Keywords

1 Introduction

Type 2 diabetes mellitus (T2DM) is defined as a permanent condition of hyperglycemia with predominant impacts on multiple metabolic pathways and physiologic functions of organs, caused by beta-cell dysfunction and insulin deficiency, tissue insulin resistance, or other metabolic alterations such as disruption of the redox balance and stress [1,2,3]. Oxidative stress has the potential to induce cell death mechanisms associated with tissue damage and multiple diabetic complications, including diabetic cardiomyopathy, retinopathy, and nephropathy [4]. This can occur via activation of nuclear factor kappa B (NF-κB), p38 MAPK, and c-jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) signaling pathways [5]. Indeed, there is an association between hyperglycemia-induced oxidative stress and local or systemic inflammation via increased pro-inflammatory cytokine production and macrophage infiltration [6]. Due to the deleterious outcomes of oxidative stress on diabetes complications, application of antioxidant therapies has been considered as a potential means of reducing T2DM pathogenesis through a decrease in free radicals and an increase in antioxidant enzyme activities [7,8,9].

Trehalose (mycose) is a carbohydrate with a disaccharide structure naturally produced by a wide range of organisms from prokaryotes to plants, except humans [10]. This sweetener molecule is frequently applied in food and drug industries and has been found to exert important biological impacts and modulate several metabolic pathways after consumption [11,12,13,14]. Experimental studies have indicated trehalose functions as an antioxidant, anti-inflammatory, and autophagy enhancer, which suppresses oxidative stress, inflammation, and autophagy-related disorders such as diabetes [15,16,17], atherosclerosis [18, 19], and Parkinson [20], Alzheimer [21, 22], and Huntington [23] diseases. Antidiabetic effects of trehalose can be linked to improving pathophysiological mechanisms such as inflammation and oxidative stress, pancreatic islet function, and lipid profile correction [24]. The role of trehalose as a natural antioxidant has been reported in in vitro and in vivo studies [25,26,27,28]. Here, we have attempted to determine the antioxidant effects of intraperitoneal (IP) and oral trehalose administration on total antioxidant capacity (TAC) and total thiols, along with the activities of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) as markers of oxidative stress in a streptozotocin (STZ)-induced diabetes rat model. In addition, antioxidant effects of trehalose were compared to those of the standard T2DM medication, metformin. The results showed that both oral and IP routes of trehalose administration suppressed oxidative stress, confirming the trehalose therapeutic potential in controlling oxidative stress-induced complications of diabetes in animal models.

2 Material and Methods

2.1 Animal

Male Wistar albino rats (8 weeks old, 180–200 g) were bred and housed in the Laboratory Animal Research Center of Medicine Faculty, Mashhad University of Medical Sciences, Mashhad, Iran. All animal experiments were approved by the Institutional Ethics Committee and Research Advisory Committee of the Mashhad University of Medical Sciences and the National Institute for Medical Research Development (NIMAD). The animals were maintained using a 12:12-h day-night cycle, at a constant 22 ± 2 °C, and humidity of 45–64%. Over the entire experimental procedure, the rats were fed with a standard rodent diet and water ad libitum. All rats were anesthetized with IP injections of thiopental sodium and blood samples collected after 4 weeks of treatment at study termination.

2.2 Induction of Rat T2DM Model

Non-insulin-dependent diabetes mellitus was induced by intravenous injection of single 60 mg/kg dose of streptozotocin in overnight-fasted rats (Masiello et al., 1998). STZ was dissolved in citrate-buffered saline (0.1 M, pH 4.5). Hyperglycemia was confirmed with blood glucose levels >180 mg/dL, determined at 72 h and then on day 7 after injection, and diabetic rats were included in this study. Two groups of diabetic rats (six rats per group) were treated daily with 45 mg/kg/day trehalose via i.p. injection and 1.5 g/kg/day via oral gavage for 4 weeks. Nondiabetic rats (n = 6) were used as the normal control (NC) group that received citrate buffer (i.p.). The diabetic (DC) and positive control groups received saline buffer and metformin (200 mg/kg/day), respectively.

