Pre-Clinical Study for the Antidiabetic Potentialof Selenium Nanoparticles

Hanaa H. Ahmed1& Mohamed Diaa Abd El-Maksoud2

& Ahmed E. Abdel Moneim3&

Hadeer A. Aglan1

Received: 22 June 2016 /Accepted: 13 October 2016 /Published online: 26 October 2016# Springer Science+Business Media New York 2016

Abstract This research was delineated to explore the efficacyof selenium nanoparticles delivered in liposomes (L-Se) in themitigation of type-2 diabetes mellitus. Adult female Wistarrats were assigned into four groups: group I, the normal con-trol group in which the rats received normal saline solutionorally; group II, the diabetic control group in which the ratswere injected intraperitoneally with a single dose ofstreptozotocin (STZ) for induction of diabetes; group III, themetformin (Met)-treated group in which the diabetic rats weretreated orally with Met; and group IV, the L-Se-treated groupin which the diabetic rats were treated orally with L-Se. Alltreatments were delivered for 21 days. Blood and pancreastissue samples were obtained for biochemical analysis, immu-nohistochemical examinations, and histopathological investi-gation. The L-Se-treated group showed significant drop inserum glucose and pancreatic malondialdehyde (MDA), nitricoxide (NO), tumor necrosis factor-α (TNF-α), and prostaglan-din F2α (PGF2α) levels associated with significant rise inserum insulin and pancreatic glutathione, superoxide dismut-ase (SOD), catalase (CAT), glutathione peroxidase (GPx), andglutathione reductase (GR) values, in addition to significantimprovement in the immunohistochemical indices (insulinand glucagon). Aforementioned results are appreciated bythe histopathological findings of pancreatic tissue. In conclu-sion, our data have brought about compelling evidence

favoring the antidiabetic potency of elemental selenium nano-particles delivered in liposomes through preservation of pan-creatic β cell integrity with consequent increment of insulinsecretion and in turn glucose depletion, repression of oxida-tive stress, potentiation of the antioxidant defense system, andinhibition of pancreatic inflammation.

Keywords Elemental selenium nanoparticles . Liposome .

Diabetes . Oxidative stress . Inflammation . Rats

AbbreviationsAGE Advanced glycosylation end productAMPK AMP-activated protein kinaseANOVA One-way analysis of varianceATF-3 Activating transcription factor-3CAT CatalaseCOX-2 Cyclooxygenase-2CREB cAMP responsive element binding proteinDM Diabetes mellitusDPPC L-α-dipalmitoyl phosphatidyl cholineDTNB 5,5′ dithiobis (2-nitrobenzoic acid)ELISA Enzyme-linked immunosorbent assayGPx Glutathione peroxidaseGR Glutathione reductaseGSSG Oxidized glutathioneIFN-γ Interferon-gammaIL-1β Interleukin-1 betaIL-6 Interleukin-6IL-10 Interleukin-10iNOS Inducible nitric oxide synthaseIK-Bα Nuclear factor of kappa light polypeptide gene

enhancer in B cell inhibitor alphaIpf1 Insulin promoter factor 1JNK1/2 c-Jun N-terminal kinase 1/2

* Hanaa H. Ahmedhanaaomr@yahoo.com

1 Hormones Department, National Research Centre, 33 EL Bohouth st.(former EL Tahrir st.) Dokki, P.O. 12622, Giza, Egypt

2 Biochemistry Department, National Research Centre, Giza, Egypt3 Zoology and Entomology Department, Faculty of Science, Helwan

University, Cairo, Egypt

Biol Trace Elem Res (2017) 177:267–280DOI 10.1007/s12011-016-0876-z

LPS LipopolysaccharidesL-Se Selenium nanoparticles delivered in liposomesMAPK Mitogen-activated protein kinaseMDA MalondialdehydeMet MetforminNa2SeO3 Sodium seleniteNBT Nitroblue tetrazoliumNF-κB Nuclear factor kappa BNO Nitric oxidePAP Peroxidase anti-peroxidasePGE2 Prostaglandin E2PGF2α Prostaglandin F2αROS Reactive oxygen speciesSe SeleniumSOD Superoxide dismutaseSPSS Statistical package for the social sciences programSTZ StreptozotocinT2DM Type 2 diabetes mellitusTEM Transmission electron microscopeTNF-α Tumor necrosis factor-α

Introduction

Diabetes mellitus (DM) is a highly prevalent disease that pos-sesses a major public health problem globally. It affects ap-proximately 382 million people worldwide in the year 2013,and this number is expected to be 592 million in the year 2035[1]. The complications of DM including cardiovascular dis-ease, nephropathy, neuropathy, and retinopathy represent ahigh burden on the quality of life [2]. Reporter assays indicat-ed that free radicals are implicated in the pathogenesis of dia-betes, and a positive correlation between oxidative stress andsecondary complications of diabetes has been evidenced [3].

Supporting evidences have suggested that the excessiveoxidative stress represents a risk factor for insulin resistance,β cell dysfunction, and impaired glucose tolerance in type 2diabetes mellitus (T2DM) [4]. Increased free radical levelscould hamper glucose-stimulated insulin secretion, downreg-ulate the expression of β cell key genes, and induce cell death[5]. Oxidative stress in diabetic patients is also correlated withthe inhibition of the antioxidant protective system [6].Therefore, it has been proposed that diabetic patients mayrequire more antioxidants compared to healthy individuals [7].

Selenium (Se) is an essential element for humans and canimprove the activity of the seleno-enzyme and glutathione per-oxidase (GPx), and can also prevent free radical insult on cellsand tissues in vivo. One of its best understood functions is thatit is present in the active center of GPx, an antioxidant enzymethat scavenges various peroxides and protects membrane lipidsand macromolecules from oxidative damage [8]. The supple-mentation of food with selenium is limited to selenium-

containing compounds, such as sodium selenite (Na2SeO3),ebselen, and other organoselenium compounds [9].

