Curcumin induced oxidative stress attenuation by N-acetylcysteine co-treatment: a fibroblast and epithelial cell in-vitro study in idiopathic pulmonary fibrosis

Abstract

Background: Idiopathic Pulmonary Fibrosis (IPF) is a fatal lung disease of unknown etiology with only two federally approved drug options. Given the complex molecular pathogenesis of IPF involving multiple cell types and multiple pathways, we explore the effects of a potential antifibrotic and antioxidant drug combination. Curcumin is a polyphenolic compound derived from turmeric with significant biological activity including a potential antifibrotic capacity. N-acetylcysteine (NAC) is a precursor to the antioxidant glutathione. To advance our understanding of these molecules, and to identify a clinical application, we present a small number of focused experiments that interrogates the effect of curcumin and NAC on pathways relevant to IPF in both fibroblasts and epithelial cells.

Methods: Primary epithelial cell and fibroblasts isolated from patients with IPF were challenged with a combination treatment of NAC and curcumin. Evaluation of the antifibrotic potential and effect on oxidative stress was performed through QPCR gene expression analysis and functional assays including scratch tests, viability assays, and measurement of induced reactive oxygen species.

Results: We demonstrate that curcumin alone does have antifibrotic potential, but that effect is accompanied by proapoptotic increases in oxidative stress. Coupled with this, we find that NAC alone can reduce oxidative stress, but that epithelial cell viability is decreased through this treatment. However, co-administration of these two molecules decreases oxidative stress and maintains high cell viability in both cell types. In addition, this co-treatment maintains an antifibrotic potential.

Conclusions: These findings suggest a novel application for these molecules in IPF and encourage further exploration of this potential therapeutic approach.

Keywords: Curcumin; N-acetylcysteine; Pulmonary fibrosis.

Fig. 1

Curcumin treatment reduces myofibroblast associated gene expression in fibroblasts: (a) 24-h 20 μM curcumin treatment significant (p < 0.05) reduces gene expression of Collagen 1a1, Smooth Muscle Actin, Proliferating Cell Nuclear Antigen, and Cyclin D in both IPF-F (n = 4) and NHLF (n = 3) (b-e) Heterogeneity of gene expression before curcumin treatment is observed for all four genes in IPF-F but not in NHLF. Additionally this heterogeneity is absent after 20 μM curcumin treatment in both cell types. * indicates p value < 0.05 ** indicates p value < 0.005

Fig. 2

Effects of 24-h 10 mM NAC challenge and 24-h combination 40 μM curcumin/10 mM NAC challenge on myofibroblast associated gene expression in fibroblasts: 10 mM NAC alone or co-treatment with 40 μM curcumin and 10 mM NAC does not induce significant changes to gene expression of Collagen 1a1 (COL1A1), Smooth Muscle Actin (ACTA2), Proliferating Cell Nuclear Antigen (PCNA), and Cyclin D in NHLF (n = 3) as compared to untreated controls. Co-treatment in IPF-F (n = 4) induces significant decreases in Smooth Muscle Actin and Proliferating Cell Nuclear Antigen, but there is no significant change in Collagen 1A1 and Cyclin D as compared to untreated controls. When comparing treatment conditions there is a significant increase in the capacity of 20 μM curcumin alone to reduce gene expression of COLA1A, ACTA2 and Cyclin D in both IPF-F and NHLF as compared to co-treatment with 10 mM NAC. This significant trend is also observed for PCNA in NHLF. * indicates p value < 0.05 ** indicates p value < 0.005

Fig. 3

Representative scratch test results in IPF-F after 24-h treatment with NAC and curcumin: 20 μM curcumin reduces wound closure in IPF-F as compared to untreated control. 10 mM NAC does not have a significant effect on wound closure rate. Combination treatment with 40 μM curcumin and 10 mM NAC results in decreased wound closure as compared to untreated control but is attenuated in comparison to 20 μM curcumin alone. Morphological changes observed in the curcumin treatment are also attenuated in the combination treatment

Fig. 4

Quantification of wound closure data in IPF (n = 4) and NHLF (n = 3) after challenge: Significant reduction in 24-h wound closure rate is observed in IPF-F after 20 μM curcumin treatment and co-treatment with NAC. This same result is observed after 20 μM curcumin in NHLF and 10 mM NAC challenge. Co-treatment with NAC and 40 μM curcumin does not induce a significant reduction in wound closure rate as compared to untreated control. ** indicates p value < 0.005

Fig. 5

Epithelial and fibroblast cell viability and DNA damage response gene expression after 24-h challenge: (a) 20 μM curcumin treatment induces reduced viability in IPF-F (n = 4) and NHLF (n = 3) with significant effect on IPF epithelial cells (IPF-E n = 3) or A549 epithelial cells (n = 3). 10 mM NAC treatment induces reduced viability in epithelial cells but no significant effect in fibroblasts. Combination 10 mM NAC and 40 μM curcumin does not induce any change in cell viability as compared to untreated controls. (b) 20 μM curcumin induces significant increases in gene expression of p53 and p21 in all cell types. 10 mM NAC alone and in combination with 40 μM curcumin does not result in significant change to this gene expression in either epithelial or fibroblast cell lines. * indicates p value < 0.05

Fig. 6

Induction of ROS and change in Oxidative Stress Associated gene expression after 24-h challenge in both epithelial and fibroblast cell lines: (a) 20 μM curcumin induces significant increases in ROS generation in all primary cell lines (IPF-F n = 3, NHLF n = 3, IPF-E n = 3), but no significant increase in A549 cells (n = 3). 10 mM NAC treatment has no significant induction of ROS, however co-treatment with 10 mM NAC and 40 μM curcumin results in a significant decrease in ROS in all cell types. (b) Challenge with 20 μM curcumin results in a decreased expression of Hypoxia inducible factor 1α (HIF1) and Superoxide Dismutase 2 (SOD2) in IPF-F (n = 4), NHLF (n = 3), IPF-E (n = 3). Expression of Catalase (CAT) is decreased in IPF derived fibroblasts while Nuclear Factor erythroid 2-related factor 2 (NRF2) expression is decreased in IPF-F and epithelial cells. A549 cells demonstrate an increase in expression of HIF1, CAT, and NRF2 after challenge with 20 μM curcumin. (c) 10 mM NAC challenge results in a significant decrease in SOD2 expression in primary IPF epithelial cells and A549s but no effect on any other gene in the assayed cells. (D) Co-treatment with 10 mM NAC and 40 μM curcumin induces decreased SOD2 expression in all cell types. * indicates p value < 0.05 and ** indicates p value < 0.005

Fig. 7

Hypothetical Molecular Model of Curcumin and NAC Co-Treatment in IPF: Curcumin induces ROS mediated apoptosis in myofibroblasts releases excess ROS into the microenvironment. As the IPF lung is an oxidative stress rich environment the excess ROS further damages epithelial cells in the lung. This propagates the wound healing response and may further induce fibrosis in a classical IPF feedforward loop. The introduction of NAC co-treatment attenuates fibroblast apoptosis and alleviates ROS induced oxidative stress in epithelial cells. In turn this prevents additional fibroblast recruitment. Deduction of optimal in-vivo co-treatment concentrations may result in significant antifibrotic potential for therapeutic application