2.3 Total Thiol (-SH) Group

Total thiol groups (-SH) were measured using the Kiazist kit according to the manufacturer’s instructions. In this assay, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) reacts with reduced sulfhydryl (-SH) groups in the serum, resulting in a yellow-colored complex, which is detectable at 405 nm.

2.4 Total Antioxidant Capacity (TAC)

The potential of samples for reducing ferric (Fe+3) to the ferrous form (Fe+2) was considered as the total antioxidant capacity (TAC) and measured by a colorimetric method. For this assay, 150 μL Kiazist TAC reagent was added to 30 μL sample or standard and incubated at room temperature for 45 min. The absorbance was read in 450 nm.

2.5 Antioxidant Enzyme Activity Assay

The levels of antiperoxidative enzymes, including GPx and SOD, were determined in the serum of diabetic rats using specific assay kits (Kiazist, Iran). The measurement of SOD and GPx activities was based on reducing free radicals produced by the xanthine/xanthine oxidase system and conversion of hydrogen peroxide to water, accompanied by glutathione oxidation, respectively.

2.6 Statistical Analysis

Statistical analysis was performed with Microsoft Excel (2019) and GraphPad Prism version 8 software. The results were analyzed using one-way analysis of variance (ANOVA) and the Tukey’s multiple comparison posttest to evaluate the significance of differences between treatment groups. Results with p < 0.05 were considered as statistically significant.

3 Results

3.1 Evaluation of Reduced (Free) Thiol (-SH) Groups and Total Antioxidant Capacity

IP and oral administration of trehalose led to an increase in TAC and thiols, with lower levels in diabetic rats than the healthy control group (nondiabetic). Although TAC alterations did not reach statistical significance (Fig. 1), total thiol groups were increased significantly (p < 0.05) in treated groups compared to nontreated diabetic control, and the effect of IP trehalose administration was more potent than the oral route (Fig. 2).

Fig. 1
figure 1

Antioxidant effect of trehalose on total antioxidant capacity (TAC) in five groups. Data are given as the mean ± standard error of the mean. *P < 0.05, ** P < 0.01, ***P < 0.001, and ****P < 0.0001

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Fig. 2
figure 2

Antioxidant effect of trehalose on total thiol groups (-SH) in five groups. Data are the mean ± standard error of the mean. *P < 0.05, ** P < 0.01, ***P < 0.001, and ****P < 0.0001

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3.2 Evaluation of SOD and GPx Antioxidant Enzyme Activities

For studying the effect of trehalose to induce enzymes that counteract free radical production, we measured the activities of SOD and GPx. These enzymes were increased in both the IP and oral trehalose-treated groups with a stronger effect of oral trehalose when compared with diabetic control rats. Differences in oral (P = 0.07) and IP trehalose (P = 0.89) groups were not significant for SOD (Fig. 3), whereas a significant increase was observed in GPx activity (P < 0.05) (Fig. 4).

