Protective effect of pregabalin on the brain tissue of diabetic rats

Abstract

Purpose

Diabetes mellitus (DM) is a metabolic disorder characterized by insulin deficiency or insulin resistance. Pregabalin (PGB) is an antiepileptic drug with proven efficacy in the treatment of epilepsy, generalized anxiety disorder, and neuropathic pain. In this study, we aimed to investigate the protective effects of PGB in brain tissue of rats with streptozotocin (STZ)-induced experimental diabetes.

Materials and methods

Twenty-eight Wistar albino male rats were randomly divided into four groups with seven rats each: (I) Control group, (II) PGB (50 mg/kg PBG), (III) DM, and (IV) DM + PGB (50 mg/kg/day PGB per orally for 8 weeks). Diabetes was induced with an intraperitoneal (i.p.) STZ injection (Sigma Chemical Co Louis Missour, USA) at a dose of 180 mg/kg. STZ was dissolved in 0.1 M phosphate-citrate tampon (pH 4.5). Paraffin sections were examined using histological and immunohistochemical analyses. To detect oxidative damage biochemically, malondialdehyde (MDA), the end product of lipid peroxidation; superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and glutathione peroxidase (GPx) which are antioxidant enzymes, levels were studied. In addition, bax, caspase-3 enzyme activities and TUNEL assay were studied to evaluate the apoptosis status.

Results

In the DM group, MDA concentrations were significantly higher and GPx and SOD activities were significantly lower compared to the control group. MDA concentrations were significantly lower and SOD activity was significantly higher in the DM + PGB group than in the DM group. The GPx activity in the DM group decreased significantly compared to the control group. In immunohistochemical examinations (Bax, Caspase-3 and TUNEL), the apoptosis rate was significantly lower in the in DM + PGB group than in the DM group.

Conclusion

Pregabalin may prevent harmful effects of oxidative damage by decreasing the MDA levels and increasing the SOD levels. In addition, it was thought that PGB may have antiapoptotic properties due to decreased bax and caspase-3 immunoreactivity and TUNEL positivity in PGB groups. Based on these findings, we think that PGB may be effective in reducing the risk of brain damage associated with DM.

Introduction

Diabetes mellitus (DM) is a metabolic disease affecting the carbohydrate, lipid, and protein metabolisms, characterized by insulin deficiency or insulin resistance [1, 2]. Recent studies have shown a linear correlation between hyperglycemia levels and development of microvascular and macrovascular complications [3, 4]. Reactive oxygen species (ROS) such as nitric oxide (NO), hydrogen peroxide (H₂O₂), hydroxyl radical (OH⁻) and superoxide (O2⁻) are primary oxidative stress indicators proliferated in the presence of DM [5]. Oxidative stress can be caused by overproduction of ROS by metabolic reactions that utilize oxygen and shift the balance between oxidant/antioxidant statuses in favor of the oxidants. Once produced, oxidative stress may damage lipids, membranes, proteins and nucleic acids [5].

