Protective role of nano-selenium-enriched Bifidobacterium longum in delaying the onset of streptozotocin-induced diabetes

Bifidobacterium longum (B. longum) could accumulate Selenium (Se) and nano-Se in the form of Se-B. longum and Nano-Se-B. longum, respectively. In this study, the effect of Nano-Se-B. longum in diabetic mice was evaluated. Physiological and metabolic parameters such as blood glucose, body weight, serum insulin level, intraperitoneal glucose tolerance test (IPGTT), food intake, water consumption and urine output were evaluated. The expression of insulin signalling pathway-related proteins was evaluated by western blotting. Haematoxylin and eosin (H&E) was used for histological examination of the liver, pancreas and kidney sections. Creatinine levels in serum (SCr) and blood urea nitrogen (BUN) were measured. Nano-Se-B. longum was the best in terms of delaying the onset of diabetes. Nano-Se-B. longum decreased blood glucose and body weight compared with those noted for the model group. IPGTT, food intake, water consumption and urine output significantly increased and serum insulin levels significantly decreased in the model group compared with those in all the Nano-Se-B. longum-treated mice. Histological results showed that the Nano-Se-B. longum-treated mice were better than the model group mice in terms of pathological changes. The expression of insulin signalling pathway-related proteins was upregulated in the Nano-Se-B. longum-treated groups. A significant increase in SCr and BUN levels was noted in the model group. This study for the first time reported the dose-dependent preventive effect of Nano-Se-B. longum on the onset of diabetes and renal damage. The mechanism may be related to changes in insulin signalling.

Shanghai, China), and 20 mg bovine serum albumin (Sangon Biotech Co., Ltd., Shanghai, China) were mixed. The pH was adjusted to 7.2 with sodium hydroxide, which led to the formation of red nano-Se and oxidized glutathione (GSSG). The red solution was dialysed against double distilled water for 96 h with the water changing every 24 h to separate GSSG from Nano-Se under magnetic stirring. The final solution containing Nano-Se and BSA was subjected to centrifugation at 13 000 r.p.m. for 10 min. The pellet thus recovered was subjected to washing by its re-suspension in deionized water followed by centrifugation at 13 000 r.p.m. for 10 min, to remove possible organic contamination present in the nanoparticles. Finally, the pellet was freeze-dried using a lyophilizer and stored at room temperature. Size measurements were performed using a Zetasizer Nano-ZSE (Malvern Instruments, Malvern, UK) with Zetasizer Software v. 7.12. The results are reported as the average of 40-44 measurements + s.d. longum was performed according to the previously established protocol [8]. Briefly, sodium selenite was purchased from Shanghai LuYuan Fine Chemical Factory, weighed, and dissolved in 200 ml TPY medium at a concentration of 25 mg ml 21 . Nano red elemental Se was dissolved in 200 ml TPY medium at 5 mg ml 21 . B. longum, Se-B. longum and Nano-Se-B. longum were cultivated overnight in TPY medium anaerobically. This overnight culture was diluted 1 : 25 in TPY medium and cultivated at 378C until the OD 600 reached about 0.2. The cultured strains were collected and then washed three times with 5% glucose saline by centrifugation at 3500 Â g for 5 min at 48C. The collected strains were resuspended in 0.1 ml of 13% milk just prior to use. Live bacteria were prepared daily for administration to each mouse.

Animals
The mice (aged between 4 and 5 weeks (w)) were maintained in a specific pathogen-free animal facility under a 12 h light-dark cycle at an ambient temperature of 218C. They were provided with water and foods ad libitum.

Induction of experimental diabetes
Male mice (C57BL/6) aged 4 -5 w were purchased from Nanjing model animal research center of Nanjing University and diabetes was induced with STZ (Merck, Darmstadt, Germany) as previously described [24]. Briefly, after overnight fasting (deprived of food for 12 h and allowed free access to water), diabetes was induced in mice by i.p. injection of STZ dissolved in 0.1 M cold citrate buffer (pH ¼ 4.5) at a dose of 50 mg kg 21 body weight for 5 consecutive days. Control mice were injected with citrate buffer alone. Diabetes was confirmed by the determination of fasting blood glucose level on the third-day post-final administration of STZ. Mice with fasting blood glucose levels greater than or equal to 11.1 mM were considered diabetic. Blood glucose levels were monitored every week after diabetes was confirmed using the glucose meter (Sinocare Inc., Changsha, Hunan, China). Middle dose group: STZ-induced diabetic mice treated with 1.5 Â 10 10 bacteria kg 21 Nano-Se-B. longum (treated); High dose group: STZ-induced diabetic mice treated with 3 Â 10 10 bacteria kg 21 Nano-Se-B. longum (treated); Toxicity test group: normal mice treated with 3 Â 10 10 bacteria kg 21 Nano-Se-B. longum

