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. 2020 Oct 1;26(1):159–172. doi: 10.1007/s12192-020-01165-2

The counteracting effects of (-)-Epigallocatechin-3-Gallate on the immobilization stress-induced adverse reactions in rat pancreas

Nermeen Mohammed Faheem 1,2,✉, Tarek Mohamed Ali 3,4
PMCID: PMC7736449  PMID: 33000400

Abstract

Many studies suggest that Epigallocatechin-3-Gallate (EGCG) has many protective effects. But little is known about its protective effects against chronic restraint stress-induced damage in rats. The aim was to demonstrate the potential protective effects of EGCG against harmful pancreatic damage to the immobilization stress in the rat model. Forty rats, 2 months old, were divided into four groups (n = 10): control group; EGCG group, rats received EGCG by gavage (100 mg/kg /day) for 30 days; stressed group, rats exposed to immobilization stress; and stressed with EGCG group, rats exposed to immobilization stress and received EGCG for 30 days. Glycemic status parameters, corticosterone, and inflammatory markers were investigated on the first day, 15th day, and the 30th day of the experiment. Pancreatic oxidative stress markers and cytokines were evaluated. Histological, immunohistological, and statistical studies were performed. On the 15th day, fasting blood glucose (FBG), fasting plasma insulin (FPI), homeostatic model assessment for insulin resistance (HOMA-IR), and fasting plasma corticosterone were significantly higher in the stressed group when compared with first and 30th day in the same group as well as when compared with control and stressed with EGCG groups. The stressed group revealed significantly higher pancreatic IL-1β, IL-6, TNF-α, MDA, and NO, serum amylase and serum lipase, and significantly lower GSH, SOD, and CAT when compared to control and stressed with EGCG groups. EGCG treatment attenuated the pancreatic stress-induced cellular degeneration, leucocytic infiltration, and cytoplasmic vacuolations; significantly decreased area percentage of collagen fibers; and significantly increased mean area percentage of insulin immunopositive cell as compared with stressed group. EGCG is a protective agent against immobilization stress because of its anti-diabetic, anti-inflammatory, and and anti-oxidative stress properties, as confirmed by biochemical and histological alterations.

Keywords: Immobilization stress, EGCG, Pancreas, Immunohistochemistry, Anti-inflammatory markers

Introduction

Stress “a state of disharmony” is a real or perceived disturbance to an organism’s homeostasis, which triggers physiological and behavioral responses (McEwen 2000). The stress response involves various mechanisms for physiological and metabolic adjustments in the body to cope with the demands of homeostasis (Bali and Jaggi 2013). Selye (1936), reprinted 1998) stated that if stress is only lasting in the short term and within our coping abilities, now it is productive and positive, enhances performance, and is defined as Eustress. Eustress truly has emotional and physical health benefits, while distress is harmful stress and can lead to many diseases. Acute stress triggers activation of the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic adreno-medullary pathways. Activation of these two pathways leads to a cascade of biological events. However, repetitive exposure of the same stressor is commonly associated with general adaptation syndrome. Nevertheless, during continuous exposure of stress, the preliminary “adaptive response” may change to “maladaptation.” Furthermore, chronic stress alerts the HPA axis, and exposure to the new, random stressor may cause prolonged deregulation of the HPA axis (Herman 2013). Stress has been assumed to be involved in the pathophysiology of a variety of diseases, including anxiety, depression, dementia, and other disorders. Adaptation can be considered as the basis of several conditions; for example, cardiovascular and psychiatric illnesses increased susceptibility to infection, autoimmune diseases, and diabetes (Godoy et al. 2018). The impact of stress has been proved to affect the endocrine pancreas causing impaired glucose metabolism leading to diabetes mellitus. Stress could potentiate inflammatory response and apoptosis in different rat organs and could induce insulin resistance in various tissues and affect insulin release from isolated islets of Langerhans (Binker et al. 2010). Stress makes the body cells utilize high energy to acclimatize to abnormal environmental conditions like an increased respiratory rate. But, increased metabolic rate causes over-production of free radicals. Additional free radicals result in oxidative damage to cellular proteins, lipids, and nucleic acids in several tissues (Yan et al. 2016). To induce physiological and psychological stress, restraint stress, a specific procedure that limits movement, is considered as one of the used classic techniques. Restraint stress either includes the limitation of the animal’s movement by restricted space, i.e., confinement or immobilization of the limbs and body of the animal by tape or plaster (Zardooz et al. 2012). An earlier study has revealed that restraint stress increases plasma lipid peroxidation and decreases plasma protection against oxidation (Salehi et al. 2018). One of the critical factors that lead to the development of many different illnesses, such as neural, renal, hepatic disease, and diabetes, is oxidative stress in long-term exposure to stress. Several natural agents could exhibit therapeutic effects against neurodegenerative diseases (Gonchar et al. 2018). The most abundant polyphenol in green tea, Epigallocatechin-3-gallate (EGCG), is a powerful antioxidant and shows extensive anti-oxidative, anti-inflammatory, and immunomodulatory pharmacological properties. (Chen et al. 2016). Studies in rodents and humans propose that EGCG may recover oxidative stress, hyperglycemia, and insulin resistance and enhance glucose tolerance in type 2 diabetes (Pham-Huy et al. 2008). Available literature revealed little about EGCG protective effects against damage induced by chronic restraint stress in rats. So, this study presented here aimed to demonstrate the protective anti-diabetic, anti-inflammatory, and anti-oxidative effects of EGCG against harmful biochemical and histological effects on the pancreas of immobilization stress rat model.

