Rosmarinic Acid Ameliorates H 2 O 2 -Induced Oxidative Stress in L02 Cells Through MAPK and Nrf2 Pathways

Abstract

Liver cells are easily damaged by oxidative stress during progression both in liver development and throughout adult life, resulting in tissue pathology that ranges from simple hepatitis to nonalcoholic fatty liver disease. In this study, we determined the attenuation of oxidative stress in liver cells with pretreatment of rosmarinic acid (RA), which is an antioxidant agent from Rosmarinus officinalis. The human liver cell line L02 was damaged by hydrogen peroxide (H₂O₂). In the RA treatment group, the viability of L02 cells increased and the intracellular reactive oxygen species levels decreased compared with the H₂O₂-induced damage group. Analysis of flow cytometry revealed that the percentage of G2/M cell cycle arrest and cell apoptosis decreased in the RA treatment group. This alteration was associated with activation of a G2/M DNA damage and oxidative stress apoptotic signal. Furthermore, we determined the redox-sensitive protein expression of mitogen-activated protein kinases (MAPKs), quinone acceptor oxidoreductase 1 (NQO1), and nuclear factor E2-related factor 2 (Nrf2), and the expression of both MAPKs and Nrf2 was activated in the RA group. Results showed that the relevant protein expression of MAPKs and Nrf2 was activated in the RA group. Thus, RA protected L02 cells from oxidative damage through suppressing cell cycle arrest and cell apoptosis with the activation of MAPK and Nrf2 signaling pathways.

Introduction

A diverse set of toxic, metabolic, and inflammatory elements causes liver damage and disease. Cell death mainly occurs by apoptosis or necrosis when the liver suffers damage and then resulting in tissue pathology such as nonalcoholic fatty liver disease (NAFLD). NAFLD ranges from simple steatosis, nonalcoholic steatohepatitis, fibrosis, liver cirrhosis to end-stage hepatocellular carcinoma (HCC).¹ In these liver pathologies, HCC is the most ordinary (70%–90%) histologic type of hepar cancer.² Besides, NAFLD has been estimated as the primary cause of liver interrelated morbidity and even an indication for liver transplantation projected.³ However, despite its high prevalence and severity, currently there is no pharmacological agent for the treatment of NAFLD.

A “Two-Hit” hypothesis has been widely proposed to explain the pathogenesis of NAFLD. The first hit involves an imbalance in the fatty acid and triglyceride metabolism in hepatocytes resulting in lipid accretion in the liver. The second hit includes oxidative stress and lipid peroxidation resulting in NASA with an increase in cytokine production and inflammation production.⁴ In the first hit, as the main store of lipids in the liver, the triglycerides are synthesized from the free fatty acids (FFA). The excessive FFA can also promote hepatocyte apoptosis through increased production of reactive oxygen species (ROS).⁵ The increase of ROS production leads to antioxidative defense decline in NAFLD. Thus, oxidative stress has been considered a key factor in the pathophysiology of NAFLD.⁶ Hydrogen peroxide (H₂O₂) is a vital cause of oxidative damage for its longer half-life than the other ROS and it can easily transform into the hydroxyl radical, one of the most destructive free radicals.⁷ Furthermore, it is generated from almost all sources of oxidative stress in many kinds of cells and tissues. In the liver, the augmented hepatic oxidative stress caused by H₂O₂ increased the progression of NAFLD.⁸ H₂O₂ has been reported to trigger apoptosis in hepatocytes.^9,10 However, the cellular and molecular mechanisms under oxidative stress are not fully understood.

There are reports that oxidative stress is intimately involved in the regulation of mitogen-activated protein kinases (MAPKs) and nuclear factor erythroid2-related factor 2 (Nrf2) signaling pathways.⁷ The close connection between oxidative stress and MAPKs and Nrf2 signaling prompted us to investigate the potential mechanism in liver pathology. The expression of MAPK, Nrf2, Keap1, and NQO1 was alternated in mouse liver after the mequindox-induced oxidative stress.¹¹ Redox-sensitive MAPKs are all serine/threonine kinases regulated by the proline residue, and MAPKs include the c-Jun N-terminal kinase (JNK), the p38 kinase (p38), and the extracellular signal-related kinases (ERK1/2).¹² Meanwhile, Nrf2 has been recognized as an important player in regulating the cellular antioxidant response. Nfr2 belongs to the cap “n” collar basic leucine zipper family. Nrf2 interacts with the Kelch-like ECH-associated protein 1 (Keap1) in the cytosol before exposure to oxidants and electrophiles. However, while under oxidative stress, Nrf2 is released and translated from the cytosol to nucleus due to modification of Cys residues of CRLKeap1.¹³