Selenium nanoparticles have gotten incredible consider-ation due to their unique biological activities and low tox-icity [10]. They display high biological activity and greatabsorptive capacity because of the interaction between –NH2, C = O, −COO, and –C–N– groups of proteins and thenanoparticles of Se [11]. Moreover, it has been detectedthat selenium nanoparticles possess antioxidant and antidi-abetic activities [12, 13].

Liposomes (small lipid spheres containing an internalaqueous phase) have long been studied as a mean of selective-ly delivering peptides and other therapeutic agents to specifictissues. Liposome technology has been exploited for manydiverse applications, ranging from delivery of vaccines, anti-microbials, cancer and enzyme therapies to use in diagnostics,cosmetics, and artificial blood [14]. The promise of liposomesas drug carriers lies in their capacity to shield their encapsu-lated contents from non-target tissues by virtue of the sur-rounding lipid membrane.

In this work, we delivered selenium nanoparticles in lipo-somes in order to obtain optimized materials for medication.The focus of our interest was to evaluate the antidiabetic po-tential of elemental selenium nanoparticles delivered in lipo-somes in the experimental model.

Materials and Methods

Selenium Nanoparticles Preparationand Characterization

Nanoseleniumwas prepared by a simple wet chemical methodaccording to Dwivedi et al. [15]. Briefly, sodiumselenosulphate precursor reacted with different organic car-boxylic acids in aqueous medium, under ambient conditions.Polyvinyl alcohol was used to stabilize selenium nanoparti-cles. Then, the synthesized nanoparticles were separated fromtheir sol by using a high-speed centrifuge (15,000 rpm) andredispersed in aqueous medium with a sonicator.

JEOL JEM-2100 high-resolution transmission electron mi-croscope (TEM) at an accelerating voltage of 200 kV wasused to image the size and shape of nanoparticles. TEM sam-ples were prepared by suspending the nanoparticles in phos-phate buffer and applying one drop of this suspension oncopper grids coated by a thin film of carbon.

Liposome Preparation

Selenium nanoparticles were used to prepare neutralmultilamellar vesicles in molar ratio of 7:2 (Se/liposome)using the method of Kim et al. [16]. Briefly, 10 mg of highpurity L-α-dipalmitoyl phosphatidyl choline (DPPC; Lipoid

268 Ahmed et al.

KG-Germany) and 1 mg of the selenium were transferred to a50-ml round bottom flask. Then, 15 ml of chloroform wasadded, and the flask was shaken until all lipids dissolved inthe chloroform. The solvent was evaporated under vacuumusing rotary evaporator until a thin dry film of lipid wasformed. The flask was then left under vacuum for 12 h toensure the evaporation of all traces of chloroform. Ten milli-liters of buffer (10 mM Trizma, pH 7) was then added to theflask which was flashed through with nitrogen stream andimmediately stopped. The flask was mechanically shaken for1 h at a temperature of 45 °C. The suspension was then cen-trifuged at 8000 rpm for 20 min and the supernatant wasdiscarded. The liposome was then re-suspended in 10 mlTrizma. Control liposome was prepared following the sameclassical method as before using only aliquots of 10 mg ofDPPC. The concentration of free selenium/milliter of bufferwas adjusted to be 1 mg/ml.

Experimental Animals

For the present study, adult female albino rats of Wistar strainweighing 180–200 g were obtained from a breeding stockmaintained in the Animal House of the National ResearchCentre, Giza, Egypt. After an adaptation period of 1 week,the animals were classified into four groups of equal averagebody weight and housed in wire-bottomed cages in a roomunder standard condition of illumination with 12 h light-darkcycle at 25 ± 1 °C. They were provided with water and rodentchow diet ad libitum. All animals received care in compliancewith the Egyptian rules for animal experiments which wereapproved by the Ethical Committee of Medical Research ofthe National Research Centre, Giza, Egypt.

Induction of Diabetes

Rats were fasted for 18–24 h before induction of diabetes byusing streptozotocin (STZ). The rats received a single intra-peritoneal (i.p.) injection of 45 mg/kg b. wt. of STZ (Sigma,St. Louis, MO, USA) which is freshly prepared and dissolvedin 0.05 M citrate buffer, pH 4.5 as described previously [17].Blood glucose levels were monitored every 2 days using anAccu-check blood glucose meter (Roche Diagnostics, Basel,Switzerland). Rats with blood glucose levels ≥15 mM(200 mg/dL) for 7 consecutive days were considered diabetic.

Experimental Set-Up

One week after the verification of diabetes, 28 rats wereassigned as (I) the normal control group in which the ratsreceived normal saline solution; (II) the untreated diabeticgroup (diabetic control group); (III) the metformin (Met)-treated group in which the diabetic rats were treated with astandard oral hypoglycemic agent, Met (100 mg/kg b. wt.)

[18]; and (IV) the selenium nanoparticles delivered in lipo-somes (L-Se)-treated group in which the rats were treated withnanoselenium stabilized in liposome (0.1 mg/kg b. wt.) orally[19].

After 21 days of daily treatment, overnight fasting animalswere euthanized under mild ether anesthesia. The blood sam-ples were immediately withdrawn from the retro-orbital ve-nous plexus in clean tubes and then centrifuged at 1800×g at4 °C for 15min to separate sera. Then, the pancreas of each ratwas promptly excised, washed in chilled saline, blotted,weighed, and processed for biochemical, immunohistochem-ical, and histological studies.

Determination of Glucose and Insulin Levels

Serum glucose level was determined by the glucose oxidasemethod [20] using kit purchased from Stanbio (TX, USA).Serum insulin concentrations were assayed by an enzyme-linked immunosorbent assay (ELISA) according to the meth-od of Temple et al. [21] using kit purchased from DRG inter-national, Inc. (NJ, USA).

