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 four 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.


Introduction
6-Mercaptopurine (6MP) was the first purine analog to prove beneficial for neoplastic diseases treatment [1]. It is used in cancer therapy to maintain the remission of acute lymphoblastic leukemia [2]. 6MP has a potent powerful immunomodulatory effect in treatment of inflammatory bowel disease, systemic lupus erythromatosus, organ transplant rejection and rheumatoid arthritis [3].
. 6MP mechanism of action is incorporation of 6-thiodeoxygoansine nucleotides into DNA during DNA synthesis process. The antagonistic effect of 6MP on purine bases could results in alteration in DNAprotein interaction or DNA mismatch repair, therefore induces cytotoxicity [1]. 25 mg of Eosin,5 gm of NaHCO3 in 100 ml water [15]. For sperm counting, 10 µl of the prepared mixture was transferred to the counting champers of homocytometer under light microscope (Sanli, China Mainland), stand for 5 minutes and count sperms at 40 X. For sperm viability determinations, equal volumes of sperm solution and Eosin-Nigrosin stain were mixed, spread thin film on clean slide and examined under light microscope to count dead (pink) sperms and live (unstained) sperms, then calculate the ratio of viable sperms in the sample [16]. Determination of sperms motility was done by addition of semen solution to Tris-buffer, and count motile sperms at 40 X in comparison with total sperms count. Abnormal forms were examined also at 40 X, and recorded to calculate its percentage [17].

Histopathological examination
Testis was fixed in 10% neutral formalin for 48 hours. And then processed according to Bancroft and Gamble (2008) [18], then washed in running water, graduated ethanol (50%, 70%, 95% then 100%) respectively, 2 h for each, cleared in xylene and embedded in paraffin wax at 70 O C, blocking in paraffin and then undermined in microtome. Tissues sections were mounted on 4 µ-6 µ on clean glass slides and stained by Hematoxylin and Eosin (H&E). Microscopically examined and photographed by using light microscope (Sanli, China Mainland). The histological sections were examined for the presence of congestion, tubular degeneration, cellular vacuolation and Pyknosis of cell lining seminephrous tubules. The histological changes were scored from 0 to 3 according to the histological findings, where 0 represents no pathological findings, and 1, 2 and 3 represent pathological findings in < 33%, 33-66% and > 66% of the testes, respectively. The scores for each parameter were calculated and the total scores were obtained and presented as means ± SEM. 2.7. Tissue preparation of the homogenate 0.5 g of testis was homogenized in 5 ml physiological saline and centrifuged at 20,000 × g for 30 min at 4 °C. The supernatants were collected and stored at − 80 °C for the assessment of reduced glutathione(GSH), glutathione reductase (GR), catalase, superoxide dismutase(SOD), lipid peroxidation (MDA), caspase-3, TNF-α 2.8. Sex hormones assay Enzyme Linked Immunosorbent Assay (ELISA) kit ( MyBioSource Company, USA ) was used for estimation of serum levels of testosterone, FSH ( follicular stimulating hormone) and LH ( leutinizing Hormone ) following the instructions of kits protocol, the principle is based on antigen antibody reaction.

