Diabetes is a chronic metabolic disease and is characterized by insulin resistance and loss of pancreatic β-cell function [1, 2]. Hyperglycemia is one of the central factors for the development of oxidative stress that induces important complications in diabetic patients [3]. These complications typically include microvascular (retinopathy, neuropathy, and nephropathy) and macrovascular (cardiovascular and cerebrovascular) diseases [4].
Diabetes affects the central nervous system (CNS) associated with cognition, especially memory and learning; moreover, it increases the risk of developing dementia by up to 60% [5]. Hyperglycemia enhances the formation of reactive oxygen species (ROS) through glucose autoxidation, polyol pathways, and non-enzymatic glycation of proteins. These free radicals can have significant effects on carbohydrates, proteins, and DNA [6]. The hippocampus is a part of the limbic system of the brain, and it is the consolidation center for cognitive functions, such as memory and learning that are affected in diabetes [7]. The gut-brain axis (GBA) has been introduced as a novel therapeutic target in neurological disorders and other diseases that function through glucagon-like peptide-1(GLP-1) [8]. Additionally, GLP-1 and its analogs can cross the blood-brain barrier and improve memory and learning [9].
Studies have indicated that probiotics can suppress the production of oxidative stress by increasing the levels of antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase
(GPx) [10,11]. Previous studies also revealed that probiotics were beneficial for brain function and had therapeutic effects for various CNS disorders [12]. Another way to treat metabolic disorders is the use of natural compounds rich in phenols and flavonoids [13-16]. Resveratrol is a polyphenol that can cross the blood-brain barrier in the areas of the brain that are involved in learning and memory
and may be involved in modulating message transmission in CNS [17]. Consequently, it has been suggested as a potential compound in the treatment of diabetes and neurodegenerative disorders [18]. Probably, the concurrent administration of these compounds can have potent neuroprotective effects.
 
Objectives
The present study aimed to investigate the therapeutic effects of probiotics and resveratrol combination on oxidative stress, memory, learning, as well as GLP-1 levels in Streptozotocin (STZ) induced diabetic rats.
 
Materials and Methods
Animals
This experimental study was conducted on 30 healthy 8-week-old Wistar rats (weight range: 250-300) that were purchased from Hamadan University of Medical Sciences, Hamadan, Iran. The rats were kept under a conditioner at 50%-60% humidity and a controlled temperature of 22±2°C within the animal room under a 12:12-h light/dark cycle. Subsequently, the rats were randomly divided into five groups of six animals per group and kept in separate cages. The experimental groups included: 1) control rats, 2) diabetic rats, 3) diabetic rats receiving probiotic supplements (50×10⁹CFU/kg), 4) diabetic rats receiving resveratrol (10 mg/kg), and 5) diabetic rats receiving the combined probiotic and resveratrol supplements.
 
Induction of Type 2 diabetes
To induce type 2 diabetes, single doses of STZ (65 mg/kg) (Sigma-Aldrich, USA) and nicotinamide (110 mg/kg) were administered intraperitoneally (dissolved in 0.1 M citrate buffer, pH=4.5)[3]. Furthermore, blood glucose levels were measured by glucometer seven days after STZ injection and regarded as type 2 diabetes if their blood glucose was above 250mg/dl.
 
Animal treatments
Probiotics contain lyophilized bacteria, including eight different strains (Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus bulgaricus) with a dose of 50×109CFU/kg in drinking water for four weeks according to previously published studies [3]. Totally, 10 mg/kg of resveratrol powder was daily mixed in 1ml sterile distilled water per rat and maintained on ice until complete gavage [19]. The animals received probiotics and resveratrol once a day for four weeks. It should be noted that the behavioral tests were performed at the end of the treatment. Furthermore, the probiotics and resveratrol were purchased from Genuine Health (Canada) and Natural Fact Companies (USA), respectively.
 