Determination of Oxidative Stress Parametersand Glutathione Content

Homogenate of pancreas was prepared in 50 mM Tris-HCland 300 mM sucrose, pH 7.4 using homogenizer to give10 % homogenate. This homogenate was used for determina-tion of malondialdehyde by reaction with thiobarbituric acid[22], nitric oxide by optimized acid reduction method [23],and glutathione contents by reduction of Elman’s reagent(5,5′ dithiobis (2-nitrobenzoic acid) BDTNB^) [24] using kitspurchased from Biodiagnostic (Egypt).

Determination of Enzymatic Antioxidants

Homogenate of the pancreas was also used for assessment ofsuperoxide dismutase (U/g tissue) by inhibition of phenazinemethosulphate-mediated reduction of nitroblue tetrazolium(NBT) dye [25], catalase (U/g tissue) by reaction with knownquantity of H2O2 [26], glutathione peroxidase (U/g tissue) byrecycling of oxidized glutathione (GSSG) to its reduced state[27], and glutathione reductase (μmol/g tissue) by catalyzingthe reduction of glutathione in the presence of NADPH [28]using kits purchased from Biodiagnostic (Egypt).

Determination of Cytokines

Prostaglandin E2 (PGE2), prostaglandin F2α (PGF2α), andtumor necrosis factor-alpha (TNF-α) were quantified in pan-creatic homogenate using ELISA kits obtained from AbcamCompany (Cambridge, UK) according to the manufacturer’sinstructions.

Antidiabetic potency of nanoselenium delivered in liposomes 269

Immunohistochemical Procedures

The harvested pancreatic tissues fixed in 10 % neutral-buffered formalin, were embedded in paraffin, and sectionedat 5 μm thickness. Immunohistochemical reactions were per-formed using the peroxidase anti-peroxidase (PAP) method[29]. Blocking of nonspecific peroxidase reactions was per-formed with methanol containing 0.1 % H2O2, and to avoidnonspecific reactions with the background, the sections wereincubated with normal goat serum prior to incubation with thespecific antibodies against insulin (dilution, 1:2000, SantaCruz, CA, USA) and glucagon (dilution, 1:2000, SantaCruz, CA, USA). After rinsing in phosphate-buffered saline(PBS; 0.01 M, pH 7.4), sections were incubated with second-ary antibodies (goat anti-rabbit IgG, dilution, 1:200; Sigma,USA). Sections were then washed in PBS and finally incubat-ed with PAP complex (dilution, 1:200). The peroxidase reac-tion was carried out using a solution 3,3′-diaminobenzidinetetrahydrochloride containing 0.01%H2O2 in Tris-HCl buffer(0.05 M, pH 7.6). The specificity of each immunohistochem-ical reaction was determined as recommended by Sternberger[29], including the replacement of specific antiserum by thesame antiserum, which had been pre-incubated with its corre-sponding antigen—insulin or glucagon. After immunostain-ing, the sections were lightly counterstained with hematoxylinand examined under light microscope.

Ten islets of Langerhans from each rat, thus 70 islets foreach group, were chosen randomly. The intensity of stainingwith anti-insulin or anti-glucagon antibodies were scoredsemi-quantitatively as 0 (absent), 1 (weak), 2 (moderate), 3(strong), and 4 (very strong).

Histological Study

The pancreatic tissues harvested from the sacrificed animalswere cut into fragments, fixed in 10 % neutral buffered for-malin, washed in tap water, and dehydrated by using ascend-ing grade of ethyl alcohol (30, 50, 70, 90 %, and absolute).Specimens were cleared in xylene and embedded in paraffin at56 °C in hot air oven for 24 h. Then, paraffin wax tissue blocks

were prepared for sectioning at 4 μ thick, collected on glassslides, deparffinized, and stained by hematoxylin and eosinstain for histopathological investigation through the electriclight microscope [30].

Statistical Analysis

Results were expressed as the mean ± standard error of themean (SEM). Data for multiple variable comparisons wereanalyzed by one-way analysis of variance (ANOVA). Forthe comparison of significance between groups, Duncan’s testwas used as post hoc test according to the Statistical Packagefor the Social Sciences program (SPSS version 17.0).

Percentage difference, representing the percentage of thevariation with respect to the corresponding control group,was also calculated using the following formula:

%Change ¼ Treated value–Control value

Control value� 100

Results

Selenium Nanoparticles Characterization

High-resolution transmission electron microscopy showedthat the prepared nanoparticles are spherical in shape withdiameter ranged between 30 and 90 nm (Fig. 1).

Biochemical Results

As shown in Table 1, blood glucose level was significantly (P< 0.05) higher in the STZ-challenged group (diabetic group)(214.3 %) than in the normal control group. Conversely, se-rum insulin level was significantly (P < 0.05) lower in thediabetic group (−45.7 %) than in the normal control group.Meanwhile, treatment with Met or L-Se elicited significantdrop (P < 0.05) in blood glucose (−57.9 and −65.1 % respec-tively) level relative to the diabetic control group. Notably, the

Fig. 1 High-resolutiontransmission electron microscopyimages of selenium nanoparticles

270 Ahmed et al.

blood glucose level remained significantly higher (P < 0.05)in the Met-treated group (32.3 %) than in normal controlgroup, whereas it showed insignificant change (P < 0.05) inthe L-Se-treated group (9.7 %) versus the normal controlgroup. While, serum insulin level revealed significant eleva-tion (P < 0.05) in the Met (54.3 %) as well as the L-Se-treated(64.3 %) groups in comparison with the diabetic controlgroup. Interestingly, serum insulin level displayed significantdecrease (P < 0.05) in the Met-treated group (−16.3 %) ascompared to the normal control group while it exhibited in-significant change (P > 0.05) in the L-Se-treated group(−10.9 %) relative to the normal control group (Table 1).Noteworthy, the L-Se-treated group showed significant drop(P < 0.05) in serum glucose level (−17.1 %) when comparedwith Met-treated one.

