Hepatoprotective properties of red betel (Piper crocatum Ruiz and Pav) leaves extract towards H2O2-induced HepG2 cells via anti-inflammatory, antinecrotic, antioxidant potency

Abstract

Background: Prolonged exposure of free radicals, or known as reactive oxygen species (ROS), in hepatic cells may cause oxidative stress. Without proper treatment, it can induce liver injury and fatal hepatic disease, including cirrhosis. Red betel (Piper crocatum Ruiz and Pav) is one of Indonesia's medicinal plants that has been known to exhibit antioxidant, anti-inflammatory activities. This study aims to determine hepatoprotective effect of red betel leaves extract (RBLE) towards liver injury.

Method: Hydrogen peroxide-induced HepG2 cells were used as liver injury model·H₂O₂-induced HepG2 cells were treated with 25 µg/mL and 100 µg/mL RBLE. Several parameters were observed, including TNF-α level through ELISA; necrotic, apoptotic, dead, live cells; and ROS level through flow cytometry analysis; and GPX gene expression through qPCR.

Result: The study showed that treatment with RBLE were able to decrease TNF-α level; necrotic and death cells percentage; as well as ROS level. On the other hand, it were able to increase apoptotic and live cells percentage; as well as GPX gene expression. Low concentration (25 µg/mL) of RBLE treatment exhibited stronger anti-inflammatory activity as it was resulted in the lower TNF-α level and were able to switched hepatic cell death pathway from necrosis to apoptosis as shown by the shifted of apoptotic cells and necrotic cells percentage. This lead to lower death cells and ultimately improve live cells percentage. Meanwhile high concentration of RBLE (100 µg/mL) exhibited stronger antioxidant properties as indicated by lower ROS level and higher GPX gene expression.

Conclusion: Overall, this study was able to demonstrate hepatoprotective effect of RBLE towards liver injury model through its anti-inflammatory and antioxidant activities.

Keywords: Anti-inflammatory; Antinecrotic; Antioxidant; HepG2; Hepatoprotective; Red betel.

Fig. 1

Effect of RBLE towards TNF-α Level of H₂O₂-induced HepG2 cells. (A) TNF-α level (pg/mg protein) on H₂O₂-induced HepG2 cells. (B) TNF-α level (pg/mL) on H₂O₂-induced HepG2 cells. *Data is presented as mean ± standard deviation. (I): Control (untreated HepG2 cells); (II): Vehicle control (HepG2 cells + DMSO 1%); (III) LI model (Liver Injury model: H₂O₂-induced HepG2 cells); (IV) RBLE25 (H₂O₂-induced HepG2 cells + RBLE 25 μg/mL); (V) RBLE100 (H₂O₂-induced HepG2 cells + RBLE 100 μg/mL). Single star sign (*) marks statistical difference between control group and Liver Injury (LI) model group at 0.05 significance level, while single hashtag symbol (#) marks statistical difference for the treatments groups compared to Liver Injury (LI) model group at 0.05 significance level.

Fig. 2

Effect of RBLE towards apoptosis inducing activity of H₂O₂-induced HepG2 cells. (A) Live cells (%); (B) Apoptotic cells (%); (C) Dead cells (%); (D) Necrotic cells (%). *Data is presented as mean ± standard deviation. (I): Control (untreated HepG2 cells); (II): Vehicle control (HepG2 cells + DMSO 1%); (III) LI model (Liver Injury model; H₂O₂-induced HepG2 cells); (IV) RBLE25 (H₂O₂-induced HepG2 cells + RBLE 25 μg/mL); (V) RBLE100 (H₂O₂-induced HepG2 cells + RBLE 100 μg/mL). Single star sign (*) marks statistical difference between control and Liver Injury (LI) model group at 0.05 significance level, single hashtag (#) marks statistical difference for treatment groups compared to Liver Injury (LI) model group at 0.05 significance level.

Fig. 3

Effect of RBLE towards ROS percentage of H₂O₂-induced HepG2 cells. *Data is presented as mean ± standard deviation. (I): Control (untreated HepG2 cells); (II) LI model (Liver Injury model; H₂O₂-induced HepG2 cells); (III) RBLE25 (H₂O₂-induced HepG2 cells + RBLE 25 μg/mL); (IV) RBLE100 (H₂O₂-induced HepG2 cells + RBLE 100 μg/mL). Single star sign (*) marks statistical difference between control group and Liver Injury (LI) model group and at 0.05 significance level, single hashtag (#) marks statistical difference in treatment groups compared to Liver Injury (LI) model group at 0.05 significance level.

Fig. 4

Effect of RBLE towards GPX gene expression of H₂O₂-induced HepG2 cells. *Data is presented as mean ± standard deviation. (I): Control (untreated HepG2 cells); (II): LI model (Liver Injury model; H₂O₂-induced HepG2 cells); (III) RBLE25 (H₂O₂-induced HepG2 cells + RBLE 25 μg/mL); (IV): RBLE100 (H₂O₂-induced HepG2 cells + RBLE 100 μg/mL). Single star sign (*) marks statistical difference for control group compared to Liver Injury (LI) model group 0.05 significance level, single hashtag (#) marks statistical difference for treatment groups compared to Liver Injury (LI) model group at 0.05 significance level.

Fig. 5

Proposed mechanism on how RBLE could act as a hepatoprotective agent in liver injury model·H₂O₂ is transformed into hydroxyl radical (HO*) through Fenton reaction with the presence of transition metal such as iron and copper. Under normal condition, H₂O₂ is neutralized by Glutathione Peroxidase (GPX) enzymes activity through oxidation of GSH into GSSG. It seemed that excessive H₂O₂ lead to down regulation of GPX gene expression. Hydroxyl radical activates NFkB that lead to TNF-α production. TNF/TNFR engagement induces production of catalase (CAT) and superoxide dismutase (SOD) and thus increasing extracellular H₂O₂. Meanwhile, TNF-α also induced JNK signaling pathways, resulting in increased mitochondrial ROS and lead to activation of Caspase 9 and apoptosis executioner, Caspase3. On the other hand, high amount of HO* triggers mitochondrial permeability transition (MPT), causing mitochondria to swell, and suppress ATP production. This pathway lead to unregulated cell death, necrosis; increase inflammation; and promote cell death. Lack of ATP also affects apoptosis pathway and switch it into necrosis instead. If this condition left untreated, liver injury could take place. Treatment with RBLE could neutralized HO* in cells and thus lowering necrosis, shifted the cells death pathway to apoptosis which induce lower inflammation response instead. Subsequently, RBLE treatment could increase the survival of hepatic cells.