Curcumin modulates the effect of histone modification on the expression of chemokines by type II alveolar epithelial cells in a rat COPD model

Figures & data

Figure 1 Histology of the rat lung tissues from control animal and COPD model.

Notes: Sections of lung parenchyma were stained with hematoxylin and eosin and photographed under a microscope. (A) control animal lung and (B) COPD model lung. Data presented were the ones representative of control and COPD models, respectively. Magnification ×200.

Figure 2 Alkaline phosphatase (AKP) assay and electronic microscopic examination of the type II alveolar epithelial cells (AEC II).

Notes: The cells were isolated and used for AKP assay (A) or electronic microscopic examination (B). Data presented were the ones representative of separate assays or examinations. Magnification (A) ×400; (B) ×9,700.

Table 1 Correlation between expression of HDAC2 and the chemokine mRNAs

Chemokine	Decreased amount of relative expression of HDAC2	Increased amount of relative mRNA expression	R
IL-8	−4.04±0.11	4.52±0.42	−0.960**
MCP-1	−4.04±0.11	4.54±0.54	−0.914*
MIP-2α	−4.04±0.11	3.94±0.95	−0.928**

Notes: The amount of relative mRNA expression was expressed by 2^−ΔCT value, and it was displayed as the mean ± SEM (n=8).

* P<0.05,

** P<0.01.

Abbreviations: HDAC2, histone deacetylase 2; IL-8, interleukin-8; MCP-1, monocyte chemoattractant protein-1; MIP-2α, macrophage inflammatory protein 2α; SEM, standard error of the mean; mRNA, messenger RNA.

Figure 3 Effect of curcumin on the expression of the chemokine mRNAs in COPD model.

Notes: Total RNA was extracted from the type II alveolar epithelial cells (AEC II), and quantitative real-time polymerase chain reaction (RT-PCR) was performed. (A) Expression of the chemokine mRNAs in control and COPD animals. Y-axis: mRNA expressed as 2^−ΔCT value; X-axis: chemokines. *P<0.05, compared to control. (B) Effect of curcumin, dexamethasone, and TSA on the expression of chemokine mRNAs. AEC II from the COPD model were treated with/without curcumin (100 µM), TSA (100 nM), or dexamethasone (100 nM) for 24 hours. Expression of mRNA in the AEC II was quantified by RT-PCR. Y-axis: comparison of 2^{−Δ CT} values; X-axis: chemokines. *P<0.05, compared to COPD model without treatment. Data represented were an average of eight separate replicates.
Abbreviations: CUR, curcumin; DEX, dexamethasone; TSA, trichostatin A; IL-8, interleukin 8; MCP-1, monocyte chemoattractant protein 1; MIP-2α, macrophage inflammatory protein 2α; mRNA, messenger RNA.

Table 2 Levels of histone (H3/H4) acetylation and H3K9 dimethylation in the promoter of IL-8, MCP-1, and MIP-2α

Site	Group	IL-8 (IP of target antigen/input)	MCP-1 (IP of target antigen/input)	MIP-2α (IP of target antigen/input)
H3	Normal	0.38±0.08	0.46±0.19	0.23±0.04
	COPD	1.06±0.05**	1.04±0.03*	0.94±0.10*
H4	Normal	0.44±0.25	0.41±0.25	0.24±0.09
	COPD	1.01±0.06*	1.02±0.15*	1.00±0.06**
H3K9	Normal	0.92±0.04	1.01±0.05	1.15±0.08
	COPD	0.23±0.06**	0.33±0.05**	0.4±0.05**

Notes: Values shown are mean ± SEM of separate experiments.

* P<0.05,

** P<0.01, compared to the normal group.

Abbreviations: IL-8, interleukin 8; MCP-1, monocyte chemoattractant protein 1; MIP-2α, macrophage inflammatory protein 2α; SEM, standard error of the mean.

Figure 4 Effect of curcumin on HDAC2 level in AEC II.

