Anti-inflammatory and anti-fibrotic treatment in a rodent model of acute lung injury induced by sulfur dioxide

Figures & data

Figure 1. Measurement of body weight in treated (DEX: dexamethasone (10 mg/kg i.p) or PFD: Pirfenidone (200 mg/kg i.p)) and untreated rats (SO₂) exposed to a 10-min exposure to 2200 ppm sulfur dioxide (SO₂). Control animals were breathing room-air while the other rats were exposed to SO₂. Statistical significant differences between initial weight and weight on termination day within each group are shown. (A) 24 h post-exposure (n = 6 rats per group) and (B) 14-days post-exposure (n = 5–6 rats per group). ^#Two animals in the 14-day SO₂ group were excluded on day 13–14 from the study due to severe decline in general health status (last time-point n = 4) Values indicate means ± SEM, **p < .01, and ***p < .001.

Figure 2. Total cell counts in bronchoalveolar lavage fluid (BALF) from rats exposed to 2200 ppm sulfur dioxide (SO₂) at 24 h post-exposure, after treatment with dexamethasone (DEX, 10 mg/kg i.p) or pirfenidone (PFD, 200 mg/kg i.p) at 1 h, 5 h and 23 h. The numbers of (A) total leukocytes, macrophages and neutrophils and (B) eosinophils and lymphocytes are shown. Values indicate means ± SEM. Statistical significant differences compared to untreated SO₂-group (vehicle) are shown, *p < .05, **p < .01 and ***p < .001 (n = 6 rats per group).

Table 1. Cytokines in bronchoalveolar lavage fluid (BALF) and serum, measured 24 h after a 10-minute exposure of female rats to 2200 ppm sulfur dioxide (SO₂), with and without post-exposure treatment (1, 5 and 23 h after SO₂-exposure).

		Control	SO2	SO2 + DEX	SO2 + PFD
BALF	CINC-1	1.00 ± 0.09	0.56 ± 0.17	0.61 ± 0.25	0.47 ± 0.08
	IL-6	1.00 ± 0.06	0.95 ± 0.05	0.76 ± 0.03	0.95 ± 0.10
	IL-1β	1.00 ± 0.04	1.12 ± 0.07	1.01 ± 0.10	0.95 ± 0.08
	MMP-9	1.00 ± 0.08**	7.23 ± 2.8	5.73 ± 1.6	5.53 ± 1. 2
	MPO	1.00 ± 0.09	1.32 ± 0.10	1.04 ± 0.10	0.98 ± 0.09
	IL-18	1.00 ± 0.07	0.96 ± 0.22	0.96 ± 0.10	1.03 ± 0.15
	TGF-β1	1.00 ± 0.09	2.00 ± 0.68	0.90 ± 0.18	1.20 ± 0.13
Serum	CINC-1	1.00 ± 0.06	0.71 ± 0.21	0.17 ± 0.02	1.19 ± 0.29
	IL-6	1.00 ± 0.02	0.99 ± 0.03	1.04 ± 0.07	1.01 ± 0.03
	IL-1β	1.00 ± 0.11	0.85 ± 0.07	0.80 ± 0.14	1.00 ± 0.18
	MMP-9	1.00 ± 0.04**	0.70 ± 0.07	0.75 ± 0.06	0.75 ± 0.08
	PAI-1	1.00 ± 0.10	0.90 ± 0.16	0.81 ± 0.03	1.36 ± 0.05*

DEX: dexamethasone (10 mg/kg i.p.), PFD: Pirfenidone (200 mg/kg i.p). Data show fold increase compared to age-matched SO₂ animals. Values indicate means ± SEM, *p < .05, **p < .01 compared to SO₂ group.

Figure 3. Respiratory mechanics in rats 24 h after exposure to 2200 ppm sulfur dioxide (SO₂) and treatment (1 h, 5 h and 23 h) with dexamethasone (DEX, 10 mg/kg i.p) or pirfenidone (PFD, 200 mg/kg i.p). Measurements of methacholine (MCh)-induced (A) respiratory resistance, R_RS (B) Newtonian resistance, R_N (C) tissue resistance, G (D) respiratory elastance, E_RS (E) tissue elastance, H and (F) hysteresivity (η) were performed using the Flexivent™. Values indicate means ± SEM. Statistical significant differences compared to untreated SO₂-group are shown, *^{, #}p < .05, **^{, # #, ¤¤}p < .01, and ***^{, # # #, ¤¤ ¤}p < .001 (*SO₂ vs. control, ^#SO₂ vs. PFD and ^¤SO₂ vs. DEX), (n = 6 rats per group).