Behavioral tests
Evaluation of passive avoidance learning
The passive avoidance learning (PAL) test was performed at the end of the experiment using a Shuttle Box. This plexiglass box is divided into both dark and light compartments with a dimension
of 30×20×30 cm. The two compartments are interconnected by a single (20×50 cm) valve connected by a guillotine door. In total, three mm metal bars with a distance of 10 mm were installed on the floor of the plexiglass box. A dark chamber was considered during testing, and the guillotine door was closed between the two compartments during the training. When the rat was placed in the light compartment, the guillotine door was opened, and the delay time was measured by a chronometer. After the entrance of the rat to the dark compartment, the guillotine door was closed, and an electric shock was applied to the animals during the training phase through bars attached to the stimulator device. At this stage, the rats were placed in the light part to be acquainted with the device, and after 30 sec, the door was opened between the two parts. After the rat entered the dark area, the door was closed and after 30 sec, the animal was taken out and returned to the cage. Subsequently, these steps were repeated after 30 min, and following the second stage, the rat was placed in a bright environment for 30 sec, and then the guillotine door was opened between the two parts. The delayed entry time (step-through latency) (STLa) was recorded for each rat at that stage. After the rat had fully entered the dark chamber, the guillotine door was closed, and an electric shock was applied through the metal rods of the chamber floor for a second. The animal was then removed from the dark chamber after 30 sec and returned to the cage. This step was repeated after 2 min, and the retention test was performed according to a previously conducted study [20]. The rats were placed in a light chamber (the same as PAL training), and 5 sec later, the guillotine door was opened, and the STLa in the retention trial (STLr), as well as the time spent in dark compartment (TDC), were recorded for up to 300 sec. If the rat did not enter the dark chamber within 300 sec, the retention test was terminated and a ceiling score of 300 s was assigned.
 
Tissue measurement of GLP-1
The ELISA Kit was used to determine the GLP-1 levels according to the manufacturer’s instructions (ZellBio, Germany).
 
Determination of total antioxidant capacity, total oxidative status, and malondialdehyde
Total antioxidant capacity (TAC) was determined by ferric-reducing antioxidant power assay [21,22]. Furthermore, the total oxidative status (TOS) of the sample was determined by the oxidation of ferrous iron to ferric in the samples with moderate acidity [21,22]. Malondialdehyde (MDA) level was measured according to the manufacturer’s instructions (ZellBio, Germany) [21,22].
 
Measurement of antioxidant enzyme activity
The activity of catalase (CAT), SOD, and GPX enzymes was measured using a kit (ZellBio, Germany).
 
Histology of hippocampus
Hippocampus were fixed in 4% formaldehyde, immersed in paraffin, and then stained with hematoxylin and eosin according to previously conducted studies [23,24].
 
Statistical analysis
The results were analyzed using SPSS software (version 20) through ANOVA and Tukey's post hoc test in case of significant results. A p-value less than 0.05 (P<0.05) was considered statistically significant.
 
Results
Findings of passive avoidance memory test using the Shuttle Box test
The result showed no significant difference among the animals regarding the latency to enter the dark box during the adaptation phase (Figure 1).
 
Comparison of the time of "first dark entry" in the step-through latency in the retention test 
A significant decrease was observed in the time of "first dark entry" in the avoidance memory test in the diabetic group, compared to the control group (P<0.001). Moreover, the time of "first dark entry" in the probiotics alone and resveratrol alone
groups indicated a significant increase (P<0.01). Furthermore, a comparison of the diabetic with the diabetic group treated with probiotics and resveratrol indicated a significant decrease in this regard (P<0.001) (Figure 1).
 
Comparison of the duration of stay in the dark room of the Shuttle Box 
After comparing the duration of stay in the dark room of the Shuttle Box in the avoidance memory test, the results indicated a significant decrease in the control group, compared to the diabetic group (P<0.001). Furthermore, the duration of stay in the dark room of the Shuttle Box in probiotics alone and resveratrol alone groups indicated a significant decrease in this regard (P<0.01). In addition, the combined probiotics and resveratrol group, compared to the diabetic group, showed a significant decrease in dark room stay (P<0.001) (Figure 1).
 
Effect of treatment with probiotics and resveratrol on malondialdehyde concentration
The level of MDA was significantly higher in the diabetic group, compared to the control group (P=0.0034). Moreover, the level of MDA was significantly decreased in the probiotic-treated diabetic group, compared to the diabetic group (39% decrease) (P=0.093). However, there was no
 

 

 
              
Figure 1. Average latency time to enter the dark compartment (A), time of "first dark entry" in the avoidance memory test (B), and duration of stay in the dark room of the Shuttle Box (C).  
Data are presented as means±SEM. **P<0.01 and ***P<0.001 were set as significant, compared to diabetes; ^###P<0.001 compared to controls. D: diabetes, RSV: resveratrol, P: probiotic, STLr: step-through latency, RSV: resveratrol, P: probiotic.
                  



Figure 2. Malondialdehyde, total oxidative status, and total antioxidant capacity levels in different treated groups.
Data are presented as means±SEM. *P<0.05 and **P<0.01 were set as significant, compared to diabetes; ^##P<0.01, compared to controls. D: diabetes, RSV: resveratrol, P: probiotic, MDA: Malondialdehyde, TOS: total oxidative status, TAC: total antioxidant capacity.
 

