < 0.05, compared with the control group; ^#P < 0.05, compared with the LPS group; ^&P < 0.05, compared with the LPS + sh-NC group showed.

Figure 5.

NRF1 knockdown alleviates LPS-triggered ROS production and inflammatory response via regulating DKK3 and the GSK-3β/β-catenin pathway. WI-38 and MRC-5 cells were transfected with sh-NRF1, or co-transfected with sh-NRF1 and sh-DKK3, and treated with or without 10 μg/ml of LPS and 5 mM of LiCl. (A) CCK-8 method evaluated the cell viability (n = 6). (B) Commercial kits revealed the ROS production treated 10 μg/ml of LPS for 12 h (n = 6). (C) ELISA assay displayed the levels of IL-6, MCP-1, and TNF-α treated at 10 μg/ml of LPS for 12 h (n = 6). Data were expressed as mean ± SD, and analyzed using one-way ANOVA with the Bonferroni test. ^*P < 0.05, compared with the control group; ^#P < 0.05, compared with the LPS group; ^&P < 0.05, compared with the LPS + sh-NRF1 group.

Figure 6.

NRF1 knockdown alleviates LPS-induced apoptosis via regulating DKK3 and the GSK-3β/β-catenin pathway. WI-38 and MRC-5 cells were transfected with sh-NRF1, or co-transfected with sh-NRF1 and sh-DKK3, and treated with or without 10 μg/ml of LPS and 5 mM of LiCl. (A) Representative flow cytometry images showed cell apoptosis in cells exposed to 10 μg/ml of LPS for 12 h (n = 6). (B) Representative western blotting images showed the protein level of Bax, Bcl-2, and c-Capase3 in cells exposed to 10 μg/ml of LPS for 24 h (n = 6). Data were expressed as mean ± SD, and analyzed using one-way ANOVA with the Bonferroni test. ^*P < 0.05, compared with the control group; ^#P < 0.05, compared with the LPS group; ^&P < 0.05, compared with the LPS + sh-NRF1 group.