Inhibitory Effect of Delphinidin on Oxidative Stress Induced by H2O2 in HepG2 Cells

Chronic liver diseases (CLDs) are correlated with oxidative stress induced by the accumulation of intracellular reactive oxygen species (ROS). In this study, we employed HepG2, a human liver carcinoma cell line containing many antioxidant enzymes, to explore the function of delphinidin against oxidative stress induced by H2O2 and to provide scientific data of the molecular mechanism. Cells were pretreated with different concentrations of delphinidin (10 μmol/L, 20 μmol/L, and 40 μmol/L) for 2 h before treatment with 750 μM H2O2 for 1 h. The results showed that H2O2 decreased the survival rate of HepG2 cells and increased the level of ROS, but delphinidin pretreatment could possess the opposite result. At the same time, the expression of Nrf2 was enhanced by the delphinidin pretreatment. This was because delphinidin promoted Nrf2 nuclear translocation and inhibited its degradation, which led to the increase expression of antioxidant protein HO-1 (Nrf2-related phase II enzyme heme oxygenase-1). Besides, we found that delphinidin could significantly alleviate the reduction of Nrf2 protein levels and the accumulation of intracellular ROS levels in Nrf2 knockdown HepG2 cells. In conclusion, our study suggested that delphinidin, as an effective antioxidant, protected HepG2 cells from oxidative stress by regulating the expression of Nrf2/HO-1.


Introduction
The death toll of chronic liver disease (CLDs) associated with cirrhosis, liver cancer, hepatitis, etc. is on the rise worldwide [1]. Increasing evidences confirmed the contributory role of oxidative stress in the pathogenesis of CLDs. As the common cause of oxidative stress [2,3], reactive oxygen species (ROS) are a collective name for molecules containing oxidative reactions, including superoxide anions (O 2 -), hydroxyl radicals (OH), hypochlorous acid (HOCl), ozone (O 3 ), and hydrogen peroxide (H 2 O 2 ) [4].
There is an adaptive and dynamic antioxidant defense system to eliminate excessive ROS and protect cells against oxidative stress in human body. Antioxidants, antioxidant enzymes, and phase II detoxifying enzymes are included in this system. Previous studies have shown that the phase II detoxification enzymes consisted of glutathione S-transferase (GST), NAD(P)H quinone oxidoreductase-1 (NQO1), and heme oxygenase-1 (HO-1) which could scavenge free radicals and electrophiles [5]. In addition, the antioxidant response element (ARE), a common nucleotide sequence in the promoter regions of phase II detoxifying enzymes, could be effectively activated by nuclear factor erythroid 2-related factor 2 (Nrf2) and could regulate the expression of its downstream genes [6].
As a key transcription factor, Nrf2 regulates the response of intracellular antioxidant and plays a crucial role in homeostasis maintenance [7]. Under normal conditions, Nrf2 is stored in the cytoplasm in an inactive state until it is coupled with Kelch-like ECH-associated protein 1 (Keap1) and rapidly degraded by the ubiquitin-protease system. Thereby the expression of Nrf2 is stable and the transcriptional activity is low at this time. While under oxidative stress conditions, Nrf2 is responsible for its separation with Keap1 and transfers into the nucleus before recognizing and binding with the ARE which regulates the transcription of downstream biphasic detoxification enzymes and antioxidant protein genes, such as GST, NQO1, and HO-1. Previous studies demonstrated that Nrf2 had protective effects against oxidative stressinduced diseases such as cancer [8], diabetes [9], respiratory diseases [10], chronic inflammation [11], cardiovascular diseases [12], and neurodegenerative diseases [13]. Other than that, several studies previously revealed that polyphenols activated the Nrf2/Keap1 pathway through different mechanisms to exert antioxidant activity such as quercetin [14,15], epigallocatechin gallate [16], baicalein [17], and resveratrol [18].
Anthocyanins, which are widely distributed in edible plants, are a benefit to health [19][20][21]. For instance, anthocyanins have an effective function in neurological diseases, hypoglycemia, cardiotoxicity, and anti-inflammation [22][23][24]. Delphinidin, a natural anthocyanin, is one of the most valuable polyphenols due to its high antioxidant activity. Kim et al. previously revealed that delphinidin possessed antiangiogenic function potential [25]. Moreover, delphinidin could inhibit the proliferation of SKOV3 cells (ovarian cancer cells) [26] and it could prevent prostate cancer via inhibiting the apoptosis mediated by p53 [27]. Lee et al. reported that delphinidin could protect chondrocytes against H 2 O 2 -mediated oxidative stress by activating NF-κB and Nrf2 [28]. However, there is a lack of data on how delphinidin regulates Nrf2 to exert the function of antioxidant stress. In this study, we intend to use H 2 O 2 to construct a cell model of oxidative damage and evaluate the antioxidant activity of delphinidin. The purpose of this study is to investigate whether delphinidin exerts antioxidant protection on cells by regulating the Nrf2 pathway.
2.3. MTT Assay. MTT assay was performed to detect the effect of delphinidin on cell viability. HepG2 cells (~5 × 10 3 cells/well) were seeded in 96-well plates for 24 h and then treated with different concentrations of delphinidin for 24 h. After changing the culture medium, 5 mg/mL MTT reagent was added and the cells were incubated for 4 h. Cell viability was calculated by measuring absorbance at 490 nm using a microplate reader. 10 μM, 20 μM, and 40 μM delphinidin with the treatment of H 2 O 2 were pretreated to assess the impact on cell viability based on the results of the above experiment.

