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Mitochondrial abnormalities are involved in periodontal ligament fibroblast apoptosis induced by oxidative stress
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Mitochondrial abnormalities are involved in periodontal ligament fibroblast apoptosis induced by oxidative stress Yuting Chen a, b, 1, Yinghui Ji a, 1, Xing Jin a, 1, Xiaoyu Sun a, Xiaorong Zhang a, Yang Chen a, Lixi Shi a, Haoran Cheng a, Yixin Mao a, c, Xumin Li a, Yubo Hou a, Dafeng Zhang a, c, Shufan Zhao a, d, ***, Jianfeng Ma a, c, **, Shengbin Huang a, c, * a
Institute of Stomatology, School and Hospital of Stomatology, Wenzhou Medical University, Wenzhou, PR China Sichuan Nursing Vocational College, Chengdu 610100, China Department of Prosthodontics, School and Hospital of Stomatology, Wenzhou Medical University, Wenzhou, PR China d Department of Oral Maxillofacial Surgery, School and Hospital of Stomatology, Wenzhou Medical University, Wenzhou, PR China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 December 2018 Accepted 19 December 2018 Available online xxx
Oxidative stress (OS)-induced apoptosis of periodontal ligament cells (PDLCs) has been suggested to be an important pathogenic factor of periodontitis. Mitochondrial abnormalities are closely linked to OS and act as the main players in apoptosis. Our aim was to investigate the potential mitochondrial abnormalities in PDLCs apoptosis induced by OS. In this study, significant reduction in viability and increased apoptosis were observed in H2O2-treated hPDLCs. H2O2 also induced mitochondrial dysfunction, judging by increased mitochondrial reactive oxygen species amounts, and decreased mitochondrial membrane potential as well as ATP levels. Furthermore, H2O2 significantly enhanced mitochondrial fission by decreasing the expression of Mfn1 and Mfn2, along with increasing the expression of Drp1, Fis1 and the cleavage of OPA1. Notably, NAC stabilized the balance of the mitochondrial dynamics, attenuated mitochondrial dysfunction, and inhibited apoptosis of hPDLCs in the presence of H2O2. In conclusion, the OSinduced apoptosis of hPDLCs may be mediated by mitochondria-dependent pathway. © 2018 Elsevier Inc. All rights reserved.
Keywords: Human periodontal ligament cells Apoptosis Oxidative stress Mitochondrial abnormalities Mitochondrial dynamics
1. Introduction Periodontitis is a highly prevalent disease and affects 2.5 billion people worldwide. Moreover, it leads to the destruction of periodontal connective tissues and the loss of alveolar bone, which is
Abbreviations: OS, Oxidative stress; PDLCs, periodontal ligament cells; Mfn1, mitofusin 1; Mfn2, mitofusin 2; Drp1, dynamin-related protein 1; Fis1, fission protein 1; OPA1, Optic atrophy 1; NAC, N-acetyl-cysteine; ROS, reactive oxygen species; TUNEL, Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling; H2O2, hydrogen peroxide. * Corresponding author. Institute of Stomatology, School and Hospital of Stomatology, Wenzhou Medical University, No. 373, Xueyuan West Road, Lucheng District, Wenzhou, PR China. ** Corresponding author. Department of Prosthodontics, School and Hospital of Stomatology, Wenzhou Medical University, No. 373, Xueyuan West Road, Lucheng District, Wenzhou, PR China. *** Corresponding author. Department of Oral Maxillofacial Surgery, School and Hospital of Stomatology, Wenzhou Medical University, No. 373, Xueyuan West Road, Lucheng District, Wenzhou, PR China. E-mail addresses: [email protected] (S. Zhao), [email protected] (J. Ma), [email protected] (S. Huang). 1 Contribute equally to this paper.
regarded as the leading factor of adult tooth loss [1,2]. PDL cells (PDLCs), the major cell type of periodontal ligament (PDL), are considered important participants of periodontal metabolism and remodeling [3]. The number of PDLCs is kept relatively stable through the balance of cell proliferation and death under physiological conditions [4]. Various pathological factors, including periodontal pathogens, cytokines, and drugs can lead to PDLCs apoptosis, accounted for periodontitis [5,6]. However, the mechanisms underlying apoptosis of PDLCs are not fully understood. Perhaps this is why the therapies available for treating periodontitis are not very efficacious. Therefore, the mechanisms related to PDLCs apoptosis must be further explored to identify effective therapeutic approaches to periodontitis. Accumulating evidence suggests that oxidative stress (OS) features overproduction of highly reactive oxygen species (ROS) owing to the disturbed pro-oxidant/antioxidant balance, playing a key role in the onset and progression of inflammation [7]. Moreover, studies indicate that periodontal-tissue destruction closely correlates with excessive ROS production and with compromised antioxidant capacity in periodontitis [8]. Notably, administration of antioxidants can effectively inhibit the development of periodontitis, partially
https://doi.org/10.1016/j.bbrc.2018.12.143 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article as: Y. Chen et al., Mitochondrial abnormalities are involved in periodontal ligament fibroblast apoptosis induced by oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.143
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through alleviation of PDLCs apoptosis induced by OS [9]. Thus, investigating the mechanisms by which OS affects the apoptosis of PDLCs represents one of the primary tasks in the search for effective treatment of periodontitis. Mitochondria are the primary source of cellular ROS and are subject to a direct attack by ROS [10]. Overproduced ROS cause oxidative damage to mitochondrial proteins and structures, thus subsequently leading to mitochondrial dysfunction [11]. The latter manifests itself as decreases in mitochondrial membrane potential and in uncoupled electron transport (in the electron transport chain) along with ATP downregulation, which affects the function of cells and subsequently results in apoptosis [12]. Our previous study has revealed that mitochondrial dysfunction is closely associated with aggravation of periodontitis caused by diabetes [13]. In addition, mitochondrial dysfunction is considered an important contributing factor of apoptosis of osteoblasts and of Hep-G2 cells under the influence of OS [14,15]. However, few studies have comprehensively examined the participation of mitochondrial dysfunction in periodontal-cell apoptosis caused by OS. Furthermore, mitochondria are organelles that dynamically alter their shape by frequent fusion and fission to continuously perform their function in the cell. Mitochondrial dynamics are not only critical for the homeostasis of mitochondria but also important for the well-being of the whole cell [16]. ROS are important for regulating the mitochondrial dynamics during several physiological and pathological processes [17]. Moreover, it is known that the loss of mitochondrial function, secondary to defects in mitochondrial dynamics, also leads to an increase of ROS generation [18]. These studies indicate that impaired mitochondrial dynamic leads to mitochondrial dysfunction and OS, which induces cellular perturbation and injury. It is still unclear, however, whether this mitochondrial fusion-and-fission machinery is active in OSaffected PDLCs. Therefore, in this study, our objectives were to investigate (1) whether mitochondrial dysfunction participates in the OS-induced apoptosis of human PDLCs (hPDLCs) and (2) whether mitochondrial dynamics act as a regulator of hPDLCs apoptosis induced by OS. We found that improvement in mitochondrial function exerted a significant protective effect against the hPDLCs apoptosis induced by OS. Thus, this result may have significant implications for therapeutic interventions in periodontitis. 2. Materials and Methods 2.1. Cell culture and treatment The study protocol was approved by the Committee on Research on Human Subjects of the School and Hospital of Stomatology, Wenzhou Medical University. hPDLCs were obtained from healthy third molars of five healthy men aged 20e30 years (mean age: 24.6) who underwent molar extraction after providing informed consent. PDL tissues were extracted from the root surface of a tooth and were cut into 1e2 mm2 pieces. The tissue was cultured in a-minimum essential medium containing 10% of fetal bovine serum and penicillin (100 U/ml) in a humidified incubator at 37 C and 5% CO2. Cells were passaged when hPDLCs reached 70%e80% confluence. Cells at passages 4e8 were used in this study.
