Protective Effect of Curcumin against Doxazosin- and Carvedilol-Induced Oxidative Stress in HepG2 Cells

Abstract

Doxazosin and carvedilol have been evaluated as an alternative treatment against chronic liver lesions and for their possible role during the regeneration of damage caused by liver fibrosis in a hamster model. However, these drugs have been reported to induce morphological changes in hepatocytes, affecting the recovery of liver parenchyma. The effects of these α/𝛽 adrenoblockers on the viability of hepatocytes are unknown. Herein, we demonstrate the protective effect of curcumin against the possible side effects of doxazosin and carvedilol, drugs with proven antifibrotic activity. After pretreatment with 1 μM curcumin for 1 h, HepG2 cells were exposed to 0.1-25 μM doxazosin or carvedilol for 24, 48, and 72 h. Cell viability was assessed using the MTT assay and SYTOX green staining. Morphological changes were detected using the hematoxylin and eosin (H&E) staining and scanning electron microscopy (SEM). An expression of apoptotic and oxidative stress markers was analyzed using reverse transcription-quantitative PCR (RT-qPCR). The results indicate that doxazosin decreases cell viability in a time- and dose-dependent manner, whereas carvedilol increases cell proliferation; however, curcumin increases or maintains cell viability. SEM and H&E staining provided evidence that doxazosin and carvedilol induced morphological changes in HepG2 cells, and curcumin protected against these effects, maintaining the morphology in 90% of treated cells. Furthermore, curcumin positively regulated the expression of Nrf2, HO-1, and SOD1 mRNAs in cells treated with 0.1 and 0.5 μM doxazosin. Moreover, the Bcl-2/Bax ratio was higher in cells that were treated with curcumin before doxazosin or carvedilol. The present study demonstrates that curcumin controls doxazosin- and carvedilol-induced cytotoxicity and morphological changes in HepG2 cells possibly by overexpression of Nrf2.

Figure 1

Cytotoxic activities of doxazosin, carvedilol, and curcumin on HepG2 cells. (a) Doxazosin reduces cell viability timeline and dose dependent in the HepG2 cell line. (b) Carvedilol induces proliferation of HepG2 cells at 24 h, while at 48 h, carvedilol is cytotoxic at low concentrations. (c) Curcumin protects HepG2 cells against the induced cytotoxicity for doxazosin. (d) Curcumin pretreatment-maintained viability in HepG2 cells treated with carvedilol. (e) Curcumin hormesis in HepG2 cells, the viability was analyzed by the MTT assay for 24 h and is presented relative to the activity at the start of the experiment in each case. The results are from three independent experiments. Data are mean ± SD; ^∗p < 0.05, ^∗∗p < 0.0025, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001 versus the control.

Figure 2

Morphological effects on the monolayer of HepG2 cells exposed to α- and β-adrenoblockers. The arrows indicate the cells' main morphological changes as alteration of the monolayer, decreased cell interaction, aggregates of eosinophilic cells, and balonization. Curcumin protects the integrity of the monolayer during an interaction with proven antifibrotic drugs. Curcumin decreases the amount of cell death, maintains the integrity of the monolayer, increases the interaction between cells, reduces cell aggregates, and decreases balonization (arrow). Treatments for 24 h were visualized by hematoxylin and eosin staining taken at 200x of magnification.

Figure 3

Ultrastructural effects of doxazosin and carvedilol on HepG2 cells. Black arrow: irregular cell surface with folds of the cell membrane. Yellow arrow: cell contraction with the formation of possible apoptotic bodies followed by fragmentation. Red arrow: balonization resulting in separation of neighboring cells. Curcumin reduces the morphological variations related to cell damage caused by α- and β-adrenoceptor blocking drugs. Cell morphology shows normal villi and intercellular junctions (blue arrow), without possible apoptotic bodies formation in HepG2 cells. Treatments for 24 h were visualized under SEM (5 μM scale bar).

