Protective eect of curcumin on histopathologya rfor induction of

Manal Abdul-Hamid *,

Department of Zoology, Faculty of

R sed 23A 013

KEYWORDS

Diabetes mellitus;

Ultrastructure

study was undertaken to investigate the protective effect against alloxan-induced pancreatic damage

samples were collected for the detection of serum glucose levels. The pancreas was prepared for his-

The electron microscopic examination of diabetic pancreas showed marked changes in pancreatic

acini represented by dilated rough endoplasmic reticulum, decrease of secretory granules, cytoplas-

endoplasmic reticulum and depletion of secretory granules. Curcumin improved the histopathology

The World Health Organization (WHO) estimates that morethan 220 million people worldwide have diabetes, and thisnumber is liable to double by 2030 (Setacci et al., 2009;

WHO, 2009). Diabetes is a disease in which the hallmarkfeature is elevated blood glucose concentrations due to aloss of insulin-producing pancreatic b-cells (type 1 diabetes)or through loss of insulin responsiveness in its target tissueslike adipose and muscle (type 2 diabetes) (Schwarz et al.,2009). It affects 5% of the world population and becomes

* Corresponding author. Address: Department of Zoology, Faculty

of Science, Beni-Suef University, Beni-Suef 65211, Egypt.E-mail address: medo_bio@yahoo.com (M. Abdul-Hamid).

Peer review under responsibility of The Egyptian German Society for

Zoology.

Production and hosting by Elsevier

The Journal of Basic & Applied Zoology (2013) 66, 169179

The Egyptian German

The Journal of Basic

www.egwww.scienceand ultrastructure changes of the islets, alpha cells and exocrine acini, which nearly reverted to their

normal structure. It increased insulin immunoreactivity and decreased the elevated glucose concen-

trations. 2013 Production and hosting by Elsevier B.V. on behalf of The Egyptian German Society for Zoology.

Introductionmic vacuolation and irregular contours of nuclei. b-Cells showed fusion of some granules, obviousvacuolation and pyknotic nuclei. a Cells revealed focal necrosis and vacuolation, dilated roughtopathological, immunohistochemical stain using anti-insulin antibody and ultrastructural studies.Curcumin;

Histopathological changes;

Immunohistochemistry;

by curcumin. Rats were divided into four groups. (1) Control group. (2) Curcumin control group.

(3) Diabetic group with a single intraperitoneal injection of 100 mg alloxan/kg b.wt. (4) Diabetic

group treated with curcumin orally at a dose of 60 mg/kg b.wt dissolved in distilled water. Blood20

hteceived 5 March 2013; revivailable online 19 August 290-9896 2013 Productiontp://dx.doi.org/10.1016/j.jobaAand hosti

z.2013.07e of pancreas in the alloxan treated ratsdiabetes

Nadia Moustafa

Science, Beni-Suef University, Egypt

June 2013; accepted 4 July 2013

bstract Alloxan has been widely used to produce experimental diabetes mellitus syndrome. Thisnd ultrastructung by Elsevier B.V. on behalf of T

.003Society for Zoology

& Applied Zoology

sz.orgdirect.comhe Egyptian German Society for Zoology.

170 M. Abdul-Hamid, N. Moustafathe third human killer following cancer and cardiovasculardisease (Taylor, 1999). Alloxan has been widely used to pro-duce experimental diabetes mellitus syndrome. It causes

necrosis of pancreatic b-cells and induces free radicals whichplay a relevant role in the etiology and pathogenesis of bothexperimental and human diabetes mellitus (Soto et al.,

2004). Moreover, widespread lipoid deposits throughoutthe exocrine tissue, and loss of b cells (Soto et al., 2004).It has been suggested that alloxan induces the production

of H2O2 and of some free radicals such as O2 and OHwhich produce cellular damage followed by cell death.Therefore, alloxan was considered adequate for the studyof pathology of diabetes mellitus (Winterbourn and

