Department of Periodontology, Dental School, University of Sevilla, SpainResearch Laboratory, Dental School, University of Sevilla, Sevilla, SpainDipartimento di Scienze Cliniche Specialistiche ed Odontostomatologiche (DISCO)-Sez. Biochimica, Facoltà di Medicina, Università Politecnica dellearche, Ancona 60131, ItalyArea de Nutrición y Salud, Universidad Internacional Iberoamericana (UNINI), Campeche C.P. 24040, MexicoFacultad de Ciencias de la Salud, Universidad Nacional de Chimborazo, Riobamba, EcuadorDipartimento di Scienze Agrarie, Alimentari e Ambientali (D3A), Università Politecnica delle Marche, Via Ranieri 65, Ancona 60131, ItalyDirector Centre for Nutrition & Health, Universidad Europea del Atlantico (UEA), Santander 39011, Spain
r t i c l e i n f o
rticle history:eceived 17 September 2014eceived in revised form 16 October 2014ccepted 23 October 2014vailable online 3 November 2014
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 patholog-ical events such as periodontal disease or sepsis. In our experiments, LPS-treated fibroblasts provokedincreased oxidative stress, mitochondrial dysfunction, reduced oxygen consumption and mitochondrialbiogenesis. After comparing coenzyme Q10 (CoQ10) and N-acetylcysteine (NAC), we observed a more sig-
nificant protection of CoQ10 than of NAC, which was comparable with other lipophilic and hydrophilicantioxidants such as vitamin E or BHA respectively. CoQ10 improved mitochondrial biogenesis by acti-vating 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 ther-apeutic strategy in pharmacology in the treatment of infectious diseases and their complications.
In general, oxidative stress can be defined as an imbalanceetween the presence of high levels of reactive oxygen speciesROS), and antioxidant defense mechanisms. These toxic molecules
re formed via oxidation–reduction reactions and are highly reac-ive since they have an odd number of electrons. ROS generatednder physiological conditions are essential for life, as they are
∗ Corresponding author at: Dipartimento di Scienze Cliniche Specialistiche eddontostomatologiche (DISCO)-Sez. Biochimica, Facoltà di Medicina, Universitàolitecnica delle Marche, Ancona 60131, Italy. Tel.: +39 0712204646;ax: +39 0712204123.∗∗ Corresponding author at: Research Laboratory, Dental School, Universidad deevilla, C/Avicena s/n, 41009 Sevilla, Spain. Tel.: +34 954 481120;ax: +34 954 486784.
involved in bactericidal activity of phagocytes, and in signal trans-duction pathways, regulating cell growth and reduction–oxidation(redox) status [1]. ROS includes free radicals, such as hydroxyl andsuperoxide radicals, and non-radicals, including hydrogen peroxideand singlet oxygen. Oxidative stress and generation of free radicals,as a primary or secondary event, have been related to a great num-ber of diseases, including infectious diseases, atherosclerosis anddiabetes [1].
Within most cells, mitochondria are the main source of reactivespecies generated as a by-product of energy production. Withinthe mitochondria the primary ROS produced is superoxide, mostof which is converted to hydrogen peroxide by the action of super-oxide dismutase. The mitochondrial production of superoxidehas been ascribed to several electron transport chain enzymes,
including Complex I and Complex III. These complexes along withCoenzyme Q10 (CoQ10) may leak electrons which in turn mayinteract with oxygen, thus forming ROS [2,3]. All conditions able toalter mitochondria efficiency can enhance ROS production, having
direct and critical effect on oxidative stress. Regarding this, aide range of antioxidants could be targeted to mitochondria
o reduce the effect of oxidative stress as a possible strategy inharmacology, however, antioxidants have had limited success
n preventing the progression of diseases involving mitochondrialxidative damage [4].
