The Protective Effect of Indole-3-Acetic Acid (IAA) on H<sub>2</sub>O<sub>2</sub>-Damaged Human Dental Pulp Stem Cells Is Mediated by the AKT Pathway and Involves Increased Expression of the Transcription Factor Nuclear Factor-Erythroid 2-Related Factor 2 (Nrf2) and Its Downstream Target Heme Oxygenase 1 (HO-1)

Kim, Daehwan; Kim, Hyewon; Kim, Kichul; Roh, Sangho

doi:https://doi.org/10.1155/2017/8639485

Oxidative Medicine and Cellular Longevity

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Abstract Introduction Materials and Methods Results Discussion Conflicts of Interest Authors’ Contributions Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2017 | Article ID 8639485 | https://doi.org/10.1155/2017/8639485

The Protective Effect of Indole-3-Acetic Acid (IAA) on H₂O₂-Damaged Human Dental Pulp Stem Cells Is Mediated by the AKT Pathway and Involves Increased Expression of the Transcription Factor Nuclear Factor-Erythroid 2-Related Factor 2 (Nrf2) and Its Downstream Target Heme Oxygenase 1 (HO-1)

Daehwan Kim,¹Hyewon Kim,¹Kichul Kim,¹and Sangho Roh¹

Academic Editor: Tullia Maraldi

Received18 Jan 2017

Revised31 Mar 2017

Accepted06 Apr 2017

Published14 Jun 2017

Abstract

Indole-3-acetic acid (IAA) is the most common plant hormone of the auxin class and is known to have many effects including cell proliferation enhancement and antioxidant property. However, no study has revealed its defensive effects against oxidative toxicity in human dental pulp stem cells (hDPSCs). In this study, we investigated the effects of IAA on hydrogen peroxide- (H₂O₂-) induced oxidative toxicity in hDPSCs. H₂O₂-induced cytotoxicity was attenuated after IAA treatment. Cell cycle analysis using FACS showed that the damaged cell cycle and increased number of apoptotic cells by H₂O₂ treatment were recovered after the treatment of IAA. The H₂O₂-mediated increased expression of the proapoptotic genes, BAX and p53, was attenuated by IAA treatment, while IAA treatment increased antiapoptotic genes, BCL-2 and ATF5 expression. The increases of cleaved caspase-3 and ROS by H₂O₂ were also decreased after treatment of IAA. To further investigate the mechanism of IAA, Nrf2-related antioxidant pathway was examined and the results showed that the level of Nrf2 and HO-1 expressions, stimulated by H₂O_2, decreased after treatment of IAA. Moreover, IAA treatment protected hDPSCs against H₂O₂-induced oxidative stress via increased expression of Nrf2 and HO-1, mediated by the AKT pathway.

1. Introduction

Dental pulp stem cells (DPSCs) are adult stem cells (ASCs) that are able to differentiate into multiple lineages [1]. Although there is one report published on teratoma-like structures from DPSCs [2], the cells in general condition are still regarded as fascinating ASCs without tumorigenesis [3]. Normally, DPSCs can be isolated from various teeth including permanent teeth and supernumerary teeth [4, 5]. The characteristics of DPSCs are similar to those of bone marrow-derived MSCs (BMSCs) [6, 7]. It has been reported that DPSCs have the potential to differentiate into mesenchymal lineages including odontoblasts, chondrocytes, myocytes, adipocytes, and osteoblasts [6, 7] as well as nonmesenchymal ectodermal lineages, which include neurons [8]. DPSCs are therefore regarded as an alternative source of BMSCs. Moreover, the isolation and cultivation of DPSCs are easier than those of BMSCs, and their proliferation rate is higher [5, 9]. The fact that nonfunctional or useless supernumerary teeth can be sources for DPSCs makes them a noninvasive alternative to BMSCs.