More recently, dietary antioxidants have been proposed as therapeutic agents to counteract oxidative stress-induced liver damage such as NAFLD.¹⁴ As a phenolic compound, rosmarinic acid (RA) extracted from Rosmarinus officinalis exhibits good antioxidant properties (Fig. 1). Certainly, some studies indicated that RA has several other biological activities, including antimemory deficits,¹⁵ antifibrosis,¹⁶ anticancer,¹⁷ antiradiation,¹⁸ antiaging,¹⁹ anti-inflammation,²⁰ anticonvulsant.²¹ Today, much is known about the chemistry and antioxidant potential of RA as a result of many studies. Reports have showed RA enhanced the activity of antioxidant enzymes in aging mice,²² and ameliorated acute liver damage in CCL4-intoxicated mice through ameliorating oxidative stress by activation of the Nrf2 and heme oxygenase-1 (HO-1) expression.²³ The current study was conducted to investigate the cytoprotective effect of RA against H₂O₂-induced oxidative stress and further elucidated its new potential mechanism of signal transduction on L02 cells.

FIG. 1.

Chemical structure of rosmarinic acid.

Materials and Methods

Rosmarinic acid and l-ascorbic acid (Vit.C [VC]) were purchased from Sigma-Aldrich (St. Louis, MO, USA). H₂O₂ (hydrogen peroxide) was from Guangzhou Chemical Reagent Factory. CCK-8 kit, ROS assay kit, cell cycle and annexin V-FITC apoptosis detection kit, RIPA, PMSF, primary antibody dilution, second antibody dilution, and BCA protein assay kit were all from Beyotime Biotechnology (Shanghai, China). The nuclear/cytosol fractionation kit was bought from Sangon Biotech (Shanghai, China). The primary antibodies for p-JNK, JNK, p-p38, p38, p-ERK1/2, ERK1/2, Nrf2, NQO1, laminB1, and β-actin, and the horseradish peroxidase (HRP)-linked second antibody were from Cell Signaling Technology (Boston, MA, USA).

Cell culture

The hepatic cell line L02 was obtained from the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 μg/mL streptomycin, 100 U/mL penicillin, and 2 mM l-glutamine at 95% air with 5% CO₂ in a humidified incubator. The culture medium was replaced with fresh medium every 2 to 3 days.

Cell viability assay

Cell viability was measured by the CCK-8 kit. L02 cells were seeded on 96-well plates (Corning) at a density of 2 × 10⁴ cells per well in 100 μL medium with three replicate wells. After 12 hours of incubation, the cells were pretreated with DMEM containing RA (0.25, 1, and 5 μM) or VC (100 μM) for 24 hours, and then stimulated with H₂O₂ (400 μM) for 6 hours. The RA and VC were washed away with phosphate-buffered saline (PBS) once, before stimulation with H₂O₂. Then, 10 μL of CCK-8 solution was added into each well and the optical densities at 450 nm were measured using an enzyme-labeled instrument (Thermo) after incubation at the cell culture incubator for 1 hour.

DCFH-DA fluorescence assay

Measurement of intracellular ROS was performed by the ROS assay kit. L02 cells were seeded on 96-well plates with 2 × 10⁴ cells per well in 100 μL medium and 6-well plates with 3 × 10⁵ cells per well in 2 mL medium with three replicate wells, respectively. After 12 hours of incubation, the cells were pretreated with DMEM containing RA (0.25, 1, and 5 μM) and VC (100 μM) for 24 hours, and then stimulated with H₂O₂ (400 μM) for 6 hours. The RA and VC were washed away with PBS once, before stimulation with H₂O₂. Then, the cells were incubated with 10 μM DCFH-DA for 30 minutes at 37°C in the dark. The green fluorescence of DCF in 96-well plates and 6-well plates was detected by Molecular Devices (Sutter Instrument Company, MD) and flow cytometry (CytoFLEX; Beckman Coulter), respectively, with an excitation wavelength of 485 nm and an emission wavelength of 525 nm.

