effect of juniper on the structure of islets of langerhans ...

TITE EGYPTIAN JOURNA L OF fIlSTOLOGY - Vol. 29. No.2, December. 193 - 204, 2006. (ISSN: 1110 - (559)

EFFECT OF JUNIPER ON THE STRUCTURE OF ISLETS OF LANGERHANS IN ALLOXAN-INDUCED DIABETIC RATS: HISTOLOGICAL AND IMM,UNOHISTOCHEMICAL STUDY SIHAM K.M. ABUNASEF Histology Department. Faculty ofMedicine, Ain Shams University

Abstract Many traditional treatments have been recommended in the alternative medicine for treatment of diabetes mellitus. Juniper procera is one ofthe known hypoglycemic agents, which is used in [olklori medicinefor improving blood glucose control and preventing long term complications in diabetes mellitus. This study was carried out to clarify the role ofJuniper on the histological structure ofislets ofLangerhans. In this study, twenty jive adult male albino rats were used. Diabetes mellitus was induced in 20 rats, using IP injection of180 mglkg body weight (EW) of alloxan. The diabetic rats were divided into four groups, three of which were fed on a diet containing 2.5%, 5% and 7.5% BW ofJuniperus procera for 6 weeks. The fourtlt group (positive control) received an ordinary diet. Another jive lion-diabetic rats reserved as (negative control group), who received neither alloxan nor the mentioned plant. Following consumption ofplants, blood glucose was measured every day and on the last day ofthe experiment serum insulin was measured. Serial sections of the pancreas were prepared and stained with H&E, Masson trichrome and immunohistochemical staining ofp-cells by anti-insulin antibody. Morphology of the pancreatic sections and the following morphometric parameters: Density {area %) of anti-insulin antibody reaction in islets, percent ofP cells and the average area of the islets in whole pancreas were studied. The quantitative results were tabulated and statistically analyzed. The microscopical results ofdiabetic group showed marked degenerative changes ofcells mostly in centers ofislets with insulinitis: The condensation ofconnective tissue around islets was evident. Also, there was marked decrease in p-cell population a/islets. The previously described effect ofalloxan on the beta cell was· not reversed completely by using juniper. Meanwhile, there was partial recovery to signs of inflammation in islets and around acini. The previously mentioned morphometric parameters detected no statistical significant change between juniper treated and untreated diabetics. In conclusion, juniper has a hypoglycemic effect hut this is not through the improvement ofp-cells structure. KeyWords: Juniper - Alloxan - Diabetes «Microscopical- Pancreas

Introduction The traditional usc of medicinal plants for the treatment of diabetes mellitus greatly declined in developed countries after the introduction of insulin in 1921. However, many of them have remained as an alternative to conventional therapy in depressed areas where insulin is not readily available(l). In the last few years serious attempts have been made to develop new easy-to-use, non-parenteral forms of insulin, but till now these have yielded poor results. On the other hand, there is a growing need for new oral antidiabetic drugs to serve as an alternative therapy in non-insulin dependent diabetes mellitusfz). The plant kingdom is a wide field to look for effective oral hypoglycaemics. More than 400 kind have been reported to be beneficial in treatment or 3( I 034 - 2006)

diabetes(3). The world health organization has recommended further investigation in this arca(4). Juniper which grows wild in different areas of the world like Spain, North Sinai and Eltaif in Kingdom of Saudi Arabia, is popularly known for its variable pharmaco-dynamic effects. Biochemical studies on normoglycemic and hyperglycemic rats and mice stated that juniper can result in a significant lowering of blood glucose level(2&5). Many traditional plant treatments for diabetes arc also used. The mechanism of most of the herbals used has not been defined. Although there arc suggestions about the mechanism of action of plants, the exact mechanism is unclear. The aim of this study is to verify the hypoglycemic effect of juniper and clarify its effect on the histological structure of islets of Langerhans. 193

Siham K.M. Abunasef

194

Material and Methods Preparation ofalloxan-induced diabetic rats: Alloxan tetra hydrate (Sigma) was dissolved in sterile distilled water. Diabetes was induced in 20 adult male albino rats by intraperitoneal injection of 180 mg/kg. The rats with blood glucose above 250 mg/dl, as well as with polydepsia, polyurea and polyphagia, which . extend for at least one week, were selected for the experiment(6). The range ofthe diabetogenic dose ofalloxan is quite narrow and even light overdosing may be generally toxic causing the loss of many animals(7). To prevent the toxic side effects, ranges of 95 to 195 mg/kg of alloxan (10 mg interval) were tested and 180 mg/kg was selected as the minimum and safest dose for induction of diabetes in this study.

