Luteolin Protects Against Acrylamide-Induced Cellular Toxicity in Mouse Leydig Cells

Abstract

Luteolin (Lut), a natural flavonoid, possesses the ability to protect the organism from reactive oxygen species (ROS) by modulating intracellular and extracellular signals. This study was conducted to investigate whether Lut exhibits a protective effect against acrylamide (Acr)-induced reproductive toxicity and to reveal the mechanisms underlying Leydig cell damage, a model cell in the male reproductive system. TM3 mouse Leydig cells were treated with Acr (1 mM) and/or Lut (1 and 5 μM) for 24 h. To determine the potential protective effects of Lut against the toxicity caused by Acr on Leydig cells, cell viability, combination index, and lactate dehydrogenase activity were examined for cytotoxicity. The effect of Lut on oxidative stress parameters in Acr-induced cellular damage was revealed by evaluating lipid peroxidation, total ROS, glutathione, and antioxidant enzymes (SOD, CAT, and GPx). In addition, we evaluated the expression levels of genes related to apoptosis (Trp53, Casp3, Bax, and Bcl2) and the apoptotic rate in the Acr and/or Lut groups. The findings indicated that Lut inhibited the cytotoxicity elevated by Acr in Leydig cells. The evaluation of the combination index in the groups treated with Lut and Acr together revealed that Lut exhibited an antagonistic effect. Furthermore, it was revealed that while Acr induced oxidative damage and apoptosis, Lut reversed these effects and protected Leydig cells from cellular damage. Also, Lut inhibited the mRNA expressions of Trp53, Casp3, and Bax, which were upregulated by Acr. Consequently, Lut may serve as a potential antioxidant molecule in promoting cell survival by mitigating oxidative damage and apoptosis induced by Acr.

Keywords: Leydig cells; acrylamide; apoptosis; luteolin; oxidative stress.

FIGURE 1

LUT improved Acr‐inhibited cell viability in Leydig cells. (A) Effects of cell viability Lut and/or Acr on Leydig cells; (B) Heat map showing the dose–response relationship of Lut and/or Acr according to % viability; (C) Lut and Acr synergy scoring (Bliss score) using minimal dose–response matrices. Data denote the mean ± SEM from three different experiments in triplicates (n = 9). *Indicate statistically different values in treated cells compared to the control group (****p < 0.0001) and ^#Statistically different values in groups versus the Acr‐alone group (^### p < 0.001).

FIGURE 2

Evidence of membrane damage by LDH release in Leydig cells after treatment with Lut and/or Acr. Data denote the mean ± SEM from three different experiments in triplicates (n = 9). *Statistically significant differences compared to control (***p < 0.001) and ^#Statistically significant differences compared to Acr‐only treated groups (^### p < 0.001).

FIGURE 3

Effects of Lut and/or Acr on total ROS production in Leydig cells. (A) Representative images of Leydig cells labeled with a DCFH‐DA probe and visualized by fluorescence microscopy; (B) Total ROS levels determined by relative fluorescence intensity and expressed in arbitrary units. Experiments were repeated three times in duplicate. The values are means ± SEM (n = 6). *Statistically significant differences compared to control (****p < 0.0001) and ^#Statistically significant differences compared to Acr‐only treated groups (^### p < 0.001).

FIGURE 4

Lut prevents Acr‐induced apoptosis. (A) Double‐fluorescent labeling images with Ho342 and PI dyes are presented to show apoptosis in Leydig cells after Lut and/or Acr treatments. Arrow: live cells, write arrowhead: apoptotic cells, empty arrowhead: dead cells. Scale bar: 100 μm. (B) Effects of Lut on the apoptotic cell rate induced by Acr. Experiments were repeated three times in duplicate. The values are means ± SEM (n = 6). *Statistically significant differences compared to control (****p < 0.0001) and ^#Statistically significant differences compared to Acr‐only treated groups (^### p < 0.001).

FIGURE 5

Effects of Lut and/or Acr on mRNA expression of apoptotic and antiapoptotic genes in Leydig cells. mRNA expression of (A) Trp53, (B) Casp3, (C) Bax, and (D) Bcl2. Experiments were repeated three times in duplicate. The values are means ± SEM (n = 6). *: Statistically significant differences compared to control (***p < 0.001) and ^#statistically significant differences compared to Acr‐only treated groups (^# p < 0.05, ^### p < 0.001).