Cell cycle assay

Cell cycle was measured by the cell cycle and apoptosis analysis kit. L02 cells were seeded on six-well plates at a density of 3 × 10⁵ cells per well in 2 mL medium with three replicate wells. After 12 hours of incubation, the cells were pretreated with DMEM containing RA (0.25, 1, and 5 μM) and VC (100 μM) for 24 hours, and then stimulated with H₂O₂ (400 μM) for 6 hours. The RA and VC were washed away with PBS once, before stimulation with H₂O₂. Then the cells were harvested by trypsin digestion, washed with ice bath precooling PBS and fixed with ice bath precooling 70% ethyl alcohol at 4°C overnight. Then the cells were further incubated with propidium iodide (PI) staining solution for 30 minutes at 37°C in the dark. The red fluorescence of PI was detected by flow cytometry (CytoFLEX; Beckman Coulter) with an excitation wavelength of 488 nm and an emission wavelength of 630 nm.

Cell apoptosis assay

Cell apoptosis was identified using the annexin V-FITC apoptosis detection kit. L02 cells were seeded on six-well plates at a density of 3 × 10⁵ cells per well in 2 mL medium with three replicate wells. After 12 hours of incubation, the cells were pretreated with DMEM containing RA (0.25, 1, and 5 μM) and VC (100 μM) for 24 hours, and then stimulated with H₂O₂ (400 μM) for 6 hours. The RA and VC were washed away with PBS once, before stimulation with H₂O₂. Then, cells were harvested, washed, and incubated in 195 μL of annexin V-FITC binding buffer, and then added to 5 μL of annexin V-FITC and 10 μL of PI for 20 minutes of incubation at room temperature in the dark. The percentage of apoptotic cells was detected using flow cytometry (Beckman Coulter, CytoFLEX) with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

Western blotting assay

L02 cells were seeded on six-well plates at a density of 3 × 10⁵ cells per well in 2 mL medium with three replicate wells. After 12 hours of incubation, cells were pretreated with DMEM containing RA (0.25, 1, and 5 μM) and VC (100 μM) for 24 hours, and then stimulated with H₂O₂ (400 μM) for 6 hours. The RA and VC were washed away with PBS once, before stimulation with H₂O₂. Then, cells were lysed with radioimmunoprecipitation assay (RIPA) buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF) for the total protein extraction in the nuclear and cytosol fractionation. The cells were lysed with buffer A, buffer B, buffer C from the nuclear/cytosol fractionation kit for the nuclear and cytosol fractionation protein extraction, respectively. Protein concentrations were quantified using bicinchoninic acid (BCA) hydrate disodium salt protein assay kit. The boiled proteins were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto the polyvinylidene fluoride (PVDF; Millipore) membrane using Western Semi-dry Transfer System (Bio-Rad). After being blocked with 5% skim milk in Tris-buffered saline Tween (TBST) buffer for 3 hours at room temperature, the membranes were incubated with primary antibodies for p-JNK, JNK, p-p38, p38, p-ERK, ERK, and β-actin (dilution 1:3000) overnight at 4°C, followed by a secondary antibody coupled to HRP (dilution 1:5000) for 1 hour at room temperature. Finally, the blots were developed using the enhanced chemiluminescent (ECL) detection system (Tanon, China).

Statistical analysis

All experiments were performed independently three times. Statistical analysis was performed by using PRISM 5.0 (GraphPad software). Group differences were assessed by the Student's t-test or ANOVA multiple comparisons. p Values of less than 0.05 were considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001).

Results

The protective effect of RA on the viability of H₂O₂-induced L02 cells

Exposure of L02 cells to various concentrations of H₂O₂ for 6 hours resulted in a dose-dependent descending in cell viability (Fig. 2A). Figure 2A shows that H₂O₂ of 400 μM could cause a 40.06% ± 5.59% decrease in cell viability. Thus, 400 μM was selected as the oxidative damage concentration for later experimentations. While the exposure of L02 cells to H₂O₂ (400 μM) for 6 hours significantly decreased cell viability, the RA-containing medium in addition significantly increased cell viability (Fig. 2B). The antioxidant VC (100 μM) also increased the viability of H₂O₂-induced L02 cells, and it was used as positive control (Fig. 2B). These results suggested that RA protected L02 cells from H₂O₂-induced oxidative damage.

