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 Table of Contents  
CASE REPORT
Year : 2014  |  Volume : 27  |  Issue : 1  |  Page : 85-92

Additive effect of ozone therapy to insulin in the treatment of diabetic rats


1 Department of Clinical Physiology, Faculty of Medicine, Menoufia University, Shebin Al Kawm, Egypt
2 Department of Anesthesia, Faculty of Medicine, Menoufia University, Shebin Al Kawm, Egypt

Date of Submission05-Jun-2013
Date of Acceptance17-Jul-2013
Date of Web Publication20-May-2014

Correspondence Address:
Safaa El-Kotb
Department of Clinical physiology, Faculty of medicine, El Menoufia University, Yassin Abd El Ghaffar street, Shebin El Kom, 32511 El Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.132759

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  Abstract 

Background
Chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the heart and blood vessels. Therapy in the past few decades was mainly aimed at reducing hyperglycemia. It became clear that ameliorating oxidative stress through treatment with antioxidants might be an effective strategy for reducing diabetic complications. Medical ozone treatment may be useful in the treatment of diabetes and its complications.
Objective
This study aims to show the additive effect of ozone to insulin in the treatment of diabetes in rats.
Materials and methods
Diabetes was induced by an intraperitoneal injection of streptozotocin (45 mg/kg) in 0.2 ml of 10 mmol/l citrate buffer. Rats were considered diabetic when fasting blood glucose was at least 113 mg/dl. Rats proved to be diabetic were isolated and subdivided into four subgroups: (a) diabetic nontreated rats (n = 8), (b) diabetic ozone-treated rats (n = 8), (c) diabetic insulin-treated rats (n = 8), and (d) diabetic insulin + ozone-treated rats (n = 8). After induction, all rats were fasted for 12 h. Systolic blood pressure (SBP) was measured. Retro-orbital blood samples were collected for estimation of fasting serum glucose, glycosylated hemoglobin, total antioxidant capacity (TAC), and malondialdehyde (MAD) level. Rats were then sacrificed; vascular reactivity to norepinephrine and acetylcholine with and without endothelial lining was estimated.
Results
The data showed that insulin reduced the elevated fasting serum glucose, glycosylated hemoglobin, MAD, and SBP significantly when compared with the diabetic nontreated group. Also, it significantly reduced TAC and vascular reactivity to norepinephrine with and without endothelium, but there was an increase in the percent of relaxation to acetylcholine. Ozone therapy potentiated the effects of insulin on SBP and vascular reactivity. Importantly, serum MAD and TAC and glycemic state were significantly improved.
Conclusion
This study shows that ozone therapy may have an additive effect in the treatment of diabetes by insulin; this may attribute to the multiprotective antioxidant effect of ozone.

Keywords: Antioxidant, diabetes, ozone, systolic blood pressure, vascular reactivity


How to cite this article:
Saleh S, El-Ridi M, Zalat S, El-Kotb S, Donia S. Additive effect of ozone therapy to insulin in the treatment of diabetic rats. Menoufia Med J 2014;27:85-92

How to cite this URL:
Saleh S, El-Ridi M, Zalat S, El-Kotb S, Donia S. Additive effect of ozone therapy to insulin in the treatment of diabetic rats. Menoufia Med J [serial online] 2014 [cited 2017 Oct 24];27:85-92. Available from: http://www.mmj.eg.net/text.asp?2014/27/1/85/132759


  Introduction Top


Diabetes mellitus is not a single disease, but a heterogeneous group of disorders of absolute or relative insulin deficiency that affects 150 million individuals worldwide [1]. The prevalence of diabetes mellitus is increasing markedly. It is projected to be 220 million by 2010 and to 300 million by 2025 [2]. The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels [3]. Macrovascular and microvascular complications are currently the principal causes of morbidity and mortality in patients with diabetes [4]. There is growing evidence that excess generation of highly reactive free radicals, largely because of hyperglycemia, causes oxidative stress [5]. There is growing evidence that excess generation of highly reactive free radicals, largely because of hyperglycemia, causes oxidative stress, which further exacerbates the development and progression of diabetes and its complications [5]. It became clear that ameliorating oxidative stress through treatment with antioxidants might be an effective strategy for reducing diabetic complications [6]. In the absence of an appropriate compensation by the endogenous antioxidant defense network, increased oxidative stress is believed to be a contributing factor in the development of endothelial dysfunction and vascular diseases that cause cellular damage and contribute toward the late complications of diabetes [6]. Nowadays, the use of complementary/alternative medicine is increasing rapidly worldwide, mostly because of the supposedly less frequent side effects when compared with modern western medicine [7]. Oxygen-ozone therapy is one of the different minimally invasive treatments that are currently available. Ozone has been used as a complementary therapeutic agent in several unrelated pathologies. Because ozone therapy can activate the antioxidant system, influencing the level of antioxidant enzymes and some markers of endothelial cell damage [8], reactive oxygen species, and lipid oxidation products, both compounds are responsible for activating several biochemical, immunological, and pharmacological mechanisms that are responsible for the biological and therapeutic effects of ozone [9]. Medical ozone treatment may be useful in the treatment of diabetes and its complications. Thus, the present study aims to show the additive effect of ozone to insulin in the treatment of diabetes in rats.