Periodontitis, an infectious disease, has also been related to apecific group of bacteria, three of which have been considereds the main periodontal pathogens: Tannerella forsythia, Aggregat-bacter actinomycetemcomitans and Porphyromonas gingivalis [5,6].everal studies have demonstrated an increase of products fromxidative damage in plasma and serum and mitochondrial dys-unction in blood cells of subjects with periodontitis compared withealthy individuals [7,8]. Moreover, there is evidence of a decreasednti-oxidant capacity in subjects with periodontitis, evaluated byifferent assays [7,8].
Because mitochondrial dysfunction and oxidative stress are twof the main factors studied which may be able to explain the patho-hysiological mechanism of inflammatory conditions occurring intherosclerosis, CVDs and periodontitis, the current study is a com-arative one evaluating the in vitro and in vivo effect of lipophilicnd hydrophilic antioxidants in mitochondrial dysfunction pro-oted by P. gingivalis lipopolysaccharide (LPS).
aterials and methods
thical statements
Written informed consent and the approval of the ethical com-ittee of the University of Seville were obtained, according to the
rinciples of the Declaration of Helsinki.Studies in mice were performed in accordance with the Euro-
ean Union guidelines (86/609/EU) and Spanish regulations for these of laboratory animals in chronic experiments (BOE 67/8509-2, 1988). All experiments were approved by the local institutionalnimal care committee.
nimals and drug administration
Four groups (control and treated) of eight six-week-old male57/BL mice weighing 25–30 g were maintained on a 12 h
ight/dark cycle. LPS, from P. gingivalis, was dissolved in saline (vehi-le) and intra-peritoneally administered at a dose of 500 ng/ml,oQ10 10 mg/kg/day and NAC 20 mg/kg/day for 15 days. Afterreatment, mice were anesthetized with CO2 and sacrificed byecapitation. Brain, liver, kidney were isolated and stored at −80 ◦Cntil analysis.
eagents and chemicals
MitosoxTM and Hoechst 3342 were purchased from Invitro-en/Molecular Probes (Eugene, OR, USA); a cocktail of proteasenhibitors from Boehringer Mannheim (Indianapolis, IN, USA);nd Immun Star HRP substrate kit from Bio-Rad Laboratories Inc.Hercules, CA, USA). Monoclonal Anti-Actin antibodies, butylated-ydroxyanisole (BHA), N-acetylcysteine (NAC), and trypsin-EDTAolution and all other chemicals were purchased from Sigma-ldrich. (St. Louis, MO, USA).
ibroblast cultures
Human gingival fibroblasts (HGF) isolated from a healthy 25-
ear-old male, were cultured in D-MEM media (4500 mg/L glucose,-glutamine, piruvate), (Gibco, Invitrogen, Eugene, OR, USA) sup-lemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen,ugene, OR, USA) and antibiotics (Sigma Chemical Co., St. Louis,
l Research 91 (2015) 1–8
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 andNAC 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 wereassessed by MitoSOXTM Red, a red mitochondrial superoxide indi-cator. MitoSOX Red is a novel fluorogenic dye recently developedand validated for highly selective detection of superoxide in themitochondria of living cells. MitoSOXTM Red reagent is live-cell per-meant and is rapidly and selectively targeted to the mitochondria.Once in the mitochondria, MitoSOXTM Red reagent is oxidized bysuperoxide and exhibits red fluorescence.
Flow cytometryApproximately 1 × 106 cells were incubated with 1 �M
MitoSOXTM Red for 30 min at 37 ◦C, washed twice with PBS, resus-pended in 500 �l of PBS and analyzed by flow cytometry in an EpicsXL cytometer, Beckman Coultier, Brea, California, USA (excitationat 510 nm and fluorescence detection at 580 nm).
Fluorescence microscopyCells grown on microscope slides in 6-well plates for 24 h were
incubated with MitoSOXTM Red for 30 min at 37 ◦C, washed twice inPBS, fixed with 4% paraformaldehyde in PBS for 0.5–1 h, and washedtwice with PBS. After that, cells were incubated for 10 min at 37 ◦Cwith anti-LC3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA,USA). Slides were analyzed by immunofluorescence microscopy(MitoSOXTM Red: excitation wavelength = 555/28; emission wave-length = 617/73).