Auxins are plant hormones that have many different functions including growth, development, and wound response [10, 11]. Recently, it has been demonstrated that auxins are able to regulate senescence in plants [12, 13]. Moreover, some auxins also have antioxidant activities in plants [14, 15]. Indole-3-acetic acid (IAA) is one of the most important members of the auxins and is synthesized naturally by plants [16]. It has been confirmed that IAA is present not only in plants but also in animals, including mammals [17, 18]. However, only a few studies have examined the functions of IAA in humans. Moreover, to date, no studies have delineated the effects of IAA on hDPSCs.

Hydrogen peroxide (H₂O₂) is a powerful inducer of oxidative stress, which causes endothelial cell dysfunction, cellular injury, and vascular disease [19–21]. H₂O₂ can also cause cell senescence and induce apoptosis [22, 23]. In the dental field, H₂O₂ is generally used for tooth whitening both professionally and in self-administered products (up to 35%) in its original form or in the form of carbamide peroxide [24, 25]. As a result of the demand for products that improve appearance, H₂O₂ tooth bleaching has become popular. However, adverse effects such as cervical root resorption, tooth sensitivity, ulceration of soft tissue, and potential tumor promotion can occur [26–28]. Moreover, it has been demonstrated that H₂O₂ can penetrate enamel and dentin, resulting in damage to dental pulp cells [29, 30]. However, little is known about the effect of H₂O₂ on hDPSCs. Moreover, the effects of IAA on H₂O₂-induced damage and the mechanism of its action in hDPSCs have not been elucidated.

In the present study, we investigated the effects of IAA on hDPSCs during H₂O₂-induced oxidative toxicity. More specifically, we determined if this compound protected hDPSCs from apoptotic and oxidative stress by assessing hDPSC morphology, proliferation, survival, cell cycle, and gene expression patterns.

2. Materials and Methods

2.1. Chemicals

Most inorganic and organic compounds were purchased from Sigma-Aldrich Korea (Yong-in, Korea), and all liquid medium and supplements were from Life Technologies (Grand Island, NY, USA) unless indicated otherwise in the text.

2.2. Human Dental Pulp Cell Culture

According to guidelines provided by the Institutional Review Board (IRB, number S-D20100005), human maxillary central supernumerary teeth () were extracted from children at the Dental Hospital of Seoul National University. Human DPSC culture process from isolation of pulp tissue to passaging culture followed our laboratory protocol [31]. Briefly, the cementoenamel junction was cut by a cutting disk to expose the pulp tissue as described previously [32] and pulp tissue was gently separated using a sterile endodontic file. After enzymatic dissociation with 1% (w/v) collagenase type I, single-cell suspensions were seeded into 24-well culture dishes. Then, the cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂ in DPSC culture medium, which consisted of α-MEM supplemented with 10% (v/v) fetal bovine serum (FBS; Life Technologies). Culture medium was replaced every three days, and they were subcultured at one-fifth dilution for later passaging when the cells were grown to 70% confluence. To avoid the use of senescent cells, all experiments were performed on cultured cells of passage number 3 to 5.

2.3. Cell Viability Assay

After H₂O₂ and/or IAA treatment, the number of viable cells was determined by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay using the commercially available CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI). hDPSCs were cultured in a 96-well plate at a cell density of 1 × 10⁴ and treated with H₂O₂ for 24 hours before performing the MTS assay.

2.4. Cell Cycle Analysis

After H₂O₂ and/or IAA treatment, cells were detached with trypsin and collected. They were resuspended and fixed in 70% ethanol at −20°C for 30 minutes. After cells were centrifuged and washed with PBS, 350 μl of propidium iodide (PI, 40 μg/ml) was added for cell staining and then 2 μl of RNase A was added. After staining, the PI-elicited fluorescence of individual cells was determined by flow cytometry (FACSAria1®; BD Biosciences, Erembodegem, Belgium). The total amount of PI fluorescence of 1 × 10⁴ cells was counted in each sample. The distribution of cells in G0/G1, S, and G2/M phase was calculated using the ModFit LT program version 3.3 (Verity Software House Inc., USA).