  Materials and methods Top


Animals

This study was carried out on 32 adult male albino rats (150-200 g). Rats were fed standard laboratory chow and water ad libitum and housed in the animal house of the Minufiya Faculty of Medicine under an artificial 12-h light/dark cycle. Rats were fasted overnight and diabetes was induced by an intraperitoneal injection of streptozotocin (STZ) (45 mg/kg) in 0.2 ml of 10 mmol/l citrate buffer (pH 5.5) [10]. Dextrose 5% (0.5 ml) was administered intraperitoneally as a protective dose 30 min before the STZ injection [11]. Rats were considered to be diabetic 24 h after the STZ injection. Fasting blood samples were taken from rat tails, and rats were considered diabetic when fasting blood glucose was found to be higher than 113 mg/dl [12]. The rats were divided into four equal groups (n = 8 each). In group I, the diabetic nontreated group (n = 8), rats were injected subcutaneously with saline at a dose of 0.2 ml/100 g body weight/day for 4 weeks. In group II, the diabetic + ozone-treated group, rats were administered an ozone/oxygen mixture intraperitoneally at doses of 1.2 mg/kg [13]. The volume of gaseous mixture administered to each animal was ∼2-2.5 ml/day, three applications weekly. In group III, the diabetic + insulin-treated group, rats were administered insulin treatment. Mixtard insulin was injected subcutaneously at a dose of 0.75 IU/100 g body weight (saline is the vehicle), once daily. In group IV, the diabetic + ozone and insulin-treated group, rats were administered ozone therapy with concomitant treatment with insulin.

Chemicals

Chemicals used for the preparation of  Krebs-Henseleit solution More Details were purchased from Sigma (St Louis, Missouri, USA); STZ was purchased from Sigma Chemical Company, St. Louis, Mo, USA. Cayman chemical San Diego, California (USA); and acetylcholine (Ach) powder and norepinephrine (NE) powder from El-Gomhoria Company (Cairo, Egypt). Kits for estimation of serum glucose were obtained from Boehringer (Mannheim, Germany), kits for estimation of serum malondialdehyde (MAD) and serum total antioxidant capacity (TAC) were obtained from Cayman Chemical (USA), and kits for estimation of glycosylated hemoglobin (HbA1c) were obtained from Riomidi (France).

Ozone injection

O 2 -O 3 mixture (2 ml) at a concentration of 100 μg/ml was used, obtained from a Longevity Ozone Generator (EXT120; Canada) from the ozone unit. Intraperitoneal ozone was administered by injecting 2 ml of one dose of O 2 -O 3 mixture. The concentration of ozone used was 100 μg/ml. The output concentration was calculated using a special table depending on the flow rate of O 2 and ozone concentration in the generator. A disposable syringe had to be used for the proper administration of the ozone mixture. Each rat received 2 ml/day, three applications weekly.

Blood pressure measurement

At the end of the experimental protocol period (4 weeks), systolic blood pressure (SBP) was measured using a rat-tail sphygmomanometer (Harvard Apparatus Ltd, UK) and a pneumatic transducer (Harvard Apparatus Ltd) [14]. The pneumatic cuff fits over the rat's tail; a four-channel recorder was used to obtain a written record of both blood flow and cuff pressure. The pulse sign had to be monitored to observe when the pulse signal became detectable and reached the maximum pulse height. The start of pulsation was viewed on the tracing and was referenced to the pressure curve signal at that point; this reading was analogous to the SBP [15].

Blood sampling and biochemical analysis

After the measurement of MSBP, retro-orbital blood samples (each 2 ml) were obtained through heparinized capillary tubes; samples were allowed to clot at room temperature in a water bath for 15 min. The supernatant serum was collected in a dry tube [16]. Serum samples were used for the estimation of fasting serum glucose (FSG), HbA1c, TAC, and MAD level.