Materials and methods

Animals

We ensured the rights of the animals as specified by the Guide of National Institutes of Health for the Care and Use of Laboratory Animals. We got an endorsement from the ethical committee guidelines of Taif University, Saudi Arabia, for our experiment. All efforts were achieved to dismiss suffering.

Forty healthy male albino Wistar rats aged 2 months, weighing from 200 to 250 g, were placed in stainless steel rat cages (five rats per cage) and had free access to food and water. Animals were kept at 12:12 hours light-dark cycles and a steady temperature of 23 ± 1 °C all the time. The animals were weighed on the 1st, 15th, and 30th days of the investigational time by a numerical scale (Tanita, Japan, 1.00 g).

Immobilization stress

Animals were subjected to stress for 6 hours a day over 30 days. The animals were immobilized inside plastic tubes dimensioned to create stress without stimulating pain (6 cm in diameter x15 cms long) individually. We avoided undesirable stress as much as possible by delicate handling (Bitgul et al. 2013).

Drug

EGCG was purchased from Sigma Aldrich (St. Louis, MO, USA). EGCG was dissolved in sterile distilled deionized water as a stock solution of 10 mM at −80 °C until dilution before use, as previously described. EGCG was administered to the rats by oral gavage at a dose of 100 mg/kg body weight (Hsieh et al. 2009) for 30 consecutive days before the induction of stress.

Grouping

Rats were haphazardly allocated into four groups (10 animals each).

Control group

Rats were not exposed to stress and allowed to move freely. Rats in the control group were treated with sterile-distilled water by oral gavage (vehicle control for EGCG).

EGCG group

Rats were not exposed to stress and allowed to move freely and received EGCG (100 mg/kg body weight) by gavage daily for 30 days.

Stressed group

Rats were exposed to immobilization stress.

Stressed with EGCG group

Rats were exposed to immobilization stress and received EGCG 30 minutes before every exposure to stress (100 mg/kg body weight) by gavage daily for 30 days.

Sample preparation

Following gentle ether anesthesia, blood samples were obtained by tail cutting on the 1st day, 15th day, and then on the 30th day of the experiment, next to overnight 16 hours fasting. Blood of 1 ml was assembled in an Eppendorf tube containing 5-μl heparin (5000 IU/ml) and then centrifuged at 3000 ×g for 5 minutes at 4 °C. Plasma was separated and reserved at −70 °C for the determination of glucose, insulin, and corticosterone levels. Twenty-four hours next to the last EGCG administration and immobilization by the end of the 30-day trial period, rats were anesthetized with diethyl ether and sacrificed by removal of blood via cardiac puncture till death (exsanguination).

Biochemical measurements

Fasting blood glucose (FBG) was determined by Glucose Oxidase assay kit (Fluorometric) (ab138884), and insulin concentration was determined using the Rat/Mouse Insulin ELISA kits (Sigma, St. Louis, MO, USA.) according to manufacturer instructions. Insulin resistance was estimated by the following equation (Wallace et al. 2004): homeostasis model assessment for insulin resistance (HOMA-IR) = [fasting glucose (mg/dL) × insulin (μU/mL)]/405. Plasma corticosterone was investigated by the rat corticosterone RIA kit (DRG, Cat. # RIA-1364, Germany). Intra-assay and inter-assay coefficients of variations for glucose were 2.04 and 9.88%, for insulin 10.6 and 10.8%, and for corticosterone measurements 7.1and 6.5%.

Light microscopic study

The specimens of each animal were collected from the pancreas and divided into two halves. The first halves of the dissected pancreas were fixed in 10% formalin and dehydrated, cleared, and paraffinized. Slices of 5 μm thickness were prepared and stained with hematoxylin and eosin (H&E) to prove histological details and Masson’s trichrome to evaluate collagen fibers (Bancroft and Gamble 2008) and immunohistochemical study for insulin detection.

Immunohistochemical staining

Paraffin sections were prepared on coated slides. They were deparaffinized in xylene then rehydrated in descending grades of alcohol. Sections were treated with 0.3% hydrogen peroxide for 30 min to hinder endogenous peroxidase activity. The sections were incubated for 1 hour with monoclonal mouse antisera against human insulin protein at a dilution of 1:100. The slides were washed in phosphate-buffered saline and then incubated with the secondary antibody (biotinylated goat anti-mouse IgG) for 1 hour and then washed in phosphate-buffered saline. Sections were counterstained with hematoxylin.

Morphometric study

Five nonoverlapping microscopic fields from five paraffin blocks were arbitrarily chosen from each group. The area percentage of a standard area equal to 125,352.4 μm2 were chosen from the anti-insulin antibody immune-stained fields and Masson’s trichrome stained fields using the Image-Pro Plus image analyzer computer system (Media Cybernetics, Rockville, MD, USA).

Measurement of lipid peroxidation

The second halves of the dissected pancreas were left in ice-cold potassium phosphate buffer (50 mM, pH 7.4) with one mM EDTA then rapidly homogenized to measure lipid peroxidation in homogenized tissue samples stored at −80 °C.