Table 2A. Acute effects of sulfur dioxide (SO₂) at 24 h post-exposure. Measurement of baseline respiratory mechanics (without methacholine) in female rats exposed for 10 minutes to 2200 ppm SO₂, with and without treatments (1, 5 and 23 h after SO₂-exposure).

	RRS cmH2O s ml^−1	ERS ml cmH2O^−1	RN cmH2O s ml^−1	G cmH2O s ml^−1	H cmH2O s ml^−1	η
Control	0.17 ± 0.01	3.00 ± 0.17	0.11 ± 0.01	0.61 ± 0.04**	3.20 ± 0.19	0.19 ± 0.01***
SO2	0.24 ± 0.05	3.43 ± 0.33	0.12 ± 0.02	0.93 ± 0.08	3.23 ± 0.41	0.30 ± 0.04
SO2 + DEX	0.17 ± 0.03	3.25 ± 0.18	0.06 ± 0.00***	0.57 ± 0.03**	3.60 ± 0.22	0.16 ± 0.00***
SO2+ PFD	0.14 ± 0.01*	2.96 ± 0.14	0.07 ± 0.01**	0.61 ± 0.01**	3.08 ± 0.16	0.20 ± 0.01***

DEX: dexamethasone (10 mg/kg i.p), PFD: Pirfenidone (200 mg/kg i.p). Values indicate means ± SEM, *p < .05, **p < .01, ***p < .001 compared to SO₂ group.

Figure 4. Lung tissue sections from (A) healthy control and (B–D) rats 24 h post-exposure to 2200 ppm sulfur dioxide (SO₂), (B) without treatment (vehicle) and with (C) dexamethasone (DEX, 10 mg/kg i.p)-treatment, or (D) pirfenidone (PFD, 200 mg/kg i.p)-treatment. Tissue sections were stained with hematoxylin-eosin and photos were taken at 20 x magnification using a light microscope. AL = airway lumen.

Table 2B. Analysis of collagen deposition in female rats at 14 days post-exposure for 10 minutes to 2200 ppm SO₂, with and without treatments (1, 5 and 23 h after SO₂-exposure).

Control	SO2	SO2 + DEX	SO2 + PFD
1.05 ± 0.08*	1.49 ± 0.16	1.00 ± 0.17*	1.09 ± 0.17

Data show fold increase compared to the SO₂ group. DEX: dexamethasone (10 mg/kg i.p), PFD: Pirfenidone (200 mg/kg i.p). Values indicate means ± SEM, *p < .05 compared to SO₂ group.

Table 3. Scoring the main histopathology features in female rats 24 h after a 10-minute exposure to 2200 ppm sulfur dioxide (SO₂), with and without post-exposure treatments (1, 5 and 23 h after SO₂-exposure).

Groups	Rat no.	Pulmonary congestion	Bronchi			Alveoli	Interstitium
			Mucosal damage	Cells	Regeneration (mitoses in epithelium)	Cells	Edema	Cells
Control	1	0	0	0	0	0	0	0
	2	0	0	0	0	0	0	0
	3	0	0	0	0	0	0	0
	4	0	0	0	0	0	0	0
SO2	1	1	4	4	1	0	3	3
	2	1	4	1	0	0	2	1
	3	1	4	3	1	2	1	3
	4	1	4	1	1	0	1	0
SO2 + DEX	1	2	4	2	0	0	0	0
	2	1	4	0	0	0	0	0
	3	0	4	1	0	0	0	0
	4	1	4	1	1	0	0	0
SO2 + PFD	1	1	3	1	0	0	0	0
	2	1	4	1	0	0	0	0
	3	2	0	1	2	0	0	0
	4	0	2	0	2	0	0	0

Grade of lesions: 4 very severe, 3: severe, 2 moderate, 1: mild or low numbers, 0: significant lesions not observed.

^aPeribronchial and perivascular spaces, and interlobular septa. Cells: leukocytes, DEX: dexamethasone (10 mg/kg i.p), PFD: Pirfenidone (200 mg/kg i.p).