Pharmacological Research 91 (2015) 1–8

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Lipophilic antioxidants prevent lipopolysaccharide-induced mitochondrial dysfunction through mitochondrial biogenesis improvement Pedro Bullón a,b , Lourdes Román-Malo b , Fabiola Marín-Aguilar b , José Miguel Alvarez-Suarez c,d,e , Francesca Giampieri f , Maurizio Battino c,g,∗ , Mario D. Cordero b,∗∗ a

Department of Periodontology, Dental School, University of Sevilla, Spain Research Laboratory, Dental School, University of Sevilla, Sevilla, Spain c Dipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche (DISCO)-Sez. Biochimica, Facoltà di Medicina, Università Politecnica delle Marche, Ancona 60131, Italy d Area de Nutrición y Salud, Universidad Internacional Iberoamericana (UNINI), Campeche C.P. 24040, Mexico e Facultad de Ciencias de la Salud, Universidad Nacional de Chimborazo, Riobamba, Ecuador f Dipartimento di Scienze Agrarie, Alimentari e Ambientali (D3A), Università Politecnica delle Marche, Via Ranieri 65, Ancona 60131, Italy g Director Centre for Nutrition & Health, Universidad Europea del Atlantico (UEA), Santander 39011, Spain b

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Article history: Received 17 September 2014 Received in revised form 16 October 2014 Accepted 23 October 2014 Available online 3 November 2014 Keywords: Porphyromonas gingivalis Lipopolysaccharide Coenzyme Q10 N-acetylcysteine Mitochondria

a b s t r a c t Oxidative stress is implicated in several infectious diseases. In this regard, lipopolysaccharide (LPS), an endotoxic component, induces mitochondrial dysfunction and oxidative stress in several pathological events such as periodontal disease or sepsis. In our experiments, LPS-treated fibroblasts provoked increased oxidative stress, mitochondrial dysfunction, reduced oxygen consumption and mitochondrial biogenesis. After comparing coenzyme Q10 (CoQ10 ) and N-acetylcysteine (NAC), we observed a more significant protection of CoQ10 than of NAC, which was comparable with other lipophilic and hydrophilic antioxidants such as vitamin E or BHA respectively. CoQ10 improved mitochondrial biogenesis by activating PGC-1␣ and TFAM. This lipophilic antioxidant protection was observed in mice after LPS injection. These results show that mitochondria-targeted lipophilic antioxidants could be a possible specific therapeutic strategy in pharmacology in the treatment of infectious diseases and their complications. © 2014 Elsevier Ltd. All rights reserved.

Introduction In general, oxidative stress can be defined as an imbalance between the presence of high levels of reactive oxygen species (ROS), and antioxidant defense mechanisms. These toxic molecules are formed via oxidation–reduction reactions and are highly reactive since they have an odd number of electrons. ROS generated under physiological conditions are essential for life, as they are

∗ Corresponding author at: Dipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche (DISCO)-Sez. Biochimica, Facoltà di Medicina, Università Politecnica delle Marche, Ancona 60131, Italy. Tel.: +39 0712204646; fax: +39 0712204123. ∗∗ Corresponding author at: Research Laboratory, Dental School, Universidad de Sevilla, C/Avicena s/n, 41009 Sevilla, Spain. Tel.: +34 954 481120; fax: +34 954 486784. E-mail addresses: [email protected] (M. Battino), [email protected] (M.D. Cordero). http://dx.doi.org/10.1016/j.phrs.2014.10.007 1043-6618/© 2014 Elsevier Ltd. All rights reserved.