2.5. Real-Time Polymerase Chain Reaction (PCR)

Total RNA from samples was extracted using the RNeasy mini kit (Qiagen, Hilden, Germany), and M-MLV Reverse Transcriptase was used to synthesize cDNA according to the manufacturer’s instructions. Real-time PCR was performed using the 7500HT system™ (Applied Biosystems Inc., Foster City, CA, USA) and SYBR Premix Ex Taq II (Takara, Otsu, Japan). The PCR volume was 20 μl, and 1 μl reverse transcript product was used. Cycling conditions were as follows: 1 cycle of 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 30 s. The ΔΔCt method was used for relative quantitation of mRNA expression in samples, and the fold change was determined as 2^−ΔΔCt. Specific primer sequences to amplify apoptotic marker genes are from previous reports and are listed in Table 1 [33, 34].

2.6. Measurement of Reactive Oxygen Species (ROS)

A DCF-DA cellular ROS detection assay (Abcam PLC, Cambridge, MA, USA) was used to measure hydroxyl, peroxyl, and other ROS activity within cells. A total of 2.5 × 10⁴ cells per well were seeded on a 96-well plate and allowed to attach for 24 h. Cells were then stained with 25 μM DCF-DA for 45 min at 37°C. After staining, cells were treated with H₂O₂ and/or IAA for 6 h. Finally, fluorescent intensity was determined by fluorescence spectroscopy with maximum excitation and emission spectra of 485 and 535 nm, respectively.

2.7. Western Blot Analysis

Cells were lysed in passive lysis buffer (Promega) and harvested with a cell scraper. Cell debris was removed by centrifuging the cell lysate at 13,000 rpm for 10 minutes at 4°C, and 30 μg of proteins were loaded on 10% SDS-PAGE gels and separated by gel electrophoresis. Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and blocked for 1 h with 10% nonfat milk in Tris-buffered saline with 0.1% Tween 20. Proteins were then blotted with antibodies against Nrf2 (C-20, Santa Cruz Biotechnology, Santa Cruz, CA), HO-1 (H-105, Santa Cruz Biotechnology), lamin B1 (A-11, Santa Cruz Biotechnology), cleaved caspase-3 (9661, Cell Signaling, Beverly, MA), and beta-actin (C4, Santa Cruz Biotechnology). Detection of the primary antibody was accomplished using HRP-conjugated anti-mouse IgG (1 : 3000, Santa Cruz Biotechnology) and anti-rabbit IgG (1 : 3000, Santa Cruz Biotechnology). Intensities of the protein bands were evaluated by densitometric analysis using GeneGnome XRQ (Syngene Corp., Cambridge, UK).

2.8. Statistical Analysis

All values are expressed as means ± SDs. To determine the significance of differences among groups, comparisons were made using Student’s t-test as implemented in GraphPad Prism V5.0 (GraphPad Software, San Diego, CA, USA). was considered significant.

3. Results

3.1. The Effect of H₂O₂ and IAA on the Viability of hDPSCs

To examine the effects of H₂O₂ on hDPSCs, cells were exposed to different concentrations of H₂O₂ in the culture medium for 24 h. As shown in Figure 1, H₂O₂ concentrations of less than 180 μM had no effect on cell viability (Figure 1(a)). However, the viability of hDPSCs significantly decreased after treatment with H₂O₂ at 180 μM and above compared with the nontreated (NT) group. The morphology of hDPSCs was also analyzed, and the results were in agreement with the cell viability findings. The shape of hDPSCs was not significantly changed by H₂O₂ at 1–150 μM concentrations (Figure 1(b)). However, many hDPSCs condensed and detached (Figure 1(b)) when cells were treated with 180 μM H₂O₂. Cells in the model group were therefore subsequently treated with 180 μM H₂O₂.