Oxidant/antioxidant assay
The testicular content of lipid peroxidation represented in Malondialdehyde (MDA) was measured according to protocol described by Ohkawa (1979), assay is based on reaction of MDA with thiobarbituric acid to form reactive colored product, absorbance of colored product can be measured at 534 nm, concentration of MDA calculated from standard curve using standard concentrations [19].
Catalase activity in testis sample was estimated using hydrogen peroxide substrate according to instructions described by Aebi, H. (1984) [20]. Super oxide dismutase was estimated according to the method described by Koracevic et al.(2001) [21]. glutathione reduced (GSH) was assayed colorimetrically, follow the method of Beutler E. (1963), the method based on the reduction of 2nitrobenzoic acid through reaction with glutathione reduced to produce colored compound, reduced chromogen absorbance can be measured at 405 nm, and its concentration is directly proportional to the concentration of GSH [22]. Glutathion reductase (GH) assay was performed according to the method of Brigelius, et al [23] 2.10. ELISA assay for measuring Caspase-3 and TNF-α Caspase-3: was assayed by rat caspase-3 ELISA kit according to instructions of manufacturer MBS1602954 MyBioSource Company, USA Tumor Necrosis Factor-alpha (TNF-α) level: was estimated as a pro-inflammatory marker using rat TNF-α ELISA kits according to manufacturer's instructions of MyBioSource Company, USA (MBS 490379) [24] 2.11. Western blotting assay for assessing PI3K, AKT, Nrf2 and HO1 Testicular samples were homogenized using RIPA lysis buffer (buffer enables protein extraction from cytoplasmic membrane and nuclear proteins), the lysate was kept on ice for 30 minutes on shaker, then centrifuged at ~ 16,000xg for 30 minutes at 4 °C, cell debris was removed and supernatant was transferred to a new tube for further protein concentration determination analysis.
Quantitative estimation of protein was assayed according to instructions of Bradford Protein Assay Kit (Markham Ontario L3R 8T4 Canada). Extracted protein in each sample was added to equal volume of loading buffer(4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.125MTrisHCl, 0.004%bromophenol blue), the mix was boiled at 95 O C for 5 minutes. Protein in loading buffer were electrophoresed using 10% SDS PAGE then electro-transferred to polyvinylidene diflouride(PVDF) membranes .The membrane was blocked in tris-buffered saline with Tween 20 buffer and 3% bovine serum albumin (BSA) for 1 hr. The membrane was incubated overnight with anti PI3K, AKT, Nrf2 and HO1 antibodies (Thermofisher. USA) separately, Beta actin was used as control. After washing 4 times for 5 min each in DPBS (tween-20), the blot was incubated with the corresponding Goat anti-rabbit IgG for 1 h, washed and then developed, finally protein band images were recorded in BioMax film, and quantified using densitometry and Molecular Analyst Software (Bio-Rad, Richmond, CA, USA).

DNA fragmentation assay
DNA was extracted from testicular tissue by using chemical method of DNA purification [25], DNA fragmentation percentage was estimated by addition of tetron-X-buffer with vortex for fragmented DNA releasing, then addition of tetrachloroacetic acid(5%) and heated at 90 O C for 15minuts. Samples were measured calorimetrically with diphenylamine stain at 600 nm. Gel electrophoresis analysis for the DNA was done according to the method described by K Weber, M Osborn [26], finally gels were illuminated with ultraviolet light at 300 nm and photographic record was made to show the fragmented DNA in comparison with DNA ladder . TLT was added to 30 mg of testis tissues, the tissue was lysed and homogenized using homogenizer for 40 seconds, the lyset was centrifuged for 3 min and the supernatant was transferred into microcenterifuge tube, 350 µl of 70 5 ethanol was added to the cleared lysat on RNeasy spin column high to washed three times using wash buffer of the kits, RNeasy spin column was placed in a new 1.5 ml collection tube. 50 µl RNase-free water was added directly to the spin column membrane, and centrifuged for 1 min at ≥ 8000 rpm to elute the RNA. The concentration of RNA was obtained using nano drop spectrophotometry. (ii)The produced total RNA (0.5-2 µg) was used as a template for cDNA synthesis with random primers using high capacity cDNA reverse transcription kit( Fermentas, USA). cDNA master mix was prepared according to kit's instructions and was added to RNA-Primers mixture to produce cDNA. (iii)Real-time qPCR reaction were performed using (Applied Biosystems, USA) with software version 3.1 (StepOne™, USA)., gene expression technique employed following the method of Pfaffl (2001) [27]. The primer sequence was shown in Table 1. PCR components include 1 µl of forward primer, 1 µl of revers primer, 5 µl of cDNA template, 5.5 µl RNAase free water and 12.5 µl syber green mix. The total volume is 25 µl. Running 40 RT-qPCR cycles according to the condition of kits then data were calculated using version 3.1 (StepOne™, USA).,software, The relative expression of P53 and androgen receptor was calculated using the comparative Ct method. The values were compared to beta actin genes and reported as fold change Table 1 Primers of targeted genes (P53 and AR ) with β-actin AR primer Forward primer; 5′-TGTGGTCAAGTGGGCCAAG-3′; Reverse primer; 5′-TGCCATCTGGTCATCCACAT-3′. P53 primer Forward primer; 5'-CGCAAAAGAAGAAGCCACTA-3 Reverse primer; 5′-TCCACTCTGGGCATCCTT-3 Β-actin Forward primer; 5′-TGTTTGAGACCTTCAACACC-3′ Reverse primer; 5′-CGCTCATTGCCGATAGTGAT-3′

Statistical analysis
Results were analyzed using one-way analysis of variance (one way ANOVA) test and presented as mean ± standard error (SEM), and followed by Tukey-Kramer methods for post hoc analysis. A value of p < 0.05 was considered statistically significant.