involved in bactericidal activity of phagocytes, and in signal transduction pathways, regulating cell growth and reduction–oxidation (redox) status [1]. ROS includes free radicals, such as hydroxyl and superoxide radicals, and non-radicals, including hydrogen peroxide and singlet oxygen. Oxidative stress and generation of free radicals, as a primary or secondary event, have been related to a great number of diseases, including infectious diseases, atherosclerosis and diabetes [1]. Within most cells, mitochondria are the main source of reactive species generated as a by-product of energy production. Within the mitochondria the primary ROS produced is superoxide, most of which is converted to hydrogen peroxide by the action of superoxide dismutase. The mitochondrial production of superoxide has been ascribed to several electron transport chain enzymes, including Complex I and Complex III. These complexes along with Coenzyme Q10 (CoQ10 ) may leak electrons which in turn may interact with oxygen, thus forming ROS [2,3]. All conditions able to alter mitochondria efficiency can enhance ROS production, having

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a direct and critical effect on oxidative stress. Regarding this, a wide range of antioxidants could be targeted to mitochondria to reduce the effect of oxidative stress as a possible strategy in pharmacology, however, antioxidants have had limited success in preventing the progression of diseases involving mitochondrial oxidative damage [4]. Periodontitis, an infectious disease, has also been related to a specific group of bacteria, three of which have been considered as the main periodontal pathogens: Tannerella forsythia, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis [5,6]. Several studies have demonstrated an increase of products from oxidative damage in plasma and serum and mitochondrial dysfunction in blood cells of subjects with periodontitis compared with healthy individuals [7,8]. Moreover, there is evidence of a decreased anti-oxidant capacity in subjects with periodontitis, evaluated by different assays [7,8]. Because mitochondrial dysfunction and oxidative stress are two of the main factors studied which may be able to explain the pathophysiological mechanism of inflammatory conditions occurring in atherosclerosis, CVDs and periodontitis, the current study is a comparative one evaluating the in vitro and in vivo effect of lipophilic and hydrophilic antioxidants in mitochondrial dysfunction promoted by P. gingivalis lipopolysaccharide (LPS). Materials and methods Ethical statements Written informed consent and the approval of the ethical committee of the University of Seville were obtained, according to the principles of the Declaration of Helsinki. Studies in mice were performed in accordance with the European Union guidelines (86/609/EU) and Spanish regulations for the use of laboratory animals in chronic experiments (BOE 67/850912, 1988). All experiments were approved by the local institutional animal care committee. Animals and drug administration Four groups (control and treated) of eight six-week-old male C57/BL mice weighing 25–30 g were maintained on a 12 h light/dark cycle. LPS, from P. gingivalis, was dissolved in saline (vehicle) and intra-peritoneally administered at a dose of 500 ng/ml, CoQ10 10 mg/kg/day and NAC 20 mg/kg/day for 15 days. After treatment, mice were anesthetized with CO2 and sacrificed by decapitation. Brain, liver, kidney were isolated and stored at −80 ◦ C until analysis. Reagents and chemicals MitosoxTM and Hoechst 3342 were purchased from Invitrogen/Molecular Probes (Eugene, OR, USA); a cocktail of protease inhibitors from Boehringer Mannheim (Indianapolis, IN, USA); and Immun Star HRP substrate kit from Bio-Rad Laboratories Inc. (Hercules, CA, USA). Monoclonal Anti-Actin antibodies, butylatedhydroxyanisole (BHA), N-acetylcysteine (NAC), and trypsin-EDTA solution and all other chemicals were purchased from SigmaAldrich. (St. Louis, MO, USA). Fibroblast cultures Human gingival fibroblasts (HGF) isolated from a healthy 25year-old male, were cultured in D-MEM media (4500 mg/L glucose, l-glutamine, piruvate), (Gibco, Invitrogen, Eugene, OR, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, Eugene, OR, USA) and antibiotics (Sigma Chemical Co., St. Louis,