Munday, 1989; Soto et al., 1994). Alloxan selectively andrapidly accumulates in b-cell (Gorus et al., 1982) and itaffects directly or indirectly on the membrane potential

and ion channels in b-cells (Carroll et al., 1994). The induc-tion of reactive oxygen species seems to be the mechanismby which alloxan damages the b-cells (Sentman et al.,1999). Alloxan potentially damages the pancreatic b-cells,because the antioxidant competencies are very low than thatof other tissues (Tiedge et al., 1997). Therefore, they are pre-

dominantly vulnerable to oxidative stress resulting in thesuppression of insulin gene transcription, glucose-stimulatedinsulin secretion and even producing apoptosis (Kanetoet al., 2001). Little is known about the effect of alloxan

on a cells. It has been assumed that alloxan toxicity is rela-tively specic for the b-cell. However, since the physico-chemical characteristics of the glucose recognition sites are

considered to be similar in a- and b-cells, it was felt logicalto investigate whether the a-cell might also be susceptible toalloxan poisoning (Pagliara et al., 1977). Aleeva et al. (2002)

reported that alloxan decreased the count of insulin-producing b-cells, but increased the number of glucagon-secreting a-cells in the pancreas (week 1 of diabetes).Moreover, immunohistochemical and morphometric investi-gation showed that antidiabetic effect of an organicderivative of vanadium (IV) produced a reliable increase inthe population of insulin-producing cells and a decrease in

the (alloxane-enhanced) population of a-cells in the pancre-atic islands (Aleeva et al., 2004).

Plants with anti-diabetic activities provide useful sources

for the development of drugs in the treatment of diabetes mel-litus. Phytochemicals isolated from plant source are used forthe prevention and treatment of cancer, heart disease, diabetes,

high blood pressure etc. (Mary et al., 2002).Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-hept-

adiene-3,5-dione), the active portion of turmeric, has beenshown to have signicant antioxidant activity, both in vitro

and in vivo (Joe et al., 2004), curcumin is a potent scavengerof reactive oxygen and nitrogen species such as hydroxyl rad-icals and nitrogen dioxide radicals (Reddy and Lokesh, 1994).

In addition curcumin has various other health-beneting prop-erties, such as anti-diabetic, antioxidants, anti-inammatory,anticarcinogenic, antiviral, hypolipidemic, and anti-infectious

effects (Araujo and Leon, 2001; Miller et al., 2001 andAggarwal et al., 2003). A broad body of evidence indicates thatcurcumin has a potential hypoglycemic effect in experimental

diabetic animal models (Pari and Murugan, 2005). Moreover,the use of curcumin is recommended for the prevention ofadvanced glycated endproduct (AGE) accumulation and theassociated complications of diabetes (Sajithlal et al., 1998).This study aims to investigate the effect of alloxan on ultra-structures of a cells since little studies were found. Further-more, it displays the hypoglycemic effects of curcumin in the

treatment of diabetes.

Materials and methods

Chemicals

All chemicals and reagents used in the present study were ob-tained from SigmaAldrich Inc. (St. Louis, MO).

Animals and experimental design

Thirty-two adult male albino rats (Rattus norvegicus) of similarage (34 months) and weight 130180 g (obtained from the

animal house of Research Institute of Opthalmology, El-Giza,Egypt) were used in the present study. All animals were keptunder observation for about 10 days before the onset of theexperiment to exclude any intercurrent infection. The chosen

animals were housed in individual cages in a temperature-and humidity-controlled room with a 12-h lightdark cycle.All the animals had free access to water and supplied daily

with a standard pellet diet. All animals included in the presentstudy were handled and received humane care in compliancewith the principles of laboratory animal care. All protocols

for their use in this investigation were approved by the Beni-Suef University.

Animals were made diabetic with a single intraperitoneal

injection of 100 mg alloxan/kg body weight in 0.1 M phos-phate citrate buffer, pH 4 (Gold, 1970).

Rats were identied as diabetic on the basis of blood glu-cose levels (ranging from 180 to 300 mg/dl at least 7 days

post-alloxan treatment). Thirty-two rats were divided into fourgroups (8 rats in each group) as required by the experiment.The rst group was regarded as normal animals which were

kept without treatment under the same laboratory conditionand regarded as the franc control group for diabetic one.The second group was made diabetic with a single intraperito-

neal injection of 100 mg alloxan/kg body weight. The third onewas treated with curcumin at a dose of 60 mg/kg b.wt dis-solved in distilled water (Sharma et al., 2006). Treatment withcurcumin was performed orally by gastric intubation and daily

between 8.00 and 10.00 a.m. for 2 months.For biochemical analyses, the animals of all tested groups

were sacriced under diethyl ether anesthesia at the end of

2 months. Blood samples were collected from each rat. Itwas allowed to coagulate at room temperature and centrifugedat 3000 rpm for 30 min.