Histopathological examination results:
The histopathological examination of rat testis from the control group ( Interstitial areas were widened with Leydig cells degeneration and presence of severe congestion (Black arrow). In RJ + 6MP group, histologically, the testicular tissue showed variable degrees of improvement to the degree to be similar to that of control group (Fig. 2d) 3.2. Effect of 6MP and RJ on testicular weights and epididymis length RJ alone gives the same results of safety such as control group without any significant differences, so they referred as control group.
Firstly we investigate the effects of 6MP alone and with RJ pre-treatment on testis weight, volume and tail of Epididymis of rats. Data in table (2) showed the significant decrease in testis weights, volume and in the length of epididymis in 6MP treated group at P < 0.05 compared to the control group. Prior administration of RJ in RJ + 6MP group evanished this decrement in compare to 6MP group by a significant degree at P < 0.05. Table 2 The weights and volumes of testes and epididymis of different treated groups. The results were expressed as means ± SE n = 10. The data were analyzed by using one way ANOVA test. a: indicates a significant difference compared to control at level p < 0.05. b: indicates a significant difference compared to 6MP at P < 0.05.

Effect of 6MP and RJ on various sperm parameters in treated rats
Administration of 6MP significantly decreased sperm motility %, sperm count and sperm viability% at P < 0.05 when compared with control group. Also 6MP caused a significant increase of sperm abnormalities % when compared to control group at P < 0.05. In RJ + 6MP group; RJ caused a significant increase of sperm motility %, sperm count and sperm viability% with a concurrent reduction of sperm abnormalities % compared to 6MP group at P < 0.05. Semen analysis of RJ group showed no significant difference compared to control at P < 0.05. Table 3 The changes in sperm motility, viability, abnormality percentage and sperm count in all examined rats The results were were analyzed by using one way ANOVA test and expressed as means ± SE, n = 10.
The data. a: indicates a significant difference compared to control at level p < 0.05. b: indicates a significant difference compared to 6MP at P < 0.05.
3.4 Effect of 6MP and RJ on testicular AR mRNA expression and serum testosterone, FSH and LH concentrations As shown in table (3); AR mRNA significantly decreased in 6MP group at P < 0.05 compared to control group, while pre-administration of RJ prevent the negative effect of 6MP on AR mRNA that revealed in significant increase of AR mRNA in RJ + 6MP group at P < 0.05 compared to 6MP group.
6MP treated rats showed a significant increase of FSH, LH and a significant decrease of testosterone level at P < 0.05, compared to control group. The levels of FSH and LH of RJ + 6MP group were significantly decreased and serum testosterone level was significantly increased at P < 0.05 when compared to 6MP group. The levels of AR mRNA expression and hormonal levels of RJ group and control are not significant at P < 0.05. The results were expressed as means ± SE n = 10. The data were analyzed by using one way ANOVA test. a: indicates a significant difference compared to control at level p < 0.05. b: indicates a significant difference at P < 0.05.

Effects of 6MP and RJ on testicular antioxidant/ oxidative redox.
The results in Fig. 3 depicted that administration of 6MP disturbed the testicular antioxidant/oxidative redox and caused a significant testicular oxidative stress. This effect was indicated by the ability of 6MP to cause a significant depletion of testicular GSH concentration, GR, Catalase and SOD activities with a concurrent increase of MDA level in 6MP group as compared with control group at P < 0.05. In RJ + 6MP group; pretreatment of RJ restored the testicular antioxidant/ oxidative redox. This protective effect was indicated by the significant increase of GSH concentration, GR, Catalase, SOD activities and the significant reduction of MDA level in RJ + 6MP group compared to 6MP group at P < 0.05. In addition, testicular antioxidant/oxidative redox of RJ group showed no significant difference compared to control group at P < 0.05.
3.6 The effect of 6MP and RJ on testicular PI3K, AKT, Nrf2 and HO1 of different treated groups Western blotting results (Fig. 3) showed that administration of 6MP significantly decreased the expression of testicular PI3K, AKT, Nrf2 and HO1 protein levels in 6MP group compared to control group at P < 0.05. RJ significantly increased PI3K, AKT, Nrf2 and HO1 protein levels in RJ + 6MP group compared to 6MP at P < 0.05. PI3K and AKT protein levels in RJ + 6MP group were significantly lower than control group at P < 0.05 while Nrf2 and HO1 protein levels of RJ + 6MP group showed no significant difference as compared to control group at P < 0.05.