Diabetes mellitus (DM) affects both the central nervous system (CNS) and the peripheral nervous system (PNS), leading to the development of various complications. The most common known neurological complication is diabetic peripheral neuropathy. In addition, cerebrovascular diseases and cognitive dysfunctions are more common in diabetic patients than in the normal population due to being an effected vascular system and thanks to hyper-hypoglycemia attacks. Pathophysiological mechanisms are similar in the development of CNS and PNS complications [6]. Possible mechanisms in neuropathy formation are known as oxidative stress, nonenzymatic glycation, polyol and hexosamine pathways, protein kinase c pathway, poly (ADPribose) polymerase, reduction of neurotrophic factors and changes in ion channels, and central excitatory mechanisms. It is accepted that these pathogenetic factors play a synergistic role in the development of neuropathy. While metabolic factors are effective in the long term, inflammation on ischemic nerve damage gains importance in severe forms of focal neuropathy. Stenosis and hyalinization of the microvascular system play a role in ischemia with a decrease in endoneurial oxygenation [6]. As in diabetic peripheral neuropathy, it has been shown in different studies that many factors are effective in the development of CNS complications of DM. These factors include the micro and macrovascular structures are affected, insulin resistance, glucose toxicity, oxidative stress, accumulation of glycation end products, hypoglycemic episodes, and changes in amyloid metabolism. Four mechanisms triggered by DM have been held responsible for the development of vascular complications. These mechanisms are increased oxidative stress and ROS damage, the formation of glycosylation end products, the entry of glucose into the aldose reductase pathway, and the activation of one or more of the protein kinase C isoenzymes. With hyperglycemia, the formation of ROS and insulin-resistant state are toxic to cells. ROS, such as superoxide anions, create NO toxic peroxynitrite ions and reduce the useability of endothelial NO. In this way, it causes a decrease in endothelium-mediated vasodilation, increased platelet activation, and proliferation and migration of vascular smooth muscles. ROS also facilitate LDL accumulation in the vascular wall by increasing oxidation. Due to excess glucose, non-enzymatic glycosylation of various proteins and lipoproteins in the vessel wall accelerates the atherosclerosis process, increases the uptake and oxidation of LDL into the cell and increases the risk of ischemic cerebrovascular disease by causing foam cell formation [7]. In DM-dementia coexistence (especially vascular dementia and Alzheimer's-type dementia), it is thought that the reasons such as cardiovascular risk factors associated with DM, glucose toxicity, insulin resistance, and inflammation, as well as demographic and socioeconomic reasons and genetic factors, trigger different pathological processes in both diseases can be considered to be effective [8]. Hyperglycemia can cause worsening in memory and attention. Chronic hyperglycemia may cause cognitive impairment and synaptic plasticity pathologies. Oxidative stress and glycosylation swarm accumulation occurs with glucose toxicity and neurodegeneration begins after brain damage occurs. This may explain the higher frequency of dementia in diabetic patients. Studies have reported that hypoglycemia that develops in patients with Type 2 DM is also effective in cognitive breakdown. It has been shown that the rate of cognitive impairment increases 1.5–2 times in patients with recurrent hypoglycemia. It has also been reported that fibrinogen formation associated with neuronal death and platelet aggregation in severe hypoglycemia may accelerate the cognitive degradation process [9]. It has been found that prolonged hyperinsulinemia impairs the insulin response due to the decrease in the blood–brain barrier and the number of insulin receptors in the brain, and inhibits the transfer of insulin to the cerebrospinal fluid (CSF) and brain tissues. These changes create defects in learning and memory formation. Impairment of insulin sensitivity of the brain was detected in Alzheimer's patients compared to healthy individuals. Insulin-degrading enzyme breaks down both insulin and amyloid. In the case of high plasma insulin levels, amyloid-beta protein degradation decreases as degradation enzymes will compete, and amyloid accumulation can trigger the development of Alzheimer's type of dementia [9, 10]. In addition, hyperglycemia promotes apoptosis by triggering the accumulation of ROS and intracellular calcium in neurons. Moreover, hyperglycemia elevates lactic acid concentration, a product of anaerobic pathway that triggers stress in the brain [11].

Pregabalin (PGB) is an antiepileptic drug approved by the Food and Drug Administration (FDA). PGB is effective in the treatment of generalized epilepsy and is also commonly used in the treatment of neuralgia caused by shingles and diabetic neuropathy [12]. Moreover, PGB potently binds the CaVα2δ-1 and after binding to voltage-gated calcium channels of neurons, it reduces the release of noradrenaline and glutamate by modulating depolarization-induced calcium flow [13]. It affects the CNS by easily crossing the blood–brain barrier. In different diabetic animal model studies, PGB’s antioxidant, immunomodulatory, antiinflammatory and antiapoptotic properties have been shown on both CNS and PNS [10, 13].

In the present study, we aimed to investigate neuroprotective effects of PGB in the brain tissue of rats with streptozotocin (STZ)-induced experimental diabetes. If the findings can be supported by different studies, it is thought that PGB may be a potential treatment agent to prevent complications due to DM such as vasculopathy in CNS and cognitive dysfunction.