2.3. Cell viability hPDLCs were cultured in 96-well plates (5 103 cells per well) under different conditions. Then, hPDLCs were incubated with MTT solution (5 mg/mL, Sigma-Aldrich). After 4 h at 37 C, the medium was replaced with 100 ml of dimethyl sulfoxide to dissolve the formazan crystals. The spectrophotometric absorbance at 490 nm was measured on a microtiter plate reader. 2.4. Detection of apoptosis by flow cytometry and Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) assays hPDLCs were collected and labeled with the Annexin V-FITC Apoptosis Detection Kit (Sigma-Aldrich). Then, cytofluorometric analysis was conducted on a FACScan laser flow cytometer. hPDLCs were preincubated with or without NAC, followed by exposure to 0.3 mM H2O2 for 24 h. Samples were processed by means of the TUNEL kit (Roche) and counterstained with 40 , 6diamidino-2-phenylindole (DAPI; Invitrogen). Image acquisition was performed under a fluorescence microscope (Leica TCS SPE). 2.5. Western blot analyses Protein levels were detected by western blot as we described before [14]. Primary antibodies were diluted in 5% defatted milk (Drp1, 1:1000; Fis1, 1:1000; OPA1, 1:1000; Mfn1, 1:1000; Mfn2, 1:1000; anti-b-actin, 1:4000; BCL-2, 1:2000; BAX, 1:2000). 2.6. Functional imaging assays hPDLCs were incubated with 2.5 mM MitoSOX for 30 min to detect the mitochondrial ROS levels. Furthermore, hPDLCs were costained with Mitotracker Green and tetramethylrhodamine methyl ester (TMRM) for 30 min to assess mitochondrial membrane potential. Mitotracker Deep Red served for detection of mitochondrial morphology alterations. A fluorescence microscope was employed to capture images. 2.7. ATP synthesis assays hPDLCs were collected after treatment with different solutions, incubated with the cell lysis buffer on ice for 30 min, centrifuged at 12,000g for 5 min, and the supernatant was collected. An ATP content detection kit was used to determine the level of ATP in the hPDLCs. 2.8. Data analysis Data were presented as the means ± SD. The Statview software (version 5.0.1, SAS Institute, USA) served for statistical analysis. Statistical significance in multiple comparisons was determined by one-way ANOVA along with individual post hoc Fisher's test. Data with P < 0.05 were regarded as statistically significant. * Additional experimental details are described in the Supplementary Materials and Methods. 3. Results
2.2. Cell treatments 3.1. H2O2-induced hPDLCs apoptosis Cells were incubated with or without hydrogen peroxide (H2O2; Sigma-Aldrich) and N-acetyl-cysteine (NAC; Sigma-Aldrich) for various periods, according to the experimental protocol. The final working concentrations were as follows: 0.3 mM H2O2 and 0.5 mM NAC.
The MTT results showed that after H2O2 treatment, cell viability significantly decreased in a time- and dose-dependent manner (Fig. 1A). The results of flow cytometry indicated that the apoptosis rate increased with treatment duration. Early apoptosis was
Please cite this article as: Y. Chen et al., Mitochondrial abnormalities are involved in periodontal ligament fibroblast apoptosis induced by oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.143
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Fig. 1. H2O2 induced apoptosis of hPDLCs. (A) Cell viability was evaluated by an MTT assay in hPDLCs in the presence of H2O2 at different concentrations and different periods. Error bars indicate SD (n ¼ 6). (BeC) Apoptosis was subjected to flow-cytometric quantification. Error bars indicate SD (n ¼ 6). (DeE) The TUNEL staining and assay. Error bars indicate SD (n ¼ 6; ***P < 0.0001). Scale bar ¼ 100 mm. (F) Western blotting of BAX and BCL-2 in hPDLCs in the presence of H2O2. (G) BAX, and (H) BCL-2 relative to b-actin. Error bars indicate SD (n ¼ 6; *P < 0.05, N.S. indicates P > 0.05).
significant after 0.3 mM H2O2 treatment for 12 h, and 0.3 mM H2O2 for 24 h induced late apoptosis and slightly increased necrosis (Fig. 1BeC). H2O2-induced DNA damage was next confirmed by obviously increased TUNEL-positive staining (Fig. 1DeE). Western blot analysis revealed that H2O2 treatment increased the protein expression of BAX but did not significantly affect BCL-2 expression (Fig. 1FeH). These experiments indicated that H2O2induced hPDLCs death was primarily apoptosis. 3.2. NAC attenuated H2O2-induced apoptosis In our preliminary experiment we found that the optimal concentration of NAC was 0.5 mM which was chosen for the present experiment. The apoptosis induced by H2O2 was significantly attenuated when hPDLCs were cotreatment with 0.5 mM NAC and 0.3 mM H2O2 for 24 h (Fig. 2B). The results of the TUNEL assay
confirmed that NAC reduced the incidence of apoptosis (Fig. 2CeD). After that, western blot analysis showed that the protein expression of BAX markedly decreased after NAC treatment (Fig. 2EeF). The above results suggested that the cytotoxic effects of H2O2-induced apoptosis were effectively attenuated by NAC. 3.3. NAC restored the mitochondrial dysfunction induced by H2O2 Compared with the cells only treated with H2O2, NAC preconditioning had significantly lower mitochondrial ROS levels, according to the lower intensity of MitoSOX staining (Fig. 3AeB). Compared with cells receiving only H2O2, NAC preconditioning had apparently more active ATP synthesis (Fig. 3C) and higher mitochondrial membrane potential (Fig. 3DeE). These results suggested that NAC antagonizes the adverse effects of H2O2 on mitochondria of hPDLCs.