Western blotting
Whole cellular lysate from fibroblasts was prepared by gentleshaking 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% acrylamideSDS/PAGE. Proteins were transferred to Immobilon membranes(Amersham Pharmacia, Piscataway, NJ). PGC-1 �, TFAM, and DNArepair enzyme 8-oxoguanine DNA glycolase-1 (OGG-1) antibod-ies were used to detect proteins by Western blotting. Proteinswere electrophoresed, transferred to nitrocellulose membranesand, after blocking over night at 4 ◦C, incubated with the respectiveantibody solution, diluted at 1:1000. Membranes were then probedwith their respective secondary antibody (1:2500). Immunolabeledproteins 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 extractsprepared from BMC was measured at 412 nm minus 360 nm(13.6 mM−1 cm−1) using 5,5-dithio-bis(2-nitrobenzoic acid) todetect free sulfhydryl groups in coenzyme A as described previously[2].
ATP levels were determined by a bioluminescence assayusing an ATP determination kit from Invitrogen-Molecular Probes(Eugene, OR, USA) according to the manufacturer’s instructions.
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xygen consumption rate (OCR)
The oxygen consumption rate (OCR) was assessed in real-timesing the 24 well Extracellular Flux Analyzer XF-24 (Seahorseioscience, North Billerica, MA, USA) according to the manufac-urer’s protocol, which allows to measure OCR changes after upo four sequential additions of compounds. Cells (5 × 104/well)ere seeded for 16 h in the XF-24 plate before the experiment
n a DMEM/10% serum medium and then incubated for 24 h with
he different compounds studied. Before starting measurements,ells were placed in a running DMEM medium (supplementedith 25 mM glucose, 2 mM glutamine, 1 mM sodium Pyruvate,
ig. 1. Effect of LPS and antioxidants in cell death and mitochondrial ROS production. (ytometry quantification of ROS production showed more protective effect of CoQ10 (C) Mitith cytochrome c in merged images, indicating that superoxide anion production occurrerrows indicate Mitosox co-localized with the cytochrome c signal present in fragmenP < 0.001, between control and LPS treated cells; **P < 0.001, between the LPS in absence
l Research 91 (2015) 1–8 3
and without serum) and pre-incubated for 20 min at 37 ◦C inthe absence of CO2 in the XF Prep Station incubator (SeahorseBioscience, Billerica MA, USA). Cells were transferred to an XF-24Extracellular Flux Analyzer and after an OCR baseline measurementa profiling of mitochondrial function was performed by sequentialinjection of four compounds that affect bioenergetics, as follows:55 �l of oligomycin (final concentration 2.5 �g/mL) at injection inport A, 61 �l of 2,4-dinitrophenol (2,4-DNP) (final concentration1 mM) at injection in port B, and 68 �l of antimycin/rotenone
(final concentration 10 �M/1 �M) at injection in port C. The bestconcentration of each inhibitor and uncoupler was obtained on thebasis of a proper titolation curve. A minimum of five wells were
A) CoQ10 induced more protection than NAC on LPS-induced apoptosis. (B) FlowoSOXTM Red stain revealed increased superoxide anion. MitoSOXTM Red colocalizedd mainly in mitochondria. (D) Magnification of a small area in LPS-treated fibroblast.ted mitochondria. Data represent the mean ± SD of three separate experiments.
or presence of CoQ10; aP < 0.005, between the LPS in absence or presence of NAC.
4 P. Bullón et al. / Pharmacological Research 91 (2015) 1–8
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 thesequential 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 basalO LPS-Nd hree sb
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CR was markedly affected in cells treated with LPS, and improved in LPS-CoQ and
ecrease which was improved by antioxidants. Data represent the mean ± SD of tetween control and LPS treated cells.
tilized per condition in any given experiment. Data are expresseds pmol of O2 consumed per minute normalized to 1000 cellspmol O2/1000 cells/min).
ipid peroxidation
Lipid peroxidation in cells was determined by analyzing theccumulation of lipoperoxides using a commercial kit from Cay-an Chemical (Ann Arbor, Michigan, USA). TBARS are expressed in
erms of malondialdehyde (MDA) levels.