Materials and methods

While planning this study, it is aimed to show the neuroprotective effect of PGB’s antioxidant and antiapoptotic effects in the central nervous system to prevent vascular complications and cognitive dysfunction, which is used in the treatment of peripheral nervous system complications of diabetes. For this purpose to detect oxidative damage biochemically, MDA, the end product of lipid peroxidation; SOD, CAT, GSH and GPx which are antioxidant enzyme levels, were studied. In addition, Bax, Caspase-3 enzyme activities and TUNEL assay were studied to evaluate the apoptosis status.

The study was conducted in Firat University Experimental Research Center in collaboration with the Histology and Embryology Department at Firat University Medical School. An approval was obtained from Firat University Laboratory Animals Ethics Committee (Approval Date: 23.12.2011, No: 145) and the study protocol was conducted in accordance with Directive 2010/63/EU regarding the protection of animals used for scientific purposes. A total of 28 male Wistar albino rats weighing 200–210 g were obtained from Firat University Animal Care Center. The rats were kept at room temperature (22–25 °C) with a 12/12-h light/dark cycle and fed standard rat diet and water ad libitum. The animals were transferred to the laboratory 1 week before the experiment.

The rats were randomly divided into four groups with seven rats each: (I) Control group, (II) PGB (50 mg/kg PGB), (III) DM, (IV) DM + PGB (50 mg/kg/day PGB per orally for 8 weeks).

Diabetes was induced with an intraperitoneal (i.p.) STZ injection (Sigma Chemical Co Louis Missour, USA) at a dose of 180 mg/kg. STZ was dissolved in 0.1 M phosphate-citrate tampon (pH 4.5). Blood samples were obtained by snipping the tails of the rats (5 μL) and blood glucose levels were measured using a glucometer (Glucostix (Myles, Ekhart, IN). Rats with a blood glucose level of ≥ 250 mg/dL were accepted as diabetic and were included in the DM and DM + PGB groups.

Blood glucose levels and body weights of the rats were measured at week 0, 4, and 8. At week 8, all the rats were decapitated after administering a combination of ketamine (75 mg/kg) + xylazine (10 mg/kg) i.p.. Following decapitation, the brain tissues were removed from the CA1 region of the hippocampus. Tissue samples were dissected into two pieces, with one piece reserved for biochemical analysis and the other for histological examination. The tissues reserved for biochemical analysis were stored at − 80 °C until analysis and the tissues reserved for histological examination were embedded in 10% formaldehyde solution and then were processed through graded alcohol series, xylene, and embedded in paraffin blocks.

Biochemical analysis

Tissues were homogenized with a glass-distilled water homogenizer. After a 10-min resting period, the supernatants were centrifuged at 2500 rpm.

Lipid peroxidation

Malondialdehyde (MDA) is an end product of lipid peroxidation and used for demonstration of the level of oxidative damage [14]. Lipid peroxidation was measured using the method that was proposed by Placer et al. [15] and modified by Matkovics et al. [16]. This method is based on the reaction between butyric acid and malondialdehyde (MDA), which is an aldehyde produced by lipid peroxidation and reacts with thiobarbituric acid (TBA), forming a pink chromogen which is measured at 532–535 nm on a spectrophotometer.

Superoxide dismutase activity

Superoxide dismutase (SOD) is an antioxidant enzyme and it destroys ROS formed due to oxidative stress [17]. SOD activity was assessed using the method proposed by Sun et al. [18]. This method is based on the reduction of nitroblue tetrazolium (NBT) by the oxygen radicals produced in the xanthine oxidase system. This reduction terminates with the formation of the blue color at the maximum absorbance of 560 nm.

Glutathione peroxidase activity

Glutathione peroxidase (GPx) is an antioxidant enzyme and it destroys ROS formed due to oxidative stress [17]. GPx activity was measured according to the method proposed by Lawrance and Burk [19]. During glutathione (GSH) oxidation, GPx uses cumene hydroperoxide and DTNB (5–5′-dithiobis [2-nitrobenzoic acid]) reacts with GSH to form yellow color chromophore, 5-thionitrobenzoic acid (TNB). GPx activity was assessed by reading absorbance levels at 412 nm using a spectrophotometer.