FIG. 2.

Cell viabilities of L02 cells were assayed by CCK-8. (A) Cell viability of L02 cells with exposure to different concentrations of H₂O₂ was measured. (B) Cell viability of L02 cells in each group was measured. Control group: normal control group; H₂O₂: oxidative damage group; Vit.C: VC at 100 μM; RA group: RA at 0.25, 1.00, and 5.00 μM. Data are represented as mean ± SD, n = 3. *p < 0.05, and ***p < 0.001 versus the H₂O₂ group. RA, rosmarinic acid.

RA reduced H₂O₂-induced ROS production

H₂O₂-induced L02 cells robustly augmented the ROS production, while RA-containing serum (5.00 μM) drastically reduced ROS levels in the damaged cells, as shown in Figure 3A–C. The antioxidant VC (100 μM) also showed the significant effects of inhibiting H₂O₂-induced ROS production, and it was used as positive control (Fig. 3A–C). These results showed that RA protected L02 cells by decreasing the ROS production. The potential cytoprotective effect might be the involvement of the induction of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), total glutathione (GSH), and malondialdehyde (MDA).²⁴

FIG. 3.

The ROS production in L02 cells was assayed in each group. (A) Flow cytometric analysis of the stained ROS in L02 cells in each group. (B) Overlapped chart of the representative ROS intensities in each group on the left and calculated DCF-stained cell intensities on the right. (C) ROS fluorescent representative microscopic images were taken with Molecular Devices. Control groups: normal control group; H₂O₂: oxidative damage group; Vit.C: Vit.C at 100 μM; RA group: RA at 0.25, 1.00, and 5.00 μM. Data are represented as mean ± SD, n = 3. ***p < 0.001 versus the H₂O₂ group. ROS, reactive oxygen species.

RA inhibited cell G2/M phase arrest progression and cell apoptosis caused by oxidative stress

To further investigate the protective effect of RA against oxidative stress in L02 cells, cell cycle was performed. As shown in Figure 4A and C, the percentage of G2/M cell cycle arrest was 19.55% ± 0.69% in the H₂O₂-induced damage group, while only 3.53% ± 0.98% in the normal control group. The result suggested a G2/M cell cycle arrest in the H₂O₂-induced damage group compared with the normal control group. In the RA treatment group, the G2/M phase was partially blocked to 12.46% ± 3.19%, 11.33% ± 3.98%, 8.6% ± 0.95% (at 0.25, 1.00, and 5.00 μM), respectively. Also, the percentage of cells in S phase increased by 15.66% ± 1.21%, 18.10% ± 3.97%, and 19.13% ± 1.92%, while the percentage of cells in G0/G1 phase decreased by 8.59% ± 3.69%, 9.88% ± 0.30%, and 8.10% ± 1.55% compared with the H₂O₂-induced damage group, which was in association with the changed percentage of the G2/M cell cycle arrest. The antioxidant VC (100 μM) showed a similarity to the effect of RA, and it was used as positive control. The results suggested that RA might decrease the percentage of the G2/M cell cycle arrest against oxidative damage in L02 cells.

FIG. 4.

Cell cycle arrest and cell apoptosis in L02 cells were assayed in each group. (A) Flow cytometric analysis of the cell cycle of L02 cells in each group. The left red area means cell intensities in G0/G1 phase, the hatched area means those in S phase, and the right red area means those in G2/M phase. (B) Flow cytometric analysis of the early and late apoptosis cells in each group. The x-axis is FITC-stained cell intensities, and y-axis is the PI-stained cell intensities. The early apoptosis cells are in Q2 and the late apoptosis cells are in Q3. (C) Fluorometric quantification of cell cycle arrest and the apoptosis cells. Control groups: normal control group; H₂O₂: oxidative damage group; Vit.C: Vit.C at 100 μM; RA group: RA at 0.25, 1.00, and 5.00 μM. Data are represented as mean ± SD, n = 3. ***p < 0.001 versus the H₂O₂ group. PI, propidium iodide.