 

significant decrease in the resveratrol-treated diabetic group, compared to the diabetic group. On the other hand, there was a significant decrease in the diabetic group treated with resveratrol and probiotics, compared to the diabetic group (44% decrease) (P=0.0185) (Figure 2).
 
Effect of treatment with probiotics and resveratrol on total oxidative status
The level of TOS as a marker of oxidant showed a remarkable increase in the diabetic group, compared to the control group (P=0.0036). The level of TOS was significantly decreased in the resveratrol-treated diabetic group, compared to the diabetic group (45% decrease; P=0.0062). Combined probiotics and resveratrol treatment reduced the level of TOS significantly (52% decrease; P=0.0015), which indicated the effect of oxidant decrease in response to combined therapies (Figure 2).
 
Effect of treatment with probiotics and resveratrol on total antioxidant capacity
The level of TAC was significantly (P=0.0192) lower in the diabetic group, compared to the control group. Moreover, the increase in the level of TAC was not significant in the diabetic group receiving resveratrol alone and probiotics alone; however, the combined probiotics and resveratrol treatment increased TAC significantly (105% increase; P=0.04) (Figure 2).
 
Effect of treatment with probiotics and resveratrol on superoxide dismutase, activity of catalase, and glutathione peroxidase activity
The level of SOD activity significantly reduced in response to the combined probiotics and resveratrol treatment (47% decrease; P <0.001). The level of enzyme activity in the combined probiotics and resveratrol treatment group was similar to that of the control group, indicating that this combination therapy was effective (Figure 3). The decrease in CAT enzyme activity was significant in the combined treatment group (45% decrease; P=0.01). Furthermore, the level of enzyme activity in the combined probiotics and resveratrol treatment group was similar to that of the control group, indicating the positive effect of the combined treatment (Figure 3). Furthermore, the combined
 

 

 

                                
Figure 3. Dismutase, catalase, and glutathione peroxidase levels in different treated groups.
Data are presented as means±SEM. **P<0.01 and ***P<0.001 were set as significant, compared to diabetes; ^##P<0.01 and  ^###P<0.001,  compared to controls. D: diabetes, RSV: resveratrol, P: probiotic, SOD:  dismutase, CAT: catalase, GPX: glutathione peroxidase.
 

 

treatment was also able to significantly decrease the activity of the GPX‎ enzyme (P<0.001). The level of enzyme activity in the treatment groups was similar to that of the control group (Figure 3). 
 
Effect of treatment with probiotics and resveratrol on glucagon-like peptide-1 ‎
The concentration level of GLP-1 in diabetic rats was significantly lower than that in the control group (P<0.001). Moreover, the level of GLP-1 significantly increased in the probiotics and resveratrol group, compared to the diabetic group (P<0.01). The combination of probiotics and resveratrol significantly increased the level of GLP-1(Figure 4).
 
Histology of hippocampus 
Hematoxylin and eosin-stained neuron densities showed that the diabetic rats showed a significant decrease in pyramidal cell densities, compared to the control group. However, treatment resveratrol
and probiotics significantly normalized these changes (Figure 5).
 



Figure 4. Glucagon-like peptide-1 levels in different treated groups.
Data are presented as means±SEM. **P<0.01 and ***P<0.001 were set as significant, compared to diabetes; ^###P<0.001 compared to controls. D: diabetes, RSV: resveratrol, P: probiotic, GLP-1: glucagon-like peptide-1.

 

 



Figure 5. Histological changes of hippocampus determined by hematoxylin and eosin (magnification 400×). Diabetic animals showing reduced thickness of pyramidal cell layer in the CA1 with elevated apoptotic neurons irregular with chromatolysis. Treatment with resveratrol and probiotics normalizing these changes.
 

 

