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The possible protective role of aloe vera extracts in pancreatic β cells of experimentally induced diabetic rats: a histological and immunohistochemical study

Salem, Mohamed Y.; El-Azab, Nahla El-Eraky

The Egyptian Journal of Histology: September 2014 - Volume 37 - Issue 3 - p 571–578
doi: 10.1097/01.EHX.0000452616.94440.ca
Original articles
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Background The increasing prevalence of diabetes mellitus in the world is of great concern. As synthetic drugs have undesirable side effects or contraindications, traditional medicinal plants are being used for treatments of diabetes.

Aim of the work This study aimed to determine the protective effect of aloe vera (AV) on β cells of diabetic rats.

Material and methods Forty adult male rats were utilized and divided equally into four groups. The first served as the control group; the second was the streptozotocin (STZ) group (single intraperitoneal dose of 65 mg/kg body weight); the third was the preventive AV group, which was given a daily oral dose of AV (300 mg/kg body weight/day) for 14 days along with a single intraperitoneal injection of STZ given after 7 days from the start of AV; the fourth group was the curative AV group, which was given a single intraperitoneal injection of STZ and after 7 days were given a daily oral dose of AV for 14 days. Pancreatic tail samples were taken 14 days after the treatment with AV. Paraffin sections were prepared for histological and immunohistochemical studies.

Results The results revealed that a single dose of STZ induced marked cytoplasmic vacuolations and pyknotic nuclei in many islet cells. The AV preventive group showed normal-appearing islet cells. The AV curative group showed cytoplasmic vacuolations and pyknotic nuclei in many islet cells. Statistical analysis revealed a significant decrease (P<0.05) in anti-insulin antibody reaction and bcl2 expression in the diabetic group and the AV curative group compared with the control group, whereas there was an insignificant increase in the preventive group.

Conclusion The present study suggests that AV when used as a preventive agent can protect against STZ-induced diabetes in rats. Therefore, AV should be given to prediabetic patients and to individuals at high risk for diabetes.

Histology and Cell Biology Department, Faculty of Medicine, Benha University, Benha, Egypt

Correspondence to Mohamed Y. Salem, MD, Histology and Cell Biology Department, Faculty of Medicine, Benha University, Benha, Egypt Tel: +20 1091567677; e-mail: mohamed_jo63@yahoo.com

Received March 25, 2014

Accepted June 1, 2014

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Introduction

According to the WHO, diabetes mellitus (DM) is one of the most common metabolic disorders prevalent worldwide. DM is a group of metabolic disorders characterized by hyperglycemia, in which alterations in the carbohydrate, fat, and protein metabolisms are accompanied by absolute or relative deficiencies in insulin secretion and/or its action 1. The WHO estimates that almost three million deaths occurring annually are a result of diabetes and that there will be 366 million cases of diabetes by the year 2030 2.

It is well known that suffering from diabetes for a long period of time may cause many complications such as diabetic nephropathy, retinopathy, neuropathy, cardiomyopathy, and hyperglycemia 3. Conventionally, insulin-dependent DM is treated with exogenous insulin and noninsulin-dependent DM with synthetic oral hypoglycemic agents like sulfonylureas and biguanides. However, the hormone fails as a curative agent for complications arising from diabetes, and synthetic oral drugs produce adverse health effects. Different medicinal systems use active plant constituents, which were discovered as natural hypoglycemic medicines and came from traditional knowledge 4.

Herbal medicines have occupied a distinct position, right from the primitive period up to the present day. In every ethnic group there exists a traditional healthcare system, which is culturally patterned. In rural communities, healthcare seems to be the first and foremost line of defense. The WHO has already recognized the contribution of traditional healthcare in tribal communities. These medicines have fewer side effects and the herbs can be obtained easily from nature 5.

Aloe vera (Syn.: Aloe barbadensis Miller) is a cactus-like perennial plant belonging to the family Liliaceae (subfamily of Asphodelaceae). It is native to North Africa and cultivated in warm climatic areas 6. The plant has elongated pointed fleshy leaves consisting of two parts – an outer skin (green rind) and an inner pulp (colorless mucilaginous gel) 7. Aloe vera (AV) consists of a high content of phenolic compounds, glycosides (aloins), 1,8-dihydroxyanthraquinone derivatives (aloe emodin), β-1,4 acetylated mannan, mannose-phosphate, and alprogen glucoprotein 8.