MDA levels were determined by consuming the thiobarbituric acid (TBA) fluorometric assay with excitation wavelength (532 nm) and emission wavelength (547 nm) and 1, 1, 3, 3-tetra ethoxy propane (MDA precursor) (Wasowicz et al. 1993). Concisely, a tissue sample (50 ml) was added to distilled water (1 ml) and then mixed with equal volumes of TBA in acetic acid (29 mM). Samples were cooled after 1 h of incubation at > 95°C, and then 25 ml of 5-mM HCl was added. The last reaction mixture was extracted with n-butanol (3.5 ml), and the butanol phase was split through centrifugation at 1500 × g for 5 min.

Measurement of pancreatic antioxidant enzyme activities and cytokines

The pancreatic tissue homogenates were centrifuged (10,000 × g, 15 min, 4 °C) to measure the activities of antioxidant enzymes and cytokines, and then supernatants were stored at −80 °C intended for advanced analysis. The activities of antioxidant pancreatic enzymes were assessed, consuming the available kits, and matched to the manufacturer’s guidelines, catalase (CAT) (ab83464; Abcam), reduced glutathione (GSH) (Abcam) assay kits, and superoxide dismutase (SOD) (Abcam). Pancreatic IL-1β, IL-2, IL-6, and TNF-α levels were measured by ELISA according to the guide of manufacturer of the available kits from Peprotech, Rocky Hill, NJ, USA. Serum amylase and serum lipase are measured by enzymatic calorimetry using the available kits from Bio-Assay, USA.

Statistical analysis

Statistical Package for the Social Sciences software (SPSS version 22, Inc., Chicago, IL, USA) was used in the evaluation of data. Data were tested for normality by the Kolmogorov-Smirnov test. Statistical differences of data within the groups were tested by one-way ANOVA and Tukey-Krammer post hoc test. P < 0.05 was statistically significant.

Results

Histological results

Hematoxylin and eosin-stained pancreatic sections

Histological examination of the control pancreas revealed the pancreatic lobules with its endocrine and exocrine portions of the pancreas. The endocrine part was represented by the islets of Langerhans embedded between the acini in clusters. They are round to oval in shape and were lightly stained with H&E. The control group showed the islets consisted of central beta and peripheral alpha cells and blood capillaries that were scattered between islet cells (Fig. 1a). The islet of Langerhans of stressed group showed cytoplasmic vacuolations in many cells. Some cells exhibit deeply stained eosinophilic cytoplasm with pyknotic nuclei, whereas other cells showed karyolitic nuclei. The blood capillaries were dilated (Fig. 1b). However, the stressed with EGCG group showed cells with granulated cytoplasm, the central beta cells with pale rounded vesicular nuclei, and the peripheral alpha cells with a darkly stained peripheral nuclei (Fig. 1c).

Fig. 1.

Fig. 1

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photomicrographs of H & E stained sections of rat’s pancreas from different groups showing an islet of Langerhans. a. Control group: rich in blood capillaries (C) surrounded by many pancreatic acini (PA), alpha cells (A) and beta cells (B). b. Stressed group: cytoplasmic vacuolation (V), deeply stained eosinophilic cytoplasm with pyknotic nucleus (arrow head), karyolitic nucleus (arrow) and dilated blood vessel (BV). c. Stressed with EGCG group: apparent normal alpha cells (A) and beta cells (B)

The lobules were separated by delicate connective tissue septa that contained blood vessels. The exocrine portions of the pancreas consisted of acini and intermingled with the pancreatic ducts (Fig. 2a). The stressed group showed many acinar cells with cytoplasmic vacuolations and pyknotic nuclei. The blood vessels were congested (Fig. 2b). Moreover, focally situated homogenous acidophilic edematous material were noticed between the acini (Fig. 2c). Furthermore, many pancreatic ducts showed homogenous acidophilic exudate in their lumina. Some blood vessels showed thickened wall. Areas of inflammatory cell infiltration were frequently observed around the congested blood vessels and between acini (Fig. 2d). On the other hand, stressed with EGCG group showed preservation of most of the acini that were formed of truncated pyramidal cells with apical acidophilic (zymogenic granules) and basal basophilic cytoplasm that contained rounded basal nuclei with prominent nucleoli. The pancreatic duct and blood vessels were apparantally normal (Fig. 2e).

Fig 2.

Fig 2.

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photomicrographs of H & E stained sections of rat’s pancreas from different groups showing pancreatic acini. a. Control group: multiple acini (PA) separated by thin connective tissue septa contained blood vessel (BV) and intermingled with the pancreatic ducts (PD). b,c,d. Stressed group: b. Many acinar cells with cytoplasmic vacuolization (V) and pyknotic nuclei (arrows). Notice the congested blood vessel (BV) between acini. c. Homogenous acidophilic edematous material in between the acini (arrows). d. Homogenous acidophilic exudate in the lumen of the pancreatic duct (red arrow), thickened blood vessel (BV) and extravasation of inflammatory cells (black arrow). e. Stressed with EGCG group: apparent normal acini with intact pancreatic duct (PD) and blood vessels (BV). Notice the truncated pyramidal cell with basal rounded nucleus with prominent nucleolus (arrow)

Masson’s trichrome-stained pancreatic sections

In the control sections, the pancreatic collagen fibers were delicate in the septa, around the pancreatic acini, and around the blood vessels and intercalated ducts (Fig. 3a). In the stressed group, the dense collagen fibers were observed between the acinar tissues of the pancreas (Fig. 3b), around the pancreatic ducts and blood vessels (Fig. 3c). Sections of the stressed with EGCG group showed few collagen fibers deposition surrounding pancreatic acini and intercalated ducts (Fig. 3d).

Fig. 3.

