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. 2017 Sep 19:8:653.
doi: 10.3389/fphar.2017.00653. eCollection 2017.

Induction of Mkp-1 and Nuclear Translocation of Nrf2 by Limonoids from Khaya grandifoliola C.DC Protect L-02 Hepatocytes against Acetaminophen-Induced Hepatotoxicity

Affiliations

Induction of Mkp-1 and Nuclear Translocation of Nrf2 by Limonoids from Khaya grandifoliola C.DC Protect L-02 Hepatocytes against Acetaminophen-Induced Hepatotoxicity

Arnaud F Kouam et al. Front Pharmacol. .

Abstract

Drug-induced liver injury (DILI) is a major clinical problem where natural compounds hold promise for its abrogation. Khaya grandifoliola (Meliaceae) is used in Cameroonian traditional medicine for the treatment of liver related diseases and has been studied for its hepatoprotective properties. Till date, reports showing the hepatoprotective molecular mechanism of the plant are lacking. The aim of this study was therefore to identify compounds from the plant bearing hepatoprotective activity and the related molecular mechanism by assessing their effects against acetaminophen (APAP)-induced hepatotoxicity in normal human liver L-02 cells line. The cells were exposed to APAP (10 mM) or co-treated with phytochemical compounds (40 μM) over a period of 36 h and, biochemical and molecular parameters assessed. Three known limonoids namely 17-epi-methyl-6-hydroxylangolensate, 7-deacetoxy-7-oxogedunin and deacetoxy-7R-hydroxygedunin were identified. The results of cells viability and membrane integrity, reactive oxygen species generation and lipid membrane peroxidation assays, cellular glutathione content determination as well as expression of cytochrome P450 2E1 demonstrated the protective action of the limonoids. Immunoblotting analysis revealed that limonoids inhibited APAP-induced c-Jun N-terminal Kinase phosphorylation (p-JNK), mitochondrial translocation of p-JNK and Bcl2-associated X Protein, and the release of Apoptosis-inducing Factor into the cytosol. Interestingly, limonoids increased the expression of Mitogen-activated Protein Kinase Phosphatase (Mkp)-1, an endogenous inhibitor of JNK phosphorylation and, induced the nuclear translocation of Nuclear Factor Erythroid 2-related Factor-2 (Nrf2) and decreased the expression of Kelch-like ECH-associated Protein-1. The limonoids also reversed the APAP-induced decreased mRNA levels of Catalase, Superoxide Dismutase-1, Glutathione-S-Transferase and Methionine Adenosyltransferase-1A. The obtained results suggest that the isolated limonoids protect L-02 hepatocytes against APAP-induced hepatotoxicity mainly through increase expression of Mkp-1 and nuclear translocation of Nrf2. Thus, these compounds are in part responsible of the hepatoprotective activity of K. grandifoliola and further analysis including in vivo and toxicological studies are needed to select the most potent compound that may be useful as therapeutic agents against DILI.

Keywords: K. grandifoliola; Mkp-1; Nrf2; acetaminophen; hepatoprotection; limonoids.