ROS Assay.
HepG2 cells (~5 × 10 3 cells/well) were seeded in 96-well plates for 24 h. After refreshing the culture medium, cells were treated with delphinidin (10 μmol/L, 20 μmol/L, and 40 μmol/L) for 2 h. And then the cells were exposed with H 2 O 2 (750 μmol/L) for 1 h. Cells were washed once with warm phosphate buffer saline (PBS) and a serum-free culture medium containing 5 μM ROS probe was added and reacted for 30 min. The fluorescence was read with a spectrofluorimeter (PerkinElmer, Waltham, MA, USA) at excitation wavelength of 485 nm.
2.5. Western Blot Analysis. Cytoplasm and nuclear protein extraction was prepared by using the Nuclear and Cytoplasmic Protein Extraction Kit. Cells were washed 3 times with ice-cold 1×PBS and lysed in 140 μL 1×SDS. Proteins were separated in a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene difluoride (PVDF) membranes for 1.5 h before blocking in a 5% skimmed milk for 1 h. Then, the PVDF membranes were incubated into the specific primary antibody (Nrf2, Keap1, HO-1, β-actin, Lamin B, and α-tubulin) at 4°C overnight. After washing 3 times with Tris-Buffered Saline Tween20 (TBS-T) solution, the PVDF membranes were incubated with the corresponding secondary antibody at room temperature. Finally, the membranes were scanned in a chemiluminescence imaging system (Tanon 5500, Shanghai, China).
2.6. Immunofluorescence Assay. HepG2 cells (~3 × 10 4 cells/well) were cultured for 24 h on coverslips in 24-well plates. Cells were treated with delphinidin (20 μmol/L) for 3 h and then H 2 O 2 (750 μmol/L) for 1 h. The cells were subsequently washed twice with warm PBS and fixed with 4% paraformaldehyde at room temperature for 30 min. After permeation with 0.1% Triton-X at room temperature for 20 min, cells were blocked with Goat Serum for 1 h. Then, cells were incubated with Nrf2 primary antibody at 4°C overnight. After washing 3 times with cold PBS, cells were incubated with 2 Oxidative Medicine and Cellular Longevity fluorescent secondary antibody for 1.5 h and stained with DAPI for 5 min in the dark at room temperature. Finally, coverslips were mounted onto glass slides, and the images were taken by fluorescence microscope (Thermo Scientific, Wilmington, USA).
2.7. Immunoprecipitation. HepG2 cells (~5 × 10 6 cells/dish) were seeded in 10 cm petri dish for 24 h. Then, cells were treated with delphinidin (20 μmol/L) for 2 h and H 2 O 2 (750 μmol/L) for 1 h. After washing twice with cold PBS, cells were lysed with cell lysis buffer containing 1 mM PMSF. The lysates were stirred for 1 h at 4°C and then centrifuged at 12,000 xg in a high-speed refrigerated centrifuge at 4°C for 15 min. After determining the protein concentrations of supernatants, the cell extracts were preincubated with 2 μg normal rabbit immunoglobulin G (IgG) and 20 μL Protein A agarose beads (Beyotime, Shanghai, China) for 2 h. The mixture was centrifuged at 2,500 rpm in a high-speed refrigerated centrifuge at 4°C for 5 min. The supernatants were added with anti-Nrf2 antibody (2 μg) and stirred at 4°C overnight and then the supernatants were mixed with 30 μL Protein A agarose beads for 2 h at 4°C. Subsequently, the mixture was centrifuged at 2,500 rpm in a high-speed refrigerated centrifuge at 4°C for 5 min to collect the beads. After washing once with cell lysis buffer, the beads were added into 1×SDS loading buffer and then heated at 100°C for 5 minutes for subsequent Western blotting experiments.