MO, USA). Cells were incubated at 37 ◦ C in a 5% CO2 atmosphere. HGF were cultured with 10 ␮g/ml LPS of P. gingivalis (Nucliber S.A., Spain). When required, CoQ10 , alpha tocopherol (␣-toc), BHA and NAC were added to the plates at a final concentration of 30 ␮M, 10 ␮M, 40 ␮M, and 10 mM, respectively. Mitochondrial ROS production Mitochondrial ROS generation in PBMCs and fibroblasts were assessed by MitoSOXTM Red, a red mitochondrial superoxide indicator. MitoSOX Red is a novel fluorogenic dye recently developed and validated for highly selective detection of superoxide in the mitochondria of living cells. MitoSOXTM Red reagent is live-cell permeant and is rapidly and selectively targeted to the mitochondria. Once in the mitochondria, MitoSOXTM Red reagent is oxidized by superoxide and exhibits red fluorescence. Flow cytometry Approximately 1 × 106 cells were incubated with 1 ␮M MitoSOXTM Red for 30 min at 37 ◦ C, washed twice with PBS, resuspended in 500 ␮l of PBS and analyzed by flow cytometry in an Epics XL cytometer, Beckman Coultier, Brea, California, USA (excitation at 510 nm and fluorescence detection at 580 nm). Fluorescence microscopy Cells grown on microscope slides in 6-well plates for 24 h were incubated with MitoSOXTM Red for 30 min at 37 ◦ C, washed twice in PBS, fixed with 4% paraformaldehyde in PBS for 0.5–1 h, and washed twice with PBS. After that, cells were incubated for 10 min at 37 ◦ C with anti-LC3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Slides were analyzed by immunofluorescence microscopy (MitoSOXTM Red: excitation wavelength = 555/28; emission wavelength = 617/73). Western blotting Whole cellular lysate from fibroblasts was prepared by gentle shaking with a buffer containing 0.9% NaCl, 20 mM Tris–ClH, pH 7.6, 0.1% triton X-100, 1 mM phenylmethylsulfonylfluoride and 0.01% leupeptine. Electrophoresis was carried out in a 10–15% acrylamide SDS/PAGE. Proteins were transferred to Immobilon membranes (Amersham Pharmacia, Piscataway, NJ). PGC-1 ␣, TFAM, and DNA repair enzyme 8-oxoguanine DNA glycolase-1 (OGG-1) antibodies were used to detect proteins by Western blotting. Proteins were electrophoresed, transferred to nitrocellulose membranes and, after blocking over night at 4 ◦ C, incubated with the respective antibody solution, diluted at 1:1000. Membranes were then probed with their respective secondary antibody (1:2500). Immunolabeled proteins were detected by using a chemiluminescence method (Immun Star HRP substrate kit, Bio-Rad Laboratories Inc., Hercules, CA). Protein was determined by the Bradford method. Measurement of citrate synthase activity The specific activity of citrate synthase in whole-cell extracts prepared from BMC was measured at 412 nm minus 360 nm (13.6 mM−1 cm−1 ) using 5,5-dithio-bis(2-nitrobenzoic acid) to detect free sulfhydryl groups in coenzyme A as described previously [2]. ATP levels ATP levels were determined by a bioluminescence assay using an ATP determination kit from Invitrogen-Molecular Probes (Eugene, OR, USA) according to the manufacturer’s instructions.

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Oxygen consumption rate (OCR) The oxygen consumption rate (OCR) was assessed in real-time using the 24 well Extracellular Flux Analyzer XF-24 (Seahorse Bioscience, North Billerica, MA, USA) according to the manufacturer’s protocol, which allows to measure OCR changes after up to four sequential additions of compounds. Cells (5 × 104 /well) were seeded for 16 h in the XF-24 plate before the experiment in a DMEM/10% serum medium and then incubated for 24 h with the different compounds studied. Before starting measurements, cells were placed in a running DMEM medium (supplemented with 25 mM glucose, 2 mM glutamine, 1 mM sodium Pyruvate,