(a)

(b)

The potential cytotoxic effects of IAA were measured after treatment of hDPSCs with different concentrations of IAA ranging from 1 to 400 μM. Cell viability of hDPSCs was not influenced by IAA treatment, and cells did not condense or detach (Figures 2(a) and 2(b)).

(a)

(b)

3.2. Protective Effect of IAA against H₂O₂-Induced hDPSC Damage

To evaluate whether IAA protected against H₂O₂-induced cytotoxicity, the cell viability and morphology of H₂O₂-damaged hDPSCs were assessed after treatment with IAA at various concentrations ranging from 1 to 300 μM. H₂O₂-induced cytotoxicity was significantly attenuated in the presence of IAA, with the maximum effect observed at 150 μM IAA (Figure 3(a)). In addition, the number of condensed and floating H₂O₂-damaged hDPSCs significantly decreased after treatment with 150 μM IAA compared with the model group (Figure 3(b)).

(a)

(b)

3.3. IAA Treatment Rescues the Cell Cycle and Prevents Apoptosis of H₂O₂-Damaged hDPSCs

To further analyze the protective effect of IAA on H₂O₂-induced cytotoxicity, cell cycle was quantified by flow cytometry after PI staining of cells (Figure 4(a)). In the model group, the normal cell cycle was notably disrupted by H₂O₂ treatment compared with the NT group. In particular, the number of apoptotic cells (sub-G1) was significantly increased compared to the NT group. Cell cycle disruption by H₂O₂-induced cytotoxicity was rescued by treatment with 150 μM IAA, and the number of apoptotic cells was also decreased by IAA treatment.

(a)

(b)

(c)

(d)

Figure 4

IAA treatment suppresses H₂O₂-induced cytotoxicity via regulation of the cell cycle and apoptosis in human dental pulp stem cells (hDPSCs). (a) Analysis of cell cycle after IAA treatment of H₂O₂-damaged hDPSCs. (b) DNA staining with DAPI. The number of cells with DNA condensation (arrow) decreased after IAA treatment of H₂O₂-damaged hDPSCs. Expression of the apoptosis-related genes BAX, p53, BCL-2, and ATF5 was evaluated by real-time PCR (c) and the expression of the apoptosis-related enzyme, cleaved caspase-3, was evaluated by Western blot (d). ,

To determine the effects of IAA on H₂O₂-induced apoptosis, additional analyses were conducted. Firstly, DAPI-stained nuclei were observed. In the model group, nuclear condensation and chromatin margination, which are typical properties of apoptotic cells (Figure 4(b)), were evident. However, the number of condensed nuclei was significantly lower in the IAA-treated group than in the model group.

The expression of apoptotic and antiapoptotic genes was also assessed by real-time PCR. Expression of the proapoptotic genes BAX and p53 was significantly increased by H₂O₂ treatment. However, the expression of BCL-2 and ATF5, which are antiapoptotic genes, was significantly lower in the model group than in the NT group (Figure 4(c)). The H₂O₂-mediated increase in BAX and p53 expression was significantly attenuated by IAA treatment, while IAA treatment increased BCL-2 and ATF5 expression. We also assessed the expression of cleaved caspase-3 by Western blot (Figure 4(d)). Levels of cleaved caspase-3 were higher in the model group than in the NT group, while the expression of cleaved caspase-3 was noticeably decreased in the IAA-treated group than in the model group.

3.4. IAA Treatment Suppresses the Generation of Reactive Oxygen Species (ROS)

To investigate the effects of IAA on the generation of ROS, we examined intracellular ROS levels in hDPSCs using DCF-DA. The fluorescence intensity of DCF-DA was significantly higher in the model group than in the NT group (Figure 5). In contrast, the fluorescence intensity of DCF-DA was dramatically lower in the IAA-treated group than in the model group.