Measurement of aortic vascular reactivity (isolated rat aortic strip technique) [17]

At the end of the experiments, rats were killed by cervical decapitation. The thoracic cavity of each rat was opened and the thoracic aorta was cut as near to the heart as possible and dissected free as far as the diaphragm. The aorta was then transferred to a  Petri dish More Details with Krebs's solution at room temperature that was aerated with carbogen (95% oxygen and 5% carbon dioxide). The aorta was cleaned from adherents and then cut into helical strips (aorta, 0.1 × 1.0 cm). Then, it was suspended in a 10 ml organ bath. The lower end of the strip was mounted to a hook fixed in the aeration tube, whereas the upper end was connected to another hook suspended from the transducer (Grass, 200 Metro center Blvd unit 8. Warwick, USA). For equilibration, the aortic strip was subjected to an initial tension of 0.5 g and was kept in an organ bath (Harvard Apparatus Ltd) for ∼90 min and washed every 15 min. The rat aortic strip preparation was used for the estimation of vascular reactivity in response to NE and Ach on their contractile activity with and without endothelial lining in different groups.

Statistical analysis

Statistical analysis was carried out using Kruskal-Wallis one-way analysis of variance for multiple comparisons, followed by Fisher's PLSD test. Values are expressed as mean ± SD. The post-hoc Scheffe test was used to identify the source of statistical significance. P values less than 0.05 were considered statistically significant.


  Results Top


At the end of the experiment, the data obtained from the present investigation indicated that STZ induced a statistically significant increased FSG in HbA1c as shown in [Table 1], MAD, MSBP, and vascular reactivity to NE with and without endothelium. Also, there was a significant reduction in TAC and decrease in the percent of relaxation to Ach.
Table 1: Fasting serum glucose (mg/dl) and glycosylated hemoglobin in percentage of normal Hb in nondiabetic nontreated and diabetic (nontreated, ozone-treated, insulin-treated, and combined ozone and insulin-treated) groups

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[Figure 1] shows the serum levels of FSG (mg/dl) and HbA1c in percentage of normal hemoglobin in all the study groups. As the figure shows, the mean value of serum FSG and the mean value of HbA1c of the ozone-treated rats were significantly lower (P < 0.001) than the corresponding values in diabetic nontreated rats. The figure also shows that the mean value of FSG and the mean value of HbA1c of the diabetic insulin-treated group were significantly lower (P < 0.001) than the corresponding values in diabetic nontreated and diabetic ozone-treated groups. The figure also shows that the mean value of FSG and the mean value of HbA1c of the diabetic ozone and insulin-treated group were significantly lower (P < 0.001) than the corresponding values in the diabetic nontreated and diabetic ozone-treated groups and insignificantly lower (P > 0.05) than the corresponding values in the diabetic insulin-treated groups.
Figure 1:

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[Figure 2] shows representative examples of MSBP (mmHg) in all study groups. The figure shows that the mean values of the MSBP of the ozone-treated rats were significantly lower (P < 0.01) than the corresponding values in diabetic nontreated rats. The figure also shows that the mean value MSBP of the diabetic insulin-treated group was significantly lower (P < 0.01) than the corresponding values in the diabetic nontreated and diabetic ozone-treated groups. The figure also shows that the mean values of MSBP in the diabetic ozone and insulin-treated group were significantly lower (P < 0.001) than the corresponding values in the diabetic nontreated and diabetic ozone-treated groups and significantly lower (P < 0.01) than the corresponding values in the diabetic insulin-treated groups.
Figure 2:

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[Figure 3] shows representative examples of the vascular reactivity of the isolated aortic strip to NE (mg tension) and Ach (% of relaxation) in all study groups. The figure shows that the mean value of vascular reactivity of aortic strips (mg tension) to NE (10 -5 mol/l) in the diabetic ozone-treated group was significantly lower (P < 0.01) than the corresponding value in the diabetic nontreated group, whereas the mean value of vascular reactivity of aortic strips to Ach solution (10 -5 mol/l) (% of relaxation) in the diabetic ozone-treated group was significantly higher (P < 0.01) than the corresponding value in the diabetic nontreated group. The figure also shows that the mean value of vascular reactivity of aortic strips (mg tension) to (10 -5 mol/l) NE in the diabetic insulin-treated group was significantly lower (P < 0.01) than the corresponding values in the diabetic nontreated and the diabetic ozone-treated group, whereas the mean value of vascular reactivity of aortic strips to Ach solution (10 -5 mol/l) (% of relaxation) in the diabetic insulin-treated group was significantly lower (P < 0.01) than the corresponding values in the diabetic nontreated and the diabetic ozone-treated groups. The figure also shows that the mean value of vascular reactivity of aortic strips (mg tension) to (10 -5 mol/l) NE in the combined ozone and insulin-treated group was significantly lower (P < 0.01) than the corresponding values in the diabetic nontreated, diabetic ozone-treated, and diabetic insulin-treated groups. The mean value of vascular reactivity of aortic strips to Ach solution (10 -5 mol/l) (% of relaxation) in the combined ozone and insulin-treated group was significantly lower (P < 0.01) than the corresponding values in the diabetic nontreated, diabetic ozone-treated, and diabetic insulin-treated groups.
Figure 3:

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[Figure 4] shows that the mean value of vascular reactivity of aortic strips without endothelium (mg tension) to (10 -5 mol/l) NE in the diabetic ozone-treated group was significantly lower (P < 0.01) than the corresponding value in the diabetic nontreated group, whereas the mean value of vascular reactivity of aortic strips without endothelium to Ach solution (10 -5 mol/l) (% of relaxation) in the diabetic ozone-treated group was 0% of relaxation; this remained unchanged compared with the corresponding value in the diabetic nontreated group. The figure also shows that the mean value of vascular reactivity of aortic strips without endothelium (mg tension) to (10 -5 mol/l) NE in the diabetic insulin-treated group was significantly lower (P < 0.01) than the corresponding values in the diabetic nontreated and the diabetic ozone-treated group, whereas the mean value of vascular reactivity of aortic strips without endothelium to Ach solution (10 -5 mol/l) (% of relaxation) in the diabetic insulin-treated group was 0% of relaxation, which remained unchanged compared with the corresponding values in the diabetic nontreated and diabetic ozone-treated groups. Finally, the mean value of vascular reactivity of aortic strips without endothelium (mg tension) to (10 -5 mol/l) NE in the combined ozone and insulin-treated group was significantly lower (P < 0.01) than the corresponding values in the diabetic nontreated, diabetic ozone-treated, and diabetic insulin-treated groups, whereas the mean value of vascular reactivity of aortic strips without endothelium to Ach solution (10 -5 mol/l) (% of relaxation) in the combined ozone and insulin-treated group was 0% of relaxation, which remained unchanged compared with the corresponding values in the diabetic nontreated, diabetic ozone-treated, and diabetic insulin-treated groups.
Figure 4:

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[Figure 5] shows that the mean value of MAD in the diabetic ozone-treated group was significantly lower (P < 0.01) than the corresponding value in the diabetic nontreated group, whereas the mean value of serum TAC in the diabetic ozone-treated group was significantly higher (P < 0.01) than the corresponding value in the diabetic nontreated group. The figure also shows that the mean value of MAD in the diabetic insulin-treated group was significantly lower (P < 0.01) than the corresponding values in the diabetic nontreated group and significantly lower (P < 0.05) than the corresponding values in the diabetic ozone-treated group, whereas the mean value of serum TAC in the diabetic insulin-treated group was significantly higher (P < 0.01) than the corresponding values in the diabetic nontreated and diabetic ozone-treated groups. Finally, the mean value of MAD in the combined ozone and insulin-treated group was significantly lower (P < 0.01) than the corresponding values in the diabetic nontreated and diabetic ozone-treated groups, and significantly lower (P < 0.05) than the diabetic insulin-treated groups, whereas the mean value of serum TAC in the combined ozone and insulin-treated group was significantly higher (P < 0.01) than the corresponding values in the diabetic nontreated, diabetic ozone-treated, and diabetic insulin-treated groups.
Figure 5:

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  Discussion Top


Administration of STZ to rats (45 mg/kg body weight) induced diabetes mellitus with a significant increase in FSG and HbA1c. Hyperglycemia of STZ-treated mice led to a marked reduction in the number of pancreatic islets, followed by a reduction in plasma insulin levels, which induced a reduction in glucose utilization by insulin-sensitive tissues such as skeletal muscle and adipose tissue and increased hepatic and renal glucose production by glycogenolysis and gluconeogenesis, also because of the unopposed action of counter-regulatory hormones for glucose homeostasis such as glucagon and catecholamines [18]. Administration of ozone (1.2 mg/kg body weight) for 4 weeks led to a significant decrease in FSG and HbA1c; this 'antidiabetic' effect produced by ozone treatment seems to be associated with the antioxidant properties of ozone. This is in agreement with Al-Dalain et al. [19], who found a direct link between the presence of oxidative stress and impaired glucose uptake. In adipocytes, glucose uptake is rapidly decreased in the presence of hydrogen peroxide (H 2 O 2 ), an effect that was reversed by ozone treatment in preclinical studies. According to Martinez et al. [20], ozone treatment could protect b-cells against STZ damage and decrease in phosphatidyl inositol 3 kinase activity and glucose transport 4 (GLUT4). Also, Martinez-Sanchez et al. [8] showed that ozone reduced STZ-induced hyperglycemia. Treatment of STZ-induced diabetic rats with insulin for 4 weeks led to a significant decrease in FSG and HbA1c. These results were in agreement with those of Fonseca [21], who found that the addition of a single basal insulin dose at bedtime led to a reduction in HbA1c.