Please cite this article as: Y. Chen et al., Mitochondrial abnormalities are involved in periodontal ligament fibroblast apoptosis induced by oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.143
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Fig. 2. NAC attenuated H2O2-induced apoptosis. hPDLCs were exposed to 0.5 mM NAC and 0.3 mM H2O2 for 24 h. (A) Samples were examined under a microscope and (B) processed for the MTT assay, (C) TUNEL assay, and (D) for measurement of the apoptosis rate of hPDLCs (***P < 0.0001). Scale bar ¼ 100 mm. (E) Western blot band of BAX and (F) the relative intensities (fold) of BAX (***P < 0.0001).
3.4. NAC ameliorated the H2O2-induced disruption of mitochondrial dynamics Mitochondria in the cells treated with H2O2 were fragmented, misshapen, and bleblike and collapsed away from the mitochondrial network (Fig. 4A). In addition, H2O2 decreased the mitochondrial density and length, which were partially recovered by NAC (Fig. 4BeC). We next evaluated the potential alteration of mitochondrial dynamics in the OS model (Fig. 4D). The results suggested that the expression of L-OPA1 and Mfn 2 was lower in the H2O2 group than in the control group (Fig. 4EeJ). NAC preconditioning group had higher expression levels of L-OPA1, Mfn1, and Mfn2 than the H2O2-stimulated alone group did. In contrast, S-OPA1, Drp1, and Fis1 expression increased in the H2O2-stimulated alone group but this increase was attenuated in the NAC preconditioning group (Fig. 4FeI). 4. Discussion Although OS-induced PDLCs apoptosis plays a critical role in the initiation and progression of periodontitis, the mechanism behind
this phenomenon is still unclear. Emerging evidence indicates that mitochondrial dysfunction is closely linked to OS. However, whether mitochondrial dysfunction is involved in PDLCs apoptosis induced by OS is poorly understood. In the current study, we clearly demonstrated that mitochondrial dysfunction and impaired mitochondrial dynamics were evident in the process of hPDLCs apoptosis induced by OS. Moreover, the reversal of OS-induced mitochondrial abnormalities using an antioxidant (NAC) significantly inhibited PDLCs apoptosis. Thus, our study uncovered mitochondrial abnormalities during OS-induced hPDLCs apoptosis, which may promote the initiation and development of periodontitis. H2O2 is a classical ROS that is produced by almost all types of OS and can infiltrate cells through their plasma membrane [19]. To PDLCs, H2O2-induced OS is inevitable because H2O2 is continuously produced by periodontal pathogens or their byproducts. Persistent OS causes apoptosis of PDLCs, thereby eventually causing malfunction of the PDL [20]. Thus, we established an H2O2-induced hPDLCs injury model according to our previous studies [13,18]. The results of flow cytometry and TUNEL assay showed that H2O2 elicited the apoptosis of hPDLCs in a dose- and time-dependent manner; this finding is coincident with the results of our past
Please cite this article as: Y. Chen et al., Mitochondrial abnormalities are involved in periodontal ligament fibroblast apoptosis induced by oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.143
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Fig. 3. NAC reversed the mitochondrial dysfunction induced by H2O2. (A) Representative images of MitoSOX staining and (B) quantification in the indicated groups. (C) ATP synthesis detected in the indicated groups (***P < 0.0001, *P < 0.05). Error bars indicate SD (n ¼ 6). (D) Representative images of TMRM staining and (E) quantification in the indicated groups (**P < 0.001, *P < 0.05). The scale bar is 100 mm.
studies [8,21]. The ratio of BCL-2 (antiapoptotic protein) to BAX (proapoptotic protein) determines the susceptibility of a given cell to apoptosis [22]. The expression of BAX in the hPDLCs was significantly increased by H2O2 in our study, in agreement with findings in Saos-2 human osteosarcoma cells [23] and human fetal lung fibroblasts [24]. In contrast to those studies, we found that BCL-2 expression was not significantly affected by H2O2; this result might be due to the difference in cell types. In addition, NAC is a glutathione precursor and regarded as an important intracellular antioxidant [25]. One study indicates that NAC activates antioxidant enzymes, which may prevent OS-induced apoptosis and the development of diseases [26]. In agreement with these results, pretreating PDLCs with NAC significantly attenuated H2O2-induced apoptosis in the present study. Therefore, application of antioxidants has a good potential to inhibit PDLCs apoptosis and postpone the progression of periodontitis. Mitochondria are organelles that not only act as a “power factory” of cells but also play vital roles in cell functions, death, and survival [27]. Mitochondrial dysfunction can trigger a variety of intracellular signaling cascades and lead to OS and apoptosis, which are involved in the progression of almost all diseases [28]. This notion is consistent with our results, namely, that H2O2 led to the mitochondrial dysfunction of hPDLCs, including increased ROS levels, greater incidence of mitochondrial membrane potential collapse, and reduced ATP levels. Notably, pretreating hPDLCs with NAC effectively counteracted ROS-induced mitochondrial dysfunction. Our current results confirmed that mitochondrial OS and dysfunction is deeply involved in apoptosis of hPDLCs. Mitochondria-targeted antioxidants, such as mitoquinone and
mitoTEMPO, have been confirmed to be effective at ameliorating OS-related diseases [29,30]. Thus, these agents may be applied to this hPDLCs injury model to further confirm the involvement of mitochondrial dysfunction in PDLCs apoptosis elicited by OS in our future research. Mitochondria are dynamic organelles that participate in repeated fusion and fission cycles [31]. The dynamic processes of mitochondria are crucial to the maintenance of their morphology, function, and distribution [32]. Moreover, growing evidence suggests that fusion and fission factors perform an important function in apoptosis [33]. In the present study, we found that when hPDLCs were treated with H2O2, the original rodlike or elongated mitochondria became misshapen or bleblike, and H2O2 caused greater fragmentation of mitochondria in hPDLCs. A growing body of evidence indicates that mitochondrial fragmentation has been accepted as an early event in the apoptosis of a variety of mammalian cells [34]. Together with our current findings, this concept highlights critical involvement of mitochondrial fission in OS-induced PDLCs apoptosis. Mitochondria actively divide (fission) and fuse with one another (fusion). Mitochondrial fusion is regulated by Mfn1 and Mfn2, outer-membrane GTPases that mediate outer-mitochondrialmembrane fusion, and by OPA1, which regulates inner-membrane fusion. Research has shown that the absence of mitofusins reduces mitochondrial fusion, impairs mtDNA maintenance and oxidative phosphorylation, and subsequently leads to apoptosis [35]. Moreover, OPA1, which exists in the L-OPA1 and S-OPA1 forms, is generated by processing at specific sites [36]. In response to a proapoptotic stimulus, L-OPA1 is proteolytically cleaved to
