Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Seoul National University Hospital, Seoul 03080, Republic of Korea.
Department of Internal Medicine, Seoul National University College of Medicine, Seoul 03080, Republic of Korea.
Abstract
Inflammation, oxidative stress, and apoptosis are thought to be important causes of chronic obstructive pulmonary disease (COPD). We investigated the effect of YPL-001 (under phase 2a study, ClinicalTrials.gov identifier NCT02272634), a drug derived from Pseudolysimachion rotundum var. subintegrum, on cigarette smoke extract (CSE)-induced inflammation, the anti-oxidative pathway, and apoptosis in human lung epithelial cells and on CSE-induced emphysema in mice. YPL-001 suppressed CSE-induced expression of IL8 mRNA and protein. This was due to the reduction in NF-κB transcriptional activity by YPL-001, which resulted from the blockade of acetylation of the NF-κB subunit p65 (Lys310). Histone deacetylases (HDACs) prevent gene transcription by condensing the DNA structure and affecting NF-κB nuclear binding. YPL-001 alone increased HDAC2 activity and enhanced CSE-induced activation of HDAC2. YPL-001-induced suppression of NF-κB transcriptional activity might be caused by increased HDAC2 activity. YPL-001 increased nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression via both degradation of its inhibitory protein, Kelch-like ECH-associated protein 1, and an increase in de novo protein synthesis. YPL-001 increased the DNA binding activity of Nrf2. Consequently, YPL-001 upregulated the expression of Nrf2-targeted anti-oxidant genes such as NAD(P)H quinone dehydrogenase 1 and heme oxygenase 1. Moreover, YPL-001 significantly suppressed CSE-induced apoptotic cell death. In vivo study showed that CSE-induced emphysematous changes, neutrophilic inflammation, protein leakage into bronchoalveolar space, and lung cell apoptosis in mice were suppressed by YPL-001 treatment. Taken together, these results suggest that YPL-001 is a good therapeutic candidate for the treatment of COPD by blocking inflammation and apoptosis and activating the anti-oxidative pathway.
Figure 1YPL-001 suppresses CSE-induced IL8 production. BEAS-2B cells were pretreated with YPL-001 for 2 h, then stimulated with CSE (1%) for 14 h (A) or 24 h (B) in the presence or absence of YPL-001. (C) Primary HBECs were pretreated with YPL-001 for 2 h, then stimulated with CSE (1%) for 24 h in the presence or absence of YPL-001. Quantitative real-time PCR for CXCL8 and GAPDH was performed (A). IL8 concentrations in the culture supernatants were determined by multiplex bead assay (B,C). Data are expressed as the mean ± SD of triplicates. ** p < 0.05. Results are representative of three separate experiments.Figure 2YPL-001 suppresses transcriptional activity of NF-κB. (A) BEAS-2B cells were pretreated with YPL-001 for 2 h, then stimulated with CSE (1%) for 1 h in the presence or absence of YPL-001. Immunofluorescent staining for p65 was performed. Hoechst dye was used as a nuclear counterstain. The cells were examined using an ECLIPSE TE300 fluorescence microscope (Nikon). (B) BEAS-2B cells were transiently co-transfected with the NF-κB–luciferase reporter construct and the control plasmid. Twenty-four hours after transfection, the cells were pretreated with YPL-001 for 2 h, then incubated with CSE (1%) for 8 h in the presence or absence of YPL-001. Luciferase activity was detected and normalized with renilla. Data are expressed as the mean ± SD of triplicates. ** p < 0.05. R.L.U. = relative luciferase units. Results are representative of three separate experiments.Figure 3YPL-001 suppresses CSE-induced acetylation of NF-κB via increase in HDAC2 activity. (A) BEAS-2B cells were pretreated with YPL-001 (50 μg/mL) for 2 h, then stimulated with CSE (1, 2%) for 6 or 13 h. Nuclear extracts (N.E.) were subjected to Western blotting analysis to detect acetyl-NF-κB p65 (Lys310). (B,C) BEAS-2B cells were pretreated with YPL-001 (50 μg/mL) for 2 h, then stimulated with CSE (1, 2%) for 13 h. Nuclear extracts were subjected to Western blotting analysis to detect HDAC2 and PARP (B). HDAC2 activity using nuclear proteins was measured with a colorimetric assay kit (C). Data are expressed as the mean ± SD of triplicates. ** p < 0.05. Results are representative of three separate experiments.Figure 4YPL-001 activates Nrf2. (A) BEAS-2B cells were treated with 50 μg/mL for the indicated times. Quantitative real-time PCR for Nrf2 and GAPDH was performed. Data are expressed as the mean ± SD of triplicates. ** p < 0.05. Both BEAS-2B cells (B) and primary HBECs (C) were treated with YPL-001 (50 μg/mL) for 4 and 8 h. Nuclear and cytoplasmic protein extracts were subjected to Western blot analysis to detect Nrf2, PARP, KEAP1, and GAPDH. (D) BEAS-2B cells were treated with YPL-001 for the indicated times. Nrf2 activity using nuclear proteins was measured using a DNA binding ELISA kit. Data are expressed as the mean ± SD of triplicates. ** p < 0.05. (E,F) BEAS-2B cells were treated with YPL-001 (50 μg/mL) for the indicated times. Quantitative real-time PCR for NQO1, HMOX1, and GAPDH was performed. Data are expressed as the mean ± SD of triplicates. ** p < 0.05. Both BEAS-2B cells (G) and primary HBECs (H) were treated with YPL-001 for 24 h. Total cell lysates were subjected to Western blot analysis for HMOX1, NQO1, and GAPDH. N.E. = nuclear extracts; C.E. = cytoplasmic extracts. Results are representative of three separate experiments.Figure 5YPL-001 suppresses CSE-induced apoptotic cell death. BEAS-2B cells (A,C) and primary HBECs (B) were pretreated with YPL-001 for 24 h, then stimulated with CSE (4%) for 24 h in the presence or absence of YPL-001. (A,B) Cell viability was determined by LDH release assay. Data are expressed as the mean ± SD of triplicates. ** p < 0.05. (C) Total cellular extracts were subjected to Western blotting analysis to detect PARP, active caspase-3, and GAPDH. S.E. = short exposure; L.E. = long exposure. Results are representative of three separate experiments.Figure 6YPL-001 reduced CSE-induced emphysematous formation via multiple mechanisms such as anti-inflammatory and anti-apoptotic effects. (A) Experimental protocols for a murine model of emphysema. C57BL/6 mice were instilled intratracheally with saline or CSE. CSE was instilled once a week for 8 weeks (saline n = 6, CSE n = 5, YPL-001 + CSE n = 5). The mice were treated orally with YPL-001 (0.5 mg/20 g mouse) once daily for 8 weeks. The mice were sacrificed on day 1 after the last CSE instillation to collect lungs and BALF. (B) Representative images of H&E staining in lungs of mice (×100). (C) The mean linear intercept (MLI) was measured. Histogram bars represent mean ± SE. ** p < 0.05. (D) Differential cells in BALF were counted. Data are expressed as the mean ± SE. (E) Protein concentrations in BALF were measured. (F) Total RNA from the lung tissues of mice was isolated and quantitative real-time PCR for KC, MIP2, IL-6, and GAPDH was performed. Data are expressed as the mean ± SD. ** p < 0.05. (G) Representative images of TUNEL staining of lung tissue sections (×40). (H) Quantitation of the TUNEL-positive cells in lung tissues. Error bars represent mean ± SE. ** p < 0.05.