Catalase activity

Catalase (CAT) is an antioxidant enzyme and it destroys ROS formed due to oxidative stress [17]. CAT activity was assessed using the method proposed by Aebi [20]. CAT converts H₂O₂ to H₂O and O₂ in a chemical reaction.

Reduced glutathione levels

Glutathione (GSH) resists damaging caused by radicals, it has a role as a substrate for antioxidant enzymes ad act as a radical holder [17]. Reduced GSH levels were measured using the method proposed by Sedlak and Lindsay [21]. This spectrophotometric method is based on the assessment of yellow color by sulfhydryl groups by the addition of DTNB into the samples.

Protein concentration

Protein concentration was measured using the method proposed by Lowry [22]. In this method, the phenol reagent is added to the mixture treated with copper to form a purple-blue color and the resulting color is read at 546 nm by spectrophotometer.

Histological examination

Brain tissues were fixed in 10% formaldehyde solution for 24 h and then rinsed with tap water. Subsequently, the tissues were processed through graded alcohol series, xylene, and embedded in paraffin blocks. Tissue sections of 5–6 µm thickness were obtained and then stained with hematoxylin–eosin. All the sections were examined under a microscope (Olympus BH–2).

Immunohistochemical study

Bax is the protein that triggers the mitochondria-mediated apoptosis mechanism of the cell that develops DNA damage. Caspase-3 is the enzyme involved in the last step of the apoptosis stage. Activity of bax and caspase-3 are used immunohistochemically as markers of apoptosis [23]. Bax and Caspase-3 activities were determined using the avidin–biotin peroxidase complex. Tissue sections of 5–6 µm thickness obtained from paraffin blocks were mounted on poly-l-lysine coated slides. After sequential dehydration of deparaffinized tissues with alcohol, the tissues were treated with H₂O₂ to prevent endogenous peroxidase activity. Ultra V Block solution was used to prevent background staining and the samples were incubated in primary antibody (Bax mouse monoclonal IgG, Santa Cruz Biotechnology, sc–7480, California, USA; caspase-3 Rabbit polyclonal IgG, Abcam, ab2302, London, UK) for 60 min.

After the application of primary antibodies, secondary antibodies (biotinylated anti-mouse/rabbit IgG, Diagnostic BioSystems, KP 50A, Pleasanton, USA), streptavidin horseradish peroxidase, and 3-amino-9-ethylcarbazole chemokine were applied and then counter staining was performed with Mayer’s hematoxylin. Phosphate buffered saline (PBS) was substituted for primary antibody and was processed in negative control tissues through the steps abovementioned. Tissues were rinsed with PBS and distilled water respectively and were covered with a suitable cover solution. Images were obtained using a Novel N-800 M microscope. The percentage of stained cells (0.1: < 25%, 0.4: 26–50%, 0.6: 51–75%, 0.9: 76–100%) and the intensity of immunoreactivity (0: none, + 0.5: weak, + 1: mild, + 2: moderate, + 3: severe) were assessed for each sample. A histoscore was calculated based on the following formula: percentage of stained cells × intensity of staining.

TUNEL assay

The TUNEL assay is a method that shows apoptosis by marking broken DNA fragments in situ [23]. Tissue sections of 5 µm thickness were obtained from paraffin blocks and then were mounted on poly L-lysine coated slides. Apoptotic cells were detected using the ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon, cat no: S7101, USA) in accordance with the manufacturer’s recommendations. The slides were examined and photographed using a Novel N-800 M microscope. In the evaluation of TUNEL staining, nuclei stained to blue with Harris hematoxylin were accepted as normal and cells presenting brown nuclear staining were accepted as apoptotic. At least 500 cells were counted in normal and apoptotic areas in randomly selected sections at tenfold magnification. Apoptotic index (AI) was calculated as the percentage of apoptotic cells to total (normal + apoptotic) cells.

