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 agreement with our results, Karawya et.al; reported that azathioprine induce testicular atrophy, histological distortion and impair spermatogenesis in male albino rats [31].
Statistical analysis of semen parameters in 6MP treated rats revealed a severe reduction of sperm count, motility and viability, as well as an increase in abnormal forms of spermatozoa. This effect of 6MP could be explained by the ability of 6MP to interfere with nucleic acid synthesis of rapidly dividing somatic and germ cells resulting in germ cell apoptosis and germ cell mutations [32].
Our data clarify that 6MP treatment significantly decreased gene expression of ARs in 6MP treated rats. ARs are nuclear receptors highly expressed in testis and play a crucial role in spermatogenesis maintenance. The reduction of ARs and their knockout alters the reproductive development and spermatogenesis [33].
Spermatogenesis is highly susceptible to reactive oxygen species (ROS) levels, which increases spermatogenic cells apoptosis and induces seminiferous epithelium damage [34], testicular dysfunction and