Recent scientific investigations on medicinal properties of AV have made it a novel valuable ingredient in food, cosmetic, and pharmaceutical industries worldwide 9. Leaf exudates and mucilaginous gel of aloe possess anti-inflammatory, antioxidant, antifungal, antibacterial, antiarthritic, antirheumatoid 10, anticancer 11, cytoprotective, cardiac stimulatory, and immunomodulatory activities 12. It is used to protect against gastric ulceration, is a remedy against a variety of skin disorders, and promotes wound healing 7. The aloe plant has stiff grayish-green lance-shaped leaves containing a clear gel in a central mucilaginous pulp. Clinical evaluations have revealed that the pharmacologically active ingredients are concentrated in both the gel and the rind of AV leaves 13.

The aim of the present study was to determine the protective effect of AV on β cells of streptozotocin (STZ)-induced diabetic rats.

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Materials and methods

In this study, 40 adult male albino rats of average weight 150–200 g were used. The animals were obtained from the animal house, Moshtohor Faculty of Veterinary Medicine, Benha University. Strict care and cleaning measures were utilized to keep the animal in a normal healthy state; the animals were kept in animal cages under the prevailing atmospheric conditions. They were kept on normal balanced diet and tap water. All ethical protocols for animal treatment were followed and were supervised by the animal facilities. All animal experiments received approval from the Institutional Animal Care Committee.

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Preparation of aloe vera extract

AV solid gel from the center of the leaf was collected and homogenized, and a mucilaginous, thick, and straw-colored homogenate was obtained and lyophilized. The lyophilized sample was extracted using 95% ethanol. The filtrate was collected and evaporated to dryness under reduced pressure in a rotary evaporator at 60°C. The residue was stored in dry, sterilized small containers at 4°C until further use. The drug solutions were prepared freshly each time. The dosing schedule used was once per day. The extracts were administered orally daily to rats at a dose of 300 mg/kg body weight 1.

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Induction of experimental diabetes

Diabetes was induced by a single intraperitoneal injection of 1% STZ (Sigma-Aldrich, St Louis, Missouri, USA) at 65 mg/kg body weight after 14 h of food deprivation. STZ was dissolved in 0.1 mol/l cold sodium citrate buffer (pH 4.5). Animals were allowed to drink 5% glucose solution overnight to overcome the drug-induced hypoglycemia. After a week, the rats were considered as diabetic rats and used for the experiment 14.

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Experimental procedure

The rats were divided into four equal groups comprising 10 animals each.

  • Group I: this group consisted of normal control rats.
  • Group II: this group comprised STZ-induced diabetic rats.
  • Group III (the preventive group): rats in this group were given AV leaf gel extract in aqueous solution daily using an intragastric tube for 14 days. A single intraperitoneal injection of 1% STZ was given after 7 days from the start of AV.
  • Group IV (the curative group): rats in this group were given AV leaf gel extract in aqueous solution daily using an intragastric tube for 14 days 7 days after a single intraperitoneal injection of 1% STZ.

Blood samples were obtained from the rats’ tail vein and their fasting blood glucose level was determined before and 7 days after STZ injection in the diabetic group using a digital glucometer (Accu-chek Advantage; Roche Diagnostic, Mannheim, Germany) to confirm diabetes induction.

The animals were anesthetized using ether and sacrificed by cervical decapitation 7 days after STZ induction (in group II) and after 14 days of the AV treatment (in groups III and IV). Small pieces of pancreatic tail were fixed in 10% formalin for H&E staining 15 and for immunohistochemical study to detect insulin antibody reaction and bcl2 expression in β cells 16.

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Immunohistochemical detection of insulin antibody

Immunohistochemical staining was carried out according to the manufacturer’s protocol. The avidin–biotin peroxidase method was used. Paraffin sections were mounted on coated slides. They were deparaffinized in xylene, rehydrated in descending grades of alcohol, and then immersed in 0.3% hydrogen peroxide for 30 min to block endogenous peroxidase activity. The sections were incubated for 1 h with monoclonal mouse antisera against human insulin protein at a dilution of 1:100 for 1 h. The slides were rinsed in PBS and then incubated with the secondary antibody (biotinylated goat anti-mouse IgG, DAKO LSAB 2 Kit; Dako, Glostrup, Denmark) for 1 h at room temperature and rinsed again in PBS. The immunoreactivity was visualized using 0.05% diaminobenzidine. Sections were counterstained with hematoxylin 17. As a negative control, the primary antibody was replaced withPBS.