Please cite this article as: Y. Chen et al., Mitochondrial abnormalities are involved in periodontal ligament fibroblast apoptosis induced by oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.143
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Fig. 4. NAC ameliorated the disruption of mitochondrial dynamics by H2O2. (A) Representative images of Mitotracker Deep Red staining in each cell group. Scale bar ¼ 10 mm. The right-hand panel is a larger image of the one on the left. (B) Detection of mitochondrial density and (C) length in each cell group (*P < 0.05). (D) The protein levels detected by western blotting: (EeF) OPA1, (G) Drp1, (H) Fis1, (I) Mfn1, and (J) Mfn2 relative to b-actin in indicated cell groups with H2O2 or NAC þ H2O2 treatment. Error bars indicate SD (n ¼ 6; ***P < 0.0001, **P < 0.001, and *P < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
form S-OPA1, and thus induces mitochondrial fragmentation and apoptosis [37]. The results of the present study revealed that H2O2 treatment significantly decreases the expression of Mfn1 and Mfn2, while promoting the rapid cleavage of L-OPA1 into S-OPA1. All these data suggest that the balance of mitochondrial dynamics in hPDLCs caused a switch to mitochondrial fission. One study suggests that a hyperfused mitochondrial reticulum is activated upon cellular stress and protects cells from death [38]. Thus, strategies promoting mitochondrial fusion may offer new insights into the attenuation of OS inducing PDLCs apoptosis; this research area
must be explored in future studies. Thus, the exact function of fusion proteins in PDLCs apoptosis should also be confirmed. Mitochondrial fission is mainly regulated and controlled by Drp1, a protein that exists in the cytoplasm and is translocated to mitochondria [39]. Fis1, a key mitochondrial complex involved in mitochondrial fission, functions by binding Drp1 [40]. In our current study, H2O2 significantly increased the expression of Fis1 and Drp1, thus further supporting the prediction that mitochondrial fission was intimately involved in the apoptosis of hPDLCs. Moreover, downregulation of Drp1 reduces the outer-mitochondrial-
Please cite this article as: Y. Chen et al., Mitochondrial abnormalities are involved in periodontal ligament fibroblast apoptosis induced by oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.143
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membrane scission related to apoptosis and attenuates mitochondrial fission [40]. Another study has proved that the decreased expression of Fis1 shifts the dynamic balance in favor of fusion, thereby reducing apoptosis [41]. Therefore, to some extent, downregulation of Fis1 or Drp1 can be an efficient way to reduce OS-induced hPDLCs apoptosis. Notably, we found that after treatment with NAC, the imbalance of mitochondrial dynamics was markedly reversed via the inhibition of mitochondrial fission and promotion of mitochondrial fusion, which favored the maintenance of mitochondrial function and cell survival in the current study. Taken together, these findings not only reveal that ROS play an irreplaceable part in the regulation of fusion and fission of PDLCs but also suggest that the regulation of mitochondrial dynamics may be a useful therapeutic strategy against periodontitis. Although our results imply that mitochondrial dysfunction is crucial for the OS-induced apoptosis of hPDLCs, this study has several limitations. First, the detailed molecular mechanisms behind mitochondrial dysfunction and abnormalities of mitochondrial dynamics were not explored. Second, the molecular mechanisms uncovered in this study were not corroborated in vivo. Conflicts of interest All authors declare that they have no competing interests. Acknowledgments This work was supported by the following grants: the Natural Science Foundation of China (No. 81500817, 81870777, and 81802230), Zhejiang Provincial Natural Science Foundation of China (No. LY15H140008), Health Science and Technology Project of Zhejiang Province (No. 2016KYB184), Zhejiang Provincial Science and Technology Project for Public Welfare (No. 2017C33081), Wenzhou Municipal Science and Technology Project for Public Welfare (No. Y20150069 and Y20170026), Wenzhou Science and Technology Bureau (No. Y20160051 and Y20140704), and Zhejiang Provincial College Students’ Science and Technology Innovation Project and Fresh Talent Program (2017R413082, 2016R413081). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.12.143. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.12.143. References [1] N.J. Kassebaum, A.G.C. Smith, E. Bernabe, et al., Global, regional, and national prevalence, incidence, and disability-adjusted life years for oral conditions for 195 countries, 1990-2015: a systematic analysis for the global burden of diseases, injuries, and risk factors, J. Dent. Res. 96 (2017) 380e387. [2] E.M. Cardoso, C. Reis, M.C. Manzanares-Cespedes, Chronic periodontitis, inflammatory cytokines, and interrelationship with other chronic diseases, Postgrad. Med. 130 (2018) 98e104. [3] D. Jonsson, D. Nebel, G. Bratthall, et al., The human periodontal ligament cell: a fibroblast-like cell acting as an immune cell, J. Periodontal. Res. 46 (2011) 153e157. [4] E. Sigalas, J.D. Regan, P.R. Kramer, et al., Survival of human periodontal ligament cells in media proposed for transport of avulsed teeth, Dent. Traumatol. 20 (2004) 21e28. [5] T. Hatai, M. Yokozeki, N. Funato, et al., Apoptosis of periodontal ligament cells induced by mechanical stress during tooth movement, Oral Dis. 7 (2001) 287e290. [6] S.W. Jee, S. Wang, Y.L. Kapila, Specific pro-apoptotic fibronectin fragments modulate proteinase expression in periodontal ligament cells, J. Periodontol.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Mitochondrial abnormalities are involved in periodontal ligament fibroblast apoptosis induced by oxidative stress Yuting Chen a, b, 1, Yinghui Ji a, 1, Xing Jin a, 1, Xiaoyu Sun a, Xiaorong Zhang a, Yang Chen a, Lixi Shi a, Haoran Cheng a, Yixin Mao a, c, Xumin Li a, Yubo Hou a, Dafeng Zhang a, c, Shufan Zhao a, d, ***, Jianfeng Ma a, c, **, Shengbin Huang a, c, * a
Institute of Stomatology, School and Hospital of Stomatology, Wenzhou Medical University, Wenzhou, PR China Sichuan Nursing Vocational College, Chengdu 610100, China Department of Prosthodontics, School and Hospital of Stomatology, Wenzhou Medical University, Wenzhou, PR China d Department of Oral Maxillofacial Surgery, School and Hospital of Stomatology, Wenzhou Medical University, Wenzhou, PR China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 December 2018 Accepted 19 December 2018 Available online xxx
