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Matsunami et al., 2009 3rd



Oxidative stress and gene expression of antioxidant enzymes in the streptozotocin-induced diabetic rats under hyperbaric oxygen exposure

Oxidative stress and gene expression of antioxidant enzymes in the streptozotocin-induced diabetic rats under hyperbaric oxygen exposure



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Matsunami et al., 2009 3rd Matsunami et al., 2009 3rd Document Transcript

  • Int J Clin Exp Pathol 2010;3(2):177-188www.ijcep.com /IJCEP911003Original ArticleOxidative stress and gene expression of antioxidantenzymes in the streptozotocin-induced diabetic ratsunder hyperbaric oxygen exposureTokio Matsunami, Yukita Sato, Takuya Sato, Satomi Ariga, Toshiko Shimomura, Masayoshi YukawaLaboratory of Biomedical Science, Department of Veterinary Medicine, College of Bioresource Sciences, NihonUniversity, Fujisawa 252-8510, Japan.Received November 17, 2009; accepted November 25, 2009; available online November 30, 2009Abstract: Diabetes mellitus (DM) causes not only hyperglycemia but oxidative stress, resulting mainly enhancedproduction of mitochondrial reactive oxygen species (ROS). Hyperbaric oxygen (HBO) treatments are appliedvarious diseases including diabetic patients with unhealing foot ulcers, however, and also increases the formationof ROS. Recently, it has been reported that oxidative stress worsens many pathological conditions including DMand obesity suggesting possible changes in regulation of genes associated with the oxidative stress, however,effects of HBO which could induce ROS on the gene expressions of oxidative stress parameters in DM animals areunknown. The purpose of this study is to investigate the effect of HBO exposure on the gene expression of threeimportant antioxidant enzymes, cytosolic superoxide dismutase (Cu-Zn SOD), cytosolic glutathione peroxidase(GPx-1), and catalase (CAT) in DM rats, respectively. We used streptozotocin-induced DM model rats andexamined both mRNA expressions and the activities of these antioxidant enzymes in the liver, skeletal muscle,and pancreas. The mRNA expressions of Cu-Zn SOD and CAT decreased significantly (p < 0.001), and GPxincreased significantly (p < 0.001) in all the studied organs of DM rats under HBO exposure compared to thosefrom DM-induced rats not exposed to HBO. Similarly, activities of these three enzymes changed in accordancewith the mRNA levels. These results suggested that DM induction and HBO exposure might synergistically affectantioxidant enzymes, resulting increase of oxidative stress state. Thus, HBO exposure seems to be an excellentmodel system for investigating oxidative stress.Key words: Diabetes mellitus, hyperbaric oxygen, oxidative stress, superoxide dismutase, glutathione peroxidase,catalaseIntroduction concentrations of oxygen can lead to an abundance of reactive oxygen species (ROS)Hyperbaric oxygen (HBO) therapy, as defined [2]. In addition, it has been demonstrated thatby the Undersea and Hyperbaric Medical HBO also increases the formation of ROS,Society (UHMS), comprises the intermittent which are known to cause cellular damageinhalation of 100% oxygen under a pressure through the oxidation of lipid, protein, and DNAgreater than one atmosphere absolute (ATA). [3]. In situations where a body’s anti-oxidantThere are various well-established therapeutic defenses are inadequate, increased freeuses of HBO which have been applied radical formation is likely to increase thesuccessfully for the treatment of 13 different damage. This situation is generally termedillnesses, such as carbon monoxide poisoning, “oxidative stress”.decompression sickness, osteomyelitis, andeven diabetic foot [1]. However, it is known The oxidative effects of HBO have beenthat exposure to oxygen at high ambient investigated in animals and humans [4-6]. Onepressure can cause damage to mammalian study in rats revealed that, after 2 h of HBOcells. It has been suggested that the exposure at 3 ATA, elevated levels of thedetrimental effects of exposure to high oxidative stress markers, thiobarbituric acid