1. Lee JY, Chan CKY, Chua SS, Ng CJ, Paraidathathu T, Lee KKC, et al. Telemonitoring and team-based management of glycemic control on people with type 2 diabetes: a cluster-randomized controlled trial. Journal of General Internal Medicine. 2020; 35(1):87-94. [DOI:10.1007/s11606-019-05316-9] [PMID] [PMCID]
2. Abbasi-Oshaghi E, Khodadadi I, Mirzaei F, Ahmadi M, Tayebinia H, Goodarzi MT. Anethum graveolens L‎. alleviates sperm‎ damage by limiting oxidative stress‎ and insulin‎ resistance‎ in‎ diabetic rats‎. The Open Medicinal Chemistry Journal. 2020; 14(1):35-44. [DOI:10.2174/
   1874104502014010035]
3. Davari S, Talaei SA, Alaei H, Salimi M. Probiotics treatment improves diabetes-induced impairment of synaptic activity and cognitive function: behavioral and electrophysiological proofs for microbiome–gut–brain axis. Neuroscience. 2013; 240:287-96. [DOI:10.1016/j.neuroscience.2013.02.055] [PMID]
4. Sastre AA, Vernooij RW, Harmand MGC, Martínez G. Effect of the treatment of type 2 diabetes mellitus on the development of cognitive impairment and dementia. Cochrane Database of Systematic Reviews. 2017; 6(6):CD003804. [DOI:10.1002/14651858.CD003804.pub2] [PMID] [PMCID]
5. Ahmed A, Zeng G, Jiang D, Lin H, Azhar M, Farooq AD, et al. Time-dependent impairments in learning and memory in Streptozotocin-induced hyperglycemic rats. Metabolic Brain Disease. 2019; 34(5):1431-46. [DOI:10.1007/s11011-019-00448-7] [PMID]
6. Malekiyan R, Abdanipour A, Sohrabi D, Jafari Anarkooli I. Antioxidant and neuroprotective effects of lycopene and insulin in the hippocampus of streptozotocin‑induced diabetic rats. Biomedical Reports. 2019; 10(1):47-54. [DOI:10.3892/br.2018.1171] [PMID] [PMCID]
7. Rababa'h AM, Mardini AN, Alzoubi KH, Ababneh MA, Athamneh RY. The effect of cilostazol on hippocampal memory and oxidative stress biomarkers in rat model of diabetes mellitus. Brain Research. 2019; 1715:182-7. [DOI:10.1016/j.brainres.2019.03.025] [PMID]
8. Foster JA, Neufeld KA. Gut–brain axis: how the microbiome influences anxiety and depression. Trends in Neurosciences. 2013; 36(5):305-12. [DOI:10.1016/j.tins.2013.01.005] [PMID]
9. Hunter K, Hölscher C. Drugs developed to treat diabetes, liraglutide and lixisenatide, cross the blood brain barrier and enhance neurogenesis. BMC Neuroscience. 2012; 13(1):33. [DOI:10.1186/1471-2202-13-33] [PMID] [PMCID]
10. D'Souza A, Fordjour L, Ahmad A, Cai C, Kumar D, Valencia G, et al. Effects of probiotics, prebiotics, and synbiotics on messenger RNA expression of caveolin-1, NOS, and genes regulating oxidative stress in the terminal ileum of formula-fed neonatal rats. Pediatric Research. 2010; 67(5):526-31. [DOI:10.1203/PDR.0b013e3181d4ff2b] [PMID]
11. Morshedi M, Saghafi-Asl M, Hosseinifard ES. The potential therapeutic effects of the gut microbiome manipulation by synbiotic containing-Lactobacillus plantarum on neurop-sychological performance of diabetic rats. Journal of Translational Medicine. 2020; 18(1):18. [DOI:10.1186/
    s12967-019-02169-y] [PMID] [PMCID]
12. Rahmati H, Momenabadi S, Vafaei AA, Bandegi AR, Mazaheri Z, Vakili A. Probiotic supplementation attenuates hippocampus injury and spatial learning and memory impairments in a cerebral hypoperfusion mouse model. Molecular Biology Reports. 2019; 46(5):4985-95. [DOI:10.1007/s11033-019-04949-7] [PMID]
13. Oshaghi EA, Khodadadi I, Tavilani H, Goodarzi MT. Effect of dill tablet (Anethum graveolens L) on antioxidant status and biochemical factors on carbon tetrachloride-induced liver damage on rat. International Journal of Applied
    and Basic Medical Research. 2016; 6(2):111-4. [DOI: 10.4103/2229-516X.179019] [PMID] [PMCID]
14. Mohammadi A, Bazrafshani MR, Oshaghi EA. Effect of garlic extract on some serum biochemical parameters and expression of npc1l1, abca1, abcg5 and abcg8 genes in the intestine of hypercholesterolemic mice. Indian Journal of Biochemistry & Biophysics. 2013; 50(6):500-4. [PMID]
15. Abbasi Oshaghi E, Khodadadi I, Saidijam M, Yadegarazari R, Shabab N, Tavilani H, et al. Lipid lowering effects of hydroalcoholic extract of anethum graveolens l. and dill tablet in high cholesterol fed hamsters. Cholesterol. 2015; 2015:958560. [DOI:10.1155/2015/958560] [PMID] [PMCID]