Oxidative stress (OS)-induced apoptosis of periodontal ligament cells (PDLCs) has been suggested to be an important pathogenic factor of periodontitis. Mitochondrial abnormalities are closely linked to OS and act as the main players in apoptosis. Our aim was to investigate the potential mitochondrial abnormalities in PDLCs apoptosis induced by OS. In this study, significant reduction in viability and increased apoptosis were observed in H2O2-treated hPDLCs. H2O2 also induced mitochondrial dysfunction, judging by increased mitochondrial reactive oxygen species amounts, and decreased mitochondrial membrane potential as well as ATP levels. Furthermore, H2O2 significantly enhanced mitochondrial fission by decreasing the expression of Mfn1 and Mfn2, along with increasing the expression of Drp1, Fis1 and the cleavage of OPA1. Notably, NAC stabilized the balance of the mitochondrial dynamics, attenuated mitochondrial dysfunction, and inhibited apoptosis of hPDLCs in the presence of H2O2. In conclusion, the OSinduced apoptosis of hPDLCs may be mediated by mitochondria-dependent pathway. © 2018 Elsevier Inc. All rights reserved.
Keywords: Human periodontal ligament cells Apoptosis Oxidative stress Mitochondrial abnormalities Mitochondrial dynamics
1. Introduction Periodontitis is a highly prevalent disease and affects 2.5 billion people worldwide. Moreover, it leads to the destruction of periodontal connective tissues and the loss of alveolar bone, which is
Abbreviations: OS, Oxidative stress; PDLCs, periodontal ligament cells; Mfn1, mitofusin 1; Mfn2, mitofusin 2; Drp1, dynamin-related protein 1; Fis1, fission protein 1; OPA1, Optic atrophy 1; NAC, N-acetyl-cysteine; ROS, reactive oxygen species; TUNEL, Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling; H2O2, hydrogen peroxide. * Corresponding author. Institute of Stomatology, School and Hospital of Stomatology, Wenzhou Medical University, No. 373, Xueyuan West Road, Lucheng District, Wenzhou, PR China. ** Corresponding author. Department of Prosthodontics, School and Hospital of Stomatology, Wenzhou Medical University, No. 373, Xueyuan West Road, Lucheng District, Wenzhou, PR China. *** Corresponding author. Department of Oral Maxillofacial Surgery, School and Hospital of Stomatology, Wenzhou Medical University, No. 373, Xueyuan West Road, Lucheng District, Wenzhou, PR China. E-mail addresses: [email protected] (S. Zhao), [email protected] (J. Ma), [email protected] (S. Huang). 1 Contribute equally to this paper.
regarded as the leading factor of adult tooth loss [1,2]. PDL cells (PDLCs), the major cell type of periodontal ligament (PDL), are considered important participants of periodontal metabolism and remodeling [3]. The number of PDLCs is kept relatively stable through the balance of cell proliferation and death under physiological conditions [4]. Various pathological factors, including periodontal pathogens, cytokines, and drugs can lead to PDLCs apoptosis, accounted for periodontitis [5,6]. However, the mechanisms underlying apoptosis of PDLCs are not fully understood. Perhaps this is why the therapies available for treating periodontitis are not very efficacious. Therefore, the mechanisms related to PDLCs apoptosis must be further explored to identify effective therapeutic approaches to periodontitis. Accumulating evidence suggests that oxidative stress (OS) features overproduction of highly reactive oxygen species (ROS) owing to the disturbed pro-oxidant/antioxidant balance, playing a key role in the onset and progression of inflammation [7]. Moreover, studies indicate that periodontal-tissue destruction closely correlates with excessive ROS production and with compromised antioxidant capacity in periodontitis [8]. Notably, administration of antioxidants can effectively inhibit the development of periodontitis, partially
https://doi.org/10.1016/j.bbrc.2018.12.143 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article as: Y. Chen et al., Mitochondrial abnormalities are involved in periodontal ligament fibroblast apoptosis induced by oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.143
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through alleviation of PDLCs apoptosis induced by OS [9]. Thus, investigating the mechanisms by which OS affects the apoptosis of PDLCs represents one of the primary tasks in the search for effective treatment of periodontitis. Mitochondria are the primary source of cellular ROS and are subject to a direct attack by ROS [10]. Overproduced ROS cause oxidative damage to mitochondrial proteins and structures, thus subsequently leading to mitochondrial dysfunction [11]. The latter manifests itself as decreases in mitochondrial membrane potential and in uncoupled electron transport (in the electron transport chain) along with ATP downregulation, which affects the function of cells and subsequently results in apoptosis [12]. Our previous study has revealed that mitochondrial dysfunction is closely associated with aggravation of periodontitis caused by diabetes [13]. In addition, mitochondrial dysfunction is considered an important contributing factor of apoptosis of osteoblasts and of Hep-G2 cells under the influence of OS [14,15]. However, few studies have comprehensively examined the participation of mitochondrial dysfunction in periodontal-cell apoptosis caused by OS. Furthermore, mitochondria are organelles that dynamically alter their shape by frequent fusion and fission to continuously perform their function in the cell. Mitochondrial dynamics are not only critical for the homeostasis of mitochondria but also important for the well-being of the whole cell [16]. ROS are important for regulating the mitochondrial dynamics during several physiological and pathological processes [17]. Moreover, it is known that the loss of mitochondrial function, secondary to defects in mitochondrial dynamics, also leads to an increase of ROS generation [18]. These studies indicate that impaired mitochondrial dynamic leads to mitochondrial dysfunction and OS, which induces cellular perturbation and injury. It is still unclear, however, whether this mitochondrial fusion-and-fission machinery is active in OSaffected PDLCs. Therefore, in this study, our objectives were to investigate (1) whether mitochondrial dysfunction participates in the OS-induced apoptosis of human PDLCs (hPDLCs) and (2) whether mitochondrial dynamics act as a regulator of hPDLCs apoptosis induced by OS. We found that improvement in mitochondrial function exerted a significant protective effect against the hPDLCs apoptosis induced by OS. Thus, this result may have significant implications for therapeutic interventions in periodontitis. 2. Materials and Methods 2.1. Cell culture and treatment The study protocol was approved by the Committee on Research on Human Subjects of the School and Hospital of Stomatology, Wenzhou Medical University. hPDLCs were obtained from healthy third molars of five healthy men aged 20e30 years (mean age: 24.6) who underwent molar extraction after providing informed consent. PDL tissues were extracted from the root surface of a tooth and were cut into 1e2 mm2 pieces. The tissue was cultured in a-minimum essential medium containing 10% of fetal bovine serum and penicillin (100 U/ml) in a humidified incubator at 37 C and 5% CO2. Cells were passaged when hPDLCs reached 70%e80% confluence. Cells at passages 4e8 were used in this study.