Figure 4

Determination of necrosis marker by fluorescence microscopy of SYTOX green following doxazosin and carvedilol treatment. Doxazosin shows dose-dependent changes in the number of necrotic cells for 24 h. Carvedilol exhibit a lower number of cells dead related to the positive control for necrosis (HepG2 cells exposed to 250 μM hydrogen peroxide for 15 min). Pretreatment with curcumin maintains the viability of the cells because the staining is impermeant for apoptotic and live cells. Staining taken at 200x of magnification.

Figure 5

Curcumin protects HepG2 cells from the possible proapoptotic effect of doxazosin and carvedilol. HepG2 cells treated with doxazosin and carvedilol showed morphological changes characteristic of apoptosis: nucleus fragmentation (red arrow) and formation of possible apoptotic bodies (blue arrow). However, the pretreatment with curcumin decreases damage in the cell line compared to the control (untreated cells), which was stained bright green. Treatments for 24 h were visualized by acridine orange staining (at 200x of magnification).

Figure 6

Oxidative stress induced by doxazosin and carvedilol. HepG2 cells were pretreated with the antioxidant curcumin (1 μM) 1 h before doxazosin and carvedilol (0.1 and 10 μM) was added, and cells were analyzed 24 h after treatment. Doxazosin and carvedilol (10 μM) induced a significant increase in ROS production compared to 0.1 μM concentration that did not increase the level of ROS. The antioxidant curcumin pretreatment decreased ROS levels induced by doxazosin and carvedilol. Hydrogen peroxide (H₂O₂, 20 mM for 15 min) was used as the positive control of oxidative stress. Flow cytometry analysis of treated cells stained with DHE. Histograms are representative of 3 independent experiments. Statistical analysis of the flow cytometry analysis. A probability value of ^∗p < 0.05 was considered statistically significant.

Figure 7

RT-qPCR analysis for Bcl-2 mRNA expression, Bax mRNA expression, and the Ratio of Bcl-2/Bax after treatment with doxazosin and carvedilol at 24 h. (a) Doxazosin upregulated the proapoptotic gene Bax, over the antiapoptotic, Bcl-2, with the doses of doxazosin treatment mainly at 10 and 25 μM. Interestingly, the Bcl-2/Bax expression ratio increased with the treatment curcumin. (b) Carvedilol represents the downregulation of Bcl-2 and upregulation of Bax. The graphic shows the Bcl-2/Bax expression ratio with a value lower than 1. Curcumin pretreatment regulates the propoptotic expression of Bax, and the graphic shows the Bcl-2/Bax expression ratio with a value higher than 1. The results are from three independent experiments. Data are mean ± SD; ^∗p < 0.05, ^∗∗p < 0.0025, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001 versus the control.

Figure 8

RT-qPCR analysis for Nrf2, Keap1, HO-1, and SOD mRNA expressions after treatment with doxazosin at 24 h in the presence and absence of curcumin. The mRNA expression of Nrf2 were increased by doxazosin treatment (a,e) in the presence and absence of curcumin followed by increase in Keap1 expression compared to the control group (b,f). The increase of Nrf2 in cells treated with doxazosin caused the increased of HO-1 and SOD (c,d) while the effect of cotreatment with curcumin regulated the expression of these genes (g,h), possibly due to the control of the oxidative stress generated. The results are from three independent experiments. Data are mean ± SD; ^∗p < 0.05, ^∗∗p < 0.0025, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001 versus the control.

Figure 9

RT-qPCR analysis for Nrf2, Keap1, HO-1, and SOD mRNA expressions after treatment with carvedilol at 24 h. Carvedilol shows mRNA expression levels of Nrf2 upregulated compared to the control with the dose 25 μM and a decrease in Keap1 compared to the control group in the presence and absence of curcumin (a,b,e,f). Curcumin pretreatment resulted in the activation of the Nrf2 gene and as result increased the antioxidant gene expression of HO-1 and SOD after the treatment (c,d,g,h). Data are expressed as the mean ± SD (n = 3). The results are from three independent experiments. Data are mean ± SD; ^∗p < 0.05, ^∗∗p < 0.0025, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001 versus the control.