  • Oxidative stress and diabetic ratsreactive substances (TBARS) and total Animals and animal welfare policysuperoxide dismutases (SOD), were found inthe lung, brain and erythrocytes [5], and Twenty adult, 8-week-old, male Wistar ratsglutathione peroxidase (GPx) and (weight range, 240–250 g), bred in ournitrate/nitrite (NOx) activities were found to be laboratory, were used for the experiment. Theyelevated in the brain [6]. Thus, HBO exposure were housed at 23 to 25 C with light fromseems to be an excellent model system for 7:00 AM to 7:00 PM and free access to waterinvestigating oxidative stress. at all times. All rats were fed a commercial diet during the experiment. All study proceduresOxidative stress occurs in some illnesses were implemented in accordance with thewithout exposure to HBO treatment. For Institutional Guidelines for Animal Experimentsexample, persistent hyperglycemia caused at the College of Bioresource Sciences, Nihonthrough diabetes induces ROS production by University under the permission of theglucose autoxidation [7, 8], activation of Committee of Experimental Animal in ourprotein kinase C, and increase flux through the college.hexosamine pathway [9]. Oxidative stress hasbeen also associated with diabetic status in Study designanimals and humans [10-14]. One of thesestudies using streptozotocin (STZ)-induced The rats were allowed to acclimatize for 1diabetic rats, a recognized model of type 1 week prior to treatment, and then at the startdiabetes mellitus (T1DM), showed that levels of the experiment they were randomly dividedof lipid peroxidation had increased, as into four groups, 5 rats per group. The groupsindicated by TBARS, an oxidative stress were as follows: non-diabetic induction andmarker [10]. Oxidative stress-related changes non-HBO group (Control), non-diabeticoccurred in the mRNA and protein expressions induction and HBO group (HBO), diabeticof Cu-Zn SOD and CAT in STZ-induced diabetic induction and non-HBO group (DM), andrat liver tissues [14]. Thus, pathological diabetic induction and HBO group (DM + HBO).conditions of diabetes mellitus could be a In this study, diabetes developed on the thirdmodel for studying oxidative stress. day after diabetic induction, and then rats in the DM + HBO group were exposed to HBOHBO therapy is used in the treatment of once daily for 7 days. Rats in the HBO groupdiabetic patients with unhealing foot ulcers were also exposed to HBO once daily for 7[1]. However, the side-effects of HBO days. The mean body weight of the animals intreatment in diabetic patients and animals all groups was measured at the start and endhave been poorly investigated. It has been of the study period.shown that the levels of TBARS and advancedoxidation protein products (AOPPs) increased Induction of diabetesin diabetic patients who received HBO therapyfor diabetic foot ulcers [15]. In our previous In two groups (DM and DM + HBO), diabetesstudy, a clinically-recommended HBO was induced by a single intraperitoneal (i.p.)treatment significantly increased TBARS levels injection of Streptozotocin (STZ: 40 mg/kgand decreased SOD activity, clearly body weight) dissolved in 0.05 M citrate bufferdemonstrating that HBO causes oxidative (pH 4.5), as described previously [17]. Controlstress in the T1DM state [16]. However, to our and HBO groups were treated with equalknowledge, there is no study with regard to the volume and concentration of STZ injectioneffects of HBO on the gene expressions of vehicle, citrate buffer. Diabetes was confirmedoxidative stress parameters in diabetic in the STZ-induced rats by measuring theanimals. In this animal study, we compared fasting plasma glucose concentration 48 hthe expression of the genes for the three post-injection. After an overnight fast, bloodimportant antioxidant enzymes, Cu-Zn SOD, was obtained from the tail vein, centrifuged atGPx, and CAT of liver, skeletal muscle, and 3,000 × g for 5 min, and then the plasma waspancreas between non-diabetic and diabetic collected. Fasting plasma glucose and insulinrats under HBO exposure. concentrations were measured every day by a commercial kit, the GLU-P Ⅲ (FUJIFILM, Tokyo,Materials and methods Japan) and Rat Insulin ELISA KIT; AKRIN-130 (Shibayagi, Gunma, Japan), respectively.178 Int J Clin Exp Pathol 2010:3(2):177-188