2.3. Cell viability hPDLCs were cultured in 96-well plates (5 103 cells per well) under different conditions. Then, hPDLCs were incubated with MTT solution (5 mg/mL, Sigma-Aldrich). After 4 h at 37 C, the medium was replaced with 100 ml of dimethyl sulfoxide to dissolve the formazan crystals. The spectrophotometric absorbance at 490 nm was measured on a microtiter plate reader. 2.4. Detection of apoptosis by flow cytometry and Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) assays hPDLCs were collected and labeled with the Annexin V-FITC Apoptosis Detection Kit (Sigma-Aldrich). Then, cytofluorometric analysis was conducted on a FACScan laser flow cytometer. hPDLCs were preincubated with or without NAC, followed by exposure to 0.3 mM H2O2 for 24 h. Samples were processed by means of the TUNEL kit (Roche) and counterstained with 40 , 6diamidino-2-phenylindole (DAPI; Invitrogen). Image acquisition was performed under a fluorescence microscope (Leica TCS SPE). 2.5. Western blot analyses Protein levels were detected by western blot as we described before [14]. Primary antibodies were diluted in 5% defatted milk (Drp1, 1:1000; Fis1, 1:1000; OPA1, 1:1000; Mfn1, 1:1000; Mfn2, 1:1000; anti-b-actin, 1:4000; BCL-2, 1:2000; BAX, 1:2000). 2.6. Functional imaging assays hPDLCs were incubated with 2.5 mM MitoSOX for 30 min to detect the mitochondrial ROS levels. Furthermore, hPDLCs were costained with Mitotracker Green and tetramethylrhodamine methyl ester (TMRM) for 30 min to assess mitochondrial membrane potential. Mitotracker Deep Red served for detection of mitochondrial morphology alterations. A fluorescence microscope was employed to capture images. 2.7. ATP synthesis assays hPDLCs were collected after treatment with different solutions, incubated with the cell lysis buffer on ice for 30 min, centrifuged at 12,000g for 5 min, and the supernatant was collected. An ATP content detection kit was used to determine the level of ATP in the hPDLCs. 2.8. Data analysis Data were presented as the means ± SD. The Statview software (version 5.0.1, SAS Institute, USA) served for statistical analysis. Statistical significance in multiple comparisons was determined by one-way ANOVA along with individual post hoc Fisher's test. Data with P < 0.05 were regarded as statistically significant. * Additional experimental details are described in the Supplementary Materials and Methods. 3. Results
2.2. Cell treatments 3.1. H2O2-induced hPDLCs apoptosis Cells were incubated with or without hydrogen peroxide (H2O2; Sigma-Aldrich) and N-acetyl-cysteine (NAC; Sigma-Aldrich) for various periods, according to the experimental protocol. The final working concentrations were as follows: 0.3 mM H2O2 and 0.5 mM NAC.
The MTT results showed that after H2O2 treatment, cell viability significantly decreased in a time- and dose-dependent manner (Fig. 1A). The results of flow cytometry indicated that the apoptosis rate increased with treatment duration. Early apoptosis was
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Fig. 1. H2O2 induced apoptosis of hPDLCs. (A) Cell viability was evaluated by an MTT assay in hPDLCs in the presence of H2O2 at different concentrations and different periods. Error bars indicate SD (n ¼ 6). (BeC) Apoptosis was subjected to flow-cytometric quantification. Error bars indicate SD (n ¼ 6). (DeE) The TUNEL staining and assay. Error bars indicate SD (n ¼ 6; ***P < 0.0001). Scale bar ¼ 100 mm. (F) Western blotting of BAX and BCL-2 in hPDLCs in the presence of H2O2. (G) BAX, and (H) BCL-2 relative to b-actin. Error bars indicate SD (n ¼ 6; *P < 0.05, N.S. indicates P > 0.05).
significant after 0.3 mM H2O2 treatment for 12 h, and 0.3 mM H2O2 for 24 h induced late apoptosis and slightly increased necrosis (Fig. 1BeC). H2O2-induced DNA damage was next confirmed by obviously increased TUNEL-positive staining (Fig. 1DeE). Western blot analysis revealed that H2O2 treatment increased the protein expression of BAX but did not significantly affect BCL-2 expression (Fig. 1FeH). These experiments indicated that H2O2induced hPDLCs death was primarily apoptosis. 3.2. NAC attenuated H2O2-induced apoptosis In our preliminary experiment we found that the optimal concentration of NAC was 0.5 mM which was chosen for the present experiment. The apoptosis induced by H2O2 was significantly attenuated when hPDLCs were cotreatment with 0.5 mM NAC and 0.3 mM H2O2 for 24 h (Fig. 2B). The results of the TUNEL assay
confirmed that NAC reduced the incidence of apoptosis (Fig. 2CeD). After that, western blot analysis showed that the protein expression of BAX markedly decreased after NAC treatment (Fig. 2EeF). The above results suggested that the cytotoxic effects of H2O2-induced apoptosis were effectively attenuated by NAC. 3.3. NAC restored the mitochondrial dysfunction induced by H2O2 Compared with the cells only treated with H2O2, NAC preconditioning had significantly lower mitochondrial ROS levels, according to the lower intensity of MitoSOX staining (Fig. 3AeB). Compared with cells receiving only H2O2, NAC preconditioning had apparently more active ATP synthesis (Fig. 3C) and higher mitochondrial membrane potential (Fig. 3DeE). These results suggested that NAC antagonizes the adverse effects of H2O2 on mitochondria of hPDLCs.
