Oxidative stress in a cellular model of alcohol-related liver disease: protection using curcumin nanoformulations

Abstract

Alcohol-related liver disease (ARLD) is a global health issue causing significant morbidity and mortality, due to lack of suitable therapeutic options. ARLD induces a spectrum of biochemical and cellular alterations, including chronic oxidative stress, mitochondrial dysfunction, and cell death, resulting in hepatic injury. Natural antioxidant compounds such as curcumin have generated interest in ARLD due to their ability to scavenge reactive oxygen species (ROS), however, therapy using these compounds is limited due to poor bioavailability and stability. Therefore, the aim of this study was to assess the antioxidant potential of free antioxidants and curcumin entrapped formulations against oxidative damage in an ARLD cell model. HepG2 (VL-17A) cells were treated with varying concentrations of alcohol (from 200 to 350 mM) and parameters of oxidative stress and mitochondrial function were assessed over 72 h. Data indicated 350 mM of ethanol led to a significant decrease in cell viability at 72 h, and a significant increase in ROS at 30 min. A substantial number of cells were in late apoptosis at 72 h, and a reduction in the mitochondrial membrane potential was also found. Pre-treatment with curcumin nanoformulations increased viability, as well as, reducing ROS at 2 h, 48 h and 72 h. In summary, antioxidants and entrapped nanoformulations of curcumin were able to ameliorate reduced cell viability and increased ROS caused by ethanol treatment. This demonstrates their potential at mitigating oxidative damage and warrants further investigation to evaluate their efficacy for ARLD therapy.

Keywords: Alcohol; Antioxidants; Curcumin; Liver; Mitochondria; Oxidative stress; Reactive oxygen species.

Fig. 1

The effect of ethanol exposure on cell viability at (A) 24 h, (B) 48 h and (C) 72 h. Data is presented as percentage from the control. Results presented as mean of replicates ± SEM (n = 9) *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001.

Fig. 2

The effect of alcohol exposure on ROS accumulation at (A) 30 min, (B) 1 h, (C) 2 h, (D) 24 h, (E) 48 h and (F) 72 h. Cells were seeded in 96-well plates and treated with 200 mM, 300 mM, and 350 mM ethanol. Data is presented as percentage from the control. Results are expressed as mean of replicates ± SEM (n = 3–6). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Fig. 3

The effect of ethanol exposure on apoptosis (A) 24 h early apoptosis (B) 48 h early apoptosis, (C) 72 h early apoptosis, (D) 24 h late apoptosis (E) 48 h late apoptosis, (F) 72 h late apoptosis. Data is presented as percentage of positive cells. Results presented as mean of replicates ± SEM (n = 3–10). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Fig. 4

The effect of ethanol on mitochondrial membrane potential at (A) 24 h, (B) 48 h and (C) 72 h. FCCP (20 μM) was used as a positive control. Data is presented as mean fluorescence values. Results presented as mean of replicates ± SEM (n = 3). *P ≤ 0.05.

Fig. 5

The effect of curcumin free drug co-treatment with and ethanol on cell viability at (A) 48 h and (B) 72 h. Cells were seeded in 96-well plates and co-treated with ethanol and curcumin. Viability was determined by MTT assay. Data is presented as percentage from the control. Results presented as mean + SEM (n = 3) *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Fig. 6

3-h pre-treatment with nanoformulated curcumin on loss of cell viability at (A) 48 h and (B) 72 h. Cells were seeded in 96-well plates and treated with 350 mM ethanol after 3 h pre-treatment of formulations. Viability of cells was determined by an MTT assay and measured at 48 h and 72 h. Data is presented as percentage from the control. Results presented as mean + SEM (n = 3) *P ≤ 0.05.

Fig. 7

3 h pre-treatment of nanoformulated curcumin on ethanol induced ROS production at (A) 30 min, (B) 1 h and (C) 2 h. Cells were seeded in 96-well plates and treated with 350 mM ethanol after  3 h pre-treatment of formulations. ROS production was determined by the DCFDA assay. Data is presented as percentage from the control. Results presented as mean + SEM (n = 3) *P ≤ 0.05, ***P ≤ 0.001.

Fig. 8

3 h pre-treatment of nanoformulated curcumin on ethanol induced ROS production at (A) 24 h, (B) 48 h and (C) 72 h. Cells were seeded in 96-well plates and treated with 350 mM ethanol after 3 h pre-treatment of formulations. ROS production was determined by the DCFDA assay. Data is presented as percentage from the control. Results presented as mean + SEM (n = 3) *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.