Physiological assessment and metabolic analysis
The protective effect of Nano-Se-B. longum in mice was studied at different doses administered for 4 w. Nano-Se-B. longum was administered during the injection of STZ. Blood glucose levels were monitored 3 days to 8 w after the final STZ injection using a glucometer via the caudal vein. Serum insulin levels were determined using Rat/Mouse Insulin ELISA (Millipore Corp, Billerica, MA, USA) at the end of the experiment. Food intake, water consumption and urine output were measured after the mice were placed in metabolic cages overnight. At the eighth week after final STZ injection, eyeball blood was collected and the mice were euthanized. At the end of the experiment, the levels of SCr and BUN were also determined using the assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). IPGTT was performed in mice on the seventh week after final STZ injection (n ¼ 3). For IPGTT, mice were subjected to an overnight fast followed by an intraperitoneal glucose injection (1.0 g kg 21 ). Blood glucose was measured at 0, 15, 30, 60, and 90 min after the injection.

Western blotting analysis
The liver samples were isolated from all the mice and then snap-frozen in liquid N 2 for subsequent protein extractions. The collected tissue samples were lysed in ice-cold lysis buffer (20 mM Tris -HCl ( pH ¼ 7.5), 150 mM NaCl, 1% Triton-X 100, 1 mM EDTA) and a protein inhibitor cocktail for 30 min. The supernatant was boiled with Laemmli sample buffer for SDS-PAGE. The following antibodies were used: anti-IRS1, anti-phospho-IRS1 (pIRS1), anti-GSK-3b, anti-phospho-GSK-3b (pGSK-3b), anti-AKT and anti-phospho-AKT ( pAKT) (Thr308) (Cell Signaling Technology, Beverly, MA); anti-b-actin monoclonal antibody, anti-a-tubulin and anti-GAPDH were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, Delaware, USA). Goat anti-rabbit IgG and goat anti-mouse IgG were from Jackson ImmunoResearch Europe Ltd. The band densities were quantified by using Image J program.

Histological analysis
To observe the morphological changes of the liver, pancreas and kidney, H&E staining was carried out as described before [25]. In brief, the liver, pancreas and kidney tissues were fixed in 4% paraformaldehyde for 16-24 h and transferred to ethanol. Then, the samples were embedded in paraffin and sectioned at 5 mm, followed by H&E staining.

Statistical analysis
Data are presented as means + SEM. The difference between two groups was analysed by a two-tailed Student's t-test using Prism software (GraphPad, San Diego, CA). Values were considered statistically significant at p , 0.05.

Effects of nano-Se-B. longum on physiological and metabolic parameters
Blood glucose testing is the gold standard for the subclinical diagnosis of diabetes. Nano-Se-B. longum-treated mice exhibited notably lower fasting blood glucose levels (figure 2a) and higher body weight (figure 2b) than model mice. Because glucose homeostasis is mainly regulated by insulin, we also detected its serum concentration (n ¼ 6). Fasting insulin levels were higher in Nano-Se-B. longum-treated mice (figure 2c) than in model mice. Twenty-four-hour food intake, water intake and urine volume were measured (n ¼ 10) and found to be decreased with an increase in the dosage of Nano-Se-B. longum (figure 3). In the IPGTT assay (n ¼ 3), the glucose levels decreased significantly in model group mice (figure 4), indicating an improved glucose clearance after Nano-Se-B. longum intervention in a dose-dependent manner.

Effects of nano-Se-B. longum on morphological changes in the liver and pancreas
Histological analysis of the liver and pancreas by H&E staining showed a notable difference between Nano-Se-B. longum-treated and control mice. As shown in figure 5a, there were no obviously harmful changes in the control mice and toxicity test group mice. A small amount of fat vacuoles was observed in part of the pancreatic section in the model, low, middle, and high dose groups (black arrow). Small amounts of inflammatory cells were only visible in the tissue in the model group (red arrow). With the increase in the dosage of Nano-Se-B. longum, the degree of lesion decreased gradually. As shown in figure 5b, no obviously harmful changes in the control mice and toxicity test group were noted. The hepatic cells were edematous and the cytoplasm was loose in the tissue (black arrow) in the STZ-treated groups, while the degree of lesion decreased gradually with an increase in the dosage of Nano-Se-B. longum. Small amounts of inflammatory cells were visible in the tissue (red arrow) in the model group and the degree of infiltrated inflammatory cells decreased with an increase in the dosage of Nano-Se-B. longum (red arrow). Overall, the progression of liver and pancreas pathological damage was slowed after Nano-Se-B. longum treatment.

Nano-Se-B. longum improved liver insulin signalling sensitivity
To investigate the molecular mechanisms underlying hypoglycaemia, we studied the insulin signalling pathway, which plays a critical role in glucose homeostasis. The mice were assessed for the presence of pIRS1, pGSK-3b and pAkt (Thr308). As shown in figure 6a,c, the expression of pIRS-1 and pAkt increased significantly in the liver from the treatment group compared with that in the control mice. pGSK-3b levels decreased markedly in the trial group compared with that in control mice (figure 6b). The expression of insulin signalling pathway proteins was upregulated, which showed that Nano-Se-B. longum improved liver insulin signalling sensitivity.

