The potential effects of pomegranate peel extract and bee venom in improving the diabetes induced damaging of spiral artery

Diabetes Mellitus is a public health condition associated with increased blood sugar level and impair insulin secretion affected nearly 184 million peoples with diabetes in 2013, reaching 288 million by 2030 [1]. It is known that early gestation is critical especially during organogenesis due to a higher susceptibility of embryonic tissues to alterations in the intrauterine environment such as diabetes and obesity [2] . The endometrium gives the demand for menstruation, implantation and placentation [3]. Uterine spiral arteries facilitated the nutrient supply for the placenta and fetus. During gestation, they undergoe remodeling directed by maternal natural killer cells and embryo-derived invasive trophoblast lineages [4].During early organogenesis, the embryo depend mainly on maternal nutrition through the Abstract

trophoblast cells, the elementary components of the placenta, at the maternal-fetal interface. Maternal blood bathes trophoblast directly and induces the reconstruction of the uterine spiral artery that facilitates the fl ow of nutrients from the distal maternal portion of the uterine spiral artery to the placenta [5]. The distal uterine spiral artery facilitated remodeling trophoblast invasion into the vascular wall and widening of the vascular lumen [6,7]. The vasculature and development of the spiral artery explains the successful fetal growth [8].
Failure differentiation of the spiral arteries is associated with uteroplacental ischemia and development of acute atherosis in the spiral arteries of the decidua parietalis or basalis leading to preeclampsia [9]. Diabetes was found to reduce the volumes of both the intervillous space and the villous membrane specifi c diffusing capacity [10]. The vascular wall distensibility was increased associated with reduction of the wall stiffness in diabetes [11,12]. Two of the 18 diabetic patients developed intramural fi brosis in the decidual portion of the spiral artery. Hyperglycaemia and abnormal level of C-peptide values in amniotic fl uid and cord blood had been infl uenced in the induction of spiral artery lesions [13] The Pomegranate fruit's peel, seeds and juice are rich in antioxidants Pomegranate (Punica granatum L.), Punica granatum Linn. (Family , Punicaceae ), is commonly known as pomegranate. It is the old native tree of the Mediterranean regions especially Egypt [14]. In vivo studies revealed that consumption of pomegranate juice improved the diabetic associated oxidative stress in placenta. Moreover, pomegranate juice reduced in vitro oxidative stress, apoptosis, and global cell death in trophoblast cultures subjected to hypoxic stress [15]. Wild type (WT, C57Bl/6J) and eNOS −/− mice supplemented orally pomegranate juice during utero life reduced relaxation of uterine arteries in response to oxidative stress [16] .
Phenolic compounds of pomegranate, have been found to show anti-oxidant activities and activated the reactivity of isolated blood vessel in rats as well as reduce the systolic blood pressure in both a hypertensive rat model [17].
Diabetic rats received interperitoneal injection of 5mg BV/kg every day for four consecutive weeks improved blood glucose and serum triglyceride and total cholesterol level [21].
In addition, BV treatment demonstrated a high therapeutic potential for improved improved wound healing in diabetic mice through increased collagen and BD-2 expression and restored Ang-1 and Nrf2 levels. This was affected by the activities of antioxidant enzymes in the damaged tissue and the elimination of chemokines [22]). The present study explored the antidiabetic capacity of either bee venom -treatment or pomegranate supplementation on the spiral artery of 14days old placenta of mother rats.

Pomegranate peel extract and applied dose-treatment
Pomegranate fruits were purchased from the market. Their

Bee venom-treatment
Bee venom was supplied from Bee Lab, Faculty of Agriculture, Assiut University. A known weight of the bee venom was dissolved in saline solution and interperitoneally administered at single dose of 0.12mg/kg body weight at the 6 th day of gestation.

Induction of diabetes
Experimental type 2 diabetes mellitus was induced by a single intraperitoneal injection of streptozotocin (60mg/kg, diluted with buffer, pH 4.5) and 100mg nicotinamide /kg body weight. Control animals were received physiological saline.
Hyperglycemia was assessed by ranging the blood sugar level within the limits of 240-280mg/dL by glucometer.