The results of pro-oxidant indicators including the pancre-atic contents of malondialdehyde (MDA), nitric oxide (NO),and the non-enzymatic antioxidant molecule, glutathione, ofdiabetic rats subjected to treatment with Met or L-Se aredepicted in Table 2. Significant rise (P < 0.05) in pancreaticMDA (24.7 %) and NO (69.1 %) in concomitant with signif-icant decline (P < 0.05) in pancreatic glutathione (−25.4 %)contents were recorded in the STZ-challenged group (diabeticgroup) relative to the normal control group. Treatment of di-abetic rats with Met or L-Se produced significant depletion (P< 0.05) in pancreatic MDA (−23.6 and −37.4 %, respectively)

and NO (−15.3 and −27.5 %, respectively) associated withsignificant elevation (P < 0.05) in pancreatic glutathione(14.2 and 41.5 %, respectively) contents with respect to thediabetic control rats. Worth noting, the pancreatic MDA con-tent showed significant suppression (P < 0.05) due to treat-ment with Met or L-Se (−4.7 and −21.9 %, respectively) indiabetic rats in comparison with the normal control ones.Whereas, the pancreatic NO content revealed significant ele-vation (P < 0.05) as a consequence of treatment withMet or L-Se (43.2 and 22.6 %, respectively) in diabetic rats in compar-ison with the normal control rats. While, treatment of diabeticrats with Met or L-Se caused significant elevation (P < 0.05)in pancreatic glutathione (14.2 and 41.5 %, respectively) con-tent versus the diabetic control rats. Worth mentioning, Mettreatment in diabetic rats induced significant drop in pancre-atic glutathione (−14.8 %) content (P < 0.05) in comparisonwith the normal control rats while treatment with L-Se indiabetic rats evoked insignificant rise (P > 0.05) in pancreaticglutathione (5.6 %) content relative to the normal control rats(Table 2). Intriguingly, treatment with L-Se gave rise to sig-nificant decline (P < 0.05) in pancreatic MDA (−18.1 %) andNO (−14.4 %) along with significant elevation (P < 0.05) inpancreatic glutathione (23.9 %) contents when compared withdiabetic rats treated with Met.

The data clarified in Table 3 revealed significant in-hibition (P < 0.05) of pancreatic activity of antioxidant

Table 1 Effect of treatment with selenium nanoparticles delivered in the liposomes on serum glucose and insulin levels of diabetic rats

Parameters Cont STZ Met L-Se

Initial serum glucose (mg/dL) 112.64 ± 3.85 246.50 ± 6.77a 238.85 ± 9.86a 253.76 ± 4.94a

Final serum glucose (mg/dL) 109.08 ± 4.56 342.83 ± 4.93a 144.36 ± 8.72ab 119.63 ± 3.74bc

Initial serum insulin (μIU/mL) 12.4 ± 1.2 8.1 ± 0.7a 8.3 ± 0.8a 8.0 ± 0.6a

Final serum insulin (μIU/mL) 12.9 ± 0.7 7.0 ± 0.6a 10.8 ± 0.9ab 11.5 ± 1.1b

Values are means ± SEM (n = 7)aP < 0.05, significant change with respect to the normal control groupbP < 0.05, significant change with respect to the STZ group (diabetic control group)cP < 0.05, significant change with respect to the Met-treated group

Table 2 Effect of treatment with selenium nanoparticles delivered in the liposomes on oxidant/antioxidant parameters of the pancreas of diabetic rats

Parameters Cont STZ Met L-Se

Malondialdehyde (nmol/g tissue) 98.96 ± 1.51 123.40 ± 1.90a 94.27 ± 3.64ab 77.24 ± 1.25abc

Nitric oxide (μmol/g tissue) 222.58 ± 9.16 376.34 ± 12.17a 318.74 ± 8.43ab 272.85 ± 4.13abc

Glutathione (mmol/g tissue) 0.481 ± 0.002 0.359 ± 0.01a 0.41 ± 0.02ab 0.508 ± 0.02bc

Values are means ± SEM (n = 7)aP < 0.05, significant change with respect to the normal control groupbP < 0.05, significant change with respect to the STZ group (diabetic control group)cP < 0.05, significant change with respect to the Met-treated group

Antidiabetic potency of nanoselenium delivered in liposomes 271

enzymes namely superoxide dismutase (−3.9 %), cata-lase (−47 %), glutathione peroxidase (−37.5 %), andglutathione reductase (−12.9 %) in the diabetic grouprelative to the normal control group. Treatment of dia-betic rats with Met or L-Se resulted in significant ele-vation (P < 0.05) in pancreatic activity of superoxidedismutase (SOD) (20.7 and 52.3 %, respectively), GPx(24.6 and 66.2 %, respectively), and glutathione reduc-tase (GR) (5.4 and 28.1 %, respectively) as compared tothe diabetic control rats. With regard to pancreatic cat-alase (CAT) activity, insignificant increase (P > 0.05)was recorded in the diabetic group treated with Met(36.1 %) relative to the diabetic control group.Whereas, significant increase (P < 0.05) in pancreaticCAT activity was detected in the diabetic group treatedwith L-Se (181.4 %) versus the diabetic control group.Treatment of diabetic groups with Met or L-Se causedsignificant increase (P < 0.05) in pancreatic SOD (16and 46.3 %, respectively) activity relative to the normalcontrol group. However, pancreatic CAT (−27.9 %),GPx (−22.2 %), and GR (−8.3 %) activity showed sig-nificant regression (P < 0.05) in the diabetic grouptreated with Met as compared to the normal controlgroup. Interestingly, the treatment of the diabetic groupwith L-Se led to significant upregulation (P < 0.05) inpancreatic CAT (49.2 %) and GR (11.5 %) activity rel-ative to the normal control group. While, pancreaticGPx (3.8 %) activity displayed insignificant increase(P > 0.05) upon treatment of the diabetic group withL-Se when compared with the normal control group(Table 3). Notably, treatment of diabetic rats with L-Seresulted in significant increase (P < 0.05) in pancreaticactivity of SOD (26.2 %), CAT (106.8 %), GPx(33.3 %), and GR (21.6 %) when compared with thosetreated with Met.