Biochemical studies

Glucose test was performed on rats of all tested groups at the

end of the experiment. Blood samples were obtained from lat-eral tail vein of overnight fasted rats (1012 h). Successiveblood samples were then taken at 30, 60, 90 and 120 min fol-lowing the oral administration of glucose solution (3 g/kg b.

wt.). Serum was then separated for the determination of glu-cose concentration according to the method of Siest and Schie-lef (1981), using reagent kits purchased from Bio Merieux

chemicals (France).

Histological preparations

After 2 months the animals were dissected. The pancreas wereremoved then xed in 10% neutral buffered formalin for 24 h;the organs were routinely processed and sectioned at 45 m

thickness. Sections of pancreas were stained with Masson tri-chrome stain (Drury et al., 1976) to demonstrate collagen -bers and also stained with the modied aldehyde fuchsinmethod (Bancroft and Stevens, 1982).

Immunohistochemistry of pancreatic islets

Immunolocalization technique for anti-insulin was performed

on 56 m thickness sections and stained with the streptavi-

dinbiotinperoxidase staining method (Cemek et al., 2008).Parafn sections were deparafnized in xylene, rehydrated indescending grades of alcohol. Endogenous peroxidase and

non-specic binding sites for antibodies were suppressed bytreating the sections with 0.3% hydrogen peroxide for20 min and 5% normal bovine serum (1:5 diluted TRIS) for

20 min at room temperature, respectively. The sections werewashed in phosphate buffered saline and 10% normal goat ser-um was applied for 30 min to reduce non-specic binding. The

sections were incubated for 1 h with anti-sera containing pri-mary antibodies for rat insulin (polyclonal antibody) suppliedby Bio Genex Cat. No. AR. 295-R. The sections were incu-bated with biotinylated secondary antibody (Dako-K0690;

Dako Universal LSAB Kit) and streptavidin horseradishperoxidase (Dako-K0690) for 30 min, and then 3,30-diam-

Table 1 serum glucose concentration values of control, alloxan diabetic and diabetic rats treated with curcumin for 2 months after

fasting and 2 h.

Parameter Group

Control Diabetic Diabetic rats treated with curcumin

Fasting 79.443 2.848e 144.960 3.180c 110.468 5.589d

After 2 h 107,161.255 5.540d 262.202 3.901a 160.982 6.784b

LSD at 5% level is: 13.942.

LSD at 1% level is: 18.776.

Data are expressed (6 rats in each group) as Mean SE.

Values, which not share the same superscript symbol within a row, are signicantly different.

LSD= Least Signicant Difference.

anc

nu

s) l

ns

led

cells

d) A

inta

Protective effect of curcumin on histopathology and ultrastructure of pancreas 171Fig. 1 Aldehyde fuchsin. (ad photomicrograph section of rat p

pancreatic acini. The acini are formed of pyramidal cells with basal

embedded within the exocrine portions (E) and alpha cells (arrow

curcumin showing nearly normal structure of Islets of Langerha

pyramidal cells with basal nuclei (arrows). (c) Diabetic rats revea

pancreas represented by vacuolation (v) and marked decrease of b-cytoplasmic vacuolation and pyknotic nuclei of some acinar cells. (

to the control and most of the island of Langerhans (I) cells werereas. (a) Control rat pancreas showing closely packed lobules of

clei and apical acidophilic cytoplasm. Islets of Langerhans (I) were

ocated on the peripher. (b) Control pancreas of rats treated with

(I) embedded within the exocrine portions which are formed of

pathological changes of both exocrine and endocrine part of the

. Some exocrine acini revealed focal acinar damage represented by

fter supplementation with curumin the pancreas appeared similar

ct with no alteration except few vacuoles (H & E; 100).

inobenzidine tetrahydrochloride (Sigma-D5905; Sigma

Fig. 2 Masson trichrome stains. Photomicrograph section of rat

pancreas showing (a and b) control pancreas showed delicate

collagen bers around islands of Langerhans islets of Langerhans

(I), delicate collagen bers around the pancreatic acini (arrows)

and blood vessels (v). (ce) Diabetic pancreas showing. (c)

Atrophy in the islands of Langerhans cells (arrow heads)

associated with dense collagen bers around the acini (arrows).