  • Oxidative stress and diabetic ratsDiabetes was considered to have been stated above. Lipid peroxidation levels wereinduced when the blood glucose level reached measured by the thiobarbituric acid (TBA)at least 14 mmol/l [18]. reaction using the method of Ohkawa et al. [19]. The activities of antioxidant enzymesHBO exposure such as Cu-Zn SOD [20], GPx [21], and CAT [22] were assayed.For all experiments, HBO was applied at aclinically-used pressure of 2.4 ATA for 90 min, Total RNA isolation and RT-PCRonce daily for 7 days, in a hyperbaric chamberfor small animals (Nakamura Tekkosho, Tokyo, RNA isolation was performed byJapan). The ventilation rate was 4–5 L/min. homogenization with a Micro Smash-100RTMEach exposure was started at the same hour (TOMY SEIKO, Tokyo, Japan) using Isogenin the morning (10 AM) to exclude any (NIPPON GENE, Tokyo, Japan) for all organs.confounding issues associated with changes in RNA purification was carried out using thebiological rhythm. RNeasy Mini kit (Qiagen, Hilden, Germany) in all organs. All samples were treated withTissue preparation DNase I (RNase Free DNase set, Qiagen). The concentrations of total RNA were measured byAfter the final HBO exposure, all rats were absorbance at 260 nm using a NanoDrop®weighed, anesthetized with sodium ND-1000 (NanoDrop, USA). The purity waspentobarbital (40 mg/kg i.p.), and estimated by the 260/280 nm absorbancedecapitated. Liver, skeletal muscle and ratio. Reverse transcription of 1 μg total RNApancreas were measured, and liver, skeletal was subjected to reverse transcription usingmuscle, and pancreas samples were collected an oligo(dT)12-18 primer and M-MuLV reversein either liquid nitrogen for analysis of transcriptase (SuperScriptTM Ⅲ First-Strandantioxidant defense enzymes and lipid Synthesis, Invitrogen Life Science, USA)peroxidation, or RNAlater solution (Qiagen, according to the manufacturer’s instructions.Hilden, Germany) for molecular, and stored at Multiplex PCR was performed to quantify the-80°C until analysis. expression of mRNA for antioxidant enzymes (Cu-Zn SOD, GPx and CAT) relative to β-actinLiver, skeletal muscle, and pancreas mRNA as an internal control. Thehomogenates were prepared as a 1:10 (w:v) oligonucleotide primer pairs used for multiplexdilution in pH 7.4, 10 mM potassium PCR are shown in Table 1. One microliter ofphosphate buffer using a Ultra-Turrax® (IKA® cDNA mixture was amplified in a 25 μl of PCRJapan, Nara, Japan). Samples were reaction mixture containing 10× PCR reactioncentrifuged at 3000 rpm for 10 min at 4°C, buffer, 25 mM MgCl2, 2.5 mM dNTPmix, 10and the supernatants were collected and μM of each primer, and 5.0 Unit Ex Taq®immediately assayed for enzyme activities. polymerase (TAKARA BIO INC, Shiga, Japan).Blood samples were collected by cardiac cDNA mixture was amplified in a PCR reaction,puncture and divided into heparinised tubes, which was carried out by using GeneAmp® PCRand separated into plasma and erythrocytes by system 9700 (Applied Biosystems, CA, USA)centrifugation at 10,000 × g for 10 min at and involved the following steps: initial4°C. The erythrocyte samples were washed denaturation at 94°C for 3 min, denaturationthree times with cold physiological saline (PS) at 94°C for 30 s, annealing at 58°C for Cu-Znand then hemolyzed by adding a fourfold SOD and GPx, and 60°C for CAT for 30 s,volume of ice-cold HPLC-grade water. extension at 72°C for 45 s (28 cycle), andHemolyzed erythrocyte samples were final extension at 72°C for 5 min. Aftercentrifuged at 10,000 × g for 10 min at 4°C, amplification, PCR products were run on 2%and the supernatants were sampled for agarose gels, and the intensity of each banddetermination of Cu-Zn SOD, GPx, and CAT was measured with Image J software [23]. Theactivity, and TBARS levels. levels of Cu-Zn SOD mRNA, GPx mRNA, and CAT mRNA were determined by calculating theBiochemical analysis intensity ratio of Cu-Zn SOD mRNA/ β-actin mRNA, GPx mRNA/ β-actin mRNA, and CATPlasma glucose and insulin concentrations mRNA/ β-actin mRNA.were measured using a commercial kit as179 Int J Clin Exp Pathol 2010:3(2):177-188