Statistical analysis

The data obtained for biochemical, histochemical and TUNEL findings were analyzed using IBM SPSS for Windows 22.0 (IBM Corp. Released 2012, Armonk, NY: IBM Corp.). Descriptives were expressed as mean ± standard deviation (SD). Two groups were compared using Kruskal–Wallis test and Mann–Whitney U test as appropriate. Comparisons among three or more groups were performed using one-way ANOVA followed by post hoc Tukey test. A p value of < 0.05 was considered significant.

Results

Biochemical findings

Blood glucose levels

Blood glucose levels in the DM and DM + PGB groups increased significantly at weeks 4 and 8 when compared to the control group (p < 0.05) (Table 1). Similarly, blood glucose levels were significantly higher in the DM and DM + PGB groups at weeks 4 and 8 compared to week 0 (p < 0.05). No significant difference was found between the PGB and control groups with regard to blood glucose levels at all three weeks (p > 0.05).

Table 1.

Initial, 4th week and final values of blood glucose in rats

	I (Control) (n = 7)	II (PGB) (n = 7)	III (DM) (n = 7)	IV (DM + PGB) (n = 7)
Initial (mg/dl)	97.40 ± 3.63	99.40 ± 3.23	100.51 ± 5.22	98.6 ± 8.86
4th week (mg/dl)	97.30 ± 7.23	98.9 ± 6.25	400.20 ± 9.11a,b	415 ± 15.21a,b
Final (8 th week) (mg/dl)	96.00 ± 8.57	97.30 ± 8.57	417.20 ± 11.57a,b	413.20 ± 13.17a,b

In DM and DM + PGB groups, blood glucose levels increased significantly at weeks 4 and 8 compared to week 0 (before the administration of STZ) (p < 0.05). There was no significant difference in the blood glucose levels of PGB and control groups at all 3 weeks

Data are expressed as mean ± standart error

^aVersus Initial, p < 0.05

^bVersus control, p < 0.05

MDA activity

The MDA levels were significantly higher in the DM and DM + PGB groups compared to the control group (p < 0.05). However, they were significantly lower in the DM + PGB and PGB group compared to the DM group (p < 0.05) and were significantly lower in the PGB group compared to the DM + PGB group (p < 0.05) (Fig. 1).

Fig. 1.

Biochemical analysis findings. The figure shows the comparison of MDA, CAT, GPx, GSH, and SOD values of experimental groups. Differences among the groups were analyzed by Kruskal–Wallis test and binary comparisons were performed using Mann–Whitney U test. a There is significance compared to control group, p < 0.05. b There is significance compared to DM group, p < 0.05. c There is significance compared to DM + PGB group, p < 0.05. The MDA levels were significantly higher in the DM and DM + PGB groups compared to the control group (p < 0.05) (a). However, they were significantly lower in the DM + PGB and PGB group compared to the DM group (p < 0.05) (b) and were significantly lower in the PGB group compared to the DM + PGB group (p < 0.05) (c). The GPx activity in the DM group decreased significantly compared to the control group (p < 0.05) (a). However, no significant difference was found among other groups with regard to GPx activity (p > 0.05). The SOD activity in the DM + PGB group increased significantly when compared to that of control (a) and DM groups (b) (p < 0.05). However, no significant difference was found among other groups with regard to SOD activity (p > 0.05)

CAT activity

No significant difference was found among the groups with regard to CAT activity (p > 0.05) (Fig. 1).

GPx activity

The GPx activity in the DM group decreased significantly compared to the control group (p < 0.05). However, no significant difference was found among other groups with regard to GPx activity (p > 0.05) (Fig. 1).

GSH activity

No significant difference was found among the groups with regard to GSH activity (p > 0.05) (Fig. 1).

SOD activity

The SOD activity in the DM + PGB group increased significantly when compared to that of DM and control groups (p < 0.05). However, no significant difference was found among other groups with regard to SOD activity (p > 0.05) (Fig. 1).