(d and e) Thickening and hypertrophy were detected in the media

with swelling in the endothelium of the intima of the congested

stromal blood vessels (V) surrounded by collagen (C), dense

collagen bers around the acini (arrows) with some vacuoles

(arrow heads). (f and g) Treatment with curcumin showed few

collagen bers around the islets (I), blood vessels (v) and

pancreatic acini compared with the diabetic group. (Masson

trichrome; 100.)

172 M. Abdul-Hamid, N. MoustafaAldrich Company Ltd., Gillingham, UK) substrate kit for10 min to obtain immunolabelling. Finally, the nuclei were

(i) stained using Harrys hematoxylin stain, (ii) dehydratedin graded alcohol, (iii) cleared in xylene, and then (iv)mounted in DPX. The binding of antibodies was evaluated

by examination under high-power light microscopy. All sec-tions were incubated under the same conditions with thesame concentration of antibodies and at the same time, so

the immunostaining was comparable among the differentexperimental groups.

Ultrastructural preparations

After 2 months specimens were taken from the pancreas of allgroups. Thin sections for transmission electron microscopywere prepared according to the method of Louis and Williams

(1995). Briey, 13 mm segment of pancreas were xed in fresh3% glutaraldehydeformaldehyde at 4 C for 1824 h. Thespecimens were then washed in phosphate buffer (pH 7.4)

and post-xed in isotonic 1% osmium tetroxide for 1 h at4 C and then processed. Semithin sections (1 lm) were stainedwith toluidine blue. Ultrathin sections (7080 nm) were stained

with uranyl acetate and lead citrate and examined on a JoelCX 100 transmission electron microscope operated at an accel-erating voltage of 60 kV, Faculty of Science, Ain ShamsUniversity.

Statistical analysis

The data were analyzed using one-way analysis of variance

(ANOVA) (PC-STAT, 1995) followed by least signicant dif-ference (LSD) analysis to compare various groups with eachother. Results were expressed as mean standard deviation

and values of P> 0.05 were considered statistically, non-sig-nicantly different, while those of P< 0.05 and P< 0.01 werestatistically signicantly and highly signicantly different,

respectively.

Results

Biochemical results

Serum glucose

Diabetic rats showed a signicant increase in fasting144.960 3.180 mg/dl and postprandial glucose concentra-

tion 262.202 3.901 mg/dl after 2 months as compared withthe control group. The present data revealed that daily admin-istration of curcumin for 2 months caused marked hypoglyce-

mic effects on fasted 110.468 5.589 mg/dl and postprandialglucose level 160.982 6.784 mg/dl as compared with the dia-betic control group (Table 1).

Light microscopic results

Aldehyde fuchsin stain

The histological structure of the control pancreas consisted ofclosely packed lobules of pancreatic acini. The acini areformed of pyramidal cells with basal nuclei and apical acido-

philic cytoplasm. Islets of Langerhans were embedded within

Protective effect of curcumin on histopathology and ultrastructure of pancreas 173the exocrine portions and a cells located on the periphery(Fig. 1a). Control pancreas of rats treated with curcumin

showing nearly normal structure of Islets of Langerhans (I)embedded within the exocrine portions which are formed ofpyramidal cells with basal nuclei (Fig. 1b).

The present light microscopic study of diabetic rats revealedpathological changes of both exocrine and endocrine part ofthe pancreas represented by vacuolation and marked decreaseof b-cells. Some exocrine acini revealed focal acinar damagerepresented by cytoplasmic vacuolation and pyknotic nucleiof some acinar cells (Fig. 1c).

After supplementation with curcumin the pancreas ap-

peared similar to the control and most of the island of Langer-hans were intact with no alteration except few vacuoles(Fig. 1d).

