Taraxerol protects the human hepatic L02 cells from hydrogen peroxide-induced apoptosis

School of Food and Biology Engineering, Bengbu University, Bengbu, China.

Abstract

Taraxerol is known to exhibit anti-inflammatory and anti-cancer activity. However, cytoprotective effect of taraxerol on hepatocytes has not been reported. In the present study, we investigated the hepatoprotective effect of taraxerol in the human hepatic L02 cells injured by hydrogen peroxide (H₂O₂). Taraxerol decreased H₂O₂-induced cell viability loss and lactate dehydrogenase release. Taraxerol also inhibited H₂O₂-induced cell apoptosis. Further, taraxerol attenuated H₂O₂-induced increase in cleaved-caspase-3 and cleaved-PARP. H₂O₂-activated p38 and JNK were also inhibited by taraxerol. These data suggest that taraxerol could protect the L02 cells against H2O2-induced apoptosis via suppression of p38 and JNK. Taraxerol may be an effective protective agent against oxidative stress-induced liver injury.

Introduction

Hydrogen peroxide (H₂O₂) is an important member of the reactive oxygen species (ROS) family of molecules that have been investigated in recent years (Brewer et al., 2015). Low levels of H₂O₂ are required for many biochemical processes, such as cell differentiation, immunity, antimicrobial infection (Veal and Day, 2011). However, excessive increase of H₂O₂ can lead to mitochondrial damage, lipid peroxidation, cytokine release, and cell death, which are involved in cancers, diabetes, rheumatisms and various neurological disorders (Gough and Cotter, 2011). Liver damage is often accompanied by apoptosis of liver cells (Guicciardi and Gores, 2010). Therefore, anti-oxidants may reverse oxidative stress-induced cell death in liver cells.

Taraxerol is a triterpenoid isolated from many medicinal plants including Mangifera indica, Taraxacum japonicum, Achillea millefolium and Acrocarpus andfraxinifolius (Sharma and Zafar, 2015). Taraxerol is also known to exhibit anti-inflammatory and anti-cancer activity (Tsao et al., 2008; Takasaki et al., 1999; Setzer et al., 2000; Jang et al., 2004). We have also reported that taraxerol significantly inhibited LPS-induced production of pro-inflammatory mediators by preventing the activation of TAK1, Akt and NF-κB (Yao et al., 2013). Taraxerol has an antidiabetic effect. It can be used as a PI3K dependent dual activator of glucose transport and glycogen synthesis and effectively restored dexamethasone-induced desensitization via restoration of PI3K and GLUT4 expression (Sangeetha et al., 2013; Sangeetha et al., 2010). However, cytoprotective effect and its mechanism of taraxerol on hepatocytes are unclear. In this study, we assessed taraxerol on H₂O₂-induced apoptotic effect in L02 cells, and determined the activation of caspases-3, PARP, Bcl-2, Bax and MAPK.

Materials and Methods

Chemicals

Taraxerol (99% purity analyzed by HPLC), 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (USA). Lactate dehydrogenase (LDH) assay kit was obtained from Nanjing Jiancheng Bioengineering Institute (China). The antibodies to Bcl-2, Bax, cleaved-caspase-3, PARP and polyclonal antibodies to p38, JNK/SAPK, and phospho-specific antibodies against JNK (Thr183/Tyr185), p38, U0126, SB203580, SP600125 were purchased from Cell Signaling Technology (USA). GAPDH antibody was from Bioworld Biotechnology (China).

Cell culture and cell treatment

The normal human hepatic cell strain, L02 obtained from Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences (China), was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone) supplemented with 10% fetal bovine serum (FBS) (Hyclone), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a 5% CO₂ humidified environment. Taraxerol was dissolved in DMSO to make a stock of 100 mM and further diluted to final concentrations of 20-200 μM with a serum-free culture medium.

Cell viability and LDH assay

L02 cells were seeded into 96-well plates (10⁵ cells/well) for 12 hours, followed by treatment with various concentrations of H₂O₂ or taraxerol. Cell viability was determined using MTT assay. The absorbance was measured at 570 nm. The LDH levels in the medium were measured with the use of commercial kits according to the manufacturer’s protocols after each treatment. The absorbance at 420 nm was recorded for the calculation of LDH activity under Mutiskan Go (Thermo, INC).