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and without serum) and pre-incubated for 20 min at 37 ◦ C in the absence of CO2 in the XF Prep Station incubator (Seahorse Bioscience, Billerica MA, USA). Cells were transferred to an XF-24 Extracellular Flux Analyzer and after an OCR baseline measurement a profiling of mitochondrial function was performed by sequential injection of four compounds that affect bioenergetics, as follows: 55 ␮l of oligomycin (final concentration 2.5 ␮g/mL) at injection in port A, 61 ␮l of 2,4-dinitrophenol (2,4-DNP) (final concentration 1 mM) at injection in port B, and 68 ␮l of antimycin/rotenone (final concentration 10 ␮M/1 ␮M) at injection in port C. The best concentration of each inhibitor and uncoupler was obtained on the basis of a proper titolation curve. A minimum of five wells were

Fig. 1. Effect of LPS and antioxidants in cell death and mitochondrial ROS production. (A) CoQ10 induced more protection than NAC on LPS-induced apoptosis. (B) Flow cytometry quantification of ROS production showed more protective effect of CoQ10 (C) MitoSOXTM Red stain revealed increased superoxide anion. MitoSOXTM Red colocalized with cytochrome c in merged images, indicating that superoxide anion production occurred mainly in mitochondria. (D) Magnification of a small area in LPS-treated fibroblast. Arrows indicate Mitosox co-localized with the cytochrome c signal present in fragmented mitochondria. Data represent the mean ± SD of three separate experiments. *P < 0.001, between control and LPS treated cells; **P < 0.001, between the LPS in absence or presence of CoQ10 ; a P < 0.005, between the LPS in absence or presence of NAC.

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Fig. 2. Oxygen consumption rate (OCR) in cells treated with LPS, CoQ10 and NAC. (A) OCR was monitored through Seahorse XF-24 Extracellular Flux Analyzer with the sequential injection of Oligomycin (1 ␮g/mL), 2,4-DNP (100 ␮M), Rotenone (1 ␮M) at the indicated time point into each well, after baseline rate measurement. (B) The basal OCR was markedly affected in cells treated with LPS, and improved in LPS-CoQ and LPS-NAC. (C) The spare respiratory capacity (SRC) treatment with LPS caused a significant decrease which was improved by antioxidants. Data represent the mean ± SD of three separate experiments. *P < 0.05, between and antioxidants treated cells; **P < 0.05, between control and LPS treated cells.

utilized per condition in any given experiment. Data are expressed as pmol of O2 consumed per minute normalized to 1000 cells (pmol O2 /1000 cells/min). Lipid peroxidation Lipid peroxidation in cells was determined by analyzing the accumulation of lipoperoxides using a commercial kit from Cayman Chemical (Ann Arbor, Michigan, USA). TBARS are expressed in terms of malondialdehyde (MDA) levels. Lipid hydroperoxides The FOX assay was carried out according to the method previously reported [9]. The FOX reagent was prepared by mixing in the following order: 90 ml methanol, 88 mg BHT, 10 ml 250 mM H2SO4, 9.8 mg ammonium ferrous sulfate hexahydrate and 7.6 mg Xylenol Orange. To 320 ␮l of sample, 680 ␮l of FOX reagent were added and the solution was incubated for 30 min at 37 ◦ C with gentle shaking. After a short high-speed centrifugation (3000 × g for 1 min at room temperature), sample absorbance was read at 560 nm against the blank (0.9% NaCl and FOX reagent). For hydroperoxide quantification, a serial standard dilution of hydrogen peroxide was used. Statistical analysis Data in figures is given as mean ± SD. Data between different groups were analyzed statistically by using ANOVA (SPSS for

Windows, 19, 2010, SPSS Inc. Chicago, IL, USA). For cell-culture studies, Student’s t test was used for data analyses. A value of p < 0.05 was considered significant. To compare the behavioral results from animals treated with vehicle alone or with drugs a two-way variance (ANOVA) analysis was used.