3.5. IAA Treatment Induces the Expression of Nuclear Factor-Erythroid 2-Related Factor 2 (Nrf2) via AKT Signaling

Nrf2 is a key transcription factor involved in the regulation of antioxidant genes; we therefore assessed expression of Nrf2 in cytosolic and nuclear fractions by Western blot. Cytosolic Nrf2 expression was significantly higher in the IAA-treated group than in the model group (Figures 6(a) and 6(b)). The pattern of nuclear Nrf2 expression was analogous to that observed for cytosolic Nrf2. Interestingly, heme oxygenase 1 (HO-1), an antioxidant enzyme regulated by Nrf2, was expressed at higher levels in the IAA-treated group than in the model group (Figures 6(a) and 6(b)).

(a)

(b)

(c)

(d)

Figure 6

Effects of IAA on nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) and the role of AKT signaling in human dental pulp stem cells (hDPSCs). (a) The expression of Nrf2 was analyzed in cytosolic (Cy) and nuclear (Nu) extracts from cells. The antioxidant enzyme, HO-1, was also profiled after treatment of H₂O₂-damaged hDPSCs with IAA. (b) Densitometric analysis of Cy-Nrf2, Nu-Nrf2, and HO-1 expression. (c) Roles of AKT signaling in IAA-induced Nu-Nrf2 and HO-1 expression. The IAA-induced increase in expression of pAKT, Nu-Nrf2, and HO-1 was significantly reduced by treatment with the AKT inhibitor LY294002. (d) Densitometric analysis of pAKT, Nu-Nrf2, and HO-1 expression. , .

To further investigate the mechanisms underlying increased Nrf2 expression in the IAA-treated group, levels of phosphorylated AKT (pAKT) after IAA treatment were evaluated. pAKT expression was remarkably higher in the IAA-treated group than in the model group (Figures 6(c) and 6(d)). Treatment of cells with LY294002, an AKT inhibitor, significantly decreased the expression of pAKT in the IAA-treated group (Figures 6(c) and 6(d)). Interestingly, Nrf2 and HO-1 expression was also significantly decreased in the IAA-treated group after treatment with LY294002, consistent with the pAKT expression pattern described above (Figures 6(c) and 6(d)).

4. Discussion

Because of the increased demand for aesthetic treatments in the dental field, H₂O₂-mediated dental bleaching procedures are commonly performed. During dental treatment, the H₂O₂ is able to permeate into dental pulp tissue through dentin [30, 35] and H₂O₂-induced oxidative stress will damage the tissue. Furthermore, it has been demonstrated that oxidative stress is significantly related to chronic apical periodontitis [36]. However, the effect of H₂O₂-induced oxidative stress on hDPSCs has received little attention.

In the present study, we confirmed that the treatment of IAA, a plant hormone, protected hDPSCs from H₂O₂-induced damage, including oxidative stress and apoptosis. In addition, the effects of IAA on the oxidative stress pathway in hDPSCs were demonstrated.

To evaluate the effects of H₂O₂ on hDPSCs, we examined cell viability and morphology. The viability of hDPSCs treated with 180 μM H₂O₂ was considerably lower than that of the NT group (Figure 1(b)). Many more condensed and detached cells were observed among hDPSCs treated with 180 μM H₂O₂ than the NT group, suggesting that 180 μM H₂O₂ is sufficient to establish an H₂O₂-damaged hDPSC model (Figure 1(b)). These findings are in agreement with a previous study [37]. In contrast to H₂O₂, IAA had no effect on the viability of hDPSCs, even at high concentrations (Figure 2). These results suggest that IAA is not toxic to hDPSCs at the concentrations evaluated.

