Role of Green Tea or Bone Marrow Mesenchymal Stem Cells against Skin Damage Induced in Male Albino Rats Exposed to Gamma Radiation

Background: Green tea intake is accompanied with a lower incidence of cancer, cardiovascular disease and neurodegenerative disorders; hence green tea extract has been included as dietary supplement along with other supplements and multivitamins. Aim of the work: This study aimed to investigate the histological and histochemical changes in the skin of male albino rats after exposure to gamma radiation and the possible preventive and therapeutic effect of green tea and Bone Marrow Mesenchymal Stem Cells (BMSCs). Material and methods: The current study was carried out on 40 adult male albino rats. They were divided into 4equal groups. Group one (G1) comprised control rats. Group two (G2) comprised of rats exposed to a single dose of gamma-radiation. Group three (G3) had rats treated with green tea extract in a dose of 35 mg/kg body weight daily one week before and one week after irradiation. Group four (G4) contained rats treated with BMSCs 5 hours after they were irradiated with gamma-radiation. Histopathological and histochemical changes in skin of albino rats were studied. Results: Rats exposed to gamma radiation showed several histological and histochemical changes in their skin. These changes were improved by using either green tea or bone marrow mesenchymal stem cells. BMSCs showed more obvious therapeutic effect than green tea extract. Conclusion: The present work showed that both green tea and BMSCs had preventive and therapeutic effect upon skins of albino rats after they were irradiated with gamma radiation.


Introduction
Th e skin is the largest organ of the body, comprising between 15 and 20% of the total body weight. It performs numerous functions, including thermo regulation, sensory perception, excretion and absorption as well as protection from insults, dehydration and infection. It consists of two tissue types; the epidermis (an external stratifi ed, non-vascularised epithelium) and dermis (underlying connective tissue, consisting largely of dense fi brous components produced by fi broblasts) [1].
Radiotherapy is one of the commonly used modality for cancer therapy. However it is usually associated with injures of the surrounding normal tissues [2,3]. In fact, radiation-induced skin injury is the most common complication of radiotherapy [2,3]. Ionizing radiation has suffi cient energy to displace the orbital electrons surrounding the nucleus. Th is displacing action in living tissues results in DNA damage through direct and indirect eff ects [4]. Direct damage occurs due to DNA strand breakdown while indirect eff ect occurs via generating reactive oxygen species (ROS) or free radicals [4]. Th e major targets for ROS include proteins, lipids, nucleic acids and DNA-protein cross-linking. ROS also induce lipid peroxide production [5,6]. Th ese toxic products disturb the balance of antioxidant defence systems of the body [7,8]. Mesenchymal stem cells are a population of adult stem cells which are a promising source for therapeutic application. Th ese cells can be isolated from the bone marrow. Th ey can be easily separated from the Hematopoietic Stem Cells (HSCs) due to their plastic adherence [9]. Adult bone marrow MSCs can diff erentiate into many mesenchymal cell types such as osteocytes, chondrocytes and adipocytes [10]. Th ey can also diff erentiate into some non-mesenchymal cells such as neural cell under appropriate experimental conditions [10].
Camellia sinensis O. Kuntze belonging to Th eaceae family (commonly Known as green tea in English) has antioxidant, anticarcinogenic, antiviral, and bactericidal properties [11]. Green tea is a rich source of polyphenols (consisting of avanol monomers (avan-3-ols) also referred to as catechins) which are antioxidants in nature [12]. Th ese natural antioxidants from green tea extracts have recently attracted considerable attention for preventing oxidativestress related diseases such as cancer, cardiovascular and degenerative conditions [13]. Th e exact mechanism of protection exerted by green tea is unclear but it has been suggested that it might be due to the antioxidant eff ect of its polyphenols and catechins [14][15][16].
Many studies have shown that the polyphenolic fractions isolated from green tea inhibit oxidant stress and possess anti-infl ammatory activity [17].
Th is study aimed to investigate the histochemical and histological changes in the skin tissue of male albino rats which exposed to gamma radiation and the possible protective role of both green tea and BMSCs.

Material and Methods
Experimental animals: A total of 40 male Swiss albino rats (Sprague dawley strain weighting 130±5 gm) were obtained from the Egyptian Organization for Biological Products and Vaccines. Th ey were kept in the laboratory for 15 days under observation to acclimatize. Th ey were housed collectively in plastic cages, maintained under standard conditions of light, ventilation, temperature and humidity and allowed free access to standard pellet diet and tap water.
Gamma-irradiation procedure: Irradiation process was performed using Gamma Cell-40 as observed by Egypt's National Center for Radiation Research and Technology, Cairo. Th e gamma cell-40 is a caesium-137 irradiation unit manufactured by Atomic Energy commission of Canada. Th e unit provides means for uniform Gamma-irradiation to small animals or biological samples while providing complete protection for operating personnel. Th e radiation dose level given was 3Gy as a single dose provided at rate of 0.54 Gy/ min. Th e radiation dose level was 3Gy single dose.
Green tea extract: was obtained as 300 mg tablets synthesized by MEPACO-Egypt with the name of Multi-Treat. Th e tablets were crushed and the required amount was dissolved in distilled water. It was given orally in a dose of 35mg/kg body weight daily from one week before to one week aft er the irradiation of rats.