ipid hydroperoxides
The FOX assay was carried out according to the method previ-usly reported [9]. The FOX reagent was prepared by mixing in theollowing order: 90 ml methanol, 88 mg BHT, 10 ml 250 mM H2SO4,.8 mg ammonium ferrous sulfate hexahydrate and 7.6 mg Xylenolrange. To 320 �l of sample, 680 �l of FOX reagent were added and
he solution was incubated for 30 min at 37 ◦C with gentle shaking.fter a short high-speed centrifugation (3000 × g for 1 min at room
emperature), sample absorbance was read at 560 nm against thelank (0.9% NaCl and FOX reagent). For hydroperoxide quantifica-ion, a serial standard dilution of hydrogen peroxide was used.
tatistical analysis
Data in figures is given as mean ± SD. Data between differentroups were analyzed statistically by using ANOVA (SPSS for
AC. (C) The spare respiratory capacity (SRC) treatment with LPS caused a significanteparate experiments. *P < 0.05, between and antioxidants treated cells; **P < 0.05,
Windows, 19, 2010, SPSS Inc. Chicago, IL, USA). For cell-culturestudies, Student’s t test was used for data analyses. A value ofp < 0.05 was considered significant. To compare the behavioralresults from animals treated with vehicle alone or with drugs atwo-way variance (ANOVA) analysis was used.
Results and discussion
CoQ10 induces a more efficient prevention of LPS-induced toxicityand ROS production than N-acetylcysteine (NAC)
Toxic effects have been described in LPS treatment character-ized by an increment of apoptotic nuclei condensation and caspase3 activation, suggesting that LPS treatment induces apoptosis bythe activation of at least the intrinsic pathway [2]. Concerningthis aspect, it has been shown that ROS plays a relevant role inmitochondrion-to-mitochondrion ROS-signaling as a positive feed-back mechanism for enhanced ROS production potentially leadingto significant mitochondrial injury [2]. In this context, cytochromec release and a consequent activation of pro-caspase 9, caspase3 and endonuclease G result in DNA degradation and apoptoticdeath. Mitochondria-targeted lipophilic antioxidants selectivelyblock mitochondrial oxidative damage and prevent some types ofcell death, so it could be appropriate to develop probes of mito-
chondrial function.
In our experiments, we studied the effect of CoQ10 in celldeath and mitochondrial ROS production compared with a wellknowledge hydrophilic antioxidant such as NAC in human gingival
P. Bullón et al. / Pharmacological Research 91 (2015) 1–8 5
Fig. 3. Effect of LPS and antioxidants in bioenergetic, mitochondrial mass and biogenesis (A) ATP levels after LPS and antioxidants were analyzed by bioluminescence asdescribed in section “Material and Methods”. (B) Citrate Synthase specific activity in fibroblasts after LPS and antioxidants was performed as described in section “Materialand 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 presenceof CoQ10; aP < 0.005, between LPS in absence or presence of NAC. (C) Mitochondrial biogenesis after LPS and antioxidant treatment was determined by protein levels ofPGC-� and TFAM by using western blotting. Protein levels were determined by densitometric analysis (IOD, integrated optical intensity) of three different western blots andn
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ormalized to the GADPH signal.
broblasts. Both antioxidants induced an important protection byeducing cell death percentage and ROS production but this wasore significant in the case of CoQ10 (Fig. 1A and B). MitoSOXTM
ed fluorescence co-localized with mitochondrial cytochrome oxidase (Fig. 1C). Fig. 1D clearly shows that in mitochondrialOS-positive cells after LPS treatment, Mitosox co-localized withhe cytochrome c signal present in fragmented mitochondriauggesting that mitochondria is responsible for ROS production.