Preparation offood: The Juniperus procera was obtained as fresh plants from AI-Taif in KSA. Then, they were dried, ground and kept in a clean sterile jar. The rats naturally were not interested in having the plants, so the ground plants were mixed with standard pellet in a percentage of2.5% of body weight (BW), 5% ofBWand 7.5% ofBW daily for 6 weeks. Rats were fed with the prepared food first, and only when they finished that, additional ordinary food was given to them.

Animal groups: Twentyfive adult male albino rats (150-200 g) were obtained from the animal house ofFaculty of Medicine, Ain Shams University and housed in an air-conditioned room. They were divided as follows:

Group I (negative control group): Animals were fed with ordinary pellet food only (5 animals). Following induction of diabetes in 20 rats, they were randomly divided into four groups, five rats each:

Group II: (positive control group): Animals were fed with ordinary pellet food (5 animals).

Group Ill: Animals were fed with the ground juniper, which is mixed with standard pellet in a percentage of2.5% ofBW (5 animals).

Group IV: Animals were fed with the ground juniper, which is mixed with standard pellet in a percentage of 5% ofBW (5 animals).

Group Jl:. Animals were fed with the ground juniper, which is mixed with standard pellet in a percentage of7.5% ofBW (5 animals).

Biochemical study: Blood glucose was tested every day. Blood was collected from the tail of fasting (lOh) animals. The tail was embedded in 45°C water bath and about one millimeter of its end was cut and a drop of blood was used for the blood glucose test with the help of glucometer GX (Ames, USA), further sampling did not need re-cutting of the tail. Level of insuliswas measured in serum using insulin radioimmunoassay kits obtained from DPC, Los Anglos, USA. The blood samples were taken after 6 weeks at the end of the experiment immediately before sacrifice.

Histological study On the last day of the experiment the rats were anesthetized and the tail part of the pancreas was removed and kept in buffered formalin. Five-microns ordinary paraffin serial sections were prepared and stained with Hematoxylin and Eosin (H/E)(8) and Masson trichrome(9). Detection of insulin secreting cells (beta cell) using immunostaining by antiinsulin polyclonal antibodies (Daco). Stained sections were assessed morphologically and morphometrically.

Morphometric study: This was done on the immunohistochemical stained slides at a magnification ofx 1000 by the aid of "Leica Q500" Image analyser computer system in histology department of Ain Shams University.

Effect of Juniper On the Structure of Islets of Langerhans in Alloxan-Induced

The following parameters were evaluated: 1- Density (area%) of anti-insulin antibody reaction in islets was measured, in 5 islets from 5 different microscopic fields from five blocks of the five animals in each group.

2- Percent of p cells (Bp) per total islet cells was calculated by using the nucleus as the counting base. The nuclei of the stained p-cells (pn) and the nuclei of total islet cells (In) per islet profile were counted. The ratio of p-cells to the total islet nuclei was expressed as the p-cell percentage per islet cells (Bp), The following equation was used to calculate Pp: Pp = (Bn/ln) x 100. This parameter was done on cells of approximately 4 islets on each tissue and 40 islets of each groupe 10). 3- The average area ofthe islets was determined by measuring the area of 4 islets in each section and totally of40 islets in each group(ll).

Statistical analysis: The results are expressed as mean ± SD. The significance of the differences in the values was performed by one-way ANOVA test using the SPSS program. P(;,

c· ,t" \-

1 f·~

(;"

t,'- l''f~ f'" ~ 1:,t' ~'l ~?~.~ I'~;' ~t t fIr,l,~ l' :t.r ret L; l'~,,~ 1: '" ,~~ ~, .c:t 1 ~ ~ ~ ,t".' ;~ 't, ~ 'I't 1 f ~' -: ;0 ::: ~~. t' 'S'" E f - E:. ~ r ~ ~,