Results and discussion CoQ10 induces a more efficient prevention of LPS-induced toxicity and ROS production than N-acetylcysteine (NAC) Toxic effects have been described in LPS treatment characterized by an increment of apoptotic nuclei condensation and caspase 3 activation, suggesting that LPS treatment induces apoptosis by the activation of at least the intrinsic pathway [2]. Concerning this aspect, it has been shown that ROS plays a relevant role in mitochondrion-to-mitochondrion ROS-signaling as a positive feedback mechanism for enhanced ROS production potentially leading to significant mitochondrial injury [2]. In this context, cytochrome c release and a consequent activation of pro-caspase 9, caspase 3 and endonuclease G result in DNA degradation and apoptotic death. Mitochondria-targeted lipophilic antioxidants selectively block mitochondrial oxidative damage and prevent some types of cell death, so it could be appropriate to develop probes of mitochondrial function. In our experiments, we studied the effect of CoQ10 in cell death and mitochondrial ROS production compared with a well knowledge hydrophilic antioxidant such as NAC in human gingival

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Fig. 3. Effect of LPS and antioxidants in bioenergetic, mitochondrial mass and biogenesis (A) ATP levels after LPS and antioxidants were analyzed by bioluminescence as described in section “Material and Methods”. (B) Citrate Synthase specific activity in fibroblasts after LPS and antioxidants was performed as described in section “Material and Methods”. Data represent the mean ± SD of three separate experiments. *P < 0.001, between control and LPS treated cells; **P < 0.001, between LPS in absence or presence of CoQ10 ; a P < 0.005, between LPS in absence or presence of NAC. (C) Mitochondrial biogenesis after LPS and antioxidant treatment was determined by protein levels of PGC-␣ and TFAM by using western blotting. Protein levels were determined by densitometric analysis (IOD, integrated optical intensity) of three different western blots and normalized to the GADPH signal.

fibroblasts. Both antioxidants induced an important protection by reducing cell death percentage and ROS production but this was more significant in the case of CoQ10 (Fig. 1A and B). MitoSOXTM Red fluorescence co-localized with mitochondrial cytochrome c oxidase (Fig. 1C). Fig. 1D clearly shows that in mitochondrial ROS-positive cells after LPS treatment, Mitosox co-localized with the cytochrome c signal present in fragmented mitochondria suggesting that mitochondria is responsible for ROS production. Impaired mitochondrial oxygen consumption by LPS is restored by CoQ10 and NAC P. gingivalis LPS has been shown to induce a mitochondrial dysfunction by inhibiting mitochondrial membrane potential ( m) and decrement of complex I and complex III in mitochondrial respiratory chain with a concomitant decrease of CoQ10 levels [2]. These compromised mitochondrial functions have several consequences including increased ROS production, described and demonstrated by us, and a deterioration of mitochondrial oxygen consumption which has never been studied before. Therefore, we investigated the protective effect of CoQ10 and NAC against the possible negative effect of LPS on mitochondrial functionality. This was assessed by measuring the Oxygen consumption rate (OCR) values in control and treated cells, exposed sequentially to each of four modulators of oxidative phosphorylation (OXPHOS) such as oligomycin (an inhibitor of F1Fo-ATPase or complex V), 2,4-DNP (uncoupling of the OXPHOS electron transport chain) and antimycin/rotenone (complex I and III inhibitors respectively) (Fig. 2A). The basal OCR was markedly affected in cells treated