  • Oxidative stress and diabetic ratsTable 1. Primer sequences and expected product size for antioxidant enzymes and internal standard β- actin Accession ProductcDNA Forward primer Reverse primer No. sizeβ-actin V01217 5-CCTGCTTGCTGATCCACA 5-CTGACCGAGCGTGGCTAC 505 bpCu-Zn SOD X05634 5-GCAGAAGGCAAGCGGTGAAC 5-TAGCAGGACAGCAGATGAGT 387 bpGPx NM_030826 5-CTCTCCGCGGTGGCACAGT 5-CCACCACCGGGTCGGACATAC 290 bpCAT AH004967 5-GCGAATGGAGAGGCAGTGTAC 5-GAGTGACGTTGTCTTCATTAGCACTG 670 bp Figure 1: Plasma glucose and insulin concentrations in non-diabetic groups and diabetic groups. Values are expressed as mean ± SEM (n = 6). a p < 0.05; b p < 0.01 compared with control value. Statistical analysis groups. At the end of the treatment, there was no difference in body weight between control The results are expressed as means ± and HBO rats (mean group weights ranged standard error of mean (SEM). An unpaired t- from 250 to 265 g). However diabetic rats test with a P value of < 0.05 was used to presented with weight loss, and the body determine statistical significance. weight of DM + HBO rats (220 ± 12 g; mean ± SEM.), in particular, decreased significantly (p Results < 0.05), when compared with the rats in the other 3 groups (control, 260 ± 5 g; HBO, 257 ± The baseline body weight of the rats at the 7 g; DM, 241 ± 8 g). beginning of the study was similar in all 180 Int J Clin Exp Pathol 2010:3(2):177-188
  • Oxidative stress and diabetic ratsHyperglycemia occurred within 3 days of STZ fasting plasma insulin concentrations of theadministration. Fasting plasma glucose DM + HBO group were significantly decreasedconcentrations of the DM + HBO group were compared with DM group (Figure 1). Nosignificantly higher at 6, 7, 8, and 9 days after significant differences were observed betweendiabetes induction (p < 0.01 - 0.05) compared any other groups (control and HBO groups) orwith the DM group (Figure 1). Moreover, experimental periods. Figure 2: Comparison of lipid peroxidation levels (TBARS) in the erythrocyte, liver, skeletal muscle, and pancreas among non-diabetic rats in the non-HBO (control), non-diabetic rats in the HBO (HBO), diabetic rats in the non-HBO (DM), and diabetic rats in the HBO (DM + HBO) groups. TBARS values are indicated as nmol/g hemoglobin in erythrocytes and as nmol/mg protein in the various organs. Values are expressed as mean ± SEM (n = 6). a Represents significance at p < 0.05 and aa represents significance at p < 0.005 compared with the control groups. b Represents significance at p < 0.05 and bb represents significance at p < 0.005 compared with the DM groups.181 Int J Clin Exp Pathol 2010:3(2):177-188
  • Oxidative stress and diabetic rats Figure 3: Comparison of the antioxidant enzyme activities (Cu-Zn SOD, GPx, and CAT) in the erythrocyte among non-diabetic rats in the non-HBO (control), non-diabetic rats in the HBO (HBO), diabetic rats in the non-HBO (DM), and diabetic rats in the HBO (DM + HBO) groups. These antioxidant enzymes values are given as U/g hemoglobin in erythrocytes. Values are expressed as mean ± SEM (n = 6). a Represents significance at p < 0.05 and aa represents significance at p < 0.005 compared with the control groups. b Represents significance at p < 0.05 and bb represents significance at p < 0.005 compared with the DM groups.Figure 4: Comparison of the antioxidant enzyme activities and mRNA expressions (Cu-Zn SOD, GPx, and CAT) in theliver (a); (Refer to Figure 4b and 4c in next page).182 Int J Clin Exp Pathol 2010:3(2):177-188
  • Oxidative stress and diabetic rats183 Int J Clin Exp Pathol 2010:3(2):177-188
  • Oxidative stress and diabetic rats Figure 4: Comparison of the antioxidant enzyme activities and mRNA expressions (Cu-Zn SOD, GPx, and CAT) in the liver (a), skeletal muscle (b), and pancreas (c) among non-diabetic rats in the non-HBO (control), non- diabetic rats in the HBO (HBO), diabetic rats in the non-HBO (DM), and diabetic rats in the HBO (DM + HBO) groups. These antioxidant enzymes values are given as U/mg protein in the various organs, mRNA expressions values are presented as the ratios of the densities of these antioxidants mRNAs and β-actin genes of all organs in all groups measured by Image J software. Values are expressed as mean ± SEM (n = 6). a Represents significance at p < 0.05 and aa represents significance at p < 0.005 compared with the control groups. b Represents significance at p < 0.05 and bb represents significance at p < 0.005 compared with the DM groups.TBARS