Masson trichrome stains

By Masson trichrome stains, the control pancreas showed del-icate collagen bers around the pancreatic acini, islands of

Langerhans and blood vessels (Fig. 2a and b).Diabetic pancreas showed atrophy in the islands of

Langerhans cells associated with dense collagen bers around

Fig. 3 Immunohistochemistry. Photomicrographs of immunohistoch

group showing pancreatic b-cells with positive immunostaining whichdeep brown color. (b) Control curcumin group of pancreatic b-cells shocontrol. (c) Diabetic group showing signicant decrease of the imm

curcumin, showing an increase in the staining particles. (Immunostainthe acini (Fig. 2c). Thickening and hypertrophy were detectedin the media with swelling in the endothelium of the intima of

the congested stromal blood vessels surrounded by collagen,dense collagen bers around the acini with some vacuoles(Fig. 2d and e).

Treatment with curcumin showed few collagen bersaround the islets, blood vessels and pancreatic acini comparedwith the diabetic group (Fig. 2f and g).

Immunohistochemical results

Immunohistochemically, b-cells of the control group stainedwith strong positive reaction for anti-insulin antibodies as

brown granules occupying the cytoplasm of great numbers ofthe b-cells (Fig. 3a). Control curcumin group of pancreaticb-cells showed positive immunostaining with deep brown colorsimilar to the control (Fig. 3b). In diabetic group, the immuno-reactivity for anti-insulin antibodies was markedly decreasedin number of insulin positive cells (Fig. 3c). After treatment

with curcumin, positive immunoreactions of b-cells for anti-insulin antibodies were obviously increased in numbers(Fig. 3d).

emical staining of insulin in pancreatic islets of rats. (a) Control

are distributed over the center of pancreatic islets and stained with

wed positive immunostaining with deep brown color similar to the

unostaining particles for insulin. (d) Diabetic rats treated with

ing for anti-insulin antibodies. 400.)

174 M. Abdul-Hamid, N. MoustafaUltrastructural results

Acinar cells

Ultrastructure of control pancreas showed acinar cells with

euchromatic nuclei, well-developed cisternae of rough endo-plasmic reticulum, mitochondria and numerous electron densesecretory granules of variable sizes in the apical part (Fig. 4a).

Electron microscopic examination of the diabetic groupshowed marked changes in pancreatic acini represented by di-lated rough endoplasmic reticulum, decrease of secretory gran-

ules, cytoplasmic vacuolation, damaged mitochondria,

Fig. 4 Exocrine part. Electron micrograph of rat pancreas showing (

cisternae of rough endoplasmic reticulum (arrow heads), mitochond

variable sizes in the apical part. Scale bar = 1 lm. 4b and c): Electronchanges in pancreatic acini represented by damaged mitochondria (

granules (arrows) and cytoplasmic vacuolation. Scale bar = 2 lm. (c)dilated rough endoplasmic reticulum (arrows) and irregular contours o

curcumin showing marked improvement represented by increase in zym

endoplasmic (arrow heads) except few vacuoles. Scale bar = 1 lm.auotophagic vacoule and irregular contours of nuclei(Fig. 4b and c).

Pancreatic acini after supplementation with curcuminshowed marked improvement represented by increase inzymogen granules, regular contours of nuclei and attened

rough endoplasmic reticulum except few vacuoles(Fig. 4d).

b-Cells

Islets of Langerhans of control rats were formed mainly ofb-cells. Their cytoplasm contains numerous electron dense

a) control acinar cells with euchromatic nuclei (N), well-developed

ria and numerous electron dense secretory granules (arrows) of

micrograph of alloxan-diabetic rat pancreas showing (b) Marked

M), dilated rough endoplasmic reticulum, decrease of secretory

Damaged mitochondria (M), auotophagic vacuole (arrow head),

f nuclei. Scale bar = 1 lm. (d) Diabetic rat pancreas treated withogen granules, regular contours of nuclei (N) and attened rough

Protective effect of curcumin on histopathology and ultrastructure of pancreas 175secretory granules surrounded by wide lucent halo, mitochon-

dria, Golgi apparatus and euchromatic nucleus (Fig. 5a and b).b-Cells of diabetic rats showed obvious vacuolation and de-

crease of secretory granules, fusion of some granules and pyk-

notic nuclei (Fig. 5c)Treatment with curcumin revealed euchromatic nucleus,

few vacuoles in b-cells and increase of secretory granules com-pared to the diabetic ones (Fig. 5d).

a-Cells

In the control a cells, the cytoplasm appeared with numeroussecretory granules of electron dense core and surrounded bynarrow halo, mitochondria and round euchromatic nucleus(Fig. 6a and b).