Mesenchymal Stem Cells (BMSCs) Transplantation:
Bone marrow mesenchymal cells were obtained from donor rats which were selected from the same breed and strain as the experimental rats. Th e donor rats were sacrifi ced, their femur bones dissected out and cleaned. Bothe ends of these bones were chipped by bone nibbling forceps. Th e marrow was blown off of the femur into saline solution under sterile conditions. Th e solution was thoroughly mixed by drawing and expelling it several times by the syringe without needle.

Experimental design
Th e experimental animals were randomly divided into 4 groups (n=10) as following.
Group 1: Control rats (G1): normal healthy rats left without any treatment.
Group 2: Irradiated group (G2): rats exposed to a single dose of gamma-radiation, 3 Gy. Group 3: Irradiated rats+green tea treatment (G3): rats of this group were treated with green tea in a dose of 35 mg /kg body weight daily one week before to one week aft er irradiation.

Group 4:
Irradiated rats+BMSCs injection (G4): rats of this group were irradiated with a single dose of gamma-radiation 3Gy and then treated with transplanted (BMSCs) 3×106 cells/ml suspension through caudal vein about 5 aft er radiation exposure.
Th e experimental rats were sacrifi ced at 7 days post irradiation.
Histological and histochemical techniques: Skin tissue were immediately excised and fi xed in 10% neutral formalin. Paraffi n sections (5 μm in thickness) were prepared for processing the histological and histochemical studies. For general histology, sections were stained with Harris' hematoxylin and eosin [18]. Collagen fi bers were detected by using Mallory's trichrome stain [18].
Polysaccharides were detected by using periodic acid Schiff 's (PAS) reagent [18]. Toluidine blue stain was used for detection of mast cells infi ltration [18]. Total proteins were detected by using the mercury bromophenol blue method and DNA material was detected by using Feulgen's method [18].

Histopathological observations of the skin
Hematoxylin & Eosin stained section: In Group 1 animals (G1 control group), observation of the skin sections stained with H & E revealed normal histological appearance of epidermis and dermis i.e. thin epithelium, regularly distributed hair follicles and glands in dermis ( Figures 1A and 1B). Epidermis was composed of stratifi ed squamous epithelium having four layers of keratinocytes. Th e underlying papillary layer of dermis had abundant capillaries and connective tissue cells whereas the inner reticular layer was composed of a denser connective tissue rich in fi bers. Dermis contained sweat and sebaceous glands and hair follicles surrounded with arrector pili muscle (Figures1A and 1B).
Observation of skin sections from group 2 animals (G2 irradiated group) showed many pathological changes. Th ere was discontinuation of epidermal cells, loss of hair follicles and sebaceous glands, dermal cell swelling as well as collagen fi ber edema ( Figure 1B). Th ere was pyknosis and karyolysis of epidermal cells, corneum detachment and disorganized papillary layer (Figures 2A and 2B).
In Group 3 (G3 irradiated+green tea treatment) animals observation of skin sections revealed partial return of the epidermal and dermal structure to normal histological pattern in (Figures 1C  and 2C). With Group 4 (G4, irradiated + BMSCs injection) animals similar observation indicated a marked return of the epidermal and dermal cellular structures to normal histological appearance ( Figures 1D  and 2D). Invasion of migratory stem cells into basal epidermal and dermal layers was also detected).

Mallory's trichrome stained section:
In Group 1 animals (G1 control group), observation of the skin sections stained with Mallory's trichrome revealed normal appearance and arrangement of collagen fi bers. Small collagen fi ber aggregates were seen just below the basal lamina of the epidermis. Th e fi bers appeared as fi ne loosely arranged network in papillary layer of dermis while in its reticular layer the fi bers became more abundant and united into thick irregular bundles ( Figure 3A).
Observation of skin sections from group 2 animals (G2 irradiated group) showed many irregularly arranged dense collagen fi bers below the basal lamina of the epidermis as well as in dermal papillary and reticular layers ( Figure 3B).
In Group 3 (G3 irradiated+green tea treatment) animals observation of skin sections revealed a moderate decrease in collagen fi ber content as compared to irradiated group ( Figure 3C).
In Group 4 (G4 irradiated+BMSCs injection) animals the collagen fi ber content showed a marked decrease in comparison to the irradiated group ( Figure 3D).