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Immunohistochemical detection of bcl2 expression

Immunohistochemical staining was carried out according to the manufacturer’s protocol. Paraffin-embedded tissue sections, 3–4-μm thick, were mounted on positively charged slides and heated at 60°C for 30 min. They were then deparaffinized and rehydrated through a series of xylene and alcohol before staining. Antigen retrieval was carried out with microwave treatment in 10 mmol/l citrate buffer (Neo-Markers; Thermo Fisher Scientific Inc., Rockford, Illinois, USA). Sections were washed three times with cold PBS after blocking with 10% normal rabbit serum. Slides were incubated for 1 h with rabbit anti-human Bax secondary antibody (dilution 1:50; Dako Cytomation Norden A/S, Glostrup, Denmark). DAB substrate chromogen solution was applied and incubated for 15–30 min until color intensity was reached. Sections were counterstained with hematoxylin. Sections treated with the same protocol in the absence of the primary antibodies served as negative controls.

The mean area percentage of insulin antibody and bcl2 expression (areas of brown cytoplasmic staining) was quantified in 10 images for each group using the Image-Pro Plus program, version 6.0 (Media Cybernetics Inc., Bethesda, Maryland, USA). Insulin antibody and bcl2 expression of groups II, III and IV compared with group I using the t-test, with P less than 0.05 as the level of statistical significance. Statistical analyses were carried out using IBM SPSS Statistics software for Windows, Version 20 (IBM Corp., Armonk, New York, USA).

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Results

H&E

Islets of Langerhans consisted of rounded cells with palely stained cytoplasm and central rounded vesicular nuclei arranged in branching and anastomosing cords intermingled with blood capillaries. Most of the peripherally situated cells appeared with darkly stained nuclei (Fig. 1). The diabetic group (affected group) showed alterations in the islet morphology with marked cytoplasmic vacuolations and pyknotic nuclei in many cells. Congested blood capillaries between the cells were distorted (Fig. 2). The AV preventive group (group III) showed normal-appearing islet cells with cytoplasmic minimal vacuolations and pyknotic nuclei in a few cells (Fig. 3). The AV curative group (group IV) showed cytoplasmic vacuolations and pyknotic nuclei in many islet cells with congested blood capillaries in between (Fig. 4).

Figure 1

Figure 1

Figure 2

Figure 2

Figure 3

Figure 3

Figure 4

Figure 4

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Immunohistochemical

Anti-insulin antibody reaction

The control group showed highly positive insulin antibody staining (areas of brown cytoplasmic staining) inside islet β cells (Fig. 5). The diabetic group showed minimally positive anti-insulin antibody reaction inside islet β cells (Fig. 6). The AV preventive group showed highly positive anti-insulin antibody reaction (Fig. 7), whereas it was mild in the AV curative group (Fig. 8).

Figure 5

Figure 5

Figure 6

Figure 6

Figure 7

Figure 7

Figure 8

Figure 8

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Bcl2 expression

Positive immunohistochemical staining of bcl2 demonstrated brown cytoplasmic staining (index for the antiapoptotic effect). The control group showed moderate bcl2 expression in the cytoplasm of islet cells (Fig. 9). The diabetic group showed minimal bcl2 expression in the cytoplasm of islet cells (Fig. 10). Bcl2 expression in the cytoplasm of islet cells of the AV preventive group was moderate (Fig. 11), whereas it was mild in the AV curative group (Fig. 12).

Figure 9

Figure 9

Figure 10

Figure 10

Figure 11

Figure 11

Figure 12

Figure 12

The mean area percentage of anti-insulin antibody reaction and bcl2 expression in all groups is presented in Tables 1 and 2 and Histograms 1 and 2. There was a significant decrease (P<0.05) in anti-insulin antibody reaction and in bcl2 expression in groups II and IV compared with group I, whereas there was insignificant increase in group III compared with group I.

Table 1

Table 1

Table 2

Table 2

Histogram 1

Histogram 1

Histogram 2

Histogram 2

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Discussion

Prevalence of diabetes and impaired glucose tolerance is rapidly increasing, and the WHO estimates a current worldwide prevalence of 346 million patients with DM 18. Oxidative stress plays a major role in the development of both microvascular and cardiovascular diabetic complications 19. Recent approaches suggest that treatment of diabetes should focus not only on insulin secretion but also on antioxidant protection of β cells. This may facilitate the repair of β cells undergoing damage by oxidative stress secondary to hyperglycemia 20. Management of diabetes without any side effect is still a challenge to the medical community. There is continuous search for alternative drugs; therefore, it is prudent to look for options in herbal medicine for diabetes as well 21.