Flow cytometric analysis

The extent of apoptosis was measured through annexin V-FITC/PI apoptosis detection kit (Nanjing Keygen Biotech, KGA108) according to the manufacture's instruction. Cells were washed twice with PBS, gently resuspended in binding buffer and incubated with annexin-V-FITC and PI in the dark for 10 min and detected by flow cytometry (BD Accuri C6). The data were analyzed using BD Accuri C6 Software.

Western blot analysis

Cells were rinsed twice with ice-cold PBS, and lysed with the RIPA lysis buffer for 30 min on ice. Lysates were centrifuged (12,000 x g) at 4°C for 15 min. Total cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes or polyvinylidene difluoride membranes. Membranes were blocked for 60 min at room temperature. Membranes were probed with specific primary antibodies overnight at 4°C and subsequently incubated with IRDye 800CW secondary antibodies for 1 hour. Membranes were visualized using Odyssey Infrared Imaging Scanner. The fluorescence intensities were analyzed using Image Studio Software (Li-Cor Biosciences) or membranes were incu-bated with horseradish peroxidase-conjugated secondary antibodies. Band quantifications were performed by ChemiScope 3400 chemiluminescence imaging systems (Clinx Science Instruments, China).

Statistical analysis

Results were expressed as mean ± SD from replicate experiments. Statistical analysis was carried out using an unpaired, two-tailed Student's t-test. Significance was defined as p<0.05 or 0.01.

Results

Effect on the cell viability

The effect of H₂O₂ and taraxerol on the viability of L02 cells was evaluated by the MTT assay. Consistent with our previous report, H₂O₂ significantly inhibited L02 cell viability (Figure 1A) (Li et al., 2011). However, as shown in Figure 1B, taraxerol alone did not significantly affect the cell viability of L02 cells even at the dose of 200 μM.

Effect on H₂O₂-induced cell viability and LDH leakage

To explore the effect of taraxerol on H₂O₂-induced cell viability loss, L02 cells were pretreated with taraxerol at different concentrations for 1 hour, followed by 0.4 mM H₂O₂ treatment for 12 hours. As shown in Figure 2A, H₂O₂-induced loss of cell viability was significantly decreased by taraxerol in a dose-dependent manner. LDH leakage which was frequently used to evaluate the degree of cellular injury was assayed. H₂O₂ stimulation significantly increased LDH leakage in L02 cells (Figure 2B). Taraxerol prevented H₂O₂-induced LDH release.

H₂O₂-induced apoptosis

We further investigated apoptosis of cells by annexin-V/PI double staining. After being incubated with 40 μM taraxerol for 24 hours, H₂O₂ could significantly increase in the early and late stages of apoptotic cells (12.1, 40.1%) compared with the control group (1.2, 4.3%) indicating that cells were significantly damaged, while cells treated with taraxerol and H₂O₂ were significantly lower in the early and late stages of apoptotic cells (4.3, 22.4%) than those in the H₂O₂-stimulated group (Figure 3). These results demonstrated that taraxerol protected H₂O₂-induced apoptosis in L02 cells.

Cleavage of caspase-3 and PARP in H₂O₂-injured cells

We examined the expression of cleaved-caspase-3 and PARP. The results showed that the protein of cleaved-caspase-3 and cleaved-PARP were up-regulated in H₂O₂-treated L02 cells, showing that the cells were undergoing apoptosis. However, pretreatment with taraxerol inhibited the increase in the two proteins induced by H₂O₂ (Figure 4). These data are in agreement with the results of annexin-V/PI double staining.

Effects on H₂O₂-induced Bcl-2 and Bax

As shown in Figure 5, Bcl-2 protein expression was down-regulated while Bax was up-regulation in H₂O₂-treated L02 cells. In contrast, taraxerol could inhibit the expression of Bax induced by H₂O₂, and increased the expression of Bcl-2. Thus, Bax/Bcl-2 ratio was significantly lower than in H₂O₂-treated L02 cells alone.