To determine whether RA reduced H₂O₂-induced cell apoptosis, cell apoptosis was performed. The percentage of apoptotic cells significantly increased in the H₂O₂-induced oxidative damage group, compared with the normal control group (39.36% ± 0.37% vs. 14.46% ± 0.87%, Fig. 5B, C). However, pretreatment of L02 cells with RA (at 0.25, 1.00, and 5.00 μM) reduced the proportion of apoptotic cells to 20.0% ± 0.58%, 16.89% ± 1.45%, and 16.03% ± 1.52%, respectively. The antioxidant VC (100 μM) also distinctly reduced cell apoptosis, and it was used as positive control (Fig. 4B, C). The results suggested that RA might decrease cell apoptosis against oxidative damage in L02 cells.

FIG. 5.

Western blotting analyses were conducted in each group. (A) Expression of JNK, p38, and ERK in MAPK signaling pathways was assessed. (B) Expression of Nrf2 and NQO1in Nrf2 signaling pathway was assessed. (C) Expression of Nrf2 translocation from the cytoplasm to the nucleus was assessed by Western blotting. (D) The quantification of immunoblotting detected by ImageJ. Control groups: normal control group; H₂O₂: oxidative damage group; Vit.C: Vit.C at 100 μM; RA group: RA at 0.25, 1.00, and 5.00 μM. Data are represented as mean ± SD, n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001. ERK, extracellular signal-related kinases; JNK, c-Jun N-terminal kinases; MAPK, mitogen-activated protein kinase; Nrf2, nuclear factor E2-related factor 2.

RA attenuated oxidative damage by the activation of MAPK and Nrf2 signaling pathways

To further determine whether RA attenuated oxidative damage in L02 cells by inducing various signaling pathways, the expression of some oxidative stress-related major genes (e.g., MAPK, Nrf2, NQO1) was evaluated using Western blotting. Exposure to H₂O₂ significantly increased the phosphorylation of JNK, while RA and VC could reverse the JNK protein expression (Fig. 5A, D). In Figure 5A and D, the p38 protein expression was downregulated in both the RA treatment group and the H₂O₂-induced damage group, but the p38 protein expression decreased more in the RA treatment group than the H₂O₂-induced damage group to protect L02 cells from oxidative stress. The ERK1/2 protein expression was upregulated in both the RA treatment group and the H₂O₂-induced damage group, but the ERK1/2 protein expression increased more in the RA treatment group than the H₂O₂-induced damage group to protect L02 cells from oxidative stress.

The expression of Nrf2 and NQO1 was significantly induced in the RA (5 μM) treatment group than the H₂O₂-induced oxidative damage group (Fig. 5B, D). To further investigate the activation role of RA on Nrf2, we examined whether RA facilitated Nrf2 translocation from the cytoplasm to the nucleus. LaminB1 was used as reference protein in the nucleus, and β-actin was used as reference protein in the cytoplasm. As shown in Figure 5C and D, Nrf2 protein expression was induced in the nuclear fraction and cytosolic fraction, and the nuclear fraction increased more than the cytosolic fraction relatively in the RA treatment group, which indicated that RA could activate Nrf2 through upregulating its phosphorylation and facilitating its nuclear translocation. The antioxidant VC (100 μM) also activated the MAPK and Nrf2 signaling pathways, and it was used as positive control. As a result, RA showed a cellular protection response to oxidative stress by regulation of the MAPK and Nrf2 signaling pathways.

Discussion

NAFLD remains a high prevalence and rising incidence worldwide. ROS generate in microsomes, peroxisomes, and mitochondria during liver damage. Oxidant formulation has been suggested to be interrelated in the pathogenesis and progression of NAFLD, and several reports highlighted the efficacy of antioxidants for its treatment. For example, the antioxidant supplementation provided a significant attenuation of insulin resistance in overweight patients with liver injury.²⁵ Also, antioxidant interventions, which include vitamin E, vitamin D, coenzyme Q, polyphenols, polyunsaturated fatty acids, and preprobiotics, have enhanced antioxidant defense in NAFLD.²⁶ In particular, RA represents a highly effective and reliable property for the treatment of liver damage than the clinical drug of silymarin and N-acetyl-l-cysteine (NAC) compounds.²³ In the present study, antioxidant intervention RA could protect L02 cells from oxidative stress-induced damage and increase cell viabilities.

