Curcumin inhibits fibrosis-related effects in IPF fibroblasts and in mice following bleomycin-induced lung injury

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive and typically fatal lung disease for which no effective therapy has been identified. The disease is characterized by excessive collagen deposition, possibly in response to dysregulated wound healing. Mediators normally involved in would healing induce proliferation of fibroblasts and their differentiation to myofibroblasts that actively secrete collagen. Curcumin, a polyphenolic compound from turmeric, has been shown to exert a variety of biological effects. Effects on IPF and associated cell types remain unclear, however. We accordingly tested the ability of curcumin to inhibit proliferation and differentiation to myofibroblasts by human lung fibroblasts, including those from IPF patients. To further examine the potential usefulness of curcumin in IPF, we examined its ability to reduce fibrosis in bleomycin-treated mice. We show that curcumin effectively reduces profibrotic effects in both normal and IPF fibroblasts in vitro and that this reduction is accompanied by inhibition of key steps in the transforming growth factor-β (TGF-β) signaling pathway. In vivo, oral curcumin treatment showed no effect on important measures of bleomycin-induced injury in mice, whereas intraperitoneal curcumin administration effectively inhibited inflammation and collagen deposition along with a trend toward improved survival. Intraperitoneal curcumin reduced fibrotic progression even when administered after the acute bleomycin-induced inflammation had subsided. These results encourage further research on alternative formulations and routes of administration for this potentially attractive IPF therapy.

Fig. 1.

Curcumin (Cur) inhibits lung fibroblast proliferation. Human lung fibroblasts were treated with varying concentrations of curcumin. Cell numbers were then counted at 24, 48, and 72 h. A and B: IMR-90 fibroblasts, unstimulated (A, left) or stimulated with 5 ng/ml PDGF (A, right) and primary human lung fibroblasts (n = 4 each group) isolated from histologically normal lungs (B, left) and lungs of patients with usual interstitial pneumonia (UIP; B, right). Representative graphs from normal and idiopathic pulmonary fibrosis (IPF) fibroblasts are shown.

Fig. 2.

Curcumin inhibits lung fibroblast cell cycle progression at G₀/G₁. A: IMR-90 fibroblasts were synchronized by serum deprivation for 48 h and treated with indicated concentrations of curcumin or with vehicle (DMSO) for 48 h and then harvested and stained with propidium iodide. Stained cells were analyzed by FACS to assess cell cycle progression in each treatment group. *P < 0.05 vs. 0 μM. B: cyclin D1 mRNA levels in the presence or absence of curcumin were measured by real-time RT-PCR and expressed relative to mRNA levels of the housekeeping gene β-actin. *P < 0.05 vs. 0 μM.

Fig. 3.

Curcumin inhibits transforming growth factor-β (TGF-β)-induced myofibroblast differentiation. Human fibroblasts obtained from histologically normal lungs (n = 4) were pretreated with varying concentrations of curcumin for 2 h and incubated in the presence of TGF-β (2 ng/ml) for 24 h. A: levels of α-smooth muscle actin (α-SMA) were analyzed by Western blotting and normalized relative to the GAPDH loading control; representative blot is shown. B: quantitative values for each condition were obtained by densitometric analysis. *P < 0.05 vs. 0 μM. C: human fibroblasts (n = 3) were either untreated (Normal), treated with TGF-β (2 ng/ml), or treated with TGF-β plus curcumin (10 μM) as before. After 24 h, immunohistochemical analysis for α-SMA (FITC) was performed, and representative images are shown. Ctrl, control.

Fig. 4.

Curcumin inhibits TGF-β-induced phosphorylation (p) of Smad2, Smad3, and extracellular signal-regulated kinase (ERK) 1/2 in lung fibroblasts. Human fibroblasts obtained from histologically normal lungs (n = 4) were treated with curcumin (20 μM) for 2 h followed by TGF-β (2 ng/ml) for varying periods. Cell lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies against pS465/467 Smad2 (A) and pS423/425 Smad3 (B). Blots were probed first with phosphospecific Smad antibodies, stripped, and reprobed with GAPDH. Mean densitometric data are also presented. *P < 0.05 vs. 0 μM at each respective time point. C: pERK and total ERK were measured by Bio-Plex, and their ratio was determined. *P < 0.05 vs. 0 μM at each respective time point.

Fig. 5.

Curcumin inhibits collagen secretion from IPF fibroblasts. Serum-starved fibroblasts from lungs of patients with IPF (n = 3) were pretreated with curcumin (5 and 10 μM) for 2 h and then incubated with fresh medium in the absence (A) or presence (B) of TGF-β (6 ng/ml) for 24 h. Soluble collagen levels were measured by Sircol assay. Values indicated were normalized to control for each experiment. *P < 0.05 vs. 0 μM.

Fig. 6.

Orally administered curcumin fails to inhibit bleomycin-induced lung injury or improve survival. C57BL/6 mice were pretreated with saline (Ctrl), carboxymethylcellulose (vehicle; Veh), or curcumin (300 mg/kg) by oral gavage (o.g.). After 72 h (day 0), mice were injected with bleomycin sulphate (BLM; 0.05 units), and daily o.g. treatment was continued. A: on day 4, mice (n = 5) were killed, and lung digests were analyzed for content of total cells (TC), mononuclear cells (MN), and polymorphonuclear neutrophils (PMN). B: on day 21, mice (n = 10) were killed, and whole lung was processed for hydroxyproline content and morphometric analysis (C; n = 5). D: mortality rate of curcumin-treated and control mice (n = 10) after bleomycin (0.075 units) instillation. n.s., Not significant.

Fig. 7.

Intraperitoneally administered curcumin suppresses bleomycin-induced lung injury and improves survival. C57BL/6 mice were intraperitoneally pretreated with saline (Ctrl), 1% DMSO (Veh), or curcumin (300 mg/kg). After 72 h (day 0), mice were injected with BLM (0.05 units), and daily intraperitoneal treatment was continued. A: on day 4, mice (n = 5) were killed and lung digests analyzed for TC, MN, and PMN. *P < 0.05 vs. Veh. B: on day 21, mice (n = 10) were killed, and whole lung was processed for hydroxyproline content and morphometric analysis (C; n = 5). *P < 0.05 vs. Veh. D: mortality rate of curcumin-treated and control mice (n = 10) after bleomycin (0.075 units) instillation. E: mice were not pretreated and began daily intraperitoneal treatments on day 10 after BLM (0.05 units) injury. Mice (n = 10) were killed on day 21, and whole lung was processed for hydroxyproline content and morphometric analysis (F; n = 5). *P < 0.05 vs. Veh.

Fig. 8.

Curcumin exhibits greater bioavailability after intraperitoneal administration than after oral administration. HPLC chromatograms were obtained from extraction of mouse plasma following no treatment (A; Vehicle), an o.g. dose of 300 mg/kg curcumin (Cur o.g.) 2 h previously (B), or injection of an intraperitoneal dose of 300 mg/kg curcumin (C; Cur i.p.) 2 h previously. Ethyl acetate was used to extract curcumin from murine plasma (200 μl). Each experiment was repeated 3 times, with a representative graph being presented.