Curcumin Nanocarriers in the Protection Against Iron- and Alcohol-Induced Oxidative Stress in a Cellular Model of Liver Disease

Abstract

During chronic alcohol misuse, hepatic iron overload occurs, leading to exacerbated oxidative stress and liver injury. The aim was to study formulations encapsulated with the antioxidant curcumin to assess their ability protect against oxidative stress in a model of alcohol-related liver disease (ALD) combined with iron. HepG2 (VL-17A) cells were treated with iron (50 µM) alone or with alcohol (200 to 350 mM) over 72 h and markers of oxidative damage, cell death, and mitochondrial function were assessed. Nanoformulations encapsulating curcumin were also studied. VL-17A cells treated with both ethanol and iron showed significant decreases in cell viability (64%, p < 0.0001) when compared to control, and a 56% decrease (p = 0.0279) when compared to iron-only treatment. Iron-alone treatment caused a 115% increase (p < 0.0001) in ROS at 48 h as well as increases of up to 118% when treated with 200 mM ethanol + 50 μM iron (p < 0.0001), compared to control DMEM. The study found that 10 µM curcumin DSPE-PEG increased cell viability by 17% and 41% when compared to control and iron treatment alone, respectively. Formulations reduced ROS by 36% (p = 0.0015) when compared to iron-alone treatment. In summary, encapsulated curcumin provided antioxidant capacity and reduced oxidative stress, demonstrating the therapeutic potential for curcumin formulations in ALD combined with iron dysregulation.

Keywords: alcohol; antioxidants; curcumin; iron; liver; mitochondria; oxidative stress; reactive oxygen species.

Figure 1

The effect of ethanol and iron exposure on cell viability over a 72 h period. Cells were seeded in 96-well plates and treated with 200 mM, 300 mM, and 350 mM ethanol as well as 50 μM iron. The viability of cells was determined by the MTT assay and measured at 24 h, 48 h, and 72 h. Data are presented as percentage from the control. Results are presented as mean ± SD (n = 3). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 2

The effect of ethanol and iron exposure on ROS accumulation over a 72 h period. (A) The percentage of ROS accumulation at 30 min, (B) percentage of ROS accumulation at 1 h, (C) percentage of ROS accumulation at 2 h, (D) percentage of ROS accumulation at 24 h, (E) percentage of ROS accumulation at 48 h, and (F) percentage of ROS accumulation at 72 h. Cells were seeded in 96-well plates and treated with 200 mM, 300 mM, and 350 mM ethanol as well as 50 μM iron. ROS accumulation was determined by the DCFDA assay and measured using fluorescence at 30 min, 1 h, 2 h, 24 h, 48 h, and 72 h. Data are presented as percentage from the control. Results are presented as mean ± SD (n = 3). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 3

The effect of ethanol and iron exposure on apoptosis over a 72 h period. (A) The percentage of cells in early apoptosis at 24 h, (B) percentage of cells in early apoptosis at 48 h, (C) percentage of cells in early apoptosis at 72 h, (D) percentage of cells in late apoptosis at 24 h, (E) percentage of cells in late apoptosis at 48 h, and (F) percentage of cells in late apoptosis at 72 h. Cells were seeded in 12-well plates and treated with 200 mM, 300 mM, and 350 mM ethanol as well as 50 μM iron. Apoptosis was assessed at 24 h, 48 h, and 72 h using the Annexin VI kit and measured using flow cytometry. Data are presented as percentage of positive cells. Results presented as mean ± SD (n = 3). * p ≤ 0.05, ** p ≤ 0.01.

Figure 4

Mitochondrial oxygen consumption rate after ethanol and iron exposure at 24 h and 48 h. (A) Oxygen consumption rate at 24 h and (B) oxygen consumption rate at 48 h. Cells were seeded in 24-well plates and treated with 200 mM, 300 mM, and 350 mM ethanol as well as 50 μM iron. Oxygen consumption rate was assessed over 48 h using Seahorse XF24 analyser. Results are presented as mean of replicates ± SD (n = 3). OCR: oxygen consumption rate.

Figure 5

The effect of ethanol and iron on mitochondrial oxidative phosphorylation parameters at 24 h. (A) Basal respiration, (B) maximal respiration, (C) proton leakage, (D) spare respiratory capacity, (E) non-mitochondrial oxygen consumption, and (F) ATP production. Cells were seeded in 24-well plates and treated with 200 mM, 300 mM, and 350 mM with 50 μM iron. Oxygen consumption rate was assessed at 24 h using the Seahorse XF24 analyser. The results are presented as mean of replicates ± SD (n = 3). OCR: oxygen consumption rate. * p ≤ 0.05.

Figure 6

The effect of ethanol and iron on mitochondrial oxidative phosphorylation parameters at 48 h. (A) Basal respiration, (B) maximal respiration, (C) proton leakage, (D) spare respiratory capacity, (E) non-mitochondrial oxygen consumption, and (F) ATP production. Cells were seeded in 24-well plates and treated with 200 mM, 300 mM, and 350 mM with 50 μM iron. Oxygen consumption rate was assessed at 24 h using the Seahorse XF24 analyser. The results are presented as mean of replicates ± SD (n = 3). OCR: oxygen consumption rate. * p ≤ 0.05.

Figure 7

The effect of a 3 h pre-treatment of nanoformulated curcumin on ethanol- and iron-induced cell damage. (A) The percentage of cell viability at 48 h and (B) percentage of cell viability at 72 h. Cells were seeded in 96-well plates and co-treated with 350 mM ethanol and/or 50 μM iron as well as 3 h pre-treatment of formulations. The viability of cells was determined by an MTT assay and measured at 48 and 72 h. Data are presented as a percentage of the control. Results are presented as mean ± SD (n = 3).

Figure 8

The effect of a 3 h pre-treatment of nanoformulated curcumin on ethanol- and iron-induced ROS production. (A) ROS production at 30 min, (B) ROS production at 1 h, (C) ROS production at 2 h, (D) ROS production at 24 h, (E) ROS production at 48 h, and (F) ROS production at 72 h. ROS production was determined by DCFDA assay. Cells were seeded in 96-well plates and co-treated with 350 mM ethanol and/or 50 μM iron as well as 3 h pre-treatment of formulations. Data are presented as a percentage of the control. Results are presented as mean + SD (n = 3) * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.