Consistent with that ROS can promote pathologic polyploidization and cell cycle arrest in the liver cells, subsequently leading to cell death by apoptosis and necrosis. The previous report showed that hepatocytes from oxidative stress-induced NAFLD mice increased the G2/M phase progression, while the antioxidant-treated NAFLD hepatocytes returned to the normal cell division.²⁷ Besides, it has been recently described that in the research models of oxidative stress-induced liver damage, cell death by apoptosis is currently occurring.²⁸ Our results showed that RA decreased G2/M phase progression and subsequent cell death, which is in agreement that cell cycle arrest increases the likelihood of cell death during ROS accumulation.²⁹

Redox-sensitive MAPKs interact with ROS generation and cell apoptosis,³⁰ and meanwhile, transcription factor Nrf2 also plays an important role against oxidative stress.³¹ As we know, oxidative stress could activate simple liver damage to the progress of NAFLD, and we further explored whether RA alleviated oxidative stress via the MAPK and Nrf2 signaling pathways in liver damage. The results suggested that RA would protect liver cells against damage by activation of MAPK and Nrf2 expression. MAPKs contribute to cell proliferation, survival, and apoptosis. In particular, the phosphorylation of p38 and JNK could induce apoptosis, while the phosphorylation of ERK1/2 protects oxidative stress-mediated cell damage.³² Our research indicated that RA may alleviate liver cell oxidative damage by inhibiting JNK and p38 expression, and inducing ERK1/2 expression. Besides, MAPKs might regulate Nrf2 in hepatic apoptosis, which is in agreement that coptisineagainst AAPH (2,2′-azobis[2-methylpropionamidine] dihydrochloride)-induced oxidative stress.³³ Oxidative modification induces the Nrf2-Keap1 complex to suppress Nrf2 degradation to bind to antioxidant response elements (ARE) and facilitates nuclear translocation. Results in the study showed that RA could protect liver cells from oxidative damage by activating expression of Nrf2, facilitating nuclear translocation, and inducing its downstream target gene expression of NQO1.

Conclusions

As previously reported, the upregulation of p38, JNK in MAPKs and the downregulation of ERK1/2 in MAPKs could induce hepatic apoptosis, while the upregulation of Nrf2 and its downstream target gene NQO1 protein expression could decrease hepatic apoptosis.^34,35 The inhibition of MAPK did not induce Nrf2 and HO-1 expression in the liver.^34,36,37 In summary, RA could decrease H₂O₂-induced oxidative damage to build a new redox homeostasis through decreasing cell cycle arrest and activating the MAPK and Nrf2 signaling pathways to suppress apoptosis (Fig. 6). These findings provided a new evidence to support RA as a potential candidate for the prevention or treatment of liver cell oxidative damage and related metabolic disorders such as NAFLD.

FIG. 6.

The proposed mechanisms of RA alleviating oxidative stress in L02 cells. ARE, antioxidant response elements; NAFLD, nonalcoholic fatty liver disease.

Footnotes

Acknowledgments

This work was supported by grants from the Special Funds of the Central Finance to Support the Development of Local Universities and Colleges, and Guangdong Province Department of Education (Grant 2015KGJHZ022).

Authors' Contributions

Y.D. was the principal investigator of the study and the writer of the original article, Z.Z. performed statistical analysis, Z.Y. and L.D. contributed to some experimental design, and H.H. and Z.H. contributed to the study design and helped in the review and editing of the original article.

Author Disclosure Statement

No competing financial interests exist.

References

Guicciardi

, Malhi

, Mott

, Gores

. Apoptosis and necrosis in the liver. Compr Physiol, 2013; 3:977–1010.

Temple

, Cordero

, Li

, Nguyen

, Oben

. A guide to non-alcoholic fatty liver disease in childhood and adolescence. Int J Mol Sci, 2016; 17:947.

Calzadilla Bertot

, Adams

. The natural course of non-alcoholic fatty liver disease. Int J Mol Sci, 2016; 17:774.

Ganji

, Kashyap

, Kamanna

. Niacin inhibits fat accumulation, oxidative stress, and inflammatory cytokine IL-8 in cultured hepatocytes: Impact on non-alcoholic fatty liver disease. Metabolism, 2015; 64:982–990.

