Hepatotoxicity prevention in Acetaminophen-induced HepG2 cells by red betel (Piper crocatum Ruiz and Pav) extract from Indonesia via antioxidant, anti-inflammatory, and anti-necrotic

Abstract

Acetaminophen (APAP) is a widely used analgesic, but it may cause liver injury (hepatotoxicity) via oxidative stress that induced by N-acetyl-p-benzoquinone imine (NAPQI) in long term usage or overdose. Multiple inflammatory mediators were also found to contribute for this effect. Many medicinal plants was known for its antioxidant and anti-inflammatory activities and one of them is Red betel (Piper crocatum Ruiz and Pav) from Indonesia. In this study, the red betel leaves extract (RBLE) protective effect against APAP-induced HepG2 cells was determined. APAP-induced HepG2 as hepatotoxicity cell model was treated with RBLE at 25 and 100 μg/mL. Protective effects of RBLE toward hepatotoxicity were evaluated by several parameters: tumor necrosis factor-α (TNF-α) concentration, reactive oxygen species (ROS) level, live cells percentage, apoptotic cells percentage, necrotic cells percentage, death cells percentage, CYP2E1 and GPX gene expression. The RBLE treatments (both 25 and 100 μg/mL) increased CYP2E1 and GPX gene expression also live cells percentage, while decreased ROS level, TNF-α concentration, also the percentage of death and necrotic cells. Red Betel leaves ethanol extract has hepatoprotective effect via anti-inflammatory, anti-necrotic, and antioxidant potency in liver injury model.

Keywords: Acetaminophen; Biochemistry; HepG2 cells; Hepatoprotective; Immunology; Inflammation; Natural product; Pharmaceutical science; Red betel leaves extract.

Figure 1

RBLE treatment effect toward TNF-α concentration in APAP-induced HepG2 cells as hepatotoxicity model. (A) TNF-α concentration (pg/mL) on hepatotoxicity model. (B) TNF-α concentration (pg/mg protein) on hepatotoxicity model. ∗Data was included as mean ± standard deviation. I) Normal cells as negative control; II) Normal cells + DMSO 1% as vehicle control; III) APAP-induced cells (Positive control); IV) Positive control + RBLE 25 μg/mL; V) Positive control + RBLE 100 μg/mL. Significance among treatments toward TNF-α concentration was presented as different letters (a,b) based on Tukey HSD post hoc test (P < 0.05).

Figure 2

RBLE effect toward apoptotic, necrotic, dead cells in hepatotoxicity model. (A) Live cells on hepatotoxicity model. (B) Early apoptotic on hepatotoxicity model. (C) Late apoptotic on hepatotoxicity model. (D) Necrotic on hepatotoxicity model. ∗Data was included as mean ± standard deviation. I) Normal cells as negative control; II) Normal cells + DMSO 1% as vehicle control; III) APAP-induced cells (Positive control); IV) Positive control + RBLE 25 μg/mL; V) Positive control + RBLE 100 μg/mL. There are significant different between all groups based on Kruskal-Wallis Test (P < 0.05) and Mann-Whitney Test (P < 0.05). It was marked as single star (∗) marks for the statistical difference between positive control and negative control while the hashtag (#) mark for statistical difference between treatment and positive control.

Figure 3

RBLE effect toward ROS level in hepatotoxicity model. ∗Data was included as mean ± standard deviation. I) Normal cells (Negative control); II) Normal cells + DMSO 1%; III) APAP-induced cells (Positive control); IV) Positive control + RBLE 25 μg/mL; V) Positive control + RBLE 100 μg/mL. Based on Kruskal-Wallis Test (P < 0.05), there are significant different among groups. It was marked as single star (∗) for statistical difference between positive control and negative control also hashtag (#) for statistical difference between treatment and positive control.

Figure 4

RBLE effect toward the expression of CYP2E1 gene in hepatotoxicity model. ∗Data was included as mean ± standard deviation. I) Normal cells as negative control; II) Normal cells + DMSO 1%; III) APAP-induced cells (Positive control); IV) Positive control + RBLE 25 μg/mL; V) Positive control + RBLE 100 μg/mL. Based on ANOVA (P < 0.05) and Games-Howell (P < 0.05), there are significant different among all groups. It was marked as single star (∗) marks for the difference between positive control and negative control also hashtag (#) mark for the difference between treatment and positive control.

Figure 5

RBLE effect toward the expression of GPX gene in hepatotoxicity model. ∗Data was included as mean ± standard deviation. I) Normal cells (Negative control); II) Normal cells + DMSO 1%; III) APAP-induced cells (Positive control); IV) Positive control + RBLE 25 μg/mL; V) Positive control + RBLE 100 μg/mL. Based on Kruskal-Wallis Test (P < 0.05) and Mann-Whitney Test (P < 0.05), there are significant different between group. It was marked as single star (∗) marks as difference between positive control and negative control while hashtag (#) mark as difference between treatment and positive control.

Figure 6

Proposed RLBE hepatoprotective mechanism in liver injury model. ∗CYP2E1 act to transform APAP to NAPQI. It induce GSH depletion then induce production of ROS. The excessive of ROS decrease the GPX gene expression leads to increase cell death. NAPQI activate the kupper cell and leads to TNF-α production that induce the JNK signaling pathways that also increase ROS level and leads to upregulate cell necrosis; increase inflammation; and induce cell death. While JNK induced the Bcl-2 down-regulation and Bax up-regulation, resulting in activation of caspase 9 and caspase 3 that leads to apoptosis cells. The RLBE treatments could inhibit the excessive ROS and TNF-α. It also could lowering the necrosis and apoptosis that leads to lowering inflammation. RBLE treatments decrease the cell death and increase the survival hepatic cells.