Toluidine blue stained section
In Group 1 animals (G1 control group), observation of the skin sections stained with Toluidine blue revealed moderate infi ltration of mast cells ( Figure 4A).
In skin sections from Group 2 animals (G2 irradiated group) the infi ltration was mild ( Figure 4B).
In Group 1 animals (G1 control group), observation of the skin sections stained with Toluidine blue revealed moderate infi ltration of mast cells ( Figure 4A).
In skin sections from Group 2 animals (G2 irradiated group) the infi ltration was mild ( Figure 4B).   Histochemical observations of the skin Polysaccharides: In Group 1 animals (G1 control group), observation of the skin sections stained with periodic acid Schiff 's (PAS) stain showed normal distribution of PAS +ve materials (magenta color). Th ere was a moderate staining affi nity of the basal lamina of epidermis as well as of papillary and reticular layers of dermis ( Figure 5A).
Th e same observation in Group 2 (G2 irradiated group) animals revealed a weak PAS reaction in the basal lamina of epidermis and dermal papillary and reticular layers ( Figure 5B).

Total protein:
In Group 1 animals (G1 control group),observation of the skin sections stained with mercury bromophenol blue stain showed normal distribution of total protein content. Th is was demonstrated by deep to moderately stained cells in epidermal basal lamina and the two layers of dermis ( Figure 6A).
In Group 2 (G2 irradiated group) animals the observation of skin sections stained with mercury bromophenol blue revealed a marked increase in amount of total proteins indicated by very deeply stained cells in the basal lamina of epidermis and dermal papillary and reticular layers ( Figure 6B).
In Groups 3 (G3 irradiated+green tea treatment) & 4 (G4 irradiated+BMSCs injection) animals the protein content was increased in basal lamina of epidermis and decreased in the papillary and reticular layers of dermis ( Figures 6C and 6D).

DNA:
In Group 1 animals (G1 control group), observation of the skin sections stained with c showed normal distribution of DNA content in the nuclei of cells of epidermis and dermal layers in the form of magenta color granules ( Figure 7A).
In Group 2 (G2 irradiated group) animals a noticeable increase in DNA content was detected in the nuclei of the epidermis and dermal layers ( Figure 7B).   In Groups 3 (G3 irradiated+green tea treatment) & 4 (G4 irradiated+BMSCs injection) animals a more or less normal appearance of DNA content was seen in the nuclei of epidermal and dermal cells ( Figures  7C and 7D).

Discussion
Experimental studies on animals have shown that exposure to ionizing radiation induces oxidative stress in diff erent tissues [19][20][21]. Many investigators have suggested that the generation of reactive oxygen species (ROS) aft er irradiation results in cyclic and long lasting up regulation of infl ammatory cytokines. It leads to the recruitment of infl ammatory cells such as neutrophils and macrophages. Th ese cells are responsible for the damage of tissues seen aft er radiation [22][23][24][25]. Long term exposure to ionizing radiation induced capillary reduction and severe atrophy in the dermis where as a short term exposure caused depigmentation of hairs and depletion of tissue stem cells [26].
Present study of ours also shows that the ionizing radiation caused oxidative stress and therefore the destructive eff ect on tissues with release of enzymes from organelles.
Our results shows an increased collagen fi ber content aft er irradiation which is in accordance to the study of Alkaabi who also detected increased collagen fi bers and glycogen content in skins of rats and those of their pups aft er radiation [27].
Our study results showed larger and irregular epidermal cells due to irradiation injury. Th is is in accordance to the work of Won et al. [28].
In our study the skins of the rats exposed to radiation and subsequently treated with green tea extract showed an improvement in the architecture of epidermal and dermal components with their appendages i.e. the hair follicles and sebaceous glands. Th ese results are in agreement to the work of Pazyar et al. who reported benefi cial eff ects of olive oil, ginseng, green tea and chamomile in the management of skin wounds [29].
An improvement of both DNA and total protein contents were noted in our results following green tea extract administration. Th is improvement may be due to the action of green tea on the skin tissue via DNA repairing system and enhancing protein synthesis. In addition, this improvement may also be due to the antioxidant activity of green tea where oleuropein stimulates endothelium formation as well as synthesis of mRNA and protein [30].
Attenuation of abnormal histological appearance of tissues following BMSCs injection as seen in our study was also reported by many authors. Th ey have attributed such recovery to the radical scavenging activities of BMSCs which prevent the accumulation of hydroxyproline in tissues [19,20,31].
In our study Bone Marrow Mesenchymal Stem Cells (BMSCs) treatment aft er irradiation improved PAS +ve materials, DNA and total protein content in the skin tissue compared to irradiated group. Similar results were reported in other studies as well [31].
Skin tissue restoration following gamma radiation exposure could be due to the therapeutic eff ect of BMSCs. As the stem cells can be transplanted to replace non functional or lost stem cells in tissues to enhance tissue healing and restore their original functions [21].

Conclusion
According to the results obtained in the current study administration of green tea or Bone Marrow Mesenchymal Stem Cells (BMSCs) provides good therapeutic eff ect against gamma radiation induced histological and histochemical alterations in skin of male albino rats. Th ese have a protective eff ect against skin tissue damage which may contribute to decrease in the risk for further skin disorders.