Low Vitamin D Status Is Associated with Inflammation in Patients with Chronic Obstructive Pulmonary Disease

Abstract

Vitamin D deficiency is associated with increased risks of chronic obstructive pulmonary disease (COPD). Nevertheless, the mechanisms remain unknown. This study analyzed the correlations between vitamin D levels and inflammation in COPD patients. One hundred and one patients with COPD and 202 control subjects were enrolled. Serum 25(OH)D level and inflammatory cytokines were detected. Serum 25(OH)D was decreased and inflammatory cytokines were increased in COPD patients. According to forced expiratory volume in 1 s, COPD patients were divided into three grades. Furthermore, serum 25(OH)D was gradually decreased in COPD patients ranging from grade 1-2 to 4. Serum 25(OH)D was inversely associated with inflammatory cytokines in COPD patients. Further analysis found that NF-κB and AP-1 signaling were activated in COPD patients. Besides, inflammatory signaling was gradually increased in parallel with the severity of COPD. By contrast, pulmonary nuclear vitamin D receptor was decreased in COPD patients. In vitro experiments showed that 1,25(OH)₂D₃ inhibited LPS-activated inflammatory signaling in A549 cells (human lung adenocarcinoma cell). Mechanically, 1,25(OH)₂D₃ reinforced physical interactions between vitamin D receptor with NF-κB p65 and c-Jun. Our results indicate that vitamin D is inversely correlated with inflammatory signaling in COPD patients. Inflammation may be a vital mediator of COPD progress in patients with low vitamin D levels.

FIGURE 1.

Serum 25(OH)D levels in patients with COPD and control subjects. Serum 25(OH)D was measured by RIA. (A) Serum 25(OH)D level was compared between COPD patients and control subjects (n = 101 for COPD patients; n = 202 for control subjects). (B) Serum 25(OH)D level was compared among patients with different grades of COPD (n = 28 for grade 1–2 of COPD patients; n = 29 for grade 3 of COPD patients; n = 44 for grade 4 of COPD patients). All data were expressed as means ± SEM. *p < 0.05, **p < 0.01.

FIGURE 2.

Correlation analysis between serum 25(OH)D and inflammation in patients with COPD. (A) Correlation analysis between serum 25(OH)D and CRP in patients with COPD. (B) Correlation analysis between serum 25(OH)D and TNF-α in patients with COPD. (C) Correlation analysis between serum 25(OH)D and MCP-1 in patients with COPD.

FIGURE 3.

NF-κB signaling in patients with COPD and control subjects. Phosphorylated IκBα, IκBα, and phosphorylated p65 were measured between patients with COPD and control subjects in the lungs. (A) Pulmonary phosphorylated IκBα, IκBα, and phosphorylated p65 were detected in COPD patients and control subjects using Western blotting. (B–D) Quantitative analysis of scanning densitometry was performed. (B) p-IκBα/IκBα. (C) IκBα. (D) p-p65. (E) Pulmonary phosphorylated IκBα, IκBα, and phosphorylated p65 were detected in the different grades of COPD patients through Western blotting. (F–H) Quantitative analysis of scanning densitometry was performed. (F) p-IκBα/IκBα. (G) IκBα. (H) p-p65. All data were expressed as means ± SEM of six different lung samples (n = 6). All experiments were repeated three times. *p < 0.05, **p < 0.01.

FIGURE 4.

The levels of nuclear translocation of NF-κB p65 and p50 in COPD patients and control subjects. Nuclear translocation of NF-κB p65 and NF-κB p50 was detected in human lung tissues. (A) Pulmonary NF-κB p65 was measured by immunohistochemistry. (B) NF-κB p65 positive nuclei were analyzed. (C) Pulmonary NF-κB p50 was determined using immunohistochemistry (blue arrows). (D) NF-κB p50 positive nuclei were calculated (blue arrows). Scale bar, 50 μm. Original magnification ×400. All data were expressed as means ± SEM of 25 different lung samples (n = 25). All experiments were repeated three times. **p < 0.01.

FIGURE 5.

AP-1 signaling in patients with COPD and control subjects. Pulmonary phosphorylated c-Fos and c-Jun were determined in human lung tissues between COPD patients and control subjects. (A) Pulmonary phosphorylated c-Fos and c-Jun were determined using Western blotting in two groups. (B and C) Quantitative analysis of scanning densitometry was performed. (B) p–c-Fos. (C) p–c-Jun. (D) Pulmonary phosphorylated c-Fos and c-Jun were determined using Western blotting in different grades of COPD patients. (E and F) Quantitative analysis of scanning densitometry was performed. (E) p–c-Fos. (F) p–c-Jun. All data were expressed as means ± SEM of six different lung samples from six cases (n = 6). All experiments were repeated three times. *p < 0.05, **p < 0.01.

FIGURE 6.

Pulmonary VDR in patients with COPD and control subjects. The protein level of VDR was detected in human lung tissues between COPD patients and control subjects. (A) Pulmonary VDR was measured using Western blotting between COPD patients and control subjects. (B) Quantitative analysis of scanning densitometry was performed. (C) Pulmonary VDR was measured using Western blotting in different grades of COPD patients. (D) Quantitative analysis of scanning densitometry was performed. All data were expressed as means ± SEM (n = 6). All experiments were repeated three times. **p < 0.01.

FIGURE 7.

Active vitamin D3 downregulates LPS-activated NF-κB and AP-1 signaling pathways in A549 cells. A549 cells were cocultured with 1,25(OH)₂D₃ (100 nM) before LPS (2 μg/ml) exposure. After 6 h of exposure, the cells were collected for Western blotting. (A) p-p65, p-IκBα, and IκBα were detected using Western blotting. (B and C) Quantitative analysis of scanning densitometry was performed. (B) p-IκBα/IκBα. (C) p-p65. (D) p–c-Jun and p–c-Fos were detected using Western blotting. (E and F) Quantitative analysis of scanning densitometry was performed. (E) p–c-Jun. (F) p–c-Fos. All data were expressed as means ± SEM of six different lung samples (n = 6). All experiments were repeated three times. *p < 0.05, **p < 0.01.

FIGURE 8.

The interaction between VDR and p65 as well as between VDR and c-Jun in A549 cells. A549 cells were cocultured with 1,25(OH)₂D₃ (100 nM) before LPS (2 μg/ml) exposure. After 6 h of exposure, the cells were collected for Western blotting. Total lysate was isolated. (A) Total lysate fractions were incubated with agarose-conjugated Ab against VDR or NF-κB p65. Co-IP (IP): anti-VDR; immunoblot: p65. (B) Quantitative analysis of scanning densitometry was performed. (C) Total lysate fractions were incubated with agarose-conjugated Ab against VDR or c-Jun. IP: anti-VDR; immunoblot: c-Jun. (D) Quantitative analysis of scanning densitometry was performed. All experiments were duplicated three times of three different samples (n = 3). *p < 0.05, **p < 0.01.