levels in the erythrocytes and all organs + HBO groups showed an increase in mRNA(liver, skeletal muscle, and pancreas) levels of GPx in all organs (p < 0.05) comparedexamined are presented in Figure 2. TBARS with the control group. The mRNA expressionslevels in the HBO, DM, and DM + HBO groups of GPx in all organs were higher in the DM +were significantly increased (p < 0.005 - 0.05) HBO group compared to the control group andcompared with the control group. In addition, these reductions were statistically significantTBARS levels in the erythrocytes and all organs (p < 0.005). In addition, mRNA levels of GPx inwere higher in the HBO and DM + HBO groups all organs were higher in the DM + HBO groupthan in the DM group (p < 0.005 - 0.05). The than in the DM group (p < 0.05). For the HBOCu-Zn SOD and CAT activities in the group, in all organs, the increases in theerythrocytes and all organs (liver, skeletal relative expression of GPx mRNA compared tomuscle, and pancreas) examined in the HBO, the DM group were about 5 – 8%, but thisDM, and DM + HBO groups were significantly difference was not statistically significant.decreased (p < 0.005 - 0.05) compared withthe control group; the same finding was not Discussionseen with the GPx activity (Figure 3, 4a, 4b,and 4c). Moreover, the Cu-Zn SOD and CAT Our study examines whether the levels ofactivities in the erythrocytes and all organs activity and mRNA expression of thewere significantly lower in the HBO and DM + antioxidant enzymes, such as Cu-Zn SOD, GPx,HBO groups than in the DM group (p < 0.005 - and CAT are differentially-affected in the liver,0.05). skeletal muscle, and pancreas of the diabetic rats receiving HBO treatment. We have shownFigure 4a, 4b, and 4c present the ratios of the for the first time that diabetic rats receivingdensities of Cu-Zn SOD, CAT, and β-actin genes HBO treatment had significantly increasedof all organs (liver, skeletal muscle, and ROS production. Hyperglycemia subsequent topancreas) in all groups measured by Image J diabetes causes oxidative stress, mainlysoftware. As shown in all figures, the HBO, DM, leading to the enhanced production ofand DM + HBO groups showed a decrease in mitochondrial ROS [9]. Oxidative stressmRNA levels of Cu-Zn SOD and CAT in all induced by hyperglycemia leads to theorgans (p < 0.05), compared with the control activation of stress-sensitive signalinggroup. The mRNA expressions of Cu-Zn SOD pathways, which worsen both insulin secretionand CAT in all organs were significantly lower and action, and promote the development ofin the DM + HBO group compared to the type 2 diabetes mellitus (T2DM) [24].control group (p < 0.005). In addition, mRNA Oxidative stress and damage to the tissueslevels of Cu-Zn SOD and CAT in all organs were and the blood in STZ-induced diabetic ratssignificantly lower in the HBO and DM + HBO enhance glucose autoxidation, and may be agroups than in the DM group (p < 0.05). contributory factor to the complications associated with diabetes [10]. Similarly, HBO,The same series of figures present the ratios which was used in our experiment, alsoof densities of GPx and β-actin genes after increases the formation of ROS that can causedensitometric analysis of the respective bands cellular damage through the oxidation of lipid,with Image J software (Figures 4a, 4b, and 4c). protein, and DNA [3]. Thus, our results ofAs seen from all figures, the HBO, DM, and DM changes in antioxidant enzymes at the mRNA184 Int J Clin Exp Pathol 2010:3(2):177-188
  • Oxidative stress and diabetic ratslevel might correlate with the oxidative stress Cu-Zn SOD and CAT, and significantlyinduced and enhanced by both diabetes increased GPx activities and mRNAinduction and HBO exposure. expressions compared with the diabetic control group (i.e., no HBO treatment). TheHBO exposure increases the amount of diabetic state caused the activity of thedissolved oxygen in the blood, resulting in the antioxidant enzymes, Cu-Zn SOD and CAT toimprovement of a variety of clinical conditions decrease and GPx to increase in liver tissuessuch as hypoxia, acute carbon monoxide of rats [28, 29]. As with the TBARS levels, theintoxication, air embolism and diabetic lower activities of Cu-Zn SOD and CAT werelimb wounds [1]. The most commonly-used significantly lower in non-diabetic rats withdoses of HBO for standard therapeutic HBO treatment than in diabetic rats withoutpurposes are 1.8–2.8 ATA for 60–90 min [25]. HBO treatment. In addition, non-diabetic ratsAlthough the oxidative effects of HBO have with HBO treatment had increased activity ofbeen investigated in animals and humans [5, GPx compared to diabetic rats without HBO6], the HBO conditions applied in previous treatment, but this difference was notstudies seemed to be higher than the usual statistically significant. Moreover, the mRNAtherapeutic levels. One study in rats revealed expressions of Cu-Zn SOD and CAT in allthat, after a single HBO exposure at 3 ATA, organs were significantly lower in non-diabeticoxidative stress markers, including TBARS rats with HBO treatment than in diabetic ratslevels and GPx, and nitrate/nitrite (NOx) without HBO treatment. These resultsactivity, were elevated in the brain [6]. In this indicated the activities of antioxidant enzymespresent study, by using an HBO exposure such as Cu-Zn SOD, CAT, and GPx wereprotocol based on clinically-used pressures associated with the expressions of eachand duration (2.4 ATA for 90 min), we showed respective gene. Recent studies reported thatthat plasma glucose concentrations were not the activities and mRNA expressions ofimproved and plasma insulin concentrations antioxidant enzymes, Cu-Zn SOD and CAT,were decreased in HBO-treated diabetic rats, were decreased [30] and those of GPx weresuggesting that even clinically-used HBO increased [14] in the liver tissue of rats, 2-3administration might enhance the functional weeks after induction of diabetes. On the otherdisorder of pancreatic cells in diabetic hand, rat liver tissue 5-6 weeks after diabetesconditions as demonstrated in our previous induction showed an increase in Cu-Zn SODstudy [26]. mRNA expression together with an increase in the total SOD activity, and an increase in CATThe primary ROS generated in the several gene expression in contrast to a decrease inorgans is superoxide anion. SOD can catalyze enzyme activity in the renal cortex [10]. Thesedismutation of superoxide anion into hydrogen results indicate that oxidative stress affectsperoxide, which is then detoxified to water and the antioxidant defense systems in themolecular oxygen by CAT or GPx. In that different organs in an organ-specific way. Therespect, evidence obtained from clinical and mRNA expressions of antioxidant enzymes inanimal studies suggested that diabetic animals and humans under HBO exposurepatients and animals have significant have been poorly investigated, however, wedecreased in antioxidant enzymes, and have shown the dynamics of mRNAincreased in lipid peroxidation [10, 13]. In the expressions of these antioxidant enzymes inpresent study, we investigated the levels of the present study.TBARS of diabetic rats with HBO treatment. Wehave showed that diabetic rats receiving HBO In this present study, we showed that Cu-Zntreatment had significantly increased TBARS SOD and CAT activities were decreased inlevels compared with both the control and DM HBO-treated diabetic rats, suggesting that HBOgroups. Moreover, we showed that the levels treatment might enhance the superoxideof TBARS were significantly higher in non- radicals under diabetic conditions. Similarly,diabetic rats treated with HBO than in diabetic HBO treatment also increased the GPx activity.rats without HBO treatment, thus there is no SOD and CAT are the major scavengingdoubt that HBO causes lipid peroxidation [27]. enzymes that remove radicals in vivo. AFurthermore, we showed that diabetic rats decrease in the activity of these antioxidantreceiving HBO treatment had significantly enzymes can lead to an excess availability ofdecreased activities and mRNA expressions of superoxide radicals, such as superoxide anion185 Int J Clin Exp Pathol 2010:3(2):177-188