Protective role on renal function
The influence of Nano-Se-B. longum on the kidney is attributable to its effects on the glomeruli. With the increase in Nano-Se-B. longum dosage, mesentery cell hyperplasia and glomerulus atrophy decreased gradually (black arrow) (figure 7a). Nano-Se-B. longum markedly decreased the levels of BUN and SCr in serum in STZ-induced mice compared to control mice ( figure 7b,c). These data suggest that Nano-Se-B. longum may improve the renal function damaged by diabetes.

Discussion
In this study, the effects of WT B. longum, Se-B. longum and Nano-Se-B. longum on glucose were measured. The results showed that Nano-Se-B. longum was the best with respect to the protective effect on high blood glucose. Normal mice were treated with the maximum dose of Nano-Se-B. longum and no significant difference was observed compared to normal mice. Sudin Bhattacharya research group had synthesized and characterized Nano-Se and found its chemoprotective (CP) activity against CPinduced hepatotoxicity, pulmonary and genotoxicity in normal Swiss albino mice [19,26] and its antitumour efficacy in the tumour-bearing Swiss albino mice [18]. The anti-genotoxic effect of Nano-Se might be due to its antioxidant and cytoprotective activity. Now, Nano-Se-B. longum showed its safety and protective effect in STZ-induced diabetes. We have expanded the functions of Nano-Se, providing further understanding and insight. Some studies found that the restorative effect of selenium on  diabetes is predominantly related to the antioxidant and insulin-like properties of selenium [14]. However, further studies are required to investigate the precise mechanisms involved in the protective effect of Nano-Se-B. longum against diabetes. The insulin signalling pathway controls glucose transport in liver cells. Insulin binds to insulin receptors on the surfaces of target cells. This binding activates insulin receptor beta (IR-b), and then activates IRS1, thereby recruiting phosphatidylinositol 3-kinase (PI3 K) to this location. An important target of PI3 K in liver cells is Akt/PKB, which has a key function in glucose uptake [27]. Previous studies have shown that pIRS1 and pAkt upregulation may have improved glucose uptake by the reduced plasma glucose levels [28]. Oral administration of Nano-Se-B. longum may give rise to elevated plasma selenium levels by enhanced hepatic secretion of selenoproteins, which may enhance insulin-induced signal transduction [16]. Therefore, we assessed the effects of Nano-Se-B. longum administration on insulin signalling pathways. In our study, Nano-Se-B. longum increased the levels of pIRS1 and pAkt proteins and decreased pGSK-3b in diabetic mice. We can reasonably speculate that an increase in the selenoproteins induced by Nano-Se-B. longum treatment enhanced insulin sensitivity by promoting the insulin signalling pathway. Diabetes mellitus can cause serious health problems including macrovascular and microvascular complications [29]. One of these is injuries to the kidney tissue that result in renal dysfunction [30]. Eight weeks after STZ diabetes induction, some indexes of renal damage such as an increase in BUN were noted [31]. There is a large amount of evidence to support the recovery effects of selenium, at suitable doses, on the cell membrane of diabetic kidneys. The beneficial effect of selenium on renal lesions can be explained with its insulin-like effect [32]. Recently, Feride Severcan et al. [14] also showed the efficiency of a low dose (1 mmol kg 21 ) of selenium administration in the prevention of diabetes-related complications in kidneys. We also investigated the renoprotective effect of Nano-Se-B. longum in STZ-induced mice. Nano-Se-B. longum can decrease renal dysfunction by lowering BUN and SCr. Our experiments in Nano-Se-B. longum-treated and STZ-induced diabetes mice revealed that Nano-Se-B. longum exerts a protective role in delaying the onset of STZ-induced diabetes as well as renal function. However, further studies are required to investigate the precise mechanisms involved in the renoprotective effect. Our findings may facilitate the understanding of the novel effects of Nano-Se-B. longum and suggest a newly recognized benefit of Nano-Se-B. longum in diabetic mice. This may provide a novel, feasible, economic protection approach for diabetes, thus deserving further investigation and development.

Conclusion
In this study, we demonstrated that oral administration of Nano-Se-B. longum can delay the onset of STZinduced diabetes, possibly via its effect on the insulin signalling pathway. It was also investigated that Nano-Se-B. longum ameliorates the damage of renal function caused by high glucose levels. Our findings may facilitate the understanding of the novel effects of Nano-Se-B. longum and suggest a newly recognized benefit of Nano-Se-B. longum in diabetic mice.
Ethics. All animal studies were performed according to approved Animal Care and Use Committee protocols of Nanjing University in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.