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Figures

FIGURE 1
FIGURE 1
High performance liquid chromatography (HPLC) chromatogram and chemical structures of purified compounds. (A) 17-epi-methyl-6-hydroxyangolensate; (B) 7-deacetoxy-7-oxogedunin and (C) 7-deacetoxy-7R-hydroxygedunin. Compounds were purified by HPLC-ACN-Standard-Purify-Method. Exact mass and chemical structures were determined by HRMS and 1H and 13C NMR, respectively.
FIGURE 2
FIGURE 2
Acetaminophen (APAP) induced cell death and loss of membrane integrity in L-02 hepatocytes. Cells were treated with various concentration of APAP (5–30 mM) for 36 h. (A) Cell viability indicating dose-response of APAP toxicity; (B) loss of membrane integrity indicated by ALT activity found in the culture medium. Values are means ± SD of three independent experiments in triplicate. ANOVA analysis: F(5,30) = 377.7, P < 0.0001 (A) and F(5,30) = 251.7, P < 0.0001 (B). Values significantly different compared to control (0 mM) (P < 0.05) using Bonferroni’s test.
FIGURE 3
FIGURE 3
Hepatoprotective screening activity of plants sub-fractions. L-02 cells were treated without or with APAP, or co-treated with APAP and KgF25 sub-fractions or silymarin for 36 h. After treatment, cell viability (A) and ALT activity (B) leakage into the incubation were determined. Values are means ± SD of three independent experiments in triplicate. ANOVA analysis: F(8,45) = 76.47, P < 0.0001 (A) and F(8,45) = 180.3, P < 0.0001 (B). ΔValues significantly different compared to control group (P < 0.05); values significantly different compared to APAP intoxicated group (P < 0.05) using Bonferroni’s test. Sil: silymarin; KgF25: methylene chloride/methanol (75:25, v/v) fraction of K. grandifoliola; KgF25sf1: sub-fraction 1 of KgF25; KgF25sf2: sub-fraction 2 of KgF25; KgF25sf3: sub-fraction 3 of KgF25; KgF25sf4: sub-fraction 4; of KgF25, KgF25sf5: sub-fraction 5 of KgF25.
FIGURE 4
FIGURE 4
Dose-dependent protective effect of active sub-fractions and isolated limonoids. Cells were treated without or with APAP (10 mM), or co-treated with APAP (10 mM) and active sub-fractions or silymarin (25; 50; and 100 μg/mL), isolated limonoids (10; 20; 30; and 40 μM) or JNK inhibitor (5; 10; 15; and 20 μM) for 36 h. (A,B) Effect of active sub-fractions on cell viability and ALT leakage, respectively. (C,D) Effect of isolated limonoids on cell viability and ALT leakage, respectively. Values are means ± SD of three independent experiments in triplicate. ANOVA analysis: F(6,14) = 33.82, P < 0.0001 (A); F(6,14) = 77.74, P < 0.0001 (B); F(5,12) = 48.96, P < 0.0001 (C); and F(5,12) = 128.3, P < 0.0001 (D). ΔValues significantly different compared to control group (P < 0.05); values significantly different compared to APAP intoxicated group (P < 0.05) using Bonferroni’s test. KgF25: methylene chloride/methanol (75:25, v/v) fraction of K. grandifoliola; KgF25sf1: sub-fraction 1 of KgF25; KgF25sf2: sub-fraction 2 of KgF25; KgF25sf3: sub-fraction 3 of KgF25; C-A: 17-epi-methyl-6-hydroxyangolensate; C-B: 7-deacetoxy-7-oxogedunin; C-C: 7-deacetoxy-7R-hydroxygedunin; SP: JNK inhibitor SP600125.
FIGURE 5
FIGURE 5
Propidium Iodide staining showing protective effect of isolated compounds against APAP-induced cell death. Cells were treated without or with APAP (10 mM), or co-treated with APAP (10 mM) and isolated compounds (40 μM) or JNK inhibitor (20 μM) for 36 h. After treatment, death cells were stained with PI (red) and DAPI (blue) was used to label the total number of cells. Each image recorded with fluorescence microscope represents one of three independent experiments in triplicate. SP: JNK inhibitor SP600125; C-A: 17-epi-methyl-6-hydroxyangolensate; C-B: 7-deacetoxy-7-oxogedunin; C-C: 7-deacetoxy-7R-hydroxygedunin.
FIGURE 6
FIGURE 6
Active sub-fractions and isolated limonoids down-regulated CYP2E1 proteins expression and rescued APAP-induced GSH depletion. Cells were treated without or with APAP (10 mM), or co-treated with APAP (10 mM) and active sub-fractions or silymarin (100 μg/mL), isolated compounds (40 μM) or JNK inhibitor (20 μM) for 6 h. After treatment, total proteins were extracted from cells and CYP2E1 expression was determined by western blotting. β-actin was used as loading control. Each blot represents one of three independent experiments. (A,C) Effect of active sub-fractions and isolated compounds on CYP2E1 expression, respectively. (B,D) Densitometry analysis of blots, respectively, for active sub-fractions and isolated compounds. (E) Effect of isolated compounds on cellular GSH. L-02 