  • Oxidative stress and diabetic ratsand hydrogen peroxide [22]. Superoxide stresses, which in turn could induce apoptosisradicals are known to inhibit CAT activity, and in the pancreatic cells [36]. Furthermore, thethen SOD activity is directly inhibited by oxidative stress caused by HBO exposure hasincreased superoxide radicals [31]. been shown to induce apoptosis via theAdditionally, in this study, we have shown that mitochondrion [37]. Future work shoulddiabetic rats receiving HBO treatment had investigate the gene expression of the insulinsignificantly increased GPx activities and receptor (IR), IRS, PI3K, and glucosemRNA expressions compared with the diabetic transporter 1 or 4 (GLUT-1 or 4), which all havecontrol group (i.e., no HBO treatment). A a wider spectrum of activity and are alsosignificant increase in GPx activities and mRNA responsible for the regulation mechanism ofexpressions could suggest that CAT activities blood glucose levels. This would help us toand mRNA expressions were inactivated by arrive at a better understanding of theROS, which are increased in diabetic rats and molecular mechanisms of the antioxidantHBO exposure. Thus, the decreasing CAT defense system of diabetes under HBOactivity and mRNA may be due to be exposure. Further studies also need tocounterbalanced by increasing GPx activities address the detailed mechanism of howand mRNA expressions in diabetic rats [14] oxidative stress caused by HBO activities isand HBO exposure. Elevated levels of cellular linked to the mitochondrial pathway ofoxidative stress caused by diabetes and HBO apoptosis. These observations suggest thatexposure contribute to the inactivation or the detection of a protection mechanismsensitive to the antioxidant enzymes, such as against oxidative stress caused by HBOCu-Zn SOD, GPx, and CAT. In addition to exposure may be beneficial in humas withcausing damage to cellular constituents, essential diabetes mellitus.recent evidence suggests that ROS can affectsignal transduction processes or the Acknowledgementstranslocation of redox-sensitive transcriptionfactors [32]. Therefore, the observed changes This study was partially supported by thein the mRNA expressions of antioxidant Academic Frontier Project “Surveillance andenzymes in diabetes under HBO exposure control for zoonoses” and Strategic Researchcould be due to oxidation of transcriptional Base Development Program "Internationalfactors responsible for the initiation machinery research on epidemiology of zoonoses andof antioxidant enzymes transcription process. training for young researchers" from Ministry of Education, Culture, Sports, Science andIn conclusion, a decrease of Cu-Zn SOD and Technology, Japan.CAT mRNA expression, and an increase of GPxmRNA expression were associated with higher Please address all correspondences to: Yukita Sato, PhD, Laboratory of Biomedical Science, Departmentplasma glucose concentrations in diabetes of Veterinary Medicine, College of Bioresourcefollowing exposure to HBO. Thus, the Sciences, Nihon University, Fujisawa 252-8510,synergistic elevation of plasma glucose Japan. Phone and Fax: +81-466-84-3445; E-mailconcentrations between STZ-induced diabetes address: sato.yukita@nihon-u.ac.jpand HBO treatment lead to glucoseautoxidation, which would increase the Referencesinduction of ROS. It has been shown thatoxidative stress disrupts insulin-induced [1] Feldmeier JJ and Undersea and Hyperbariccellular redistribution of insulin receptor Medical Society: Hyperbaric oxygen 2003:substrate-1 (IRS-1) and phosphatidylinositol 3- indications and results: the Hyperbaric Oxygen Therapy Committee report. Edited by Feldmeierkinase (PI3K) in adipocytes [33]. Oxidative JJ. North Carolina, Durham, 2003, pp. 141stress produced via glucose autoxidation [2] Jamieson D, Chance B, Cadenas E and Boverisworsens many pathological conditions and A. The relation of free radical production toinduces diabetic complications [9]. Recent hyperoxia. Annu Rev Physiol 1986;48:703-studies have reported that STZ-induced β-cell 719.death was associated with oxidative stress [3] Speit G, Dennog C, Radermacher P andcaused by the production of excess Rothfuss A. Genotoxicity of hyperbaric oxygen.intracellular ROS [34, 35], and that STZ could Mutat Res 2002;512:111-119.damage the pancreatic tissue through [4] Eken A, Aydin A, Sayal A, Ustündağ A, Duydu Y and Dündar K. The effects of hyperbaric oxygenimposing oxidative as well as nitrosative treatment on oxidative stress and SCE186 Int J Clin Exp Pathol 2010:3(2):177-188
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