Royal Jelly ameliorates 6-mercaptopurine induced spermatogenesis impairment and testicular apoptosis by regulating PI3K/AKT pathway in male rats
Testicular apoptosis is an obvious adverse effect of many chemotherapeutic agents.one of these chemotherapeutic drugs is 6-mercaptopurine (6MP) which has a powerful anticancer effect. On the contrary, it has an adverse effect on the male reproductive system. This study aimed to evaluate the prospective ameliorative effects of Royal Jelly (RJ) on 6MP induced testicular apoptosis and investigate the mechanistic pathway of protection. For this aim, forty male adult albino rats were divided into
... r equal groups (n= 10): control rats, RJ group (200 mg/kg.b.wt. of RJ for 30 day P.o.), 6MP group (5 mg/kg.b.wt of 6MP for 20 day P.o.), and RJ+6MP group pretreated with RJ (200 mg/kg.b.wt. for 10 day P.o.), and continued with 6MP (5 mg/kg.b.wt, P.o) for 20 day. After 30 days blood samples, epididymis and testis were collected to investigate sex hormones, sperm parameters, histological and molecular changes of testicular tissues, that include anti-oxidants activity, caspase-3, TNF-α, gene expression of Androgen receptors (AR) and P53 also protein concentration of PI3K, AKT, Nrf2 and HO1were estimated. The results of our study revealed that Pretreatment of Royal Jelly (RJ) abrogated 6MP induced spermatogenesis impairment by ameliorating sperm count, motility and morphology, regulating AR mRNA expression and sex hormones levels. RJ ameliorated testicular damage of 6MP exposed rats through restoring testicular antioxidant/oxidative redox, inhibiting caspase-3 activity and P53 mRNA expression as well as regulation of PI3K, AKT, Nrf2 and HO1 protein levels. Our data concluded that RJ protected testicular tissue from 6MP induced apoptosis by regulation PI3K/AKT pathway. Efficacy of 6MP is associated with various adverse effects on body organs induced toxicity as hepatotoxicity , bone marrow suppression and infertility  . 6MP induced male reproductive organ toxicity include testicular atrophy, Leydig cell failure and spermatogenesis impairment  . Recent trends in treating diseases promote uses of natural products to protect the body against the adverse effects of some chemotherapeutic drugs  . Royal Jelly (RJ) is one of the natural products which considered an effective adjuvant to chemotherapies. RJ is secreted by hypo-pharyngeal glands of worker honeybee and consider a principal food of queen honeybee, RJ components are important for cell growth and tissue repair, it is consists of water (50-60%), proteins (18%), carbohydrates (15%), lipids (3-6%), mineral salts (1.5%), and vitamins . Previous studies reports that RJ act as anti-inflammatory, antioxidant and lower serum cholesterol and glucose levels when used as adjuvant to certain drugs [9, 10]. RJ enhanced spermatogenesis and ameliorated the adverse effects some drugs on the reproductive organs [11, 12] . The underlying protective mechanism of RJ against 6MP induced testicular apoptosis and oxidative stress is not clearly studied. Therefore, our study focused on the impact of RJ to prevent reproductive complications in male albino rats when used as adjuvant to 6MP, and following the mechanistic pathway of protection through measuring biochemical, molecular, and histological changes occurred in the testis. Material And Methods Chemicals Royal jelly was obtained from GlaxoSmithKline Company, Egypt. RJ capsule (1 gm) was opened and dissolved in physiological saline (0.9% Nacl). 6-mercaptopurine (5 gm package) were purchased from Sigma-Aldrich Company, USA, 6MP powder was grinding then suspended in physiological saline (0.9% Nacl). MDA and antioxidant parameters were estimated using commercial kits (Bio-diagnostic for Research Kits, Egypt). Molecular kits were obtained from QIAGEN Company, USA. Other kits and tools were purchased from high quality sources Experimental animals Forty adult male albino rats, 8 weeks old, weighting an average 180-200 g were transferred to the animal house of biochemistry laboratory two weeks prior to the experiment initiation for acclimatization. The rats were housed under standard laboratory conditions maintained at 12-hour 4 light / day cycle and ambient temperature of 25 ± 2 O C. All experiments were conducted in accordance with the criteria of the investigations and Ethics Committee of the Community Laws governing the use of experimental animals of Beni-suef University. Experimental design: After the adaptation period, rats were randomly assigned to 4 groups (n = 10 rats): group I (control group); rats received saline orally by gastric gavage tube for 30 days, Group II (RJ group); rats received RJ (200 mg/kg B.W.) for 30 days , Group III (6MP group); rats received saline (0.9% Nacl) for 10 successive days followed by 6MP (5 mg/kg B. W.) daily for 20 day. Group IV (RJ + 6MP): Rats received RJ (200 mg/kg B.W.) daily for 30 days, at the 10th day rats administered 6MP (5 mg/kg B. W.) for 20 days 2.4. Blood sampling and tissue collection: After 24 hours from the last administered dose, under ether anesthesia, blood was collected from the medial canthus blood capillaries of the eye by capillary tube  in labeled dry centrifuge tubes which were leaved for coagulation then were centrifuged at 1000 g for 20 minutes. Clear sera were separated in labeled Eppendorf tubes and preserved in deep freezer (-80ᵒC) until analysis for detection of sex hormones. At the time of sacrifice, the animals were euthanized by cervical dislocation (dislocation of spinal cord column from brain by applying pressure to neck). Testis were dissected out and weighed. The right testis was immediately fixed in 10% neutral formalin for histomorphological study and the left testis was washed in physiological saline then silicate into two parts, the first part was homogenized by using homogenizer (Ortoalresa, Spain) for biochemical analysis. The second part was used for molecular investigation (real time PCR and western blotting techniques). Weighting testes, epididymis and Semen evaluation After scarification of rats, the testes were removed along with its epididymis and weighted separately. Also, The Testes and epididymis were separately immersed in 5 ml normal saline in a measuring cylinder and recording its volumes. The cauda epididymis were isolated and immersed immediately in physiological saline (0.9% NaCl ),then incubated to allow spermatozoa separation from epididymis tubules, supernatant was added by percentage (1:100) to solution contains 1 ml formalin (35%), performed the experiments. KS, AZ and NW assisted in the data analysis and helped prepare the manuscript. All authors read and approved the final manuscript.