Protective effects of Silibinin and cinnamic acid against paraquat-induced lung toxicity in rats: impact on oxidative stress, PI3K/AKT pathway, and miR-193a signaling

Abstract

Levels of reactive oxygen species (ROS) are the primary determinants of pulmonary fibrosis. It was discovered that antioxidants can ameliorate pulmonary fibrosis caused by prolonged paraquat (PQ) exposure. However, research on the precise mechanisms by which antioxidants influence the signaling pathways implicated in pulmonary fibrosis induced by paraquat is still insufficient. This research utilized a rat model of pulmonary fibrosis induced by PQ to examine the impacts of Silibinin (Sil) and cinnamic acid (CA) on pulmonary fibrosis, with a specific focus on pro-fibrotic signaling pathways and ROS-related autophagy. Lung injury induced by paraquat was demonstrated to be associated with oxidative stress and inflammation of the lungs, downregulated (miR-193a), and upregulated PI3K/AKT/mTOR signaling lung tissues. Expression levels of miR-193a were determined with quantitative real-time PCR, protein level of protein kinase B (Akt), and phosphoinositide 3-Kinase (PI3K) which were determined by western blot analysis. Hydroxyproline levels (HYP) and transforming growth factor-β1 (TGF-β1) were measured by ELISA, malondialdehyde (MDA), total antioxidant capacity (TAC), glutathione peroxidase (GSH), and catalase and were measured in lung tissue homogenates colorimetrically using spectrophotometer. Long-term exposure to paraquat resulted in decreased PI3K/AKT signaling, decreased cell autophagy, increased oxidative stress, and increased pulmonary fibrosis formation. Silibinin and cinnamic acid also decreased oxidative stress by increasing autophagy and miR-193a expression, which in turn decreased pulmonary fibrosis. These effects were associated by low TGF-β1. Silibinin and cinnamic acid inhibited PQ-induced PI3K/AKT by stimulating miR-193-a expression, thus attenuating PQ-induced pulmonary fibrosis.

Keywords: AKT; Cinnamic acid; MiRNA 193a; PI3K; Paraquat; Silibinin.

Fig. 1

Representative images of hematoxylin and eosin (H&E)-stained lung tissues treated with paraquat (PQ), Silibinin with paraquat (Sil +PQ) and cinnamic with paraquat (CA+PQ). a Control (normal) group (H&E ×200 magnification). b PQ group, rats were administered PQ (30 mg/kg, intraperitoneal) single dose on the 7^th day (H&E ×200 magnification). c Sil + PQ group, pre-treated rats with Sil (200 mg/kg/day, orally for 7 days) then PQ (30 mg/kg) single dose, intraperitoneal on the 7^th day (H&E ×100 magnification). d CA + PQ group, pretreated rats with CA (50 mg/kg/day, orally for 7 days) then PQ (30 mg/kg) single dose intraperitoneal on the 7^th day (H&E ×100 magnification). Black arrows indicate the alveolar septa, red arrow indicates the peribronchial inflammations and epithelial desquamation

Fig. 2

Graphical illustration of the effect of Silibinin and cinnamic acid on PQ-toxified rats on oxidative stress markers in the lung tissue of rats. a Comparison of catalase level (CAT) in lung tissue homogenate. b Comparison of hydroxyproline level (HYP) in lung tissue homogenate. c Comparison of glutathione peroxidase level (GPx) in lung tissue homogenate. d Comparison of malonaldehyde level (MDA) in lung tissue homogenate. e Comparison of total antioxidant capacity level (TAC) in lung tissue homogenate. CA (Cinnamic acid 50 mg/kg/day) and Sil (Silibinin 200 mg/kg/day) were given orally for 7 days prior to PQ (Paraquat 30 mg/kg, intraperitoneal, single dose on the 7^th day). Data were expressed as mean ± SD (n = 10). Data were analyzed statistically utilizing ANOVA and Tukey post hoc tests. ** differs significantly from the normal control group (p < 0.001), and ## differs significantly from the paraquat group (p < 0.001)

Fig. 3

Effect of cinnamic acid and Silibinin on PQ-toxified rats by measuring TGF-β1 level in tissue homogenate using ELISA. CA (Cinnamic acid 50 mg/kg/day) and Sil (Silibinin 200 mg/kg/day) were given orally for 7 days prior to PQ (Paraquat 30 mg/kg, intraperitoneal, single dose on the 7^th day). Data were expressed as mean ± SD (n = 10). Data were analyzed statistically utilizing ANOVA and Tukey post hoc tests. ** differs significantly from the normal control group (p < 0.001), and ## differs significantly from the paraquat group (p < 0.001)

Fig. 4

Effect of cinnamic acid and Silibinin on PQ-toxified rats by measuring phosphatidyl inositol-3-kinase (PI3K) protein expression level using western blot. a A representation of western blot of lung tissue PI3K protein expression. b Graphical illustration showing comparison of lung PI3K protein expression. CA (Cinnamic acid 50 mg/kg/day) and Sil (Silibinin 200 mg/kg/day) were given orally for 7 days prior to PQ (Paraquat 30 mg/kg, intraperitoneal, single dose on the 7^th day). Data were expressed as mean ± SD (n = 10). Data were analyzed statistically utilizing ANOVA and Tukey post hoc tests. ** differs significantly from the normal control group (p < 0.001), and ## differs significantly from the paraquat group (p < 0.001)

Fig. 5

Effect of cinnamic acid and Silibinin on PQ-toxified rats by measuring protein kinase B (AKT) protein expression level in lung tissues of rats using western blot. a A representation of western blot of lung tissue AKT protein expression. b Graphical illustration showing comparison of lung AKT protein expression level. CA (Cinnamic acid 50 mg/kg/day) and Sil (Silibinin 200 mg/kg/day) were given orally for 7 days prior to PQ (Paraquat 30 mg/kg, intraperitoneal, single dose on the 7^th day). Data were expressed as mean ± SD (n = 10). Data were analyzed statistically utilizing ANOVA and Tukey post hoc tests. ** differs significantly from the normal control group (p < 0.001), and ## differs significantly from the paraquat group (p < 0.001)

Fig. 6

Effect of cinnamic acid and Silibinin on PQ-toxified rats by measuring miRNA 193-a gene expression by RT-PCR. CA (Cinnamic acid 50 mg/kg/day) and Sil (Silibinin 200 mg/kg/day) were given orally for 7 days prior to PQ (Paraquat 30 mg/kg, intraperitoneal, single dose on the 7^th day). Data were expressed as mean ± SD (n = 10). Data were analyzed statistically utilizing ANOVA and Tukey post hoc tests. ** differs significantly from the normal control group (p < 0.001), and ## differs significantly from the paraquat group (p < 0.001)

Fig. 7

A scattered chart showing a the correlation between miRNA 193-a and AKT ( protein kinase B), r = −0.94 at p < 0.001. b The correlation between miRNA 193-a and HYP (hydroxyproline), r = −0.94 at p < 0.001. c The correlation between miRNA 193-a and TGF β1 (transforming growth factor β1), r = −0.96 p < 0.001

Fig. 8

Illustrated scheme for the mechanism of each of paraquat, cinnamic acid, and Silibinin on lung toxicity and fibrosis. CA (Cinnamic acid 50 mg/kg/day) and Sil (Silibinin 200 mg/kg/day) were given orally for 7 days prior to PQ (Paraquat 30 mg/kg, intraperitoneal (I.P), single dose on the 7^th day)