Notes: Total protein was extracted from the AEC II isolated from control animals and COPD model and subjected to immunoblotting with HDAC2 and GAPDH antobodies. (A) Levels of HDAC2 in normal (n=8) versus COPD model (n=8). Top insert: one representative image data of immunoblotting. Bottom bar graph: semiquantitative data of HDAC2 density. Y-axis: ratio of density of HDAC2 versus GAPDH; X-axis: groups, **P<0.01. (B) Levels of HDAC2 in normal and COPD models treated with curcumin (100 µM), trichostatin A (100 nM), or dexamethasone (100 nM). Top insert: one representative image data of immunoblotting. Bottom bar graph: semiquantitative data of HDAC2 density. Y-axis: ratio of density of HDAC2 versus GAPDH; X-axis: treatment. Data presented were an average of eight samples for each group. *P<0.05 versus COPD group; ^##P<0.01, ^###P<0.001, versus normal group.
Abbreviations: HDAC2, histone deacetylase 2; AEC II, type II alveolar epithelial cells; CUR, curcumin; DEX, dexamethasone; TSA, trichostatin A; GADPH, glyceraldehyde-3-phosphate dehydrogenase.

Table 3 Levels of histone (H3/H4) acetylation and H3K9 dimethylation in the promoter regions of IL-8, MCP-1, and MIP-2α in response to curcumin

Different sites of promoter	Normal	COPD	CUR	DEX	CUR + DEX	TSA	CUR + TSA
IL-8 H3	0.7±0.19	1.86±0.59	1.4±0.49*	1.81±0.53	0.82±0.23*	3.2±0.88*	3.4±0.74*
IL-8 H4	0.88±0.19	2.0±0.47	1.5±0.4*	1.98±0.44	0.93±0.32*	3.21±0.8*	3.34±0.7*
IL-8 H3K9	1.39±0.33	0.76±0.21	1.0±0.26	0.93±0.1	1.4±0.2*	0.77±0.15	0.57±0.5
MCP-1 H3	0.78±0.21	2.8±0.37	2.3±0.4*	2.7±0.44	0.9±0.1**	3.8±0.1**	3.4±0.11*
MCP-1 H4	1.16±0.21	1.87±0.19	1.6±0.1	1.7±0.1	0.94±0.5**	2.9±0.2**	2.6±0.4**
MCP-1 H3K9	3.5±0.54	1.5±0.16	2.5±0.5*	2.1±0.5	2.9±0.8**	1.3±0.1	1.9±0.5
MIP-2α H3	0.74±0.1	1.84±0.14	1.4±0.1*	1.7±0.12	0.71±0.2**	2.34±0.3*	2.2±0.4*
MIP-2α H4	0.8±0.15	2.17±0.45	1.7±0.6*	2.1±0.58	0.99±0.1**	3.1±0.1**	2.9±0.17*
MIP-2α H3K9	1.8±0.14	0.98±0.1	1.2±0.13	1.1±0.17	1.7±0.12*	0.94±0.1	0.88±0.4

Notes: Values shown are mean ± SEM of separate experiments.

* P<0.05,

** P<0.01, compared to the COPD group.

Abbreviations: IL-8, interleukin 8; MCP-1, monocyte chemoattractant protein 1; MIP-2α, macrophage inflammatory protein 2α; CUR, curcumin; DEX, dexamethasone; TSA, trichostatin A; SEM, standard error of the mean.

Figure 5 Effect of cigarette smoke on interleukin 8 (IL-8) promoter binding to histones.

Notes: Chromatin immunoprecipitation (ChIP) assay was performed. (A) DNA marker ranged between 300 and 800 bp. (B) Targeting of IL-8 promoter binding to H3. (C) Targeting of IL-8 promoter binding to H4. (D) Targeting of IL-8 promoter binding to H3K9. Lanes 1 and 5: input samples (positive control); lane 2: IP of target antigen in normal group; lane 6, IP of target antigen in COPD group; lanes 3 and 7: negative control (IgG); lanes 4 and 8: ddH₂O. Data presented were the ones representative of separate experiments.

Abbreviations: M, maker; IgG, immunoglobulin G; ddH₂O, double distilled water.

Figure 6 Effect of curcumin on interleukin 8 (IL-8) promoter binding to histones.

Notes: Chromatin immunoprecipitation (ChIP) assay was performed. Panels A, B, and C: Effect on the IL-8 promoter binding to H3, H4, and H3K9 sites, respectively. Lane 1: DNA marker; lanes 2–8: Input samples (positive control) for normal, COPD, COPD + curcumin, COPD + dexamethasone, COPD + curcumin + dexamethasone, COPD + TSA, COPD + TSA + curcumin, respectively; lanes 9–15: IP of target antigen in normal, COPD, COPD + curcumin, COPD + dexamethasone, COPD + curcumin + dexamethasone, COPD + TSA, COPD + curcumin + TSA group, respectively. Data presented were the ones representative of separate experiments.
Abbreviations: CUR, curcumin; DEX, dexamethasone; TSA, trichostatin A.