Diabetes mellitus causes a significant increase in plasma MAD and a significant decrease in serum TAC. The high glucose level causes an increase in a product of oxidative damage, namely, MAD because of accelerated metabolism by the thermic effect of food and increased mitochondrial respiration and release of superoxide [22],[23]. Glucose auto-oxidation and protein glycation are important additional sources of free radicals during hyperglycemia [24]. Vessby et al. [25] and Keenoy et al. [24] reported a decrease in total antioxidant status in diabetes mellitus, thus indicating the delicate balance between oxidants and antioxidants, whereas hyperglycemia can result in the generation of free radicals through several biochemical pathways such as nonenzymatic glycation, the polyol pathway, and glucose auto-oxidation [26]. Free radicals can result in the destruction of antioxidant defenses and enhanced susceptibility to lipid peroxidation. In our study, ozone has an antioxidant effect so it play this role through lowering the MAD level so ozone tends to bring the peroxide back to near normal levels, which indicates that it may enhance antioxidant endogenous systems. These results were in agreement with those of Martinez et al. [20], who showed that ozone treatment reduced oxidative stress in STZ-induced diabetic rats and decreased MAD production. These results were also supported by Al-Dalain et al. [19], who showed that with ozone treatment, there was a reduction in total peroxides and the concentrations of MAD and an increase in antioxidant systems. Insulin therapy alone resulted in a significant reduction in plasma MAD. These results were also supported by Sindhu et al. [27], who showed that insulin therapy normalizes the activities and protein expression of all antioxidant enzymes. According to Monge et al. [28], insulin infusion could maintain the plasma antioxidant defenses. Insulin is involved in the regulation of fatty acid metabolism. It exerts inhibitory effects on lipolysis and leads to a reduction in free fatty acid production. Free fatty acid metabolism maintains the generation of free oxygen species as their conjugated double bonds can interact with hydroxyl radicals and hydrogen peroxide [29], increases MSBP, and increases vascular reactivity of aortic strips - with and without endothelium - to NE, and decreases vascular reactivity of aortic strips - with and without endothelium - to Ach in STZ-induced diabetes mellitus. These results were in agreement with those of Pacher and Szabo [30], who reported that hyperglycemia in diabetes results in increased production of reactive oxygen species in the cell, leading to oxidative stress. Free radicals play a major role in endothelial dysfunction during hyperglycemia [31]. Endothelium-dependent vasodilation is markedly reduced and the myogenic tone of resistance arteries is increased in animal models of diabetes mellitus [32]. The bioactivity of nitric oxide (NO) is particularly sensitive to oxidative stress. Superoxide combines readily with NO to form peroxynitrite, a compound with considerably less bioactivity than NO itself [33]. This poor sequelae might play a role in the mechanism of the increase in the contractile response to NE in these rats and increase arterial blood pressure [34]. These results are in agreement with those of Naowaboot et al. [35], who reported that vascular responses of diabetic rats to vasodilators and Ach were significantly suppressed, whereas those to vasoconstrictor and phenylephrine were significantly increased compared with normal rats. These results were also supported by Wang et al. [36], who concluded that maximum contraction to NE increased significantly in diabetic aortas, and also observed a significant decrease in relaxation to Ach in the diabetic group compared with the controls. There are two possible mechanisms for reductions in Ach-induced relaxation: First, NO-dependent vasodilatation, a decrease in NO release from the endothelium, and a decreased reactivity of vascular smooth muscle to NO in diabetic animals. Another possible mechanism of reduced responses to Ach in diabetic animals is that oxidative degradation and inactivation of NO may be increased in vessels of diabetic rats [4]. Ach-induced endothelium-dependent relaxation is impaired in diabetic rats probably because of the dysfunction of Ach receptors [37]. In terms of the decrease in MSBP after ozone therapy on the basis of the mechanism of action, ozone therapy can improve blood circulation and oxygen delivery to tissue owing to the concerted effect of NO and an increase in the intraerythrocytic 2,3-DPG level; by improving oxygen delivery, it enhances the general metabolism and upregulates the cellular antioxidant enzymes [38]. These vascular effects suggest that coadjuvant ozone therapy could decrease vasoconstriction [39]. The administration of ozone decreases the vascular reactivity of aortic strips - with and without endothelium - to NE and increases the vascular reactivity of aortic strips - with and without endothelium - to Ach. These results were in agreement with those of Clavo et al. [39], who showed that ozone therapy could decrease vasoconstriction. These results were also supported by Al-Dalain et al. [19], who showed that with ozone treatment, there was an improvement in aortic relaxation and a decrease in microvessel reactivity. According to Martinez et al. [20], catalase enzyme showed a slight increase after ozone therapy, and there was a decrease in vascular smooth muscle damage. Also, H 2 O 2 can activate the transcription factor, nuclear factor-kB, which promotes the generation of cell adhesion molecules, cytokines, and procoagulant tissue factor, mediators of the vascular complications present in diabetic patients [40]. According to Martinez-Sanchez et al. [8], the NO 2- /NO 3- ratios, a measure of NO, reverted to normal values after ozone therapy. Thus, ozone may protect against the imbalance in NO-ROS species interactions, improve NO-mediated relaxation, and decrease microvessel reactivity in an STZ experimental model of diabetes [19]. Administration of insulin decreases MSBP, thus decreasing the blood glucose level. Insulin receptors are found in endothelial and vascular smooth muscle cells (VSMCs) and insulin has been shown to modulate vascular tone and tissue blood flow [41]. Insulin causes an increase in blood flow; this effect of insulin on the endothelium is mediated by its own receptor and insulin signaling pathways, resulting in the increased release of NO [42]. Vascular reactivity of aortic strips - with and without endothelium - to NE in the present investigation showed a significant decrease and a significant increase in vascular reactivity of aortic strips - with and without endothelium - to Ach; insulin has the ability to modulate the vascular contractile response induced by various vasoactive substances. The analysis of the concentration-contraction curves to NE showed a significant insulin-mediated vasorelaxation. Several studies have clarified that insulin, besides exerting metabolic effects on the target tissues, also has a vasorelaxant action. This latter action could be because of both a direct effect on the VSMC and an indirect endothelium-mediated effect [43]. Insulin attenuates VSMC contractility by regulating agonist-induced increases in cytosolic calcium through voltage-sensitive calcium channels and altering the activity of myosin light-chain phosphatases. Also, the vascular actions of insulin are mediated chiefly through the regulation of endothelium-derived factors. In this respect, insulin can stimulate the production of NO [44]. Also, insulin plays an important antiatherogenic role in both VSMCs and endothelial cells. It maintains the quiescent or the differentiated phenotype of VSMCs, counteracts the proatherogenic effects of platelet-derived growth factor in VSMC, and also counteracts the proatherogenic effects of vascular endothelial growth factor in endothelial cells [45].

The combination of metabolic actions of insulin and the ability of ozone to enhance antioxidant endogenous systems, in addition to oxidative stress by oxidative preconditioning mechanisms, resulted in upregulation of NOS isoforms and reduction in lipid and glucose oxidation, and increased NO oxidation caused by insulin alone plus antioxidant supplementation of ozone can normalize all parameters. According to Sindhu et al. [27], diabetic animals showed marked weight loss, decreased activities of Cu, Zn, superoxide dismutase (SOD), and catalase (CAT), and normal glutathione peroxidase (GPX) activity, but combined therapy with insulin and antioxidants normalized all the antioxidant enzyme protein expression and activities measured. Thus, diabetes-associated reductions in the antioxidant state can be ameliorated by insulin combined with antioxidant therapy. These results indicate that the combination of insulin treatment with ozone - in calculated doses - leads to a more significant enhancement in metabolic, oxidative states and vascular complications in STZ-induced diabetic rats.


  Acknowledgements Top


Conflicts of interest

None declared.



 
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