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Fig. 2. NAC attenuated H2O2-induced apoptosis. hPDLCs were exposed to 0.5 mM NAC and 0.3 mM H2O2 for 24 h. (A) Samples were examined under a microscope and (B) processed for the MTT assay, (C) TUNEL assay, and (D) for measurement of the apoptosis rate of hPDLCs (***P < 0.0001). Scale bar ¼ 100 mm. (E) Western blot band of BAX and (F) the relative intensities (fold) of BAX (***P < 0.0001).
3.4. NAC ameliorated the H2O2-induced disruption of mitochondrial dynamics Mitochondria in the cells treated with H2O2 were fragmented, misshapen, and bleblike and collapsed away from the mitochondrial network (Fig. 4A). In addition, H2O2 decreased the mitochondrial density and length, which were partially recovered by NAC (Fig. 4BeC). We next evaluated the potential alteration of mitochondrial dynamics in the OS model (Fig. 4D). The results suggested that the expression of L-OPA1 and Mfn 2 was lower in the H2O2 group than in the control group (Fig. 4EeJ). NAC preconditioning group had higher expression levels of L-OPA1, Mfn1, and Mfn2 than the H2O2-stimulated alone group did. In contrast, S-OPA1, Drp1, and Fis1 expression increased in the H2O2-stimulated alone group but this increase was attenuated in the NAC preconditioning group (Fig. 4FeI). 4. Discussion Although OS-induced PDLCs apoptosis plays a critical role in the initiation and progression of periodontitis, the mechanism behind
this phenomenon is still unclear. Emerging evidence indicates that mitochondrial dysfunction is closely linked to OS. However, whether mitochondrial dysfunction is involved in PDLCs apoptosis induced by OS is poorly understood. In the current study, we clearly demonstrated that mitochondrial dysfunction and impaired mitochondrial dynamics were evident in the process of hPDLCs apoptosis induced by OS. Moreover, the reversal of OS-induced mitochondrial abnormalities using an antioxidant (NAC) significantly inhibited PDLCs apoptosis. Thus, our study uncovered mitochondrial abnormalities during OS-induced hPDLCs apoptosis, which may promote the initiation and development of periodontitis. H2O2 is a classical ROS that is produced by almost all types of OS and can infiltrate cells through their plasma membrane [19]. To PDLCs, H2O2-induced OS is inevitable because H2O2 is continuously produced by periodontal pathogens or their byproducts. Persistent OS causes apoptosis of PDLCs, thereby eventually causing malfunction of the PDL [20]. Thus, we established an H2O2-induced hPDLCs injury model according to our previous studies [13,18]. The results of flow cytometry and TUNEL assay showed that H2O2 elicited the apoptosis of hPDLCs in a dose- and time-dependent manner; this finding is coincident with the results of our past
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Fig. 3. NAC reversed the mitochondrial dysfunction induced by H2O2. (A) Representative images of MitoSOX staining and (B) quantification in the indicated groups. (C) ATP synthesis detected in the indicated groups (***P < 0.0001, *P < 0.05). Error bars indicate SD (n ¼ 6). (D) Representative images of TMRM staining and (E) quantification in the indicated groups (**P < 0.001, *P < 0.05). The scale bar is 100 mm.
studies [8,21]. The ratio of BCL-2 (antiapoptotic protein) to BAX (proapoptotic protein) determines the susceptibility of a given cell to apoptosis [22]. The expression of BAX in the hPDLCs was significantly increased by H2O2 in our study, in agreement with findings in Saos-2 human osteosarcoma cells [23] and human fetal lung fibroblasts [24]. In contrast to those studies, we found that BCL-2 expression was not significantly affected by H2O2; this result might be due to the difference in cell types. In addition, NAC is a glutathione precursor and regarded as an important intracellular antioxidant [25]. One study indicates that NAC activates antioxidant enzymes, which may prevent OS-induced apoptosis and the development of diseases [26]. In agreement with these results, pretreating PDLCs with NAC significantly attenuated H2O2-induced apoptosis in the present study. Therefore, application of antioxidants has a good potential to inhibit PDLCs apoptosis and postpone the progression of periodontitis. Mitochondria are organelles that not only act as a “power factory” of cells but also play vital roles in cell functions, death, and survival [27]. Mitochondrial dysfunction can trigger a variety of intracellular signaling cascades and lead to OS and apoptosis, which are involved in the progression of almost all diseases [28]. This notion is consistent with our results, namely, that H2O2 led to the mitochondrial dysfunction of hPDLCs, including increased ROS levels, greater incidence of mitochondrial membrane potential collapse, and reduced ATP levels. Notably, pretreating hPDLCs with NAC effectively counteracted ROS-induced mitochondrial dysfunction. Our current results confirmed that mitochondrial OS and dysfunction is deeply involved in apoptosis of hPDLCs. Mitochondria-targeted antioxidants, such as mitoquinone and
mitoTEMPO, have been confirmed to be effective at ameliorating OS-related diseases [29,30]. Thus, these agents may be applied to this hPDLCs injury model to further confirm the involvement of mitochondrial dysfunction in PDLCs apoptosis elicited by OS in our future research. Mitochondria are dynamic organelles that participate in repeated fusion and fission cycles [31]. The dynamic processes of mitochondria are crucial to the maintenance of their morphology, function, and distribution [32]. Moreover, growing evidence suggests that fusion and fission factors perform an important function in apoptosis [33]. In the present study, we found that when hPDLCs were treated with H2O2, the original rodlike or elongated mitochondria became misshapen or bleblike, and H2O2 caused greater fragmentation of mitochondria in hPDLCs. A growing body of evidence indicates that mitochondrial fragmentation has been accepted as an early event in the apoptosis of a variety of mammalian cells [34]. Together with our current findings, this concept highlights critical involvement of mitochondrial fission in OS-induced PDLCs apoptosis. Mitochondria actively divide (fission) and fuse with one another (fusion). Mitochondrial fusion is regulated by Mfn1 and Mfn2, outer-membrane GTPases that mediate outer-mitochondrialmembrane fusion, and by OPA1, which regulates inner-membrane fusion. Research has shown that the absence of mitofusins reduces mitochondrial fusion, impairs mtDNA maintenance and oxidative phosphorylation, and subsequently leads to apoptosis [35]. Moreover, OPA1, which exists in the L-OPA1 and S-OPA1 forms, is generated by processing at specific sites [36]. In response to a proapoptotic stimulus, L-OPA1 is proteolytically cleaved to
