Protective effects of curcumin against amiodarone-induced pulmonary fibrosis in rats

Abstract

(1) We have studied whether curcumin prevents amiodarone-induced lung fibrosis in rats. Intratracheal instillation of amiodarone (6.25 mg kg(-1) on days 0 and 2, and then killed on day 3, day 5, week 1, week 3 and week 5 after amiodarone administration) induced increases in total protein and lactate dehydrogenase (LDH) activity on days 3 and 5 in bronchoalveolar lavage fluid (BALF). Total cell counts, alveolar macrophages, neutrophils and eosinophils recovered by BAL, and lung myeloperoxidase (MPO) activity were significantly higher in amiodarone rats. (2) Tumor necrosis factor-alpha (TNF-alpha) release after lipopolysaccharide (LPS) stimulation and superoxide anion generation after phorbol myristate acetate (PMA) stimulation were higher in the alveolar macrophages of amiodarone rats at 3 and 5 weeks postamiodarone instillation than in controls. Amiodarone also induced increases in transforming growth factor-beta1 (TGF-beta1) expression, collagen deposition, type I collagen expression and c-Jun protein in lungs. (3) Curcumin (200 mg kg(-1) body weight after first amiodarone instillation and daily thereafter for 5 weeks)-treated amiodarone rats had reduced levels of protein, LDH activity, total cell numbers and differential cell counts in BALF. LPS-stimulated TNF-alpha release and PMA-stimulated superoxide generation were significantly suppressed by curcumin. Furthermore, curcumin inhibited the increases in lung MPO activity, TGF-beta1 expression, lung hydroxyproline content, expression of type I collagen and c-Jun protein in amiodarone rats. Our results have important implications for the treatment of amiodarone-induced lung fibrosis.

Figure 1

Time-course analysis of lung hydroxyproline content in amiodarone-treated rats (a) and the protective effects of curcumin on amiodarone-induced increase in lung hydroxyproline content (b). Rats were killed at several time points after i.t. administration of amiodarone and lung hydroxyproline levels were measured as described under the Methods section. All data are expressed as mean±s.d. (n=6 rats group⁻¹ time point⁻¹). Since there was no statistical difference between water- and curcumin-treated control groups, we show the values of water-treated groups as controls. ^*Significantly (P<0.01) higher than control groups (dotted line); ^†significantly (P<0.01) lower than CR(0)+AMD group; hydroxyproline values in CR(50)+AMD and CR(100)+AMD groups are not statistically different from CR(0)+AMD group; CR: curcumin; AMD: amiodarone; d: day; w: week.

Figure 2

Effects of curcumin on amiodarone-induced increases in bronchoalveolar lavage BALF total protein (a) and LDH activity (b). BALF was performed at various time points after i.t. administration of amiodarone, and protein concentration and LDH levels in BALF were measured. All data are expressed as mean±s.d. (n=6 rats group⁻¹ time point⁻¹). Since there was no statistical difference between water- and curcumin-treated control groups, we show the values of water-treated groups as controls. ^*Significantly (P<0.01) higher than control groups (dotted line); ^†significantly (P<0.01) lower than amiodarone group. CR: curcumin; AMD: amiodarone; d: day; w: week.

Figure 3

Effects of curcumin on amiodarone-induced increases in lung inflammation as reflected by influx of inflammatory cells into airspaces. BALF was performed at several time points after i.t. administration of amiodarone, and changes in total cells (a) alveolar macrophages (b) neutrophils (c) and eosinophils (d) were measured as described under the Methods section. Since there was no significant difference in total and differential cell counts recovered by BALF at latter time points between control and amiodarone groups, we present the data for 3 and 5 days postamiodarone administration. All data are expressed as mean±s.d. (n=6 rats group⁻¹ time point⁻¹). ^*Significantly (P<0.01) higher than control groups; ^†significantly (P<0.01) lower than the amiodarone group. CON: water-treated controls; CR: curcumin; AMD: amiodarone; d: day; w: week.

Figure 4

Effects of curcumin on amiodarone-induced increases in lung MPO activity. The lung MPO levels were measured at several time points after i.t. administration of amiodarone as described under the Methods section. All data are expressed as mean±s.d. (n=6 rats group⁻¹ time point⁻¹). Since there was no statistical difference between water- and curcumin-treated control groups, we show the values of water-treated groups as controls. ^*Significantly (P<0.01) higher than controls (dotted line); ^†significantly (P<0.01) lower than the amiodarone group. CR: curcumin; AMD: amiodarone; d: day; w: week.

Figure 5

Effects of curcumin on amiodarone-induced increases in alveolar macrophage release of TNF-α (a) and superoxide anion (b). BALF was performed at several time points after i.t. administration of amiodarone, and the recovered alveolar macrophages were stimulated either by LPS (1 μg ml⁻¹) or PMA (0.1 μg ml⁻¹) for the determination of TNF-α and superoxide anion production, respectively. All data are expressed as mean±s.d. (n=6 rats group⁻¹ time point⁻¹). Since there was no statistical difference between water- and curcumin-treated control groups, we show the values of water-treated groups as controls. ^*Significantly (P<0.01) higher than control groups (dotted line); ^†significantly (P<0.01) lower than the amiodarone group. CR: curcumin; AMD: amiodarone; d: day; w: week.

Figure 6

Inhibitory effects of curcumin on amiodarone-induced increases in type I collagen (a), TGF-β1 (b) and c-Jun protein (c) expression in the lung tissue. Lung tissue proteins were separated by SDS–PAGE, transferred onto nitrocellulose membranes and then probed using antibodies against type I collagen (a) or TGF-β1 (b) or c-Jun protein (c). Both type I collagen and c-Jun expression were analyzed 5 weeks after amiodarone administration, while TGF-β1 analysis was carried out on 1 week postamiodarone instillation. Quantitative analysis of the respective immunoblots was accomplished by an image analysis system as described under the Methods section. Findings are representative of three separate observations from three different rats. ^*Significantly (P<0.01) higher than control groups; ^†significantly (P<0.01) lower than the amiodarone group at the corresponding time. CON: water-treated control; CR: curcumin; AMD: amiodarone.