The results represented in Fig. 2 indicated that the pancre-atic contents of pro-inflammatory modulators, PGE2 (15 %),

PGF2α (70 %), and TNF-α (96 %), were significantly in-creased (P < 0.05) in the diabetic group versus the normalcontrol group. On the other side, significant decrease (P <0.05) in pancreatic PGF2α and TNF-α was observed in dia-betic rats treated with Met (−14.7 and −15.3 %, respectively)or L-Se (−25 and −33.7 %, respectively) relative to the dia-betic control group. Of note, insignificant change (P > 0.05)was detected in pancreatic PGE2 of diabetic groups treatedwith Met (6.5 %) or L-Se (−2.2 %) in comparison with thediabetic control group. Noteworthy, significant elevation (P <0.05) in pancreatic PGF2α and TNF-α was noted in diabeticgroups treated with Met (45 and 66 %, respectively) or L-Se(27.5 and 30 %, respectively) with respect to the normal con-trol group. As well, significant elevation (P < 0.05) in pancre-atic PGE2 of the diabetic group treated withMet (22.5 %) wasrecorded relative to the normal control group. Whereas, insig-nificant change (P > 0.05) was demonstrated in pancreaticPGE2 of the diabetic group treated with L-Se (12.5 %) versusthe normal control group (Fig. 2). Interestingly, the L-Se-treated group showed significant drop (P < 0.05) in pancreaticPGF2α (−12.1%) and TNF-α (−21.7%) levels in comparisonwith the Met-treated one.

Immunohistochemical Findings

The immunohistochemical staining of pancreatic tissue sec-tions of rats in the normal control group showed strong insulinantigen positivity in the β cells of the islets of Langerhans(Fig. 3a). The immunohistochemical staining of pancreatictissue sections of rats in the diabetic group revealed weakinsulin immunoreactivity in a few β cells in the islets ofLangerhans (Fig. 3b). The immunohistochemical staining ofpancreatic tissue sections of rats in the Met-treated groupshowed moderate insulin antigen positivity in some β cellso f t h e i s l e t s o f L a n g e r h a n s ( F i g . 3 c ) . T h eimmunohistochemical staining of pancreatic tissue sectionsof rats in the L-Se-treated group showed strong insulin antigen

Table 3 Effect of treatment with selenium nanoparticles delivered in the liposomes on pancreatic antioxidant enzyme activity of diabetic rats

Parameters Cont STZ Met L-Se

Superoxide dismutase (U/g tissue) 1924.30 ± 37.92 1848.76 ± 21.58a 2231.25 ± 13.86ab 2814.87 ± 7.71abc

Catalase (U/g tissue) 1.83 ± 0.03 0.97 ± 0.02a 1.32 ± 0.11a 2.73 ± 0.05abc

Glutathione peroxidase (U/g tissue) 3422.54 ± 203.58 2137.55 ± 125.56a 2663.60 ± 109.59ab 3551.57 ± 247.20bc

Glutathione reductase (μmol/g tissue) 2462.09 ± 41.60 2143.47 ± 59.91a 2258.52 ± 58.92ab 2746.32 ± 61.32abc

Values are means ± SEM (n = 7)aP < 0.05, significant change with respect to the normal control groupbP < 0.05, significant change with respect to the STZ group (diabetic control group)cP < 0.05, significant change with respect to the Met-treated group

272 Ahmed et al.

positivity in the majority of β cells of the islets of Langerhans(Fig. 3d).

The immunohistochemical staining of pancreatic tis-sue sections of rats in the normal control group revealedmedium glucagon-positive cells of the islets ofLangerhans (Fig. 4a). The immunohistochemical stainingof pancreatic tissue sections of rats in the diabetic group

showed strong glucagon immunoreactivity of pancreaticcells of the islets of Langerhans (Fig. 4b). The immu-nohistochemical staining of pancreatic tissue sections ofrats in the group treated with Met showed mild gluca-gon antigen positivity in the majority of pancreatic cellso f t h e i s l e t s o f L a n g e r h a n s ( F i g . 4 c ) .Immunohistochemical staining of pancreatic tissue

Fig. 2 Effect of treatment withselenium nanoparticles deliveredin the liposomes on pancreaticpro-inflammatory cytokines ofdiabetic rats. Values are means ±SEM (n = 7). aP < 0.05, signifi-cant change with respect to thenormal control group; bP < 0.05,significant change with respect tothe STZ group (diabetic controlgroup); cP < 0.05, significantchange with respect to the Met-treated group.

Fig. 3 Immunohistochemicalexamination of insulin expressionin pancreatic tissues of the studiedgroups. a Normal control. bDiabetic control. c Met-treated. dL-Se-treated

Antidiabetic potency of nanoselenium delivered in liposomes 273

sections of rats in the L-Se-treated group revealed mod-erate glucagon antigen positivity in some of the pancre-atic cells of the islets of Langerhans (Fig. 4d).

Table 4 illustrated the percentage of the area of insulin andglucagon immunoreactive β cells. STZ induced remarkabledecrease in the area of insulin immunoreactive β cells in con-comitant with observable increase in the area of glucagonimmunoreactive cells. Treatment of diabetic rats with Metelicited appreciable increase in the area of insulin immunore-active β cells paralleled by detectable decrease in the immu-noreactivity of glucagon cells. Treatment of diabetic rats withL-Se evoked a marked increase in the area of insulin immu-noreactiveβ cells associated with sharp decrease in the area ofglucagon immunoreactive cells (Table 4).