Quantitative
Real-Time PCR. Total RNA was extracted from cells with trizol reagent, and 1% agarose gel was used to assess RNA integrity by using a UV gel imaging system. Total RNA was reverse transcribed into cDNA according to the reverse transcription kit instructions. Moreover, realtime PCR analysis was performed using a qPCR SYBR Green Master Mix and a LightCycler 96 Instrument (Roche, Basel, Switzerland). The primers used in the study were synthesized by Sangon Biotech (Sangon Biotech Co, Shanghai, China) and shown in Table 1. Finally, the data were calculated using the 2-ΔΔCq method.
2.9. Transfection of shRNA. The design of knockdown short hairpin ribonucleic acid (shRNA) (ccggacTGACAGAAGTT GACAATTActcgagTAATTGTCAACTTCTGTCAgttttt-tg) targeting Nrf2 was commissioned by GeneChem (Shanghai, China), and subsequently, the shRNA was cloned into double-marked lentiviral vector GV248 (GeneChem, Shanghai, China). Lipofectamine 3000 was used to transfect sh-con (negative control) and sh-Nrf2 plasmids into HepG2 cells. After puromycin treatment, nonspecific cells were eliminated and cells with green fluorescence were sorted using flow cytometry. Transfection cells were cultured and detected using Western blot and ROS methods.
2.10. Statistical Analysis. The SPSS18.0 statistical (Chicago, IL, USA) software was used for statistical analysis. All the data was repeated at least 3 times, and the results were expressed as the mean ± SD. The data was estimated the normality distribution and homogeneity of variance. Statistical differences between two groups were assessed using a twotailed Student's t-test. The one-way analysis of variance was used between the multiple sample means (the pairwise comparison used the LSD test). Otherwise, the Kruskal-Wallis H test was used for data analysis (Student-Newman-Keuls test was used for pairwise comparison). Statistical significance was considered at P < 0:05.

Activation of Nrf2-Related Proteins Regulated by
Delphinidin. Compared with the control group, the protein expression of Nrf2 increased 1.2-fold at 3 h (P < 0:05) and that of HO-1 promoted at 3 h, 6 h, and 9 h (P < 0:05) after treatment with 20 μM of HepG2 cells (Figures 2(a) and 2(b)). The results showed that cells treated with 20 μM delphinidin had upregulated the level of Nrf2 and HO-1 protein at 3 h. Besides, the protein expression of Nrf2 and HO-1 was upregulated after 20 μM and 40 μM delphinidin treatment (P < 0:05). On the other hand, delphinidin did not affect the protein expression of Keap1 (Figures 2(c) and 2(d)). Therefore, 3 h processing time and 20 μM delphinidin were chosen for the following experiment.  (Figure 3(a)). As shown in Figures 3(b) and 3(c), we found that at concentrations of 20 μM and 40 μM also upregulated the protein expression of Nrf2 and HO-1. However, the level of Keap1 protein remained unchanged in each group (P > 0:05). This result showed that delphinidin could increase Nrf2 and HO-1 mRNA and protein levels, but had no effect on Keap1.     Figure 4(a)). In Figure 4(b), the results showed that few Nrf2 could be detected in the cytoplasm of the control group. Conversely, compared with the H 2 O 2 -treated group, Nrf2 protein expression was increased in the nucleus of the delphinidin intervention group (10 μM, 20 μM, and 40 μM) (P < 0:05). These data indicated that delphinidin could promote Nrf2 to enter and accumulate in the nucleus.

Inhibition of Nrf2 Degradation by Delphinidin and H 2 O 2 .
Nrf2 in the cytoplasm was degraded by the 26S proteasome. The 26S proteasome inhibitor MG132 was employed to pretreat cells and then the ubiquitination of Nrf2 was detected by coimmunoprecipitation and Western blot. After treatment with MG132, the levels of ubiquitinated protein and Nrf2 protein were higher in each group than those without MG132 ( Figure 5(a)). In the IP experiment, whether MG132 (10 μM) was added or not, the expression of ubiquitinated Nrf2 was higher in cells supplemented with delphinidin (20 μM) and H 2 O 2 (750 μM) than other groups ( Figure 5(b)). The above results might indicate that delphinidin could attenuate the degradation of Nrf2 by inhibiting the function of the 26S proteasome.