Machado

, Cortez-Pinto

. Non-alcoholic fatty liver disease: What the clinician needs to know. World J Gastroenterol, 2014; 20:12956–12980.

Abdelmegeed

, Banerjee

, Yoo

S-H

, Jang

, Gonzalez

, Song

B-J

. Critical role of cytochrome P450 2E1 (CYP2E1) in the development of high fat-induced nonalcoholic steatohepatitis. J Hepatol, 2012; 57:860–866.

Schieber

, Chandel

. ROS function in redox signaling and review oxidative stress. Curr Biol, 2014; 24:453–462.

Koliaki

, Szendroedi

, Schlensak

, Roden

. Adaptation of hepatic mitochondrial function in humans with non-alcoholic fatty liver is lost in steatohepatitis. Cell Metab, 2015; 21:739–746.

Trinh

, Ngo

, Tran

, et al. Prevention of H₂O₂-induced oxidative stress in Chang liver cells by 4-hydroxybenzyl-chitooligomers. Carbohydr Polym, 2014; 103:502–509.

10.

Chen

, Ye YY

G, Liu

. hesperidin upregulates heme oxygenase-1 to attenuate hydrogen peroxide-induced cell damage in hepatic L02 cells. J Agric Food Chem, 2010; 58:3330–3335.

11.

Liu

, Lei

, Huang

, et al. Toxic metabolites, MAPK and Nrf2/Keap1 signaling pathways involved in oxidative toxicity in mice liver after chronic exposure to Mequindox. Sci Rep, 2016; 7:41854.

12.

Shin

H-J

, Kwon

H-K

, Lee

J-H

, Anwar

, Choi

. Etoposide induced cytotoxicity mediated by ROS and ERK in human kidney proximal tubule cells. Sci Rep, 2016; 6:34064.

13.

Dayoub

, Vogel

, Schuett

, et al. Nrf2 activates augmenter of liver regeneration (ALR) via antioxidant response element and links oxidative stress to liver regeneration. Mol Med, 2013; 19:237–244.

14.

Nunes

, Madureira

, Campos

, et al. Therapeutic and nutraceutical potential of rosmarinic acid—Cytoprotective properties and pharmacokinetic profile. Crit Rev Food Sci Nutr, 2015; 19:1799–1806.

15.

Fonteles

, de Souza

, Julliana de Sousa Neves

, et al. Rosmarinic acid prevents against memory deficits in Ischemic mice. Behav Brain Res, 2016; 297:91–103.

16.

Yang

, Chiang

Y-M

, Higashiyama

, et al. Rosmarinic acid and baicalin epigenetically de-repress Pparγ in hepatic stellate cells for their anti-fibrotic effect. Hepatology, 2012; 55:1271–1281.

17.

Moore

, Yousef

, Tsiani

. Anticancer effects of rosemary (Rosmarinus officinalis L.) extract and rosemary extract polyphenols. Nutrients, 2016; 8:731.

18.

, Yang

, Zhang

, Shen

. Protective effects of rosmarinic acid against radiation-induced damage to the hematopoietic system in mice. J Radiat Res, 2016; 57:356–362.

19.

Shan

, Wang

D-D

, Xu

Y-X

, et al. Aging as a precipitating factor in chronic restraint stress-induced Tau aggregation pathology, and the protective effects of Rosmarinic Acid. J Alzheimers Dis, 2016; 49:829–844.

20.

Liang

, Xu

, Wen

, et al. Rosmarinic acid attenuates airway inflammation and hyperresponsiveness in a murine model of asthma. Molecules, 2016; 21:769.

21.

Grigoletto

, Oliveira

, Grauncke

, et al. Rosmarinic acid is anticonvulsant against seizures induced by pentylenetetrazol and pilocarpine in mice. Epilepsy Behav, 2016; 62:27–34.

22.

Zhang

, Chen

, Yang

, Zu

, Lu

. Effects of rosmarinic acid on liver and kidney antioxidant enzymes, lipid peroxidation and tissue ultrastructure in aging mice. Food Funct, 2015; 6:927–931.

23.

Domitrović

, Škoda

, Vasiljev Marchesi

, Cvijanović

, Pernjak Pugel

, Stefan

. Rosmarinic acid ameliorates acute liver damage and fibrogenesis in carbon tetrachloride-intoxicated mice. Food Chem Toxicol, 2013; 51:370–378.