H₂O₂-induced activation of p38 and JNK

In our previous study, taraxerol markedly repressed LPS-induced p38, ERK and JNK phosphorylation in RAW264.7 cells (Yao et al., 2013). To verify that MAPKs are involved in the apoptosis, cells were pretreated with 30 μM inhibitors (U0126, an ERK inhibitor; SB203580, a p38 inhibitor or SP600125, a JNK inhibitor) for 1 hour, followed by addition of H₂O₂. As shown in Figure 6A, increase in cleaved-caspase-3 and cleaved-PARP were prevented by SB203580 and SP600125, while they were no significant change in the U0126-pretreated cells when compared with H₂O₂-treated cells alone, indicating that p38 and JNK are involved in H₂O₂-induced cell apoptosis in L02 cells.

We further explored whether taraxerol affected the activation of p38 and JNK induced by H₂O₂. Cells were treated with H₂O₂, JNK and p38 activation increased, while cells were pretreated with taraxerol and H₂O₂, p38 and JNK activation reduced in L02 cells compared with the H₂O₂-treated group (Figure 6B).

Discussion

In the present study, we showed that taraxerol dose-dependently decreased H₂O₂-induced cell viability loss and lactate dehydrogenase (LDH) release. Taraxerol inhibited H₂O₂-induced cell apoptosis. Further, taraxerol attenuated H₂O₂-induced increase in cleaved-caspase-3 and cleaved-PARP. H₂O₂-activated p38 and JNK were also inhibited by taraxerol. These data suggest that taraxerol could protect L02 cells against H₂O₂-induced apoptosis via suppression of p38 and JNK. Taraxerol might potentially be useful in the prevention and treatment of liver diseases.

Bax/Bcl-2 ratio plays an important role in cell apoptosis (Cory and Adams, 2005). Some studies have shown that H₂O₂-induced apoptosis is usually related to the ratio of Bax/Bcl-2 (Liu et al., 2013; Huang et al., 2015). In this study, we found that taraxerol significantly inhibited the expression of Bax and promotion of Bcl-2 in L02 cells after H₂O₂ stimulation, indicating that taraxerol inhibited H₂O₂-induced apoptosis which is associated with decreasing the ratio of Bax/Bcl-2.

It is well known that increased ROS production leads to the activation of ERK, JNK, or p38 (McCubrey et al., 2006). ERK signaling promotes cell survival under mild oxidative stress, whereas JNK and p38 can induce apoptosis by involving direct phosphorylation of pro/anti-apoptotic Bcl-2 family members, transactivation of transcription factor AP-1, and stabilization of p53(Groeger et al., 2009). We showed taraxerol protects L02 cells from H₂O₂-induced apoptosis by inhibiting JNK and p38. Our previous study demonstrated that L-theanine could protect L02 cells against H₂O₂-induced apoptosis via suppression of p38 (Li et al., 2011). However, propofol protects hepatic L02 cells from H₂O₂-induced apoptosis via activation of ERK pathway (Wang et al., 2008). These data support the view that the MAPK pathway in H₂O₂-induced apoptosis is complex. Fisetin, a dietary flavonoid induces apoptosis via modulating the MAPK and PI3K/Akt signalling pathways in human osteosarcoma cells (Li et al., 2015).

Furthermore, hesperidin augmented cellular anti-oxidant defense capacity through the induction of HO-1 via ERK/Nrf2 signaling (Chen et al., 2010). Hyperoside attenuates H₂O₂-induced L02 cell damage via MAPK (p38 and ERK)-dependent Keap/Nrf2/ARE signaling pathway (Xing et al., 2011). Thus, whether taraxerol-stimulated cytoprotective effect is involved in MAPK-dependent Keap/Nrf2/ARE signaling pathway remains elusive. On the other hand, Mantzaris reported that iron determines H₂O₂-induced apoptotic signaling by sustaining the ASK1/JNK-p38 axis (Mantzaris et al., 2016). Further investigation is needed to identify up-stream regulatory proteins such as ASK1 in protective mechanism of taraxerol in H₂O₂-induced apoptosis.

Conclusion

Taraxerol protects human hepatic L02 cells from hydro-gen peroxide-induced cell injury including promotion of cell viability, the inhibition of apoptosis and LDH release by the activation of caspases-3 and PARP, down-regulation of Bax/Bcl-2 ratio. It inhibits p38 and JNK signal pathway activated by H₂O₂. Taraxerol may be an effectively protective agent against oxidative stress-induced liver injury.

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Available online on April 20, 2017