16. Goodarzi MT, Khodadadi I, Tavilani H, Abbasi Oshaghi E. The role of Anethum graveolens L. (Dill) in the management of diabetes. Journal of Tropical Medicine. 2016; 2016:1098916. [DOI:10.1155/2016/1098916] [PMID] [PMCID]
17. Abbasi Oshaghi E, Goodarzi MT, Higgins V, Adeli K. Role of resveratrol in the management of insulin resistance and related conditions: Mechanism of action. Critical Reviews in Clinical Laboratory Sciences. 2017; 54(4):267-93. [DOI:10.1080/10408363.2017.1343274] [PMID]
18. Tian X, Liu Y, Ren G, Yin L, Liang X, Geng T, et al. Resveratrol limits diabetes-associated cognitive decline in rats by preventing oxidative stress and inflammation and modulating hippocampal structural synaptic plasticity. Brain Research. 2016; 1650:1-9. [DOI:10.1016/j.brainres.2016.08.032] [PMID]
19. Palsamy P, Subramanian S. Resveratrol, a natural phytoalexin, normalizes hyperglycemia in streptozotocin-nicotinamide induced experimental diabetic rats. Biomedicine & Pharmacotherapy. 2008; 62(9):598-605.
    [DOI:10.1016/j.biopha.2008.06.037] [PMID]
20. Mirzaei F, Khazaei M, Komaki A, Amiri I, Jalili C. Virgin coconut oil (VCO) by normalizing NLRP3 inflammasome showed potential neuroprotective effects in Amyloid-β induced toxicity and high-fat diet fed rat. Food and Chemical Toxicology. [DOI:10.1016/j.fct.2018.04.064] [PMID]
21. Abbasi-Oshaghi E, Mirzaei F, Mirzaei A. Effects of ZnO nanoparticles on intestinal function and structure in normal/high fat diet-fed rats and Caco-2 cells. Nanomedicine (London, England). 2018; 13(21):2791-816. [DOI:10.2217/nnm-2018-0202] [PMID]
22. Abbasi-Oshaghi E, Mirzaei F, Pourjafar M. NLRP3 inflammasome, oxidative stress, and apoptosis induced in the intestine and liver of rats treated with titanium dioxide nanoparticles: in vivo and in vitro study. International Journal of Nanomedicine. 2019; 14:1919-36. [DOI: 10.2147/IJN.S192382] [PMID] [PMCID]
23. Ravan AP, Bahmani M, Ghasemi Basir HR, Salehi I, Oshaghi EA. Hepatoprotective effects of Vaccinium arctostaphylos against CCl4-induced acute liver injury in rats. Journal of Basic and Clinical Physiology and Pharmacology. 2017; 28(5):463-71. [DOI:10.1515/jbcpp-2016-0181] [PMID]
24. Shahryari J, Poormorteza M, Noori-Sorkhani A, Divsalar K, Abbasi-Oshaghi E. The effect of concomitant ethanol and opium consumption on lipid profiles and atherosclerosis in golden syrian Hamster’s aorta. Addiction & Health. 2013; 5(3-4):83-9. [PMID] [PMCID]
25. Tuzcu M, Baydas G. Effect of melatonin and vitamin E on diabetes-induced learning and memory impairment in rats. European Journal of Pharmacology. 2006; 537(1-3):106-10. [DOI:10.1016/j.ejphar.2006.03.024] [PMID]
26. Isik AT, Soysal P, Yay A, Usarel C. The effects of sitagliptin, a DPP-4 inhibitor, on cognitive functions in elderly diabetic patients with or without Alzheimer’s disease. Diabetes Research and Clinical Practice. 2017; 123:192-8. [DOI:10.1016/j.diabres.2016.12.010] [PMID]
27. Gandhi S, Abramov AY. Mechanism of oxidative stress
    in neurodegeneration. Oxidative Medicine and Cellular Longevity. 2012; 2012:428010. [DOI:10.1155/2012/428010] [PMID] [PMCID]
28. Franco JG, de Moura EG, Koury JC, Trotta PA, Cordeiro A, Souza LL, et al. Resveratrol reduces lipid peroxidation and increases sirtuin 1 expression in adult animals programmed by neonatal protein restriction. The Journal of Endocrinology. 2010; 207(3):319-28. [DOI:10.1677/JOE-10-0124] [PMID]
29. Mazloom Z, Yousefinejad A, Dabbaghmanesh MH. Effect of probiotics on lipid profile, glycemic control, insulin action, oxidative stress, and inflammatory markers in patients with type 2 diabetes: a clinical trial. Iranian Journal of Medical Sciences. 2013; 38(1):38-43. [PMID] [PMCID]
30. Wang H, Lee IS, Braun C, Enck P. Effect of probiotics on central nervous system functions in animals and humans: a systematic review. Journal of Neurogastroenterology and Motility. 2016; 22(4):589-605. [DOI:10.5056/jnm16018] [PMID] [PMCID]