Histological findings

Bax immunoreactivity

The examination of Bax immunoreactivity indicated mild immunoreactivity in the control group and the PGB group, with no significant difference found between the two groups (p = 0.724) (Fig. 2a, b; Table 2). Severe immunoreactivity was detected in the DM group, which established a significant difference when compared to that of control group (p = 0.013) (Fig. 2c; Table 2). Moderate immunoreactivity was detected in the DM + PGB group, which was significantly lower than that of DM group (p = 0.035) and was similar to that of control group (Fig. 2d; Table 2).

Fig. 2.

Findings of Bax immunoreactivity. The examination of Bax immunoreactivity indicated mild immunoreactivity in the control group and the PGB group, with no significant difference found between the two groups (p = 0.724) (a, b; Table 2). Severe immunoreactivity was detected in the DM group, which established a significant difference when compared to that of control group (p = 0.013) (c; Table 2). Moderate immunoreactivity was detected in the DM + PGB group, which was significantly lower than that of DM group (p = 0.035) and was similar to that of control group (d; Table 2)

Table 2.

Bax and Caspase-3 Histoscores, apoptotic index (%)

	Bax	Caspase	Apoptotic index (%)
Control	0.54 ± 0.15	0.49 ± 0.22	2.16 ± 0.75
PRG	0.39 ± 0.12	0.46 ± 0.17	2.66 ± 1.21
DM	1.03 ± 0.42a	1.15 ± 0.50a	11.50 ± 2.73a
DM + PRG	0.60 ± 0.16b	0.56 ± 0.25b	2.83 ± 1.32b

The examination of Bax immunoreactivity indicated mild immunoreactivity in the control group and the PGB group, with no significant difference found between the two groups (p = 0.724). Severe immunoreactivity was detected in the DM group, which established a significant difference when compared to that of control group (p = 0.013). Moderate immunoreactivity was detected in the DM + PGB group, which was significantly lower than that of DM group (p = 0.035) and was similar to that of control group. In the examination of Caspase-3 immunoreactivity, mild immunoreactivity was detected in the PGB group, which established no significant difference when compared to that of control group (p = 0.999). Severe immunoreactivity was detected in the DM group, which was significantly higher than that of control group (p = 0.009). Moderate immunoreactivity was detected in the DM + PGB group, which was significantly lower than that of DM group (p = 0.035) and was similar to that of control group. In the TUNEL assay, the number of TUNEL-positive cells was similar in the PGB and control groups (p = 0.954). However, the DM group had a higher number of TUNEL-positive cells compared to the control group (p = 0.001). Conversely, the DM + PGB group had a lower number of TUNEL-positive cells compared to the DM group (p = 0.0001)

Values are given as mean ± standard deviation

^aCompared to the control group,

^bCompared to the diabetes group, (p < 0.05)

Caspase-3 immunoreactivity

In the examination of Caspase-3 immunoreactivity, mild immunoreactivity was detected in the PGB group, which established no significant difference when compared to that of control group (p = 0.999) (Fig. 3a, b; Table 2). Severe immunoreactivity was detected in the DM group, which was significantly higher than that of control group (p = 0.009) (Fig. 3c; Table 2). Moderate immunoreactivity was detected in the DM + PGB group, which was significantly lower than that of DM group (p = 0.035) and was similar to that of control group (Fig. 3d; Table 2).

Fig. 3.

Findings of Caspase-3 immunoreactivity. In the examination of caspase-3 immunoreactivity, mild immunoreactivity was detected in the PGB group, which established no significant difference when compared to that of control group (p = 0.999) (a, b; Table 2). Severe immunoreactivity was detected in the DM group, which was significantly higher than that of control group (p = 0.009) (c; Table 2). Moderate immunoreactivity was detected in the DM + PGB group, which was significantly lower than that of DM group (p = 0.035) and was similar to that of control group (d; Table 2)

TUNEL assay

In the TUNEL assay, the number of TUNEL-positive cells was similar in the PBG and control groups (p = 0.954) (Fig. 4a, b; Table 2). However, the DM group had a higher number of TUNEL-positive cells compared to the control group (p = 0.001) (Fig. 4c; Table 2). Conversely, the DM + PGB group had a lower number of TUNEL-positive cells compared to the DM group (p = 0.0001) (Fig. 4d; Table 2). However, no TUNEL positivity was found in the negative control (Fig. 5a).