Fig. 3

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photomicrographs of Masson’s trichrome stained sections of rat’s pancreas from different groups showing. a. Control group: delicate collagen fibers in the septa, around the pancreatic acini and around the blood capillaries (BV). b and c. Stressed group: b. Dense collagen fibers between the acinar tissues of the pancreas (arrow). c. Dense collagen fibers around the pancreatic duct (PD) and blood vessels (BV). d. Stressed with EGCG group: few collagen fibers inbetween the acinar pancreatic tissue and around the pancreatic duct (PD)

Anti-insulin-stained pancreatic sections

Sections of the control group revealed dark brown granular cytoplasmic immunoreactivity in most of the insulin-secreting cells of the pancreatic islets (Fig. 4a). Sections of stressed group revealed that most of islet cells showed negative immunoreaction (Fig. 4b). Others showed weak positive immunoreaction (Fig. 4c). Sections of stressed with EGCG group revealed that most of the islet cells were strongly positive for insulin immunostaining (Fig. 4d).

Fig 4.

Fig 4.

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photomicrographs of immunohistochemical staining for insulin antibodies of rat’s pancreas from different groups showing. a. Control group: pancreatic b-cells with dark brown granular cytoplasmic immunoreactivity. b and c. Stressed group: b. negative immunoreaction in the islet of Langerhans. c. weak positive immunoreaction in the islet of Langerhans. d. Stressed with EGCG group: strong positive immunoreaction in the islet of Langerhans

The histological results of the EGCG group were similar to the control group.

Morphometric results

Mean area percent of collagen fiberes and insulin immunopositive cells

Stressed with EGCG group revealed a significant decrease in area percentage of collagen fibers and a significant increase in the mean area percentage of insulin immunopositive cells when compared with stressed group (Table 1).

Table 1.

The mean values of area percent of collagen fibers and insulin immunopositive cells

Group Area percent of collagen fibers Area percent of insulin immunopositive cells Significance
Control group 3.6 ± 1.7 14.3 ± 1.4
EGCG group 3.8 ± 0.9 14.7 ± 1.2
Stressed group 11.4* ± 2.1 2.1* ± 0.72 Versus any of the other studied groups (P < 0.05)
Stressed with EGCG group 8.2* ± 1.3 9.2* ± 1.2 Versus control group P < 0.05
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*Statistically significant difference

Biochemical results

Effects of stress on body weight changes in different rat groups

The body weight increased significantly (P < 0.05) on the 15th and 30th day in the control group when compared to the 1st day. In spite of the increase in body weight in EGCG group in the 15th and 30th day, only the weight gain on the 30th day was significant (P < 0.05). The stressed group showed a significant (P < 0.05) decrease in body weight on the 15th day when compared with 1st day and when compared with control and EGCG group, but the weight was significantly (P < 0.05) increased on the 30th day. In the stressed with EGCG group, the body weight increased significantly (P < 0.05) on the 15th and 30th day in comparison with the 1st day, and the increase on the 15th day was significant in comparison with stressed group. On the 30th day, the body weight revealed a significant (P < 0.05) decrease in the three study groups as compared with the control group on the same day (Table 2).

Table 2.

Effects of stress on body weight changes in different rat groups

Day 1 Day 15 Day 30
Control group 267.32+/−7.37 290.22+/−12.07* 320.94+/−14.37*#
EGCG group 274.22+/−9.37 280.72+/−10.89 292.32+/−12.97*a
Stressed group 270.34+/−8.97 259.02+/−7.67*ab 290.02+/−11.07*#a
Stressed with EGCG group 269.32+/−7.79 279.72+/−9.77*c 284.02+/−9.87*a
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Data are expressed as mean ± SD. n = 10 rats. a = significantly different from control group, b = significantly different from EGCG group, c = significantly different from stressed group in the same day, * = significantly different from 1st day, # = significantly different from 15th day of the same group

The impact of stress on fasting plasma glucose, insulin, HOMA-IR and corticosterone concentrations

On the 1st day, there was no significant difference in FBG between the four groups. However, on the 15th day, there was no significant difference in FBG between control and EGCG groups. But, in the stressed group, FBG was significantly (P < 0.05) higher when compared with the control group and EGCG group. Treatment of stressed with EGCG group reduced significantly (P < 0.05) FBG level compared with the stressed group.

The stressed with EGCG group showed an insignificantly (P < 0.05) higher FBG compared with control and EGCG (Fig. 5a). On the 30th day, FBG in the stressed and stressed with EGCG groups returned back toward normal values (P < 0.05) compared with control and EGCG on the same day. In the control group, there was no significant difference shown in FBG on the 1st day, on the 15th day, and the 30th day. Also, in the EGCG group on the 1st day, on the 15th day, and the 30th day, there were no significant differences shown in FBG. In the stressed group, FBG level on the 15th day was significantly higher than on the 1st day. However, the FBG level on the 30th day was significantly lower than the level on the 15th day of the same group.

Fig. 5.

Fig. 5

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a-d: a = significantly different from control group, b = significantly different from EGCG group, c = significantly different from stress group, * = significantly different from 1st day, # = significantly different from 15th day. e: a = significantly different from control group, b = significantly different from EGCG group, c = significantly different from stress group

In the stressed with EGCG group, there was no significant difference shown in FBG on the 1st day, on the 15th day, and the 30th day.