Histological Changes

The microscopic examination of pancreatic tissue sections ofrats in the normal control group showed normal histologicalfeature of pancreatic islet cells (Fig. 5a). Whereas, microscop-ic investigation of pancreatic tissue sections of rats in thediabetic group revealed degenerative and necrotic changes,and the islets of Langerhans appeared to be shrunk. The de-generation was mostly hydropic and the degranulation in thecytoplasm of the degenerative and necrotic cells was noticedin addition to the presence of lymphocyte infiltration (Fig. 5b).Microscopic examination of pancreatic tissue sections of ratsin the group treated withMet showed slight improvement inβcells of the islets of Langerhans with the presence of hydropic

Table 4 Semi-quantitativeanalysis of the area ofimmunohistochemical staining ofinsulin and glucagon ofpancreatic cells in the islets ofLangerhans of the differentstudied groups

Groups Number 0(absent)

1(weak)

2(moderate)

3(strong)

4 (verystrong)

% of the area ofimmunoreactive cells in theislets of Langerhans

Insulin

Cont 70 – – – 87 13 44.7

STZ 70 74 26 – – – 3.7

Met 70 – 13 73 14 – 30.1

L-Se 70 – – – 31 69 52.7

Glucagon

Cont 70 – 43 45 12 – 24.1

STZ 70 27 10 8 27 28 31.3

Met 70 – 49 44 7 – 22.6

L-Se 70 – 47 37 16 – 24.1

Fig. 4 Immunohistochemicalexamination of glucagonexpression in pancreatic tissues ofthe studied groups. a Normalcontrol. b diabetic control. cMet-treated. d L-Se-treated

274 Ahmed et al.

degeneration, cytoplasmic degranulation, and necrotic cells(Fig. 5c). Microscopic investigation of pancreatic tissue sec-tions of rats in the group treated with L-Se revealed repairmentin the structural organization of the majority of β cells of theislets of Langerhans. Nevertheless, there were some cells withlight hydropic degeneration, degranulation, and necrosis(Fig. 5d).

Discussion

According to the results, treatment of diabetic rats with Met orL-Se caused a significant drop in blood glucose paralleled bysignificant increase in serum insulin levels. Metformin is awell-known antidiabetic agent affecting blood glucose lev-el via three ways: suppression of basal hepatic glucoseproduction, limitation of glucose entry from the gastroin-testinal tract, and enhancement of insulin action in theperipheral tissues [31]. Also, Met was found to stimulatebeta cells to release insulin [32]. However, the studies ofFazilati et al. [33] and Al-Quraishy et al. [13] documentedthat treatment of diabetic rats with selenium nanoparticlescaused a significant drop in blood glucose level (−67.9 %)and significant rise in serum insulin level (5.6 %). Themechanism behind the anti-hyperglycemic activity of L-Se may involve insulin-like action by increasing peripheralglucose consumption [34]. Also, selenium could restorebeta cells activity and increase insulin release with conse-quent decline in blood glucose level [35]. Earlier studies

that have been performed on isolated rat adipocytes dem-onstrated that sodium selenate stimulates glucose uptakethrough translocation of glucose transporters to the plasmamembrane and activates serine/threonine kinases includingthe p70 S6 kinase [36]. In addition, the depletion in bloodglucose level of the diabetic group treated with L-Se mayalso be ascribed to the renewal of β cells following L-Seadministration. The renewal of β cells in diabetes hasbeen studied in several animal models, and it has beenreported that the regeneration of islet cells after the useof drug may be the primary cause of the recovery of STZ-induced diabetic animals [37].

The implication of oxidative stress in the pathogenesis ofdiabetes and diabetic complications have been extensivelystudied for years based on animal models of diabetes anddiabetic patients. Experimental animal models as well as dia-betic patients exhibited high oxidative stress as a result ofpersistent and chronic hyperglycemia, which thereby depletedthe activity of the anti-oxidative protective system and thuspromoted de novo free radical generation [38]. Circumstantialevidences have indicated an increase in lipid peroxides andreactive oxygen species (ROS) as markers of oxidative stressin different animal models of diabetes [39]. Oxidative stresshas been shown to be responsible for the β cell dysfunctioncaused by glucose toxicity. Under hyperglycemia, the produc-tion of various reducing sugars such as glucose-6-phosphateand fructose is increased through glycolysis and the polyolpathway. During this process, ROS are produced, causing tis-sue damage [40]. In vitro and in vivo studies have proposed

Fig. 5 Photomicrograph of the pancreatic tissue section of a the normalcontrol group showing normal histological feature of pancreatic isletcells; b the diabetic control group showing degenerative and necroticchanges, shrunken in the islets of Langerhans and lymphocytesinfiltration; c the Met-treated group showing slight improvement in the

cells of the islets of Langerhans with the presence of hydropic degenera-tion, cytoplasmic degranulation, and necrotic cells; and d the L-Se-treatedgroup showing improvement in the structural organization of the majorityof cells of the islets of Langerhans (H&E ×80)

Antidiabetic potency of nanoselenium delivered in liposomes 275

the involvement of oxidative stress in the progression ofβ celldysfunction in type 2 diabetes [41].

Streptozotocin causes type-2 diabetes via the generation ofROS, causing oxidative damage of pancreatic β cells. In viewof our data, Met and more likely L-Se could ameliorate oxi-dative stress induced by STZ in diabetic rats as indicated bythe significant reduction in pancreatic MDA and NO contents.Metformin had the ability to reduce the damaging impactcaused by free radicals including lipid peroxidation product(MDA) formation [42] and NO radical generation [43].Selenium with its well-known antioxidant activity had a pro-tective effect on pancreatic β cells via scavenging the reactivefree radicals. It could prevent pancreatic lipid peroxidationwith the recovery of β cells function [44]. Zeng et al. [45]proved the ability of selenium to decrease mRNA expressionand activity of inducible nitric oxide synthase (iNOS) andcontent of pancreatic NO in diabetic mice.