with LPS, with values approximately 2.5-fold (p < 0.05) lower than in controls. On the contrary, basal OCR considerably improved (p < 0.05) in cells incubated together with LPS-CoQ10 and LPS-NAC (1.7-fold and 1.6-fold, respectively) compared to cells treated only with LPS (Fig. 2B). The spare respiratory capacity (SRC) of cells was obtained by calculating the mean of OCR values after injection of 2,4-DNP minus the basal respiration and could be used as an indicator of how close a cell is operating to its bioenergetic limit. Treatment with LPS caused a significant decrease (2.9-fold, p < 0.05) of SRC compared to control cells, while treatment together with LPS-CoQ10 and LPS-NAC caused a significant improvement (p < 0.05) approximately 2.9-fold and 2.3-fold respectively, when compared with cells treated only with LPS (Fig. 2C). Mitochondrial biogenesis impairment induced by LPS is restored by CoQ10 Mitochondria are highly dynamic organelles in cells which need to maintain very specific levels. For this, mitochondrial biogenesis and mitophagy are two pathways that regulate mitochondrial content and metabolism preserving homeostasis. Recently, we described reduced levels of CoQ10 and mitochondrial chain complex induced by LPS [2] and active autophagic processes in blood cells from periodontitis patients and activated in fibroblasts after P. gingivalis LPS [10]. To assess the functional consequences of decreased respiratory chain enzyme activities and CoQ10 levels, we determined ATP levels as an indicator of cellular bioenergetics and well-being status. As shown in Fig. 3A, LPS treatment provoked a significant contraction in ATP levels, suggesting that

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Fig. 4. Effect of LPS and Vitamin E or BHA in cell death and mitochondrial function. (A) Vitamin E induced more protection than BHA on LPS-induced apoptosis. (B) Flow cytometry quantification of ROS production showed more protective effect of Vitamin E. (C) ATP levels after LPS and antioxidants were analyzed by bioluminescence as described in section “Material and Methods”. (D) Citrate Synthase specific activity in fibroblasts after LPS and antioxidants was performed as described in section “Material and Methods”. Data represent the mean ± SD of three separate experiments. *P < 0.001, between control and LPS treated cells; **P < 0.001, between LPS in absence or presence of Vitamin E; a P < 0.005, between LPS in absence or presence of BHA.

mitochondrial power of energy production may be diminished. Furthermore, a reduced mitochondrial mass was confirmed by decrease of citrate synthase (Fig. 3B). To elucidate the mechanism of lowered mitochondrial mass after LPS treatment, the expression levels of proteins involved in mitochondrial biogenesis were determined. Protein expression levels of phosphorylated PGC-1␣ and TFAM were found to be diminished (Fig. 3C). This is the first time that a down regulation of mitochondrial biogenesis by P. gingivalis LPS has been demonstrated. Interestingly, CoQ10 induces a more significant restoration of mitochondrial biogenesis, ATP and mitochondrial mass than NAC. According to these in vitro data, CoQ10 causes a significant reduction of LPS-induced ROS and a more significant improvement of ATP and mitochondrial mass than NAC. Both antioxidants, however, induced similar restoration of oxygen consumption rate. This is interesting because NAC has a very evident antioxidant effect which has been described to be involved in the positive effect of mitochondrial respiration compared with CoQ analogs [4]. In this regard, a possible explanation of the more significant effect in the other parameters of CoQ10 compared with NAC is the induction of mitochondrial biogenesis of CoQ10 observed and demonstrated by us after oral CoQ10 treatment [3]. This effect on mitochondrial biogenesis was found in NAC treatment. Because CoQ10 , a lipophilic antioxidant, has been shown to be more effective than NAC, a hydrophilic antioxidant, we compared two well-known lipophilic and hydrophilic antioxidants, vitamin E and BHA respectively. Preliminary data about vitamin E showed a protective effect in LPS-induced cytotoxicity [11]. Therefore, according to our expectations, vitamin E induced a more significant