Effect of Delphinidin and H 2 O 2 on Nrf2 Knockdown Cells.
In order to further verify the cytoprotective effect of Nrf2 and delphinidin on oxidative stress, HepG2 cells were transfected with sh-Nrf2 plasmid and subsequently pretreated with 20 μM delphinidin before adding 750 μM H 2 O 2 . The expression of Nrf2 in the control group was significantly reduced after transfecting with sh-Nrf2 plasmid, indicating that the transfection was effective. In addition, the expression of Nrf2 in HepG2 cells treated with delphinidin alone increased in both sh-con and sh-Nrf2 cells, which indicated that delphinidin could upregulate the expression of Nrf2 (Figures 6(a) and 6(b)). Compared with the control group, the results of ROS assay showed that H 2 O 2 treatment significantly increased the fluorescence intensity of sh-con cells by   Oxidative Medicine and Cellular Longevity 0.77 times, while that of sh-Nrf2 cells increased by 1.55 times (P < 0:05). Furthermore, the fluorescence intensity of sh-con cells and sh-Nrf2 cells in the delphinidin intervention group was significantly reduced by 32.5% and 64.9%, respectively, compared with that in the H 2 O 2 -treated group (Figure 6(c)). These results indicated that silencing Nrf2 gene enhanced H 2 O 2 -induced oxidative stress in HepG2 cells, while delphinidin reduced oxidative stress back to normal.