cells were treated without or with APAP (10 mM), or co-treated with APAP (10 mM) and isolated compounds (40 μM) or JNK inhibitor (20 μM) for 6, 12, 24, and 36 h. After each time point, total proteins were extracted and GSH contents were quantified. Values are means ± SD of three independent experiments in triplicate. ANOVA analysis: F(6,14) = 200.9, P < 0.0001 (B); F(5,12) = 219.6, P < 0.0001 (D); and F(5,12) = 59.88, P < 0.0001 (D). ΔValues significantly different compared to control group (P < 0.05); values significantly different compared to APAP intoxicated group (P < 0.05) using Bonferroni’s test. Lane1: control; Lane2: APAP; Lane3: silymarin+APAP; Lane4: KgF25+APAP; Lane5: KgF25sf1+APAP; Lane6: KgF25sf2+APAP; Lane7: Kgf25sf3+APAP. Sil: silymarin; KgF25: methylene chloride/methanol (75:25, v/v) fraction of K. grandifoliola; KgF25sf1: sub-fraction 1 of KgF25; KgF25sf2: sub-fraction 2 of KgF25; KgF25sf3: sub-fraction 3 of KgF25; SP: JNK inhibitor SP600125; C-A: 17-epi-methyl-6-hydroxyangolensate; C-B: 7-deacetoxy-7-oxogedunin; C-C: 7-deacetoxy-7R-hydroxygedunin.
FIGURE 7
FIGURE 7
Limonoids from K. grandifoliola attenuated APAP-induced oxidative stress in L-02 cells. Cells were treated without or with APAP (10 mM), or co-treated with APAP (10 mM) and isolated compounds (40 μM) or JNK inhibitor (20 μM) for 36 h. After treatment, cellular ROS level (A) and MDA concentrations (B) in the incubation medium were measured. Values are means ± SD of three independent experiments in triplicate. ANOVA analysis: F(5,12) = 28.25, P < 0.0001 (A) and F(5,12) = 303.0, P < 0.0001 (B). ΔValues significantly different compared to control group (P < 0.05); values significantly different compared to APAP intoxicated group (P < 0.05) using Bonferroni’s test. SP: JNK inhibitor SP600125; C-A: 17-epi-methyl-6-hydroxyangolensate; C-B: 7-deacetoxy-7-oxogedunin; C-C: 7-deacetoxy-7R-hydroxygedunin.
FIGURE 8
FIGURE 8
Active sub-fractions and isolated limonoids prevented APAP-induced JNK activation. Cells were treated without or with APAP (10 mM), or co-treated with APAP (10 mM) and active sub-fractions or silymarin (100 μg/mL), isolated limonoids (40 μM) or JNK inhibitor (20 μM) for 6 h. After treatment, total proteins were extracted from cells and the lysates were probed for JNK2 and p-JNK by western blotting. β-actin was used as loading control. Each blot represents one of three independent experiments. (A,C) Effect of active sub-fractions and isolated compounds on JNK activation, respectively. (B,D) Densitometry analysis of blots, respectively, for active sub-fractions and isolated compounds. Values are means ± SD of three independent experiments in triplicate. ANOVA analysis: F(6,14) = 322.0, P < 0.0001 (B) and F(5,12) = 1162.0, P < 0.0001 (D). ΔValues significantly different compared to control group (P < 0.05); values significantly different compared to APAP intoxicated group (P < 0.05) using Bonferroni’s test. Lane1: control; Lane2: APAP; Lane3: silymarin+APAP; Lane4: KgF25+APAP; Lane5: KgF25sf1+APAP; Lane6: KgF25sf2+APAP; Lane7: Kgf25sf3+APAP. Sil: silymarin; KgF25: methylene chloride/methanol (75:25, v/v) fraction of K. grandifoliola; KgF25sf1: sub-fraction 1 of KgF25; KgF25sf2: sub-fraction 2 of KgF25; KgF25sf3: sub-fraction 3 of KgF25; SP: JNK inhibitor SP600125; C-A: 17-epi-methyl-6-hydroxyangolensate; C-B: 7-deacetoxy-7-oxogedunin; C-C: 7-deacetoxy-7R-hydroxygedunin.
FIGURE 9
FIGURE 9
Limonoids from K. grandifoliola prevented mitochondrial p-JNK translocation. Cells were treated without or with APAP (10 mM), or co-treated with APAP (10 mM) and isolated limonoids (40 μM) or JNK inhibitor (20 μM) for 6 and 12 h. After treatment, p-JNK level was detected into the cytosolic and mitochondrial fractions by western blotting. JNK2 was used as internal control. Each blot represents one of three independent experiments. (A,B) Effect of isolated compounds on JNK activation, respectively, at 6 and 12 h after treatment. (C) densitometry analysis of blots. Values are means ± SD of three independent experiments in triplicate. ANOVA analysis: F(5,12) = 1119.0, P < 0.0001 and F(5,12) = 1841.0, P < 0.0001 (C). ΔValues significantly different compared to control group (P < 0.05); values significantly different compared to APAP intoxicated group (P < 0.05) using Bonferroni’s test. SP: JNK inhibitor SP600125; C-A: 17-epi -methyl-6-hydroxyangolensate; C-B: 7-deacetoxy-7-oxogedunin; C-C: 7-deacetoxy-7R-hydroxygedunin.
FIGURE 10
FIGURE 10