3.7
The effect of 6MP and RJ on TNF-α, DNA fragmentation%, P 53 mRNA expression and caspase-3 of different treated groups.
Results in Fig. 4 showed that 6MP significantly increased TNF-α, DNA fragmentation %, P53 mRNA expression and caspase-3 in 6MP group compared to control group at P < 0.05. RJ administration reduced testicular inflammatory marker, DNA damage and apoptosis. This effect was indicated by the significant reduction of TNF-α, DNA fragmentation %, P53 and caspase-3 respectively in RJ + 6MP group compared to 6MP group at P < 0.05.TNF-α and DNA fragmentation % of RJ + 6MP group were higher than those in control group at P < 0.05. There was no significant difference between P53 and caspase-3 of RJ + 6MP and control group at P < 0.05.

Discussion
6MP is an active metabolite of azathioprine, clinically used in chemotherapy and immunosuppression.
Despite, the efficacy of 6MP in treatment, it has cytotoxic effect on normal body tissues [28, 29]. Our study insight into 6MP gonadotoxicity and infertility problems and the effect of RJ as a prophylactic agent through studying changes in sperm parameters and seminiferous tubules.
It is reported that the active metabolites of 6MP damages rapidly dividing cells, such as those in the bone marrow, intestinal epithelium, and reproductive organs of adults [30]. The reduction of the testicular and epididymal weight in 6MP treated rats indicated its obvious gonadotoxic effect. This effect was confirmed by the histological study of testicular tissues which showed a severe atrophy of many seminiferous tubules with wide interstitial spaces, few numbers of spermatozoa and spermatogenic cells, also morphology distortion and atrophy of Leydig cells. . In consistent with these reports our results showed that reduction of antioxidants was associated with DNA fragmentation and was accompanied by significant increased percentages of abnormal, dead and immotile sperms in 6MP treated rats Elevation of reactive oxygen species (ROS) is associated with Testicular inflammation, 6MP induced inflammation was evidenced through histomorphological changes of testicular tissues and elevated levels of TNF-α in testicular homogenate. TNF-α, produced by testis germ cells and Sertoli cells, is an essential cytokine that regulates multiple cellular processes in testis [38]. The susceptibility of spermatogenesis to TNF-α has been reported [39], our data clarify that significant increase in TNF-α in 6MP treated rats has associated with increasing abnormal forms of sperms and reduction in sperm viability. TNF-α in addition of being mediator of inflammation it also induces apoptosis [40][41][42].
To insight into 6MP-induced testicular apoptosis, we evaluated PI3K and AKT protein expression, proapoptotic caspase-3 activity and mRNA expression of P53. PI3K/AKT signaling pathway plays a pivotal role in cell apoptosis, proliferation and differentiation [43]. In addition, PI3K/AKT is related to the growth and development of testicular tissue and is involved in the interaction between FSH and Sertoli cells [44]. PI3Ks are a family of important lipid kinases. PI3K can be activated by cell surface receptor [45] and it can act as a messenger molecule involved in signal transduction which is the first regulator of AKT activation. The activated PI3K1 can make AKT transpose to the cell membrane and activate it. AKT, a serine-threonine kinase, is an important downstream protein of PI3K. AKT mainly exists in three forms: AKT1, AKT2 and AKT3. AKT1 is the main form in testis which located in spermatogenic cells and Sertoli cells. In accordance with our findings, Reggio et al., (2017), report that 6MP down regulated PI3K/AKT pathway [46].
Down-regulation of PI3K/AKT pathway can results in activation of caspase-3 and trigger apoptosis [47].
In support to the previous report this study illustrated that administration of 6MP for 20 days induced down-regulation of PI3K and AKT which was associated with high activity of caspase-3 and over expression of P53 in 6MP treated rats. In confirm to our result Kanemitsu, et al.,(2009) reported that 6MP induced apoptosis through P53, caspase-3 pathway [48].
It is reported that the overexpression of caspase-3 is associated with Leydig cell atrophy in 6MP treated rats [6]. Leydig cells are decisive to production of testosterone, which can effect on function of Sertoli cells. Atrophy of Leydig cells results in reduction of testosterone levels that also associated with elevated values of FSH and LH. Therefore premature detachment of spermatids may occur due to decrement in testosterone levels. Due to the essential role of testosterone in spermatogenesis, reduction of its level was associated with sperm abnormalities and reduced sperm count that was associated with Sertoli cell dysfunction, and impairment of spermatogenesis [31,49]. Leydig cells atrophy in 6MP treated rats was accompanied by significant decrease in serum testosterone levels in compare to other groups Herein, we evidenced the ameliorative effects of RJ in 6MP-induced testicular dysfunction and the ability of RJ to improve spermatogenesis process in RJ pretreated rats. The data of RJ + 6MP clearly revealed a significant increase in testicular weight, sperm count, motility and viability as well as reduction in abnormal forms of sperms which reversed the impairment occurred in 6MP treated rats.