Fig. 4.

Findings of TUNEL assay. In the TUNEL assay, the number of TUNEL-positive cells was similar in the PGB and control groups (p = 0.954) (a, b; Table 2). However, the DM group had a higher number of TUNEL-positive cells compared to the control group (p = 0.001) (c; Table 2). Conversely, the DM + PGB group had a lower number of TUNEL-positive cells compared to the DM group (p = 0.0001) (d; Table 2)

Fig. 5.

TUNEL assay negative and positive controls. No TUNEL positivity was found in the negative control (a). Breast tissue was used as positive control (b)

Discussion

Previous studies have demonstrated advanced structural and functional abnormalities in central and peripheral nerve fibers in STZ-induced diabetic rats [24–27]. Accordingly, the present study evaluated the effect of PGB as an antioxidant and brain-protective agent in rats with STZ-induced diabetes.

Neurodegeneration associated with diabetes can be triggered by various pathophysiological mechanisms. Accordingly, it has been reported that increasing extracellular glutamate levels after a trauma to the CNS lead to intracellular calcium overload, thereby triggering ROS formation and lipid peroxidation, ultimately leading to neurodegeneration [28].

Some other studies also demonstrated that PGB has a potential to reduce neuronal excitotoxicity [29]. Ha et al. indicated that PGB showed a neuroprotective effect in rats after spinal cord injury [30]. Additionally, Andre et al. investigated the pathogenesis and pharmacology of epilepsy in a lithium-pilocarpine model in rats and showed that PGB reduced neuronal excitotoxicity [31].

Products of lipid and membrane peroxidation impair membrane permeability and micro-viscosity by damaging the structure and components of the cell membrane [32, 33]. Cosar et al. demonstrated the protective effects of omega-3 fatty acids in the brain tissue of STZ-induced diabetic rats and reported that the MDA, SOD and CAT levels and the number of apoptotic cells were increased in the diabetic group [34].

Malondialdehyde (MDA) is an indicator of lipid peroxidation and is generally used for this purpose [35, 36]. Nader et al. indicated the implementation of PGB decreased the hippocampal MDA level, which was significantly higher than that of control group [12]. In the present study, MDA concentration was significantly higher in the DM group compared to the control group (p < 0.05), whereas it was significantly lower in the DM + PGB group than in the DM group (p < 0.05). The high levels of MDA in DM and DM + PGB groups might support the idea that DM increases lipid peroxidation due to oxidative damage. The low levels of MDA in the PGB and PGB + DM groups compared to the DM group may suggest that PGB can have a preventive effect on oxidative damage in the brain tissue.

Glutathione peroxidase (GPx) is a well-known indicator of oxidative stress. In the present study, GPx activity was significantly lower in the DM group than in the control group (p < 0.05), whereas no significant difference was found between the DM + PGB and control groups. Our study, in a similar way to Seghrouchni et al. [37], showed that PGB reduced the GPx concentration in diabetic rats. The low levels of GPx, an antioxidant enzyme, in the DM group compared to the control group may support the presence of oxidative damage in the DM group.

In a recent study, pretreatment of cerebral ischemia with PGB decreased the ischemic damage by reducing oxidative stress and lipid peroxidation and promoting GPx and CAT activity. Based on the results, the authors indicated that PGB has anti-apoptotic, antioxidant, and anti-inflammatory effects [28]. Nader et al. demonstrated that the brain tissues of the rats administered with PGB showed a significant increase in SOD and CAT activity and no significant change in GSH and GSP-x activity when compared to the brain tissues of pentylenetetrazole-induced epileptic rats [12]. Asci et al. evaluated brain tissues of rats induced with experimental ischemia/reperfusion (I/R) and reported that GPx activity, which is an antioxidant marker, was significantly higher in the PGB + I/R group than in the I, I/R, and control groups. The authors also noted that CAT activity, which is another antioxidant marker, was significantly higher in the PGB + I and PGB + I/R groups compared to the I and I/R groups [13]. In this study, unlike other studies, administration of PGB in diabetic rats led to a significant increase in SOD activity, which suggests that PGB may have a protective effect against oxidative stress. However, no significant difference was among other groups with regard to GSH and CAT activity.