Isolated compounds attenuated mitochondrial Bax translocation and release of AIF from mitochondria to cytosol. Cells were treated without or with APAP (10 mM), or co-treated with APAP (10 mM) and limonoids (40 μM) or JNK inhibitor (20 μM) for 12 h. After treatment, AIF and Bax protein levels (A) were detected into the cytosolic and mitochondrial fractions by western blotting. β-actin and COX IV (cytochrome oxidase IV) were used as loading control, respectively, for the cytosolic and mitochondrial fraction, respectively. Each blot represents one of three independent experiments. (B) Densitometry analysis of blots. Values are means ± SD of three independent experiments in triplicate. ANOVA analysis: F(5,12) = 671.2, P < 0.0001 and F(5,12) = 317.5, P < 0.0001 (B). ΔValues significantly different compared to control group (P < 0.05); values significantly different compared to APAP intoxicated group (P < 0.05) using Bonferroni’s test. SP: JNK inhibitor SP600125; C-A: 17-epi-methyl-6-hydroxyangolensate; C-B: 7-deacetoxy-7-oxogedunin; C-C: 7-deacetoxy-7R-hydroxygedunin.
FIGURE 11
FIGURE 11
Active sub-fractions and isolated Limonoids up-regulated Mkp-1 protein expression. Cells were treated without or with APAP (10 mM), or co-treated with APAP (10 mM) and active sub-fractions or silymarin (100 μg/mL), isolated compounds (40 μM) or JNK inhibitor (20 μM) for 6 h. After treatment, total proteins were extracted from cells and Mkp-1 expression was determined by western blotting. β-actin was used as loading control. Each blot represents one of three independent experiments. (A,B) Effect of active sub-fractions and isolated compounds on Mkp-1 expression, respectively. (C,D) Densitometry analysis of blots, respectively, for active sub-fractions and isolated compounds. Values are means ± SD of three independent experiments in triplicate. ANOVA analysis: F(6,14) = 307.1, P < 0.0001 (C) and F(5,12) = 270.3, P < 0.0001 (D). ΔValues significantly different compared to control group (P < 0.05); values significantly different compared to APAP intoxicated group (P < 0.05) using Bonferroni’s test. Lane1: control; Lane2: APAP; Lane3: silymarin+APAP; Lane4: KgF25+APAP; Lane5: KgF25sf1+APAP; Lane6: KgF25sf2+APAP; Lane7: Kgf25sf3+APAP. Sil: silymarin; KgF25: methylene chloride/methanol (75:25, v/v) fraction of K. grandifoliola; KgF25sf1: sub-fraction 1 of KgF25; KgF25sf2: sub-fraction 2 of KgF25; KgF25sf3: sub-fraction 3 of KgF25; SP: JNK inhibitor SP600125; C-A: 17-epi-methyl-6-hydroxyangolensate; C-B: 7-deacetoxy-7-oxogedunin; C-C: 7-deacetoxy-7R-hydroxygedunin.
FIGURE 12
FIGURE 12
Isolated limonoids activated Nrf2-Keap-1 antioxidant defense system. Cells were treated without or with APAP (10 mM), or co-treated with APAP (10 mM) and isolated limonoids (40 μM) or JNK inhibitor (20 μM) for 12 and 24 h. After treatment, Nrf2 level was detected into the nuclear fraction and Keap-1 level was detected at 24 h into the cytosolic fractions by western blotting (A). Lamin-B and β-actin were used as loading control, respectively, for the nuclear and cytosolic fraction. Each blot represents one of three independent experiments. (B) Densitometry analysis of blots. Values are means ± SD of three independent experiments in triplicate. ANOVA analysis: F(5,12) = 370.8, P < 0.0001 and F(5,12) = 48.80, P < 0.0001 (B). ΔValues significantly different compared to control group (P < 0.05); values significantly different compared to APAP intoxicated group (P < 0.05) using Bonferroni’s test. SP: JNK inhibitor SP600125; C-A: 17-epi-methyl-6-hydroxyangolensate; C-B: 7-deacetoxy-7-oxogedunin; C-C: 7-deacetoxy-7R-hydroxygedunin.
FIGURE 13
FIGURE 13
Limonoids from K. grandifoliola increased mRNA levels of CAT, SOD1, GST, and MAT1A genes. Cells were treated without or with APAP (10 mM), or co-treated with APAP (10 mM) and isolated limonoids (40 μM) or JNK inhibitor (20 μM) for 36 h. After treatment, RNA was extracted from cells and relative mRNA expression level of CAT (A), SOD (B), GST (C), and MAT1A (D) were determined by qRT-PCR. GAPDH was used as internal control. Values are means ± SD of three independent experiments in triplicate. ANOVA analysis: F(5,12) = 70.43, P < 0.0001 (A); F(5,12) = 45.72, P < 0.0001 (B); F(5,12) = 113.0, P < 0.0001 (C); and F(5,12) = 20.65, P < 0.0001 (D). ΔValues significantly different compared to control group (P < 0.05); values significantly different compared to APAP intoxicated group (P < 0.05) using Bonferroni’s test. SP: JNK inhibitor SP600125; C-A: 17-epi-methyl-6-hydroxyangolensate; C-B: 7-deacetoxy-7-oxogedunin; C-C: 7-deacetoxy-7R-hydroxygedunin.

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