mpaired mitochondrial oxygen consumption by LPS is restored byoQ10 and NAC
P. gingivalis LPS has been shown to induce a mitochondrial dys-unction by inhibiting mitochondrial membrane potential (�� m)nd decrement of complex I and complex III in mitochondrialespiratory chain with a concomitant decrease of CoQ10 levels2]. These compromised mitochondrial functions have severalonsequences including increased ROS production, described andemonstrated by us, and a deterioration of mitochondrial oxygenonsumption which has never been studied before. Therefore, wenvestigated the protective effect of CoQ10 and NAC against theossible negative effect of LPS on mitochondrial functionality.his was assessed by measuring the Oxygen consumption rateOCR) values in control and treated cells, exposed sequentially toach of four modulators of oxidative phosphorylation (OXPHOS)
uch as oligomycin (an inhibitor of F1Fo-ATPase or complex V),,4-DNP (uncoupling of the OXPHOS electron transport chain)nd 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 thanin 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 treatedonly with LPS (Fig. 2B). The spare respiratory capacity (SRC) ofcells was obtained by calculating the mean of OCR values afterinjection of 2,4-DNP minus the basal respiration and could be usedas an indicator of how close a cell is operating to its bioenergeticlimit. Treatment with LPS caused a significant decrease (2.9-fold,p < 0.05) of SRC compared to control cells, while treatment togetherwith LPS-CoQ10 and LPS-NAC caused a significant improvement(p < 0.05) approximately 2.9-fold and 2.3-fold respectively, whencompared with cells treated only with LPS (Fig. 2C).
Mitochondrial biogenesis impairment induced by LPS is restoredby CoQ10
Mitochondria are highly dynamic organelles in cells which needto maintain very specific levels. For this, mitochondrial biogenesisand mitophagy are two pathways that regulate mitochondrialcontent and metabolism preserving homeostasis. Recently, wedescribed reduced levels of CoQ10 and mitochondrial chain com-plex induced by LPS [2] and active autophagic processes in bloodcells from periodontitis patients and activated in fibroblasts afterP. 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 bioener-getics and well-being status. As shown in Fig. 3A, LPS treatmentprovoked a significant contraction in ATP levels, suggesting that
6 P. Bullón et al. / Pharmacological Research 91 (2015) 1–8
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) Flowcytometry quantification of ROS production showed more protective effect of Vitamin E. (C) ATP levels after LPS and antioxidants were analyzed by bioluminescence asdescribed in section “Material and Methods”. (D) Citrate Synthase specific activity in fibroblasts after LPS and antioxidants was performed as described in section “Materialand 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 presenceo
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f Vitamin E; aP < 0.005, between LPS in absence or presence of BHA.
itochondrial power of energy production may be diminished.urthermore, a reduced mitochondrial mass was confirmed byecrease of citrate synthase (Fig. 3B). To elucidate the mechanismf lowered mitochondrial mass after LPS treatment, the expressionevels of proteins involved in mitochondrial biogenesis wereetermined. Protein expression levels of phosphorylated PGC-1�nd TFAM were found to be diminished (Fig. 3C). This is the firstime that a down regulation of mitochondrial biogenesis by P.ingivalis LPS has been demonstrated. Interestingly, CoQ10 induces
more significant restoration of mitochondrial biogenesis, ATPnd mitochondrial mass than NAC.
According to these in vitro data, CoQ10 causes a significant reduc-ion of LPS-induced ROS and a more significant improvement ofTP and mitochondrial mass than NAC. Both antioxidants, how-ver, induced similar restoration of oxygen consumption rate. Thiss interesting because NAC has a very evident antioxidant effect
hich has been described to be involved in the positive effect ofitochondrial respiration compared with CoQ analogs [4]. In this
egard, a possible explanation of the more significant effect in thether parameters of CoQ10 compared with NAC is the inductionf mitochondrial biogenesis of CoQ10 observed and demonstratedy us after oral CoQ10 treatment [3]. This effect on mitochondrialiogenesis was found in NAC treatment.