To clarify the effect of IAA on H₂O₂-damaged hDPSCs, H₂O₂-damaged hDPSCs were treated with various concentrations of IAA. The viability of 150 μM IAA-treated hDPSCs was significantly higher than that of the model group (180 μM H₂O₂) (Figure 3(a)). Moreover, the number of condensed and detached cells was lower in the IAA-treated group (150 μM IAA) than in the model group (Figure 3(b)). Moreover, living cells in the 150 μM IAA-treated group had similar morphologies to those in the NT group. Together, these results indicate that IAA treatment can protect hDPSCs from H₂O₂-induced damage.

Previously, it was reported that the exposure of cells to H₂O₂ was able to trigger cell cycle arrest and apoptosis [38, 39]. We therefore hypothesized that IAA may prevent the H₂O₂-induced disruption of cell cycle and apoptosis in hDPSCs. In the model group, the cell cycle was completely disrupted compared to that in the NT group (Figure 4(a)). However, after treatment of cells with 150 μM IAA, the cell cycle was rescued, suggesting that H₂O₂-induced damage of the cell cycle was restored by IAA. Interestingly, the population of sub-G1 cells was significantly higher in the NT group than in the model group. Cells in the sub-G1 range are considered to be dead cells, including necrotic and apoptotic cells [40]. Because H₂O₂ not only disrupts the cell cycle but also induces apoptosis [38, 39], we hypothesized that H₂O₂-induced apoptosis could be involved in disruption of the cell cycle, implying that the protective effect of IAA was also associated with apoptosis. As expected, hDPSCs with condensed and fragmented nuclei, suggesting apoptotic cells, were observed in the model group (Figure 4(b)). However, there were far fewer cells with condensed and fragmented nuclei in the IAA-treated group than in the model group. Expression of apoptosis-related genes was also evaluated; expression of proapoptotic genes was decreased whereas that of antiapoptotic genes was increased in the IAA-treated group compared to the model group (Figure 4(c)). Moreover, cleaved caspase-3 expression in the model group was restored after IAA treatment (Figure 4(d)). Treatment of hDPSCs with H₂O₂ therefore disrupted the cell cycle and induced apoptosis. However, IAA protected against H₂O₂-induced damage by rescuing the cell cycle and preventing apoptosis. To the best of our knowledge, this is the first study to demonstrate that IAA is able to protect hDPSCs from H₂O₂-induced damage through cell cycle- and apoptosis-related pathways.

Oxidative stress leads to cellular damage and death via generation of ROS [41]. We therefore hypothesized that IAA treatment would regulate the ROS pathway. To test this hypothesis, we evaluated the effects of IAA treatment on ROS generation. ROS levels were significantly higher in the model group than in the NT group (Figure 5). In contrast, ROS generation was significantly lower in the IAA-treated group than in the model group. These results suggest that IAA treatment influences ROS and oxidative stress pathways. However, IAA alone did not have any significant effects on the generation of ROS.

Nrf2 is a key transcription factor that regulates expression of endogenous antioxidant enzymes. Under normal conditions, Nrf2 is bound to Kelch-like epichlorohydrin-associated protein 1 (Keap-1), and this complex is located in the cytoplasm [42]. Under oxidative stress conditions, Nrf2 is released from Keap-1 and translocates to the nucleus, where it recognizes the antioxidant response element (ARE) and regulates the expression of antioxidant enzymes, including HO-1. Thus, the Nrf2-ARE pathway is crucial for protecting against oxidative stress. In previous studies, the Nrf2-ARE pathway has been shown to be highly activated to protect against oxidative damage [43]. In the present study, we also investigated whether IAA is able to regulate the Nrf2-ARE pathway. Treatment of hDPSCs with IAA resulted in enhanced expression of both Nrf2 and HO-1, suggesting that IAA treatment activates the Nrf2-ARE pathway, resulting in protection against H₂O₂-induced oxidative damage and enhanced cell viability (Figures 6(a) and 6(b)).