Fig. 5 Beta cells. Electron micrograph of rat pancreas showing (a a

Their cytoplasm contains numerous electron dense secretory granule

head), Golgi apparatus (G) and euchromatic nucleus (N). Scale bar =

cells of diabetic rats with obvious vacuolation and decrease of secreto

Scale bar = 2 lm. (d) Electron micrograph of diabetic rat pancreas treain b-cells and increase of secretory granules (arrows) compared to thebar = 1 lm.a cells of the diabetic group revealed focal necrosisand vacuolation, dilated rough endoplasmic reticulum,depletion of secretory granules and pyknotic nuclei(Fig. 6c and d).

Supplementation with curcumin resulted in obviousimprovement in the ultrastructure of a-cells including increaseof zymogen granules (Fig. 6e).

Discussion

The present diabetic rats showed a signicant increase in fast-

ing and postprandial glucose concentration compared with thecontrol group. This result is in agreement with Dahecha et al.(2011) and Ramar et al. (2012).

nd b) control Islets of Langerhans were formed mainly of b-cells.s surrounded by wide lucent halo (arrow), mitochondria (arrow

2.1 lm respectively. (c) Alloxan-diabetic rat pancreas showing b-ry granules, fusion of some granules (F) and pyknotic nuclei (N).

ted with curcumin showing euchromatic nucleus (N), few vacuoles

diabetic one appeared nearly similar to the control group. Scale

176 M. Abdul-Hamid, N. MoustafaCurcumin showed an anti-hyperglycemic effect as re-ported by Peeyush et al. (2009), El-Moselhy et al.(2011). Moreover, curcumin antagonizes the decit of glu-

Fig. 6 Alpha cells. Electron micrograph of rat pancreas showing (a)

(arrows) of electron dense core and surrounded by narrow halo. N

Mitochondria (arrows) of control a cells and numerous secretory granupancreas showing a cells of diabetic group revealed focal necrosis andsecretory granules and pyknotic nuclei (N). Scale bar = 2.1 lm respeobvious improvement in the ultrastructure of a-cells including increasegroup. Scale bar = 2 lm.cose energy metabolism or oxidative stress related to cog-nitive impairment associated with diabetes. (Shishodiaet al., 2005).

the cytoplasm of control a cells with numerous secretory granulesotice the round euchromatic nucleus (N). Scale bar = 2 lm. (b)les (arrow head). Scale bar = 1 lm. (c and d) Alloxan-diabetic ratvacuolation (V), dilated rough endoplasmic reticulum, depletion of

ctively. (e) Diabetic rat pancreas treated with curcumin showing

of zymogen granules which appeared nearly similar to the control

The present study showed histopathological changes after Degirmenci et al. (2005) reported a decrease in secretorygranules of b-cells, vacuolization and swelling of mitochon-

Protective effect of curcumin on histopathology and ultrastructure of pancreas 177alloxan injection represented by destructed b-cells and vacuo-lated pancreatic acini. Similar results were observed by Jelodar

et al. (2005) and Hamden et al., (2009). Moreover, alloxanadministration elicited signicant morphological changes indiabetic rats with severe injury of pancreatic b-cells, such asdecreasing the islets cell numbers, cell damage, and cell death(Dahecha et al., 2011). The thickened and hyalinized bloodvessels causing not enough oxygen reach the tissue which re-

sulted in degenerative changes and necrosis. (Pushparajet al., 2000).

The present immunoreactivity for anti-insulin antibodieswas markedly decreased in number of insulin positive cells

after alloxan injection. Mendez and Haro Hernandez (2005),Ao et al. (2008) and Simsek et al. (2012) reported similar re-sults. Furthermore, alloxan selectively destroyed and rapidly

accumulates in b-cell (Gorus et al., 1982; Jelodar et al.,2005). Alloxan experimental models of pancreatic damagehave been demonstrated with structural and functional altera-

tions such as disorganization of pancreatic architecture, anddepletion of insulin producing cells (Davidson et al., 1989;Waguri et al., 1997; Hashemi et al., 2009). The cytotoxic action

of alloxan is mediated by reactive oxygen species, with a simul-taneous massive increase in cytosolic calcium concentrationleading to rapid destruction of b-cells (Szkudelski, 2001). Inthe present study after treatment with curcumin, positive

immunoreactions of b-cells for anti-insulin antibodies wereobviously increased in numbers. Previous reports showed thatcurcumin increases plasma insulin level in diabetic mice (Seo

et al., 2008) and in diabetic rat (Peeyush et al., 2009). Further-more, the previous results of immunohistochemical and mor-phometric investigation showed that antidiabetic effect of

organic derivative of vanadium (IV) oxide produced a reliableincrease in the population of insulin-producing cells (Aleevaet al., 2004).