All these biochemical findings suggest that DM increases oxidative damage in the CNS, and PGB may have a protective effect against DM-related oxidative damage, so PGB may be a potential therapeutic agent in vasculopathy and cognitive dysfunction due to oxidative damage in the CNS in DM.

There is an inverse correlation between the duration of DM and neuronal volume in type 1 DM and previous studies indicated that apoptosis-related neuronal loss increases in long durations of DM [38, 39]. If excitotoxic neurotransmitters, such as glutamate, are overexpressed, they can trigger apoptosis by means of the direct damage to the mitochondria and the induction of apoptosis inducing enzymes named of caspases [29]. TUNEL assay is used for detecting the in situ staining of DNA strand breaks. Bcl-2/Bax gene is responsible for the regulation of apoptosis. Xue et al. demonstrated increased Bax immunoreactivity in STZ-induced diabetic rats [40]. Zhen-guo et al. showed increased neuronal apoptosis in diabetic rats and the authors indicated that the causes of apoptosis include intracytoplasmic calcium accumulation, mitochondrial dysfunction with or without receptor mechanisms, and ischemia with the suppression of insulin-like growth factor (IGF) activation [39]. ROS play a role in the mechanism of apoptosis by reducing the expression of Bcl-2 gene and increasing the activity of Caspase-3 [41, 42]. Ha et al. showed that PGB can prevent brain injury through its antiapoptotic and neuroprotective effects. PGB decreases the proliferation of astrocytes in case of spinal cord injury and reduces the production of Caspase-3 and phosphorylated p38 MAPK [30]. A recent study reported that PGB had histopathologically demonstrable anti-edema, anti-inflammatory, and neuroprotective effects in rats when it was administered to limit diffuse brain damage during the acute phase of experimental brain injury [43]. Additionally, Nader et al. demonstrated that the use of PGB in the treatment of an experimental epileptic mice model showed a significant increase in autophagy markers and a significant decrease in apoptotic markers (Caspase-3, Bax) compared to pentylenetetrazole-injected rats [12]. In our study, the number of apoptotic cells was significantly lower in the DM + PGB group compared to the DM group. If these findings are supported by cognitive and behavioral tests, the use of PGB can be beneficial treatment agent to reduce the incidence of cognitive dysfunction due to DM and DM-dementia coexistence by preventing neuronal apoptosis.

Limitations

Our study was limited since apoptotic cells could have been assessed by a more objective test, such as electron microscopy, and also no behavioral and cognitive tests were performed in the study.

Conclusion

The results indicated that oxidative stress increased as a result of lipid peroxidation and that brain tissue was damaged in STZ-induced diabetic rats. PGB was administered to protect tissues against oxidative stress caused by DM and it was revealed that PGB reduced lipid peroxidation, improved the antioxidant capacity of the rats and protected the cells against apoptosis. Based on these findings, we consider that PGB probably reduced these neurotransmitters and it may have the ability to inhibit the activation of catalytic enzymes by reducing intracellular Ca + 2. On the other hand, both Bax and Caspase-3 immunohistochemical studies indicated lower apoptosis rates in diabetic rats treated with PGB. PGB may be effective in reducing the risk of brain damage associated with DM. However, further studies are needed to find out the preventive and therapeutic effects of PGB and its mechanisms of action.

Author contributions

All autors confirm that this work is original and has not been published elsewhere nor is it currently under consideration for publication elsewhere.

Compliance with ethical standards

Conflict of interest

All authors declare that there is no conflict of interest.

Ethical approval

All institutional and national guidelines for the care and use of laboratory animals were followed.