It has been evidenced that overexpression of non-enzymatic and enzymatic antioxidants could efficiently pre-vent the damaging impact of ROS [46]. Met and L-Se admin-istration in diabetic rats induced significant increase in pan-creatic glutathione content as shown in the present results.Metformin has been found to enhance total thiol in type 2diabetic patients [47]. On the experimental level, Met couldsignificantly increase blood and liver reduced glutathione indiabetic rats [48]. Concerning the effect of L-Se on pancreaticglutathione content, it has been reported that selenium is anessential component for the generation of glutathione and sul-fur amino acids such as methionine and cysteine [35] as theproduction of glutathione depends on the availability of sev-eral amino acids, along with available iron and selenium.Wang et al. [49] stated that dietary selenium supplementationin broilers leads to increased concentration of glutathione invarious organs.

Both selenium supplementation and metformin showedbeneficial effect against the development of diabetes byexhibiting antioxidant properties in the experimental modelof type 2 diabetes in the present work. This finding was doc-umented by the increased pancreatic antioxidant enzymes ac-tivity (SOD, CAT, GPx, and GR) in diabetic rats treated witheither Met or L-Se. Metformin with its antioxidant propertiescould reduce ROS by suppressing mitochondrial respiration[48] and repressing advanced glycosylation end product(AGE) indirectly through reduction of hyperglycemia and di-rectly through an insulin-dependent mechanism [50]. In ac-cordance of our results, Majithiya and Balaraman [42] men-tioned that Met treatment significantly increases antioxidantenzymes and reduces lipid peroxidation in STZ-diabetic rats.In general, one probable reason for the observed increase inthese antioxidant enzymes with Met was the improvement inthe glycemic level [51]. This is because of hyperglycemia isassociated with a chain reaction-breaking antioxidant [52],and hyperglycemia has been found to inhibit the ability of

lipids to resist oxidation [53]. In consequence, the hypothesisof Tessier et al. [51] proposed that by decreasing the glycemiclevel, the level of oxidative stress is diminished and a relativesparing of the antioxidants is ensued. Thus, Met could im-prove the antioxidant/oxidant status of the diabetic subjects.

Regarding the enhancing effect of L-Se on the pancreaticantioxidant enzymes activity, our findings echo those of aprevious study [49] which have demonstrated that dietary se-lenium supplementation elevates GPx and SOD activity in thepancreas of broilers. Selenium is an important component ofthe selenoprotein enzyme GPx in animal tissues [54].Moreover, selenium has been found to retrieve the antioxidantproperty of broiler tissue by potentiating the activities of theantioxidant enzymes and concentrations of non-enzymatic an-tioxidant to protect against deteriorative reactions during lipidperoxidation [49]. Recently, it has been found that dietarynanoselenium supplementation increases the activity of GPx,SOD, and CAT in erythrocytes of layered chicks. These find-ings indicated that nanoselenium has higher selenium reten-tion in the liver, pancreas, and breast muscle [55] than dietaryselenium. This may be explained by the different absorptionprocess and metabolic pathways as nanoparticles exhibit newcharacteristics of transport and uptake and display high ab-sorption efficiency [56]. Therefore, it has been suggested thatthe superior performance of nanoparticles may be allied totheir small particle size and large surface area, increased mu-cosal permeability and improved intestinal absorption as wellas tissue deposition [55].

The association of inflammation and diabetes is a topic ofactive research. The results of Pradhan and his colleaguesproved the role of inflammation in the development of diabe-tes [57]. As well, Esposito et al. [58] reported that hypergly-cemia acutely increases circulating interleukin-6 (IL-6) andTNF-α. The increased cytokines in diabetes appears to beoriginated from non-circulating cells [59] and the likely can-didates are adipocytes and endothelial cells [60].

In the current set-up, treatment of diabetic rats with Met orL-Se produced significant reduction in the pancreatic contentof PF2α and TNF-α. Metformin has been detected to decreasemarkers of inflammation and contribute to the reduction ofoxidative stress in diabetic patients [61]. Apart from its con-ventional hypoglycemic effects, Met could act as an inhibitorof pro-inflammatory responses through direct inhibition ofnuclear factor kappa B (NF-κB) by blocking the PI3K-Aktpathway [62]. Metformin dose-dependently diminished theproduction of NO and PGE2 and suppressed the mRNA andprotein levels of iNOS and cyclooxygenase-2 (COX-2) inlipopolysaccharides (LPS)-activated macrophages [63].Recent study of Kim et al. [64] demonstrated that Met inhibitsLPS-induced production of TNF-α and IL-6 in parallel toinduction of activating transcription factor-3 (ATF-3), a tran-scription factor and member of the cAMP responsive elementbinding protein (CREB) family. Finally, Met suppressed

276 Ahmed et al.

mitogen-activated protein kinase (MAPK) phosphorylationand NF-κB production in association with AMP-activatedprotein kinase (AMPK) phosphorylation in LPS-stimulatedmurine macrophages. Anyhow, that study suggested thatMet exhibits anti-inflammatory action, at least in part, viapathways involving AMPK activation and ATF-3 induction.