protection than BHA regarding the effect elicited by P. gingivalis LPS (Fig. 4). Mitochondrial dysfunction provoked by P. gingivalis LPS is restored by CoQ10 in mice In order to study the role of lipophilic and hydrophilic antioxidants in the LPS-induced mitochondrial dysfunction, and according to in vitro results, we exposed mice to intra-peritoneally administered LPS. LPS induces high levels of oxidative stress in brain, liver, and kidney compared with vehicle showing an increment of hydroperoxides and malondialdehyde levels (Fig. 5A and B). We also observed an important decrement regarding ATP and citrate synthase (Fig. 5C and D) and, interestingly CoQ10 generated a more significant restoration regarding LPS effect than NAC, according to in vitro results. As LPS has a very evident effect on mitochondria, antioxidants that act preferentially on mitochondria reduce mitochondrial damage and organ dysfunction [12]. The results described in this article may serve as a new way for designing experiments to better understand the influence of oxidative stress on the development of infectious diseases and generate new therapeutic strategies. NAC acts as an essential precursor to many endogenous antioxidants involved in the decomposition of peroxides and attenuates oxidative stress from various underlying causes by replenishing intracellular glutathione stores. However, CoQ10 can prevent lipid peroxidation by itself or by biochemical reduction of other antioxidants such as alpha-tocopherol (vitamin E) and ascorbate (vitamin C), transfer of electrons from complexes I and II to complex III

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Fig. 5. Effects of LPS, CoQ10 and NAC in brain, liver and kidney from mice. (A) Hydroperoxides levels in brain, liver and kidney were determined by FOX assay. (B) Lipid peroxidation in brain, liver and kidney was determined in terms of malondialdehyde (MDA) levels. (C) ATP levels after LPS and antioxidants were analyzed by bioluminescence as described in section “Material and Methods”. (D) Citrate Synthase specific activity in fibroblasts after LPS and antioxidants was performed as described in section “Material and Methods”. Data represent the mean ± SD of three separate experiments. *P < 0.001, between control and LPS treated cells; **P < 0.001 or ***P < 0.01, between the LPS in absence or presence of CoQ10 ; a P < 0.005 or aa P < 0.05, between the LPS in absence or presence of NAC.

in the oxidative phosphorylation with subsequent ATP generation and induce mitochondrial biogenesis [4,13]. Furthermore, kidney, brain and liver contain the highest endogenous levels of CoQ10 [14]. The findings of the present study show that lipophilic antioxidants such as CoQ10 and vitamin E ameliorated mitochondrial dysfunction, oxidative stress and reduced cell death with a more specific interaction with mitochondrial biogenesis, which is not observed in hydrophilic antioxidants such as NAC or BHA. Since CoQ10 is a pivotal element in mitochondrial respiratory chain and, at the same time, is an important antioxidant, these results suggest that mitochondrial dysfunction is crucial in the pathophysiology of periodontal disease and CoQ10 could be a therapeutic option in bacterial LPS infection, sepsis or other similar conditions. In this respect, new antioxidants based on CoQ or vitamin E such as MitoQ and MitoE, both lipohilic antioxidants with a protective role of mitochondria, have been shown to induce an important protection in a sepsisinduced organ failure model by LPS treatment [15]. Furthermore,

CoQ10 has been shown to have beneficial effects in periodontal diseases under experimental conditions in clinical trials [16–18]. Conclusions Previous work has shown that P. gingivalis, one of the key etiological factors for periodontal pathology, induces oxidative stress and mitochondrial dysfunction with bacterial LPS playing a major role in the pathogenesis of periodontal pathology which has been related to cardiovascular diseases, risk of developing cerebrovascular incidents and, in particular, non-hemorrhagic stroke. This is an interesting issue that warrants consideration when designing new experiments in pharmacology in order to gain further insight into its potential therapeutic applications, given that systemic antioxidant status is an exogenously modifiable factor. The results described in this article may serve as a novel