It has been found that oxidative stress is associated with themolecular mechanism of the decreased insulin biosynthesis andsecretion, which is the main etiology of glucose toxicity. In-deed, it was suggested that the pancreas may be more suscep-

tible to oxidative stress than other tissues and organs, becausepancreatic islet cells show extremely weak manifestation ofantioxidative enzymes (Evans et al., 2003; Robertson, 2006).

It is evident that oxidative stress plays a key role in causinginsulin resistance and b-cell dysfunction by their ability to acti-vate stress-sensitive signaling pathways (Evans et al., 2003).

Increases in intracellular glucose lead to an abundance ofelectron donors generated in the Krebs cycle which drive theinner mitochondrial membrane potential upward, a state thatis associated with mitochondrial dysfunction and increased

ROS production (Korshunov et al., 1997).The present ultrastructural study of the diabetic group

showed marked changes in pancreatic acini represented by di-

lated rough endoplasmic reticulum, decrease of secretory gran-ules, cytoplasmic vacuolation, damaged mitochondria.Moreover, b-cells and a cells showed obvious vacuolationand decrease of secretory granules and pyknotic nuclei. Allox-an, like glucose, affects a- and b-cells directly, stimulating theb-cell and inhibiting the a-cell. So alloxan might have directtoxic actions on both cell types, transiently stimulating andthen permanently blocking the b-cells and irreversibly inhibit-ing the a-cells (Pagliara et al., 1977).dria. The semiquantitative evaluation of islet ultrastructure

after alloxan exposure in vitro demonstrated that mouse b-cellsshowed signs of both necrotic and apoptotic cell death, distur-bances in the insulin secretory pattern during and after an al-

loxan perfusion (Tyrberg et al., 2001). Alloxan induces damageto b-cell DNA (Yamamoto et al., 1981), mitochondria (Bo-quist and Ericsson, 1984), lysosomes (Zhang et al., 1992),

and plasma membrane (Watkins et al., 1964). Bogolepov(1983) stated that vacuolation was one of the structural indica-tions of permeability disorders of the membranes, which re-sults in an enhanced transport of water and electrocytes into

the cell. The permeability disorder could be attributed to manycellular membrane insults caused by reactive oxygen speciesmediated formation of lipid peroxides, which ultimately gener-

ate self-sustaining lipid peroxidation (Halliwell and Chirico,1993).

It has been reported that curcumin scavenges oxygen free

radicals and inhibits lipid peroxidation and protects cellularmacromolecules, including DNA from oxidative damage (Kal-pana and Menon, 2004; Polasa et al., 2004). It is known to be a

multi-functional agent, such as powerful anti-oxidant, anti-diabetic, anti inammatory and anti-cancer agent (Milleret al., 2001).

In conclusion, this study investigated the effect of alloxan

on a cells and threw light on the potential of curcumin inthe prevention or treatment of diabetes due to the importanceof human diabetes mellitus as a world health problem as well

as the increasing demand by patients to use the natural prod-ucts with antidiabetic activity, because insulin and oral hypo-glycemic drugs possess undesirable side effects. Further study

will be needed to study the effect of alloxan on a cells.

Conict of interest

The authors declare that there are no conicts of interest.

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Protective effect of curcumin on histopathology and ultrastructure of pancreas 179

Protective effect of curcumin on histopathology and ultrastructure of pancreas in the alloxan treated rats for induction of diabetesIntroductionMaterials and methodsChemicalsAnimals and experimental designBiochemical studiesHistological preparationsImmunohistochemistry of pancreatic isletsUltrastructural preparationsStatistical analysis

ResultsBiochemical resultsSerum glucose

Light microscopic resultsAldehyde fuchsin stainMasson trichrome stains

Immunohistochemical resultsUltrastructural resultsAcinar cells-Cells-Cells

DiscussionConflict of interestReferences