The improvement of histomorphological data of RJ pretreated rats evidenced the ability of RJ for tissue repair and protection against tissue damage, in accordance with our finding Temamoğulları, et.al, (2018) report the protective effect of RJ against mice testicular degeneration and spermiotoxicity induced by flunixin [11].
Pre-administration of RJ increase serum testosterone and reduced the concentration of FSH and LH in RJ + 6MP group, RJ ability to increase serum testosterone levels might be due to Zn containing which has an essential role in spermatogenesis [50] Antioxidant activity of RJ has been reported [51][52][53]. In agreement with previous reports we evidenced that RJ pretreatment restored levels of SOD, catalase, GR and GSH to approach normal control levels and scavenged ROS, therefore reduced lipid peroxidation (low MDA level). The ability of RJ to scavenge the ROS might be due to RJ up-regulation of Nrf2 expression which increased significantly in RJ pretreated rats with concurrent increase in HO-1 protein levels in compare with 6MP treated rats. in consistence with our data Almeer, et al (2018) illustrate that RJ abrogated the testicular oxidative stress induced by cadmium through up-regulation of Nrf2 [54]. Nrf2 up regulation stimulates the expression of HO-1, up-regulation of Nrf2/HO1 pathway increases antioxidants activity and protects against tissue [55] damages, in agreement with this report our histopathological finding of RJ + 6MP revealed markedly improvement in seminiferous tubules appearance as well as Leydig cell numbers in compare with 6MP group. In addition to antioxidant activity of RJ, it has anti-inflammatory effects, Kohno, et al reported the inhibitory effect of RJ on pro-inflammatory cytokines by activation of macrophages [56], also report of Aslan, Z, et al who illustrated that RJ abrogated renal inflammation induced by ethylene glycol [57]. In the present study we found that rats which pretreated with RJ revealed markedly decreased TNF-α activity when compared with 6MP treated rats, this result evidenced the inhibitory effect of RJ on TNF-α which was associated with spermatogenesis enhancement.
Anti-apoptotic effect of RJ is supported by report of Karadeniz, A., et al., (2011) who suggest that RJ acts as anti-apoptotic agent and antioxidant when used in co-administration with cisplatin in kidney and liver [58]. In addition Valiollahpoor Amiri, et al (2016) report that RJ inhibit apoptosis by increasing the expression of BCL2 [59]. In present study we insight into the mechanistic pathway of RJ anti-apoptotic effect through following PI3K/AKT pathway and pro-apoptotic caspase-3 with deepen in P53 mRNA gene expression. Pre-administration of RJ induced up-regulation of PI3K/AKT pathway and caspase-3 inhibition. Data of RJ pretreated rats revealed that high protein levels of PI3K and AKT was associated with decrement in caspase-3 activity and P53 mRNA expression. In addition, histopathological data of RJ + 6MP appeared to restore normal number of Leydig cells and seminiferous tubule appearance that approach control data. Normal size and number of Leydig cells was observed in RJ pretreatment also testosterone levels was restored to approach normal control levels. The positive impact of RJ on Leydig cells could be due to suppression of caspase-3 activity, [6] reported that caspase-3 activity induced Leydig cell death .The possible mechanism of RJ protection against 6MP-induced apoptosis is the up-regulation of PI3K/AKT pathway and inhibition of caspase-3 activity. A recent study proved that PI3K/AKT pathway correlates with Nrf2 expression in testicular tissue and regulates the level of Nrf2-dependent inducible expression of HO-1 [60]. In support with the previous report, we reported that RJ up-regulation of PI3K/AKT pathway was concurrent with increasing in Nrf2 protein levels in RJ pretreated rats.
Finally, the present study proves that Royal Jelly is a safe product for albino rats and can be used in  Summary of the experimental design shows the different treated groups, specific dose and duration of each treatment.  Values are means ± SE, n = 10. a indicates a significant difference compared to control group at P<0.05. b indicates a significant difference compared to 6MP group at P<0.05.