Because CoQ10, a lipophilic antioxidant, has been shown to beore effective than NAC, a hydrophilic antioxidant, we compared
wo well-known lipophilic and hydrophilic antioxidants, vitamin End BHA respectively. Preliminary data about vitamin E showed
protective effect in LPS-induced cytotoxicity [11]. Therefore,ccording 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 isrestored by CoQ10 in mice
In order to study the role of lipophilic and hydrophilic antioxi-dants in the LPS-induced mitochondrial dysfunction, and accordingto in vitro results, we exposed mice to intra-peritoneally admin-istered LPS. LPS induces high levels of oxidative stress in brain,liver, and kidney compared with vehicle showing an increment ofhydroperoxides and malondialdehyde levels (Fig. 5A and B). Wealso observed an important decrement regarding ATP and citratesynthase (Fig. 5C and D) and, interestingly CoQ10 generated a moresignificant restoration regarding LPS effect than NAC, according toin vitro results.
As LPS has a very evident effect on mitochondria, antioxidantsthat act preferentially on mitochondria reduce mitochondrial dam-age and organ dysfunction [12]. The results described in this articlemay serve as a new way for designing experiments to betterunderstand the influence of oxidative stress on the developmentof infectious diseases and generate new therapeutic strategies.NAC acts as an essential precursor to many endogenous antioxi-dants involved in the decomposition of peroxides and attenuatesoxidative stress from various underlying causes by replenishing
intracellular glutathione stores. However, CoQ10 can prevent lipidperoxidation by itself or by biochemical reduction of other antioxi-dants such as alpha-tocopherol (vitamin E) and ascorbate (vitaminC), transfer of electrons from complexes I and II to complex III
P. Bullón et al. / Pharmacological Research 91 (2015) 1–8 7
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) Lipidperoxidation in brain, liver and kidney was determined in terms of malondialdehyde (MDA) levels. (C) ATP levels after LPS and antioxidants were analyzed by bioluminescenceas described in section “Material and Methods”. (D) Citrate Synthase specific activity in fibroblasts after LPS and antioxidants was performed as described in section “Materialand 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 ina r pres
iabTstiiasmoLabhi
bsence or presence of CoQ10; aP < 0.005 or aaP < 0.05, between the LPS in absence o
n the oxidative phosphorylation with subsequent ATP generationnd induce mitochondrial biogenesis [4,13]. Furthermore, kidney,rain and liver contain the highest endogenous levels of CoQ10 [14].he findings of the present study show that lipophilic antioxidantsuch as CoQ10 and vitamin E ameliorated mitochondrial dysfunc-ion, oxidative stress and reduced cell death with a more specificnteraction with mitochondrial biogenesis, which is not observedn hydrophilic antioxidants such as NAC or BHA. Since CoQ10 is
pivotal element in mitochondrial respiratory chain and, at theame time, is an important antioxidant, these results suggest thatitochondrial dysfunction is crucial in the pathophysiology of peri-
dontal disease and CoQ10 could be a therapeutic option in bacterialPS infection, sepsis or other similar conditions. In this respect, new
ntioxidants based on CoQ or vitamin E such as MitoQ and MitoE,oth lipohilic antioxidants with a protective role of mitochondria,ave been shown to induce an important protection in a sepsis-
nduced organ failure model by LPS treatment [15]. Furthermore,
ence of NAC.
CoQ10 has been shown to have beneficial effects in periodon-tal diseases under experimental conditions in clinical trials[16–18].
Conclusions
Previous work has shown that P. gingivalis, one of the keyetiological factors for periodontal pathology, induces oxidativestress and mitochondrial dysfunction with bacterial LPS playinga major role in the pathogenesis of periodontal pathology whichhas been related to cardiovascular diseases, risk of developingcerebrovascular incidents and, in particular, non-hemorrhagicstroke. This is an interesting issue that warrants consideration
when designing new experiments in pharmacology in order to gainfurther insight into its potential therapeutic applications, giventhat systemic antioxidant status is an exogenously modifiablefactor. The results described in this article may serve as a novel
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onflict of interest statement
All the authors declare that no conflict of interest exists for anyf them.
cknowledgements
Authors are indebted with Ms. Monica Glebocki for exten-ive editing of the manuscript. This work has been supported byroyecto de Investigación de Excelencia de la Junta de AndalucíaTS113.
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