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Fig. 4. NAC ameliorated the disruption of mitochondrial dynamics by H2O2. (A) Representative images of Mitotracker Deep Red staining in each cell group. Scale bar ¼ 10 mm. The right-hand panel is a larger image of the one on the left. (B) Detection of mitochondrial density and (C) length in each cell group (*P < 0.05). (D) The protein levels detected by western blotting: (EeF) OPA1, (G) Drp1, (H) Fis1, (I) Mfn1, and (J) Mfn2 relative to b-actin in indicated cell groups with H2O2 or NAC þ H2O2 treatment. Error bars indicate SD (n ¼ 6; ***P < 0.0001, **P < 0.001, and *P < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
form S-OPA1, and thus induces mitochondrial fragmentation and apoptosis [37]. The results of the present study revealed that H2O2 treatment significantly decreases the expression of Mfn1 and Mfn2, while promoting the rapid cleavage of L-OPA1 into S-OPA1. All these data suggest that the balance of mitochondrial dynamics in hPDLCs caused a switch to mitochondrial fission. One study suggests that a hyperfused mitochondrial reticulum is activated upon cellular stress and protects cells from death [38]. Thus, strategies promoting mitochondrial fusion may offer new insights into the attenuation of OS inducing PDLCs apoptosis; this research area
must be explored in future studies. Thus, the exact function of fusion proteins in PDLCs apoptosis should also be confirmed. Mitochondrial fission is mainly regulated and controlled by Drp1, a protein that exists in the cytoplasm and is translocated to mitochondria [39]. Fis1, a key mitochondrial complex involved in mitochondrial fission, functions by binding Drp1 [40]. In our current study, H2O2 significantly increased the expression of Fis1 and Drp1, thus further supporting the prediction that mitochondrial fission was intimately involved in the apoptosis of hPDLCs. Moreover, downregulation of Drp1 reduces the outer-mitochondrial-
Please cite this article as: Y. Chen et al., Mitochondrial abnormalities are involved in periodontal ligament fibroblast apoptosis induced by oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.143
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membrane scission related to apoptosis and attenuates mitochondrial fission [40]. Another study has proved that the decreased expression of Fis1 shifts the dynamic balance in favor of fusion, thereby reducing apoptosis [41]. Therefore, to some extent, downregulation of Fis1 or Drp1 can be an efficient way to reduce OS-induced hPDLCs apoptosis. Notably, we found that after treatment with NAC, the imbalance of mitochondrial dynamics was markedly reversed via the inhibition of mitochondrial fission and promotion of mitochondrial fusion, which favored the maintenance of mitochondrial function and cell survival in the current study. Taken together, these findings not only reveal that ROS play an irreplaceable part in the regulation of fusion and fission of PDLCs but also suggest that the regulation of mitochondrial dynamics may be a useful therapeutic strategy against periodontitis. Although our results imply that mitochondrial dysfunction is crucial for the OS-induced apoptosis of hPDLCs, this study has several limitations. First, the detailed molecular mechanisms behind mitochondrial dysfunction and abnormalities of mitochondrial dynamics were not explored. Second, the molecular mechanisms uncovered in this study were not corroborated in vivo. Conflicts of interest All authors declare that they have no competing interests. Acknowledgments This work was supported by the following grants: the Natural Science Foundation of China (No. 81500817, 81870777, and 81802230), Zhejiang Provincial Natural Science Foundation of China (No. LY15H140008), Health Science and Technology Project of Zhejiang Province (No. 2016KYB184), Zhejiang Provincial Science and Technology Project for Public Welfare (No. 2017C33081), Wenzhou Municipal Science and Technology Project for Public Welfare (No. Y20150069 and Y20170026), Wenzhou Science and Technology Bureau (No. Y20160051 and Y20140704), and Zhejiang Provincial College Students’ Science and Technology Innovation Project and Fresh Talent Program (2017R413082, 2016R413081). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.12.143. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.12.143. References [1] N.J. Kassebaum, A.G.C. Smith, E. Bernabe, et al., Global, regional, and national prevalence, incidence, and disability-adjusted life years for oral conditions for 195 countries, 1990-2015: a systematic analysis for the global burden of diseases, injuries, and risk factors, J. Dent. Res. 96 (2017) 380e387. [2] E.M. Cardoso, C. Reis, M.C. Manzanares-Cespedes, Chronic periodontitis, inflammatory cytokines, and interrelationship with other chronic diseases, Postgrad. Med. 130 (2018) 98e104. [3] D. Jonsson, D. Nebel, G. Bratthall, et al., The human periodontal ligament cell: a fibroblast-like cell acting as an immune cell, J. Periodontal. Res. 46 (2011) 153e157. [4] E. Sigalas, J.D. Regan, P.R. Kramer, et al., Survival of human periodontal ligament cells in media proposed for transport of avulsed teeth, Dent. Traumatol. 20 (2004) 21e28. [5] T. Hatai, M. Yokozeki, N. Funato, et al., Apoptosis of periodontal ligament cells induced by mechanical stress during tooth movement, Oral Dis. 7 (2001) 287e290. [6] S.W. Jee, S. Wang, Y.L. Kapila, Specific pro-apoptotic fibronectin fragments modulate proteinase expression in periodontal ligament cells, J. Periodontol.
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Please cite this article as: Y. Chen et al., Mitochondrial abnormalities are involved in periodontal ligament fibroblast apoptosis induced by oxidative stress, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.143