Histopathological observations
In placenta of control and pomegranate extract treatment, the spiral artery is characterized by dilated lumen. The wall is composed of tunica intima line with endothelial cells and thin layer of elastic fi ber.
It is surrounded by intima tunica formed by a thin layer of smooth muscle fi bers and accompanied by a thin adventitia tunica ( Figure 1A).
In bee venom-treated mother's placenta, the spiral artery's tunica media and adventitia showed dense cellularity.
In placenta of diabetic mother, the spiral artery attained In placenta of diabetic mother and received bee venomtreatment, there was a dense collection of infl ammatory cells in both the tunica media and adventitia. The lumen of the spiral artery showed congestion associated with protrusion of the tunica media inside the luminal cavity ( Figure 1E ).
However, there was apparent improvement of the spiral artery of diabetic mother received pomegranate-extract treatment. The blood vessel dilated and the endothelium become regenerated ( Figure 1F ).

Immunohistochemistry of caspase 3
Following examining placenta of control, pomegranateextract treatment and bee venom-treatment mother, the spiral arteries showed missing immunohistochemical reaction ( Figure 2A,B). However, bee venom-treated placenta possessed mild dark-brown immunohistochemical reaction in the tunica media and adventitia ( Figure 2C).
In diabetic placenta, there was a detected dense darkbrown immunohistochemical reaction in the tunica intema and endothelial layer ( Figure 2D). Image analysis showed marked increased intensity of caspase 3 in spiral artery of diabetic mother compared to less degree in those of diabetic mother and bee venom-treatment.
However, there was a detected negative immunostaining activity in the other studied groups (Figure 3 ).

Discussion
The placenta is a main structural organ involved for fetal growth. It supplied the nutrients, oxygen and steroids required for differention of fetal organs [23]. Uterine spiral arteries allowed placenta and fetus nutrient supply.
They undergo remodeling during gestation, directed by natural maternal killer cells and invasive trophoblast lineages derived from embryos [14]. The present fi ndings were consistent with Bjork, et al., [13], whom reported two of the 18 diabetic patients showed Abnormal spiral artery was also detected in 69/203 of obese cases (33.9%) [24].
High fat-fed dams showed delayed invasion of trophoblast due to over expression of smooth actin around the placental spiral arteries. This led to impairment of remodeling of the spiral artery and consequently deformed the placent and increased fetal mortality [25] .
It is known that diabetes was associated with altering maternal environment and increased oxidative stress [26], resulting in retarding their remodeling and impaired uteroplacental function leading to preeclampsia [21] .
The diabetic associated deformation of the spiral artery associated damage of endothelium and vascular dysfunction predicted the early sign of preeclampsia . These may result from reduction of nutrients like calcium, protein, vitamins and essential fatty acids and consequently impair the trophoblast invasion [27].
On the other hand, two trials of treatments were carried out by either oral administration of pomegranate peel extract and or intraperitoneal injection of therapeutic dose of bee venom. The observed improvement of pomegranate peel extract may result from pomegranate ellagic polyphenols which manage the blood glucose within almost the normal level. Also, they impair the diabetes associated infl ammation through decrease of tumor necrosis factor-alpha , interleukin-6 and C-reactive protein [28]. Diabetic patients received daily administration of three capsules containing 1g pomegranate seed oil showed improved fasting blood sugar level, Insulin concentration, HbA1C, and increased the GLUT-4 gene expression [29]. In vivo studies revealed that consumption of pomegranate juice improved the diabetic associated oxidative stress in placenta. Moreover, pomegranate juice reduced in vitro oxidative stress, apoptosis, and global cell death in trophoblast cultures subjected to hypoxic stress [15]. Wild type (WT, C57Bl/6J) and eNOS −/− mice supplemented orally pomegranate juice during utero life reduced relaxation of uterine arteries in response to oxidative stress [16].
The capacity of improvement of pomegranate extract exceed bee venom treatment. The tunica media and adventitia of the spiral artery of bee venom-treated placenta possessed densely grouping infl ammatory cells. Although bee venom-treatment was found to improve diabetes via managing blood sugar, triglyceride and total cholesterol level [30], it possessed sign of cytotoxicity manifested by a detected sign of infl ammation.
The author fi nally concluded that administration of pomegranate juice as a natural product may give suffi cient