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way in designing experiments to better understand and generate new, more specific, therapeutic strategies. Conflict of interest statement All the authors declare that no conflict of interest exists for any of them. Acknowledgements Authors are indebted with Ms. Monica Glebocki for extensive editing of the manuscript. This work has been supported by Proyecto de Investigación de Excelencia de la Junta de Andalucía CTS113. References [1] Rajendran P, Nandakumar N, Rengarajan T, Palaniswami R, Gnanadhas EN, Lakshminarasaiah U, et al. Antioxidants and human diseases. Clin Chim Acta 2014;S0009-S8981:00262–9. [2] Bullon P, Cordero MD, Quiles JL, Morillo JM, del Carmen Ramirez-Tortosa M, Battino M. Mitochondrial dysfunction promoted by Porphyromonas gingivalis lipopolysaccharide as a possible link between cardiovascular disease and periodontitis. Free Radic Biol Med 2011;50:1336–43. [3] Cordero MD, Alcocer-Gómez E, de Miguel M, Culic O, Carrión AM, AlvarezSuarez JM, et al. Can coenzyme q10 improve clinical and molecular parameters in fibromyalgia. Antioxid Redox Signal 2013;19:1356–61. [4] Murphy MP. Targeting lipophilic cations to mitochondria. Biochim Biophys Acta 2008;1777:1028–31. [5] Armitage GC. Comparison of the microbiological features of chronic and aggressive periodontitis. Periodontol 2000 2010;53:70–88.

[6] Wade WG. The oral microbiome in health and disease. Pharmacol Res 2013;69:137–43. [7] Nibali L, Donos N. Periodontitis and redox status: a review. Curr Pharm Des 2013;19:2687–97. [8] Bullon P, Newman HN, Battino M. Obesity, diabetes mellitus, atherosclerosis and chronic periodontitis: a shared pathology via oxidative stress and mitochondrial dysfunction? Periodontol 2000 2014;64:139–53. [9] Jiang ZY, Hunt JV, Wolff SP. Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein. Anal Biochem 1992;202:384–438. [10] Bullon P, Cordero MD, Quiles JL, Ramirez-Tortosa Mdel C, Gonzalez-Alonso A, Alfonsi S, et al. Autophagy in periodontitis patients and gingival fibroblasts: unraveling the link between chronic diseases and inflammation. BMC Med 2012;10:122. [11] Nishio K, Horie M, Akazawa Y, Shichiri M, Iwahashi H, Hagihara Y, et al. Attenuation of lipopolysaccharide (LPS)-induced cytotoxicity by tocopherols and tocotrienols. Redox Biol 2013;1:97–103. [12] Fink BD, Herlein JA, Yorek MA, Fenner AM, Kerns RJ, Sivitz WI. Bioenergetic effects of mitochondrial-targeted coenzyme Q analogs in endothelial cells. J Pharmacol Exp Ther 2012;342:709–19. [13] Navas P, Villalba JM, de Cabo R. The importance of plasma membrane coenzyme Q in aging and stress responses. Mitochondrion 2007;7(Suppl.):S34–40. [14] Dallner G, Sindelar PJ. Regulation of ubiquinone metabolism. Free Radic Biol Med 2000;29:285–94. [15] Lowes DA, Webster NR, Murphy MP, Galley HF. Antioxidants that protect mitochondria reduce interleukin-6 and oxidative stress, improve mitochondrial function, and reduce biochemical markers of organ dysfunction in a rat model of acute sepsis. Br J Anaesth 2013;110:472–80. [16] Chatterjee A, Kandwal A, Singh N, Singh A. Evaluation of Co-Q10 anti-gingivitis effect on plaque induced gingivitis: a randomized controlled clinical trial. J Indian Soc Periodontol 2012;16:539–42. [17] Hans M, Prakash S, Gupta S. Clinical evaluation of topical application of perio-Q gel (Coenzyme Q(10)) in chronic periodontitis patients. J Indian Soc Periodontol 2012;16:193–9. [18] Yoneda T, Tomofuji T, Ekuni D, Azuma T, Endo Y, Kasuyama K, et al. Anti-aging effects of co-enzyme Q10 on periodontal tissues. J Dent Res 2013;92:735–9.

Lipophilic antioxidants prevent lipopolysaccharide-induced mitochondrial dysfunction through mitochondrial biogenesis improvement.

Oxidative stress is implicated in several infectious diseases. In this regard, lipopolysaccharide (LPS), an endotoxic component, induces mitochondrial...
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