24.

Bacanlı

, Aydın

, Taner

, et al. Does rosmarinic acid treatment have protective role against sepsis-induced oxidative damage in Wistar Albino rats?. Hum Exp Toxicol, 2016; 35:877–886.

25.

Abenavoli

, Greco

, Milic

, et al. Effect of Mediterranean diet and antioxidant formulation in non-alcoholic fatty liver disease: A randomized study. Nutrients, 2017; 9:870.

26.

Dongiovanni

, Lanti

, Riso

, Valenti

. Nutritional therapy for nonalcoholic fatty liver disease. J Nutr Biochem, 2016; 29:1–11.

27.

Gentric

, Maillet

, Paradis

, et al. Oxidative stress promotes pathologic polyploidization in nonalcoholic fatty liver disease. J Clin Invest, 2015; 125:981–992.

28.

Brenner

, Galluzzi

, Kepp

, Kroemer

. Decoding cell death signals in liver inflammation. J Hepatol, 2013; 59:583–594.

29.

Matés

, Segura

, Alonso

, Márquez

. Intracellular redox status and oxidative stress: Implications for cell proliferation, apoptosis, and carcinogenesis. Arch Toxicol, 2008; 82:273–299.

30.

Chowdhury

, Chatterjee

, Giri

, Mandal

, Chaudhuri

. Arsenic-induced cell proliferation is associated with enhanced ROS generation, Erk signaling and CyclinA expression. Toxicol Lett, 2010; 198:263–271.

31.

Pilar Valdecantos

, Prieto-Hontoria

, Pardo

, et al. Essential role of Nrf2 in the protective effect of lipoic acid against lipoapoptosis in hepatocytes. Free Radic Biol Med, 2015; 84:263–278.

32.

Ling

, Zhang

, Tan

, et al.: Protective effects of Oviductus Ranae on oxidative stress-induced apoptosis in rat ovarian granulosa cells. J Ethnopharmacol. 2017; 208:138–148.

33.

Y-R

, Ma

, Zou

Z-Y

, et al. Activation of Akt and JNK/Nrf2/NQO1 pathway contributes to the protective effect of coptisine against AAPH-induced oxidative stress. Biomed Pharmacother, 2017; 85:313–322.

34.

Lee

I-C

, Ko

J-W

, Lee

S-M

, et al. Time-course and molecular mechanism of hepatotoxicity induced by 1,3-dichloro-2-propanol in rats. Environ Toxicol Pharmacol, 2015; 40:191–198.

35.

Zhang

, Ni

, Kong

, et al. Ligustrazine attenuates oxidative stress-induced activation of hepatic stellate cells by interrupting platelet-derived growth factor-β receptor-mediated ERK and p38 pathways. Toxicol Appl Pharmacol, 2012; 265:51–60.

36.

Zhang

, Yuan

, Huang

, Qu

, Yang

, Zeng

. Gastrodin induced HO-1 and Nrf2 up-regulation to alleviate H₂O₂-induced oxidative stress in mouse liver sinusoidal endothelial cells through p38 MAPK phosphorylation. Braz J Med Biol Res, 2018; 51:7439.

37.

Choi

, Lee

, Jegal

, Cho

, Kim

. Oxyresveratrol abrogates oxidative stress by activating ERK–Nrf2 pathway in the liver. Chem Biol Interact, 2016; 245:110–121.

Rosmarinic Acid Ameliorates H 2 O 2 -Induced Oxidative Stress in L02 Cells Through MAPK and Nrf2 Pathways

Abstract

Introduction

Materials and Methods

Cell culture

Cell viability assay

DCFH-DA fluorescence assay

Cell cycle assay

Cell apoptosis assay

Western blotting assay

Statistical analysis

Results

The protective effect of RA on the viability of H2O2-induced L02 cells

RA reduced H2O2-induced ROS production

RA inhibited cell G2/M phase arrest progression and cell apoptosis caused by oxidative stress

RA attenuated oxidative damage by the activation of MAPK and Nrf2 signaling pathways

Discussion

Conclusions

Footnotes

Acknowledgments

Authors' Contributions

Author Disclosure Statement

References

The protective effect of RA on the viability of H₂O₂-induced L02 cells

RA reduced H₂O₂-induced ROS production