Discussion
According to several reports [29], anthocyanins are watersoluble flavonoids, and their skeleton is a 2-phenylbenzopyran ring structure (Figure 1(a)). They have an antioxidant activity which is associated with the diorthohydroxyl functional moiety. Among various kinds of anthocyanins, delphinidin (Dp) has attracted much attention due to its largest number of hydroxyl groups on the B ring and high antioxidant activity [30,31]. In this study, the HepG2 cells, which possessed many biological characteristics of hepatocytes and the activity of many phases I and II antioxidant enzymes, were selected to evaluate the antioxidative property effect of delphinidin treatment [32,33]. So far, there are few reports on the antioxidant activity of delphinidin in HepG2 cells. As a small molecule that easily crosses the cell membrane, H 2 O 2 is the main component of intracellular ROS produced in many physiological and pathological processes, and it can cause oxidative damage to cells [34,35]. Therefore, H 2 O 2 is often used to investigate the mechanism of oxidative stress [34,36]. In our previous experiments, we treated HepG2 cells with 500 μM H 2 O 2 and found that although the intracellular ROS level increased, it could not meet the requirements for a stable oxidative stress environment. In this experiment, we selected 750 μM H 2 O 2 to treat HepG2 cells for 1 h, based on the results of our previous experiments and others' studies [37][38][39]. In our study, the treatment with 750 μM H 2 O 2 resulted in decreased cell viability and accumulated ROS in HepG2 cells. Similarly, our study also showed that 750 μM H 2 O 2 could cause the changes of cells morphology ( Figure S1). It was significantly alleviated after pretreatment with delphinidin, which was consistent with the researches of NI [40] and Lee et al. [28]. At present, the toxicity of delphinidin to HepG2 cells has not been studied. In our study, we obtained that the IC50 value of delphinidin was 65.58 μM by using MTT assays (Figure 1(b)). The cells treated with different concentrations (10 μM, 20 μM, and 40 μM) of delphinidin did not cause any damage to the cells (Figure 1(c)). In some other studies, varied concentrations of delphinidin were used to treat with   Oxidative Medicine and Cellular Longevity different cells, but it was generally believed that the dose of delphinidin under of 40 μM had no impact to cell proliferation [28,41]. Compared with the delphinidin intervention group (40 μM), the delphinidin intervention group (20 μM) had a stronger antioxidant capacity (Figures 1(d) and 1(e)). It was suggested that the antioxidant activity of delphinidin in HepG2 cells might be associated with the dose dependence. As we all known, delphinidin has a strong antioxidant capacity, but the molecular mechanism by which delphinidin exerts its antioxidant activity remains elusive.
Polyphenols, such as delphinidin, quercetin, and kaempferol, could exert its cytoprotective properties due to its ability to increase the activity of phase II detoxification enzymes and the ability to directly clear ROS [42][43][44]. Among multiple phase II detoxification enzymes, HO-1 and NQO1 are considered to produce beneficial responses to oxidative stress in a variety of cells [16,45]. In fact, that is because heme-derived metabolites produced by HO-1 have powerful antioxidant and cytoprotective activities [46,47]. In addition, according to the related researches, it has been found that HO-1 and NQO1 could be effectively activated 7 Oxidative Medicine and Cellular Longevity by Nrf2 [30]. In our study, delphinidin treatment significantly upregulated the protein expression of Nrf2 and HO-1 ( Figure 3). Unlike the petunidin [48], delphinidin also increased the mRNA level of Nrf2. Moreover, according to several previous reports, we found that polyphenols might activate the Nrf2 pathway by applying slightly oxidative stress [49]. However, in our study, compared with the control group, there were no significant differences in ROS levels when cells were treated with delphinidin (20 μM) alone (Figure 1(e)). Thus, delphinidin did not promote Nrf2 activation in a way that increased slightly ROS levels.
In physiological conditions, Nrf2 binds to Keap1 in the cytoplasm and it is continuously ubiquitinated under the synergistic effect of ub-activating enzyme E1, ub-conjugating enzyme E2, and ub-ligating enzyme E3. After that, the ubiquitinated Nrf2 is rapidly degraded by the 26S proteasome. However, if the body is exposed to poisons, drugs, carcinogens, or other electrophiles, Nrf2 will be isolated from Keap1 and transferred into nucleus, where Nrf2 combines with small Maf proteins to form heterodimers before binding to the ARE; finally, the downstream phase II detoxification enzyme is activated [7,50] (Figure 7). In this study, the immunofluorescence and ubiquitination experiments were used to investigate whether delphinidin exerted an antioxidant effect through this mechanism. The results showed that after delphinidin treatment, the expression of green fluorescence in the nucleus of HepG2 cells was enhanced (Figure 4(a)). In addition, according to the results of Nrf2 protein expression in cytoplasm and nucleus, we could see that delphinidin promoted the nuclear translocation of Nrf2 (Figure 4(b)). Recently, it has been reported that polyphenols such as geraniin [51] and hyperoside [49] could stimulate the expression of Nrf2 in the nucleus, and our results were consistent with these compounds, but different from the quercetin in NB4 leukemia cells [52]. Especially after the pretreatment of MG132, we found that the expression of Nrf2 and ub-Nrf2 was higher in the group which treated with delphinidin (20 μM) and H 2 O 2 (750 μM) than in other groups ( Figure 5). Zhang et al. previously demonstrated that the fisetin, a dietary flavonoid that had the same function as delphinidin in upregulating the expression of Nrf2, prolonged the half-life of Nrf2 protein [2]. These results suggested that delphinidin might reduce the degradation of Nrf2 by inhibiting the function of the 26S proteasome, resulting in a higher expression of the total Nrf2 protein. Actually, Chen et al. previously revealed that the dietary flavonoids with OH groups on the B ring and/or unsaturated C ring could act as proteasome inhibitors [53], which was further supported by our finding. In addition, Qin et al. previously revealed that myricetin [54] and baicalein [17] could inhibit the ubiquitination of Nrf2 and downregulate the expression of Keap1, but this change was not observed after delphinidin intervention. After analyzing the structures of those compounds, we speculated that it might be associated with a 4-carbonyl group on the C ring, which needed further experimental verification. Based on the above results, we considered that delphinidin elevated Nrf2 activation probably by upregulating Nrf2 mRNA  Oxidative Medicine and Cellular Longevity expression and promoting Nrf2 nuclear translocation, rather than by inducing Keap1 degradation. According to the above study, we must consider whether delphinidin could still protect cells from oxidative stress when Nrf2 was knocked down. Although the ROS level of the H 2 O 2 -treated group in the sh-Nrf2 cells was higher than that of the sh-con cells, delphinidin could still decrease the ROS in the sh-Nrf2 cells back to normal level (Figure 6(c)),  9 Oxidative Medicine and Cellular Longevity and we also found that the delphinidin could promote the expression of Nrf2 protein after knocking down Nrf2. The above results indicated the important role of delphinidin in alleviating intracellular oxidative damage in Nrf2 knockdown cells (Figures 6(a) and 6(b)). Considering that there were no significant differences in ROS levels which treated with delphinidin (20 μM) among the sh-con and sh-Nrf2 cells, and the fluorescence intensity of the delphinidin intervention group was lower than that of the control group in sh-Nrf2 cells, we speculated that delphinidin might also play a role in cytoprotective effect by activating other antioxidant pathways, such as phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway [55] or inhibiting phosphorylation of ERK, JNK, and p38 [56], which needed further experimental verification.

Conclusion
As an antagonist of the 26S proteasome, delphinidin could upregulate the expression of Nrf2 mRNA, promote the accumulation of Nrf2 in the nucleus, and inhibit the degradation of ub-Nrf2, which activated the expression of the downstream HO-1. In summary, this study demonstrated that delphinidin proposed an antioxidant protective effect of alleviating the toxic effect induced by H 2 O 2 in HepG2 cells, and the mechanism was through the Nrf2 signaling pathway. It was suggested that delphinidin could be used as a new type of antioxidant to prevent diseases related to oxidative stress.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare no conflict of interest.