Fig. 3
figure 3

Antioxidant effect of trehalose on SOD activity

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Fig. 4
figure 4

Antioxidant effect of trehalose on GPx activity. *P < 0.005

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4 Discussion

Diabetes is a chronic disease characterized by hyperglycemia resulting from deficiency of insulin secretion or insulin resistance, leading to microvascular and macrovascular complications that can damage different organs and tissues [29]. Hyperglycemia causes oxidative stress through multiple pathways, which is considered as a trigger for developing vascular complications of T2DM [30, 31]. High glucose levels promote the activity of some enzymes, including protein kinase C and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, leading to augmentation of reactive oxygen species (ROS) and oxidative stress, which in turn promote cell damage and tissue injuries [15]. Free radicals may attack cell membranes resulting in lipid peroxidation and an increase in MDA as a sensitive index of the systemic redox status and potential disease progression [32]. Besides lipid oxidation effects, ROS can oxidize free thiols and decrease circulating sulfhydryl (SH) concentrations, leading to a reduction in total antioxidant capacity [33]. Moreover, the alterations of antioxidant enzyme patterns are a characteristic feature of the uncontrolled diabetic state associated with a higher incidence of diabetic complications [34]. Since oxidative stress is a critical pathogenic factor for secondary complications of diabetes, the antioxidant therapy approach may be a useful strategy to treat diabetes by controlling free radical production; increasing intracellular antioxidant defenses, along with protective mechanisms against oxidative stress-induced apoptosis; and preserving β-cell function [35,36,37]. This study aimed to evaluate the antioxidant effects of trehalose as a natural antioxidant compound in T2DM . The changes in antioxidant markers such as serum thiol levels, and TAC, as well as the activity of GPx and SOD, were determined following 4 weeks of trehalose administration in STZ-induced diabetic rats.

Trehalose is a nonreducing disaccharide consisting of two glucose units in an α,α-1,1-glycosidic linkage, synthesized in numerous organisms from plants and bacteria to invertebrates and yeast [38]. Recent studies indicate that trehalose may decrease blood glucose and ameliorate insulin sensitivity and, thereby, may serve as a potential non-pharmacological agent for the management of diabetes [24]. We evaluated this possibility in our previous animal study and confirmed trehalose antidiabetic effects in a rat model of type 2 diabetes. The antioxidant effects of trehalose have also been assessed in different in vitro and in vivo studies [39, 40]. Treatment with trehalose in preclinical studies revealed that this antioxidant molecule significantly decreased the amount of ROS and H2O2 levels in a dose-dependent manner [15, 25] and upregulated antioxidant gene expression of SOD , glutathione (GSH), and catalase (CAT) via promotion of nuclear translocation of Nrf2 [25, 41]. Although antioxidant enzyme-dependent defenses play a crucial role in scavenging free radicals produced under oxidative stress [42, 43], there have been conflicting reports on SOD and GPx activity in diabetes mellitus. Both increased and decreased antioxidant enzyme activities have been reported [44,45,46,47,48,49], while some studies have shown no change in comparison to nondiabetic healthy controls [50, 51]. In diabetes, impaired pancreatic β-cells may express low physiological levels of the antioxidant enzymes SOD and GPx [52,53,54]. On the other hand, elevated ROS levels and increased production of O2− may increase the total antioxidant enzyme activity, suggesting a possible adaptive response to oxidative status [55]. Our results indicated a marked decrease in GPX activity in the diabetic rats, whereas this activity was significantly increased in both trehalose-treated groups compared with the DC group. A similar trend was found for SOD activity after 4 weeks of trehalose intervention, though the differences were not statistically significant. Experimental models have determined that antioxidant compounds can change TAC in serum or plasma; therefore, monitoring plasma TAC may be a valuable index for oxidative burden [56, 57]. However, no prior study has investigated the effects of trehalose on plasma TAC levels; our research reported that TAC and the amount of free thiol increased during the treatment process. Differences in TAC marker was significant between the IP-treated trehalose group and DC group. Intraperitoneal administration of trehalose had greater potential efficacy than oral administration, which could be due to the higher bioavailability of trehalose in the IP route.

As mentioned earlier, previous studies displayed in vitro antioxidant activities of trehalose, and here we carried out the in vivo experimental study to support an antioxidant effect of trehalose in T2DM model during 4 weeks of treatment. The obtained results suggest that trehalose might be regarded as a safe antioxidant supplement for diabetic subjects in clinical studies over a longer timeframe.

In conclusion, regarding the importance of oxidative stress in activating intracellular signaling pathways and the pathogenesis of multiple disorders, natural antioxidant products could be a potential therapeutic strategy to manage and reduce oxidative damage. The findings of our study demonstrated that trehalose administration could enhance antioxidant capacities, and protect antioxidant enzyme activity slightly; however, a clear and comprehensive understanding of the effect of trehalose on antioxidant enzymes needs further investigation.

Conflict of Interests

None.