AKT signaling is involved in activation of the Nrf2-ARE pathway in response to oxidative stress [44]. AKT signaling also regulates cell survival via antioxidant and antiapoptotic roles. We found that pAKT expression was remarkably higher in the IAA-treated group than in the model group, while pAKT expression was noticeably decreased by treatment with LY294002 (Figures 6(c) and 6(d)). In addition, the pattern of Nrf2 and HO-1 expression was analogous to that of pAKT expression, implying that Nrf2 and HO-1 induced by IAA may be regulated by AKT signaling in H₂O₂-damaged hDPSCs. Together, these results suggest that IAA activates Nrf2 and HO-1 expression through AKT signaling, thereby protecting against oxidative damage.

In conclusion, we demonstrated that IAA treatment protected hDPSCs against H₂O₂-induced oxidative stress via increased expression of Nrf2 and HO-1, mediated by the AKT pathway (Figure 7). To the best of our knowledge, this is the first report of the protective effect of the natural plant hormone IAA, suggesting that application of this plant hormone may have therapeutic value in the treatment of human dental diseases associated with oxidative stress.

Conflicts of Interest

The authors declare that there is no conflict of interest that would prejudice the impartiality of this work.

Authors’ Contributions

Daehwan Kim and Hyewon Kim isolated and cultured human dental pulp stem cells. They also performed proliferation, real-time PCR, and ROS level analyses. Kichul Kim performed Western blot analysis. Daehwan Kim and Sangho Roh designed the experiments and wrote the manuscript.

Acknowledgments

This work was supported by the National Research Foundation of Korea Grants (2016R1D1A1B03931864 and no. 2016-929358 through the Oromaxillofacial Dysfunction Research Center for the Elderly at Seoul National University), Republic of Korea. The authors also thank Eura Roh for assisting in correcting the report’s grammatical errors.

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Copyright

Copyright © 2017 Daehwan Kim et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Oxidative Medicine and Cellular Longevity

The Protective Effect of Indole-3-Acetic Acid (IAA) on H2O2-Damaged Human Dental Pulp Stem Cells Is Mediated by the AKT Pathway and Involves Increased Expression of the Transcription Factor Nuclear Factor-Erythroid 2-Related Factor 2 (Nrf2) and Its Downstream Target Heme Oxygenase 1 (HO-1)

Abstract

1. Introduction

2. Materials and Methods

2.1. Chemicals

2.2. Human Dental Pulp Cell Culture

2.3. Cell Viability Assay

2.4. Cell Cycle Analysis

2.5. Real-Time Polymerase Chain Reaction (PCR)

2.6. Measurement of Reactive Oxygen Species (ROS)

2.7. Western Blot Analysis

2.8. Statistical Analysis

3. Results

3.1. The Effect of H2O2 and IAA on the Viability of hDPSCs

3.2. Protective Effect of IAA against H2O2-Induced hDPSC Damage

3.3. IAA Treatment Rescues the Cell Cycle and Prevents Apoptosis of H2O2-Damaged hDPSCs

3.4. IAA Treatment Suppresses the Generation of Reactive Oxygen Species (ROS)

3.5. IAA Treatment Induces the Expression of Nuclear Factor-Erythroid 2-Related Factor 2 (Nrf2) via AKT Signaling

4. Discussion

Conflicts of Interest

Authors’ Contributions

Acknowledgments

References

Copyright

The Protective Effect of Indole-3-Acetic Acid (IAA) on H₂O₂-Damaged Human Dental Pulp Stem Cells Is Mediated by the AKT Pathway and Involves Increased Expression of the Transcription Factor Nuclear Factor-Erythroid 2-Related Factor 2 (Nrf2) and Its Downstream Target Heme Oxygenase 1 (HO-1)

3.1. The Effect of H₂O₂ and IAA on the Viability of hDPSCs

3.2. Protective Effect of IAA against H₂O₂-Induced hDPSC Damage

3.3. IAA Treatment Rescues the Cell Cycle and Prevents Apoptosis of H₂O₂-Damaged hDPSCs