Zeng et al. [45] mentioned that the administration of apharmacological dose of selenium improves blood glucoselevel of the STZ-induced diabetic mice. Additionally, it couldsuppress pancreatic mRNA expression of pro-inflammatorycytokines interleukin-1 beta (IL-1β), TNF-α, and interferon-gamma (IFN-γ); inhibit the pancreatic mRNA expression andactivity of iNOS; and reduce the pancreatic level of NO. Theobservations of Zeng and his co-workers indicated that thehypoglycemic influence of selenium may be related with theprevention of inflammation from overshooting via suppress-ing the production of pro-inflammatory cytokines and ROS/RNS induced by STZ in the pancreas of diabetic mice. Thetrace mineral selenium functions primarily as a component ofthe antioxidant enzyme glutathione peroxidase and this affectsall aspects of immunity. Glutathione peroxidase is especiallyimportant in reducing inflammatory cytokines. Seleniumnanoparticles have been found to exhibit anti-inflammatoryeffect as manifested by their inhibitory action on TNF-α inthe brain and kidney of acetaminophen-challenged rats [65].Recent report by Wang et al. [66] discussed the anti-inflammatory capacity of selenium nanoparticles against mu-rine Raw 264.7 macrophage cells induced by LPS. Seleniumnanoparticles were shown to significantly abrogate LPS-stimulated NO production against RAW 264.7 macrophages.Moreover, they could downregulate mRNA gene expressionfor pro-inflammatory cytokines (iNOS, IL-1, and TNF-α) in adose-dependent manner. The opposite was observed regardingthe anti-inflammatory cytokine (interleukin-10 (IL-10)). In theNF-κB signal pathway, selenium nanoparticles significantlyblocked the phosphorylation of nuclear factor of kappa lightpolypeptide gene enhancer in B cells inhibitor alpha (IK-Bα).Retraction of phosphorylation of c-Jun N-terminal kinase 1/2(JNK1/2) and P38mitogen-activated protein kinase (MAPKs)was also observed after in vitro treatment with selenium nano-particles. These findings indicated that selenium nanoparticleshave anti-inflammatory potency modulating pro/anti-inflammatory cytokine secretion profiles, and the underlyingmechanism is partially explained by blocking the activation ofNF-κB, JNK1/2, and P38 MAPKs [66].

Our immunohistochemical findings showed weakinsulin-positive cells in the islets of Langerhans of ratsin the diabetic group. However, there was a strong glu-cagon immunoreactivity of pancreatic cells of the isletsof Langerhans of diabetic rats. As well, a remarkabledecline in the area of insulin immunoreactive β cellsin concomitant with the marked increase in the area ofglucagon immunoreactive cells have been detected in

the islets of Langerhans in the diabetic group. Our re-sults are in parallel to those obtained previously by Itoet al. [67] who explained these observations by theability of STZ to induce a non-insulin-dependent diabet-ic mouse model which is characterized by aberrant in-sulin response to glucose stimulation.

According to our results, immunohistochemical examina-tion of pancreatic tissue sections of diabetic rats treated withMet showed moderate insulin antigen positivity in some βcells. Also, mild glucagon antigen positivity in the majorityof pancreatic cells of the islets of Langerhans has been notedin Met-treated rats. Appreciable increase in the area of insulinimmunoreactive β cells accompanied with detectable de-crease in the immunoreactivity of glucagon cells of islets ofthe pancreas have been recorded in diabetic rats treated withMet. These observations are in keeping with those of Hassanet al. [68] who interpreted these findings by the capability ofMet to prevent the destruction of β cells of the islets ofLangerhans or its partial effect in triggering the recovery ofthe destroyed β cells. Thus, Met showed a pivotal role inincreasing the number of insulin-positive cells in the pancreas.Yu et al. [69] cited that Met suppresses glucagon-inducedgluconeogenesis in diabetic hepatocytes in vitro, and thismay contribute in its glucose-lowering effect in diabeticconditions.

In our experimental setting, treatment of diabetic rats withL-Se resulted in the presence of strong insulin antigen posi-tivity in the majority of β cells of the islets of Langerhans asshown in the immunohistochemical investigation. Whereas,moderate glucagon antigen positivity in some of the pancre-atic cells of the islets of Langerhans of diabetic rats treatedwith L-Se was detected. Additionally, marked increase in thearea of insulin immunoreactive β cells accompanied withsharp decrease in the area of glucagon immunoreactive cellsof islets of the pancreas of diabetic rats treated with L-Se havebeen detected. Campbell et al. [70] reported that selenium isimplicated in the regulation of specific β cell target genes as itspecifically upregulates insulin promoter factor 1 (Ipf1) geneexpression and amplifies insulin content and secretion in iso-lated primary rat islets of Langerhans. While, Roden et al. [71]stated that selenium exerts a potent insulin-like effect on he-patic glycogenolysis in vitro by counteracting glucagonaction.

The microscopic investigation of pancreatic tissue sectionsof rats in the diabetic group revealed degenerative and necroticchanges, and the islets of Langerhans appeared to be shrunk.The degeneration was mostly hydropic, and the degranulationin the cytoplasm of the degenerative and necrotic cells wasnoticed in addition to the presence of lymphocytes infiltration.These findings are in harmony with those of Kanter et al. [72].Cuncio et al. [73] reported that high glucose level increasesthe production of markers of cell damage related to free rad-icals, such as MDA and conjugated dienes.

Antidiabetic potency of nanoselenium delivered in liposomes 277

On the opposite side, the microscopic examination of pan-creatic tissue sections of rats in the group treated with Metshowed slight improvement in the cells of the islets ofLangerhans. Hull et al. [74] mentioned that Met protects βcells through alleviating the progressive loss of β cell massand function in type 2 diabetes.

In the same direction, the microscopic investigation ofpancreatic tissue sections of rats in the group treated withL-Se showed improvement in the structural organizationof the majority of cells of the islets of Langerhans. Thisfinding could be explained by the ability of seleniumnanoparticles to scavenge free radicals. Tong et al. [44]reported that selenium can promote the recovery of β cellfunct ion through prevent ion of pancreat ic l ipidperoxidation.

In conclusion, the current findings justify the antidi-abetic potential of elemental selenium nanoparticles de-livered in liposomes in the experimental model of type2 DM. This preferable effect could be ascribed to theirability to preserve pancreatic β cell integrity, amplifyinsulin secretion with consequent reduction of glucoselevel, restore oxidant/antioxidant homeostasis, anddownregulate pancreatic inflammation. Thus, seleniumnanoparticles delivered in liposomes may be clinicallybeneficial to proceed for further clinical trials on DMtype 2.

Compliance with Ethical Standards All animals received care incompliance with the Egyptian rules for animal experiments which wereapproved by the Ethical Committee of Medical Research of the NationalResearch Centre, Giza, Egypt.

Conflict of Interest The authors declare that they have no conflict ofinterest.

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