There was no significant difference in fasting plasma insulin (FPI) levels between the four groups on the 1st day. However, on the 15th day, in the stressed group, FPI was significantly (P < 0.05) raised when compared with the control group and EGCG group. Treatment of stressed rats with EGCG significantly decreased (P < 0.05) FPI level compared with the untreated stressed rats but insignificantly (P < 0.05) changed when compared with control and EGCG groups (Fig. 5b).

On the 30th day, in the stressed group, FPI showed insignificant differences (P > 0.05) when compared with the control group and EGCG group. However, the treatment of stressed rats with EGCG insignificantly decreased (P > 0.05) FPI level compared with the untreated stressed rats, but it showed an insignificantly (P > 0.05) higher FPI when compared with the control and EGCG groups (Fig. 5b).

No significant differences were shown in plasma insulin levels between the control group on the 1st day, on the 15th day, and the 30th day. Also, in the EGCG group on the 1st day, on the 15th day, and the 30th day, there were no significant differences shown in plasma insulin level.

In the stressed group, plasma insulin level on the 15th day and the 30th day was significantly higher on the 1st day. However, the plasma insulin level on the 30th day was significantly lower than the level on the 15th day.

In the stressed with EGCG group, plasma insulin level on the 15th day was significantly lower than on the 1st day. However, the plasma insulin level on the 30th day did not change significantly when compared with the 1st and the 15th day.

There was no significant difference in HOMA-IR between the four groups on the 1st day. However, on the 15th day, in the stressed group, HOMA-IR was significantly (P < 0.05) raised when compared with the control group and EGCG group. Treatment of stressed rats with EGCG significantly decreased (P < 0.05) HOMA-IR compared with the untreated stressed rats but insignificantly (P > 0.05) changed when compared with control and EGCG groups (Fig. 5c).

On the 30th day, in the stressed group, HOMA-IR showed insignificant differences (P > 0.05) when compared with the control group and EGCG group. However, the treatment of stressed rats with EGCG insignificantly decreased (P > 0.05) HOMA-IR level compared with the untreated stressed rats, but it showed an insignificantly (P > 0.05) higher HOMA-IR when compared with the control group and EGCG group (Fig. 5c).

No significant differences were shown in HOMA-IR between the control group on the 1st day, on the 15th day, and the 30th day. Also, in the EGCG group on the 1st day, on the 15th day, and the 30th day, there were no significant differences shown in HOMA-IR.

In the stressed group, HOMA-IR on the 15th day and the 30th day was significantly higher on the 1st day. However, the HOMA-IR on the 30th day was significantly lower than the level on the 15th day.

In the stressed with EGCG group, HOMA-IR on the 15th day was significantly lower than on the 1st day. However, the HOMA-IR on the 30th day did not change significantly when compared with the 1st and the 15th day.

There was no significant difference in fasting plasma corticosterone levels between the four groups on the 1st day. However, on the 15th day, there were no significant differences in fasting plasma corticosterone levels in the EGCG group when compared with the control group. While in the stressed group, fasting plasma corticosterone levels were significantly (P < 0.05) higher when compared with the control group and EGCG group. Treatment of stressed rats with EGCG reduced considerably (P < 0.05) fasting plasma corticosterone levels compared with the untreated stressed rats. However, the stressed with EGCG group showed insignificantly higher fasting plasma corticosterone levels compared with control and EGCG groups (Fig. 5d).

On the 30th day, there was no significant difference in fasting plasma corticosterone levels between the 4 groups. No significant differences were shown in fasting plasma corticosterone levels between the control group on the 1st day, on the 15th day, and the 30th day. Also, in the EGCG group on the 1st day, on the 15th day, and the 30th day, there were no significant differences shown in fasting plasma corticosterone levels. In the stressed group, fasting plasma corticosterone levels on the 15th day were significantly higher compared with its value on the 1st day. However, fasting plasma corticosterone levels on the 30th day were significantly lower than the level on the 15th day. In the stressed with EGCG group, there were no significant differences in fasting plasma corticosterone levels on the 1st day, 15th day, and the 30th day.

There was no significant difference in pancreatic IL-1β, IL-6, and TNF-α levels between the EGCG group when compared with the control group. While in the stressed group, pancreatic IL-1β, IL-6, and TNF-α levels were significantly (P < 0.05) higher when compared with the control group and EGCG group. Treatment of stressed rats with EGCG reduced significantly (P < 0.05) pancreatic IL-6 and TNF-α levels compared with the untreated stressed rats. However, the stressed with EGCG group showed significantly higher pancreatic TNF-α levels when compared with the control and EGCG groups (Fig. 5e).

The impact of stress on oxidative and anti-oxidative stress parameters of the pancreatic tissues

The EGCG group showed no significant differences in oxidative/anti-oxidative stress parameters as compared with the control group. The stressed group showed significantly elevated (P < 0.05) pancreatic MDA and NO levels. However, it revealed a significantly lower (P < 0.05) content of GSH and lower activities of SOD, CAT when compared to the control and EGCG groups. (Table 3). Moreover, treatment with EGCG has significantly lowered the pancreatic MDA and NO levels and elevated the content of GSH and the activities of SOD and CAT.

Table 3.