The STZ-induced diabetes (group II) in the present study showed alterations in the islet morphology with marked cytoplasmic vacuolations and pyknotic nuclei in many cells and significant decrease (P<0.05) in insulin antibody reaction and bcl2 expression compared with the control group (group I). In agreement with these findings, a previous study showed that in STZ-induced diabetic rats the most consistent findings were characterized by the presence of pyknotic cells, cytoplasmic degranulation, and vacuolization, with increase (P<0.05) in the numbers of insulin-negative cells 22. In addition, it was reported that STZ selectively destroys pancreatic β cells, which cause inhibition of synthesis and release of insulin, thereby leading to the onset of DM 14,23, accompanied by inhibited expression of antiapoptotic Bcl-2 24. The diabetogenic effect of STZ was attributed to the fact that STZ methylates proteins and DNA methylation is ultimately responsible for beta cell death. Some minor generation of reactive oxygen species, including superoxide and hydroxyl radicals originating from hydrogen peroxide dismutation during hypoxanthine metabolism, may accompany the effect of STZ and accelerate the process of β-cell destruction 25, and persistent hyperglycemia causes increased production of free radicals, especially reactive oxygen species, in tissues because of glucose auto-oxidation and protein glycosylation 3.

The use of herbs and medicinal plants is a universal phenomenon. Every culture has relied on the huge variety of natural chemistry found in medicinal plants for their therapeutic properties 26. AV is a medicinal plant used traditionally in diverse therapeutic applications 27. AV supplementation can help prevent oxidative stress and might also be useful in the treatment of oxidative stress-related human disorders by virtue of its antioxidant activity 28.

The AV preventive group (group III) in the present study showed normal-appearing islet cells with cytoplasmic minimal vacuolations and pyknotic nuclei in a few islet cells and insignificant increase (P<0.05) in insulin antibody reaction and bcl2 expression compared with the control group (group I). In agreement with these findings, some researchers reported that AV was given to mice 30 days before STZ injections over a period of 73 days, after STZ injections were found to be significantly effective in inhibiting the destruction of islets of Langerhans in the pancreas 29. Previous studies have reported that in STZ diabetes oxidative stress is a confounding factor, and it contributes to the pathogenesis and complications of this disease. β-Cells are especially vulnerable to oxidative insult in that they possess a relatively poor complement of antioxidants 30. It was also suggested that the medicinal properties of AV can be investigated from the point of view of their potent antioxidant activities, absence of side effects, and economic viability 31. In addition, AV is used as a beneficial therapeutic agent that, along with other antioxidant properties, has been shown to have a protective role as a free radical scavenger in diabetic patients, as proven by its control of elevated anions in STZ-induced diabetic animal models 1. Previous studies have suggested many explanations for the antidiabetic effect of AV. The first explanation is the potent antioxidant effect of the aloe extract. Aloe has been long known to have antioxidant potential through the suppression of free radical formation and enhancement of cellular thiol status. It has also been reported to stimulate glutathione-S-transferase enzyme activity 32.

AV in group IV (curative group) in the present study failed to improve the alterations in the islet morphology (cytoplasmic vacuolations and pyknotic nuclei in many islet cells), as well as insulin antibody reaction and bcl2 expression induced by STZ. These findings were in agreement with those of a previous study 33, which reported that treatment of diabetic rats with AV extracts has no beneficial influence on the pancreas and thus may not be appropriate for the treatment of diabetes in alternative medicine. In contrast, another study has reported the potential of AV as an antidiabetic drug in AV-treated diabetic rabbits 21. Previous studies have explained these controversial results by reporting that the beneficial effect of AV is through stimulation of synthesis and release of insulin from the remnant pancreatic β cells, by studying STZ-induced diabetic rats 34,35.

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Conclusion

The present study suggests that AV when used as a preventive agent can protect against STZ-induced diabetes in rats. Therefore, AV should be given to prediabetic patients and to individuals at high risk for diabetes.

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

Table

Table

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Keywords:

aloe vera; diabetes; pancreatic β cells

© 2014 The Egyptian Journal of Histology