Effects of stress on oxidative/antioxidative stress parameters of the pancreatic tissues

Parameters
Control group EGCG group Stressed group Stressed with EGCG group
MDA (nmol/mg protein) 1.26 +/−0.11 1.31 +/−0.13 4.45+/−0.44 ab 1.36+/−0.16c
NO (μmols/L) 5.00 +/−1.75 5.16 +/−1.34 20.88+/−4.66 ab 5.96+/−1.92c
GSH (μg/mg protein) 6.92 +/−1.07 6.37 +/−1.65 3.12+/−0.59 ab 7.01+/−1.08c
SOD (units/mg protein) 4.79 +/−0.46 4.91 +/−1.50 2.24+/−0.70 ab 4.90+/−1.13c
CAT (μmoles of H2O2) 6.47 +/−1.52 6.31 +/−1.65 2.98+/−0.55 ab 6.14+/−1.16c
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Data are expressed as mean ± SD. n = 10 rats. a = significantly different from control group, b = significantly different from EGCG group, c = significantly different from stressed group

Serum markers of exocrine pancreatic injury

In the stressed group, there was a significant increase in the exocrine pancreatic injury markers, serum amylase, and serum lipase, when compared with the control group. In EGCG group, there was no significant change in serum amylase and serum lipase levels compared with the control group, while the stressed with the EGCG group reversed the effect of stress and produced a significant decrease in their serum levels if compared with the stressed group (P < 0.05) (Table 4).

Table 4.

Effects of stress on parameters of exocrine pancreatic tissue damage

Parameters
Control group EGCG group Stressed group Stressed with EGCG group
Serum amylase level (U/L) 13.9 ± 1.2 14.8 ± 1.9 38.9 ± 3.1ab 21.7 ± 1.8abc
Serum lipase level (U/L) 497.8 ± 19.6 512.4 ± 30.6 1450.5 ± 63.2ab 770.3 ± 24.8abc
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Data are expressed as mean ± SD. n = 10 rats. a Significantly different from normal control. b Significantly different from EGCG group. c Significantly different from stressed group

Discussion

Both human and animal studies propose that EGCG could correct oxidative stress, hyperglycemia, insulin resistance, and dyslipidemia and improve glucose tolerance in type 2 diabetes (Roghani and Tourandokht 2010). However, little is known about the effect of EGCG on pancreatic adverse effects of chronic stress in rats. So, the current study aimed to explore these potential protecting properties of EGCG against the harmful effects of chronic restraint stress in rats.

In the current study, the untreated stressed rats showed a significant (P < 0.05) decrease in body weight on the 15th day when compared with 1st day and when compared with control and EGCG group, but the weight was significantly (P < 0.05) increased on the 30th day. The weights of the stressed group were significantly less than the control group on the 15th and 30th days. In the agreement with our results, In the agreement with our results, the weights of the animals of stressed and control groups increased significantly, but the increase in the control group was significantly bigger on 15th day and on 30th day (Zardooz et al. 2006b). The “negative energy balance” following stress exposure is suggested to be related to the activity of corticotropin-releasing hormone (CRH) and subsequent corticosterone increment. CRH acts as an anorexigenic neuropeptide which causes a reduction of food intake and body weight in the stressed rats (Leibowitz and Wortley 2004). This result agrees with our results and may explain the decrease body weight of the stressed rat on the on the 15th day. On the 30th day, the body weight revealed a significant (P < 0.05) decrease in the three study groups as compared with the control group on the same day. Green tea polyphenol has its beneficial effects on reducing body weight through regulating anti-inflammation, anti-oxidant capacity, estrogen related actions, and obesity-related genes in high-fat-induced obese rats (Lu et al. 2012). In the present study, on the 15th day of stress exposure, FBG, FPI, HOMA-IR, and fasting plasma corticosterone were significantly higher (P < 0.05) in the stressed group when compared with 1st and 30th day of the same group as well as when compared with control and EGCG groups.

A previous study revealed that 15 days of stress exposure significantly increased plasma level of glucose as compared with day 1 with no significant effect on plasma glucose concentration on 30th day. In the same study, acute and chronic psychological stress did not change fasting plasma glucose, insulin levels, HOMA-IR index, and basal (before stress) plasma corticosterone concentrations, but only FPI and plasma corticosterone concentrations levels (after stress exposure) were significantly elevated on the 1st day of the experiment as compared with the corresponding control group and as compared with values before exposure (Rostamkhani et al. 2012).

However, in another study, chronic stress exposure showed an increment in blood glucose and insulin reduction in the stressed group on the 15th day of the experiment with increased fasting plasma corticosterone (before stress) as compared to their 1st day values and as compared to the controls which return to the values approximately similar to those previous to stress exposure on the 30th day (Zardooz et al. 2006a).

The elevated glucose levels observed during stress might be explained by the hyperglycemic effect of stress hormones, catecholamines, and glucocorticoids. Glucocorticoids stimulate pancreatic β cells, possibly by increasing sensitivity to glucose, in addition to their indirect stimulation of insulin secretion by induction of insulin resistance (Nakamuta et al. 2005). The secretion of the catecholamines stimulates glycogenolysis and increases the basal metabolic rates and productions of glucose and insulin as well (Teague et al. 2007).

The diverse-detected results may imitate different threshold responses of stress level, variance rates of glucose as well as insulin production or further intrinsic metabolic alterations between animals used in the different studies. In the chronically stressed rats of the present study, FBG, FPI, HOMA-IR, and fasting plasma corticosterone were significantly lower on the 30th day when compared with 15th day of stress exposure. By increasing the days of exposure to stress, the catecholamines’ response may be adapted and consequently may cause the plasma glucose level on the 30th day not to change considerably. It is remarkable that the designs of corticosterone and catecholamine of sympathoadrenal response and adaptation to intermittent stress are dissimilar (de Boer et al. 1989).

The stressed with EGCG group significantly lowered FBG, FPI, HOMA-IR, and fasting plasma corticosterone on the 15th day of the experiment in comparison with the stressed group. EGCG treatment in the present study was accompanied with enhancing of the function of beta-cell as verified by strong positive immunoreaction and increased mean area percentage of insulin immunopositive cells in the islets of Langerhans. This finding suggests the anti-apoptotic, cytoprotective, anti-inflammatory, and antioxidant effects of EGCG on the islets improving their mass and function. Similar effects of EGCG were noticed when the administration of EGCG significantly reduced blood glucose levels and significantly increased pancreatic insulin content in high-fat rats and increased islet number, islet area, and beta-cell area compared with high-fat controls (Pathak et al. 2017).

The EGCG treatment in the present study decreased the elevated fasting plasma corticosterone levels in stressed rats, especially on the 15th day. The corticosterone lowering effect of EGCG coincides with that observed in a previous study where mice treated with EGCG and displayed reduced corticosterone concentrations and significantly decreased 11 β-Hydroxysteroid dehydrogenase type 1 staining in the pancreas compared with high-fat groups (Hintzpeter et al. 2014). In accordance with the present study, EGCG reversed significantly elevated circulating plasma corticosterone concentrations in vitro (Hintzpeter et al. 2014).

The restraint stress is accompanied by disturbed glucose homeostasis, which can be attributed to dysfunction of the pancreatic cell following their degenerative changes, particularly the β-cell, caused by oxidative stress. Stress increased ROS ultimately results in decreased expression of glucose transporters in the cellular membrane (Hintzpeter et al. 2014).

The current study revealed that the stressed group showed degenerative changes in the Langerhans islets as vacuolated cytoplasm, deeply stained eosinophilic cytoplasm, pyknotic nuclei, and karyolitic nuclei. Moreover, insulin immunostaining cells were markedly decreased compared with the control group.

The main event and the possible cause of stress-induced degenerative changes in the pancreatic islets could be mediated by the increased production of ROS due to oxidative stress confirmed by the significantly elevated MDA level in the pancreas tissue, decreased SOD and CAT activities, and decreased GSH content. The pancreatic β-cells are highly subjected to oxidative stress due to diminished expression of their antioxidant enzymes. Moreover, damage and apoptosis could be due to the direct destruction of DNA by ROS (Lutgendorff et al. 2008). In two previous studies, male Fischer rats (Oishi et al. 1999) and male Wistar rats (Zaidi et al. 2003), there was a significant increase in plasma lipid peroxidation during and after stress when immobilized with stainless wire gauze for 6 hours.

In the present study, the observed cytoplasmic vacuolation of the islet and pancreatic acinar cells in the untreated stress rats could be attributed to the endoplasmic reticulum (ER) stress that causes extensive ER dilatation. Pancreatic acinar cells and β cells are more vulnerable to ER stress for the reason that they have high secretory functions and little antioxidant capacities (Pandol et al. 2011). The homogenous acidophilic edematous material in the parenchyma of the pancreas of the stressed rats in this study could be explained by Reilly’s phenomenon where sympathetic activation causes a disturbance in the microcirculation of many organs. This leads to alternating periods of tissue ischemia and reperfusion, which instantly damages capillary endothelium, causing an increase of venous permeability, stasis, and slugging of blood cells (Crestani 2016). Moreover, the degenerative changes and necrosis could also be due to the ischemia because of the thickened and hyalinized blood vessels (Pushparaj et al. 2000). In the current study, EGCG treatment preserved the architecture of the pancreas. This may be attributed to the anti-oxidative and anti-inflammatory effect of EGCG and its ability to decrease lipid peroxidation. Besides, a previous study concluded that the anti-oxidative and anti-inflammatory actions are the underlying protective mechanisms of the green tea catechins (Shimizu et al. 2015).

The EGCG antioxidant action is mediated directly by scavenging ROS and chelation of metal ions and indirectly by enhancing the production of antioxidant enzymes, e.g., SOD, CAT, and glutathione peroxidase and hindering pro-oxidant enzymes, e.g., nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase (Youn et al. 2006). A previous study revealed that EGCG enhances the expression of catalase and superoxide dismutase antioxidant enzymes in the pancreas of non-obese diabetic mice consuming 0.2% EGCG daily, probably in part through MAPK pathway signaling (Dickinson et al. 2014). Administration of EGCG significantly decreased the level of serum nitric oxide and the MDA content and increased the activities of SOD, CAT, and the content of GSH in sodium arsenite-treated mice. EGCG improved the function of mitochondria, reduced mitochondrial respiration inhibition, and conserved the NADH pool leading to high ATP (Yu et al. 2017).

It was strongly suggested that the few ROS produced by EGCG is essential for its antioxidant efficacy as those ROS can create several signal pathways enhancing cellular protective mechanism (Elbling et al. 2010).

Not only is the endocrine part affected by immobilization stress but also the exocrine part, as evidenced by elevated pancreatic enzymes and atrophy of exocrine tissue and dense collagen fibers between the acinar tissues of the pancreas and around the pancreatic duct and blood vessels. This might be explained by the absence of trophic action of insulin (Czakó et al. 2009). The treated stressed group in the present study showed minimal deposition of collagen fibers around pancreatic acini and intercalated ducts. EGCG could significantly diminish the collagen distribution and inhibit cell proliferation and inhibited the synthesis of collagen in the liver of a rat model (Nakamuta et al. 2005).

The two pancreatic enzymes, amylase and lipase, were considered consistent biomarkers of laboratory diagnosis for the exocrine pancreatic injury in both humans and animals; their serum concentrations rise within hours of the pancreatic injury (Carroll et al. 2007; Smith et al. 2005). There was a great evidence that the destruction of the pancreatic acinar cells resulted in increased serum pancreatic enzyme levels as in pancreatitis and pancreatic cancer (Muniraj et al. 2015).

A recent study revealed that in stressed rats, there was a significant increase in serum amylase and lipase levels compared with the control group indicating the marked stress-induced injury in the exocrine pancreatic tissue. (Elbassuoni and Abdel Hafez 2019). Pancreatic lipase (PL) is an enzyme secreted into the duodenum that plays a key role in digestion and absorption of fats and is a target for intervention (Birari and Bhutani 2007; Torgerson et al. 2004). The enzyme first rapidly hydrolyzes triglycerides to diglycerides and then slowly to a monoglyceride (Beck 1973).

α-Amylase, a major pancreatic protein and starch hydrolase, is essential for energy acquisition. Mammalian pancreatic α-amylase binds specifically to glycoprotein N-glycans in the brush-border membrane to activate starch digestion, whereas it significantly inhibits glucose uptake by Na+/glucose cotransporter 1 (SGLT1) at high concentrations (Date et al. 2015). However, a previous study on the effect of green tea catechins against PL found catechins have a relatively weak inhibition on PL (effective concentrations >1 mmol/l) (Ikeda et al. 2005). It is well known that starch is primarily metabolized by α-amylase resulting in the formation of glucose and maltose. A previous study examined the inhibitory effect of EGCG and (−)-epigallocatechin (EGC) on α-amylase activity in a cell-free system. Both EGCG and EGC inhibited α-amylase activity with EGCG being the more potent inhibitor (Forester et al. 2012).

A prominent outcome of the present study is that stress significantly augmented pancreatic IL-1β, IL-6, TNF-α, MDA, and NO and significantly lowered GSH, SOD, and CAT when compared with control and EGCG groups. Moreover, the histological results showed interstitial edema, vascular congestion, and mononuclear cellular infiltration in the stress group. A recent study revealed that stressed rats displayed a significant increase in the pancreatic MDA and TNF-α, as compared with the control group (Elbassuoni and Abdel Hafez 2019). Furthermore, stress-induced oxidative stress and generation of ROS, proved in the current study and described in previous studies, might also be involved in the increase of all inflammatory parameters (Soliman 2012). In the agreement with our results, severe stress over activates the immune system, leading to the imbalance of inflammation and anti-inflammation (Miller et al. 2009). It was stated that ROS release from exposure to stress could be implicated in the enhancement release of almost all inflammatory mediators in pancreatic tissue (Fonseca et al. 2010). Also, ROS might directly damage DNA with subsequent enhanced apoptotic process. Furthermore, stress-induced increase in TNF-α was strongly suggested to be the cause of apoptosis in pancreatic cells (Binker et al. 2010). Chronic stress appeared as a risk factor to develop pancreatitis by sensitizing the exocrine pancreas through TNF-α (Binker and Cosen-Binker 2014). It is reported that experiencing chronic mild stress influences immune signaling, as demonstrated by changes in cytokine composition. Earlier studies revealed that stress increases IL-6 in rodents (Voorhees et al. 2013) and humans (Rohleder et al. 2012). Moreover, TNF-α, a pro-inflammatory cytokine released from pancreatic cells, explains the inflammation and degeneration of these cells (Binker et al. 2010). EGCG treatment showed a significant decrease in pancreatic IL-1β, IL-6, and TNF-α as compared with the untreated stressed group. Accumulating evidence has revealed that EGCG could inhibit the overexpression of pro-inflammatory mediators and inhibits the release of inflammatory factors in rats (Aneja et al. 2004). EGCG treatment showed a noticeable improvement of the vascular congestion and mononuclear cellular infiltration that may be attributed to the reduced release of IL-6 and TNF-α and, therefore, the inhibition of neutrophil aggregation, so minimizing the endothelial injury induced by ROS (Cho et al. 2008). EGCG can scavenge the ROS/RNS, so decreasing the production of inflammatory factors. Moreover, EGCG regulates the expression of the pro-inflammatory genes in vascular endothelial cells (Babu and Liu 2008). In addition, it was found that EGCG attenuated the inflammatory response in the respiratory passage by its ability to reduce the IL-8 release by the balance of vasodilation and vasoconstriction substances (Kim et al. 2006).

In the current study, the immobilization stress-induced oxidative stress increased the generation of ROS and enhanced the release of almost all inflammatory mediators in pancreatic tissue together with influenced immune signaling, as demonstrated by changes in cytokine levels. The antidiabetic effect of EGCG treatment which may be attributed to the significant decrease in corticosterone level and the enhancement of the beta-cell function suggests the cytoprotective, anti-inflammatory, and antioxidant effects of EGCG on the islets improving their mass and function. Also the noticeable improving effect of EGCG on the vascular congestion and mononuclear cellular infiltration reflects its minimizing effect on the pancreatic damage induced by ROS.

Conclusions

The presented data investigated the effects of immobilization stress on the pancreas and suggested the promising role of EGCG as a protective agent by its anti-diabetic, anti-inflammatory, and anti-oxidative stress properties. EGCG might be recommended for immobilized patients.

Compliance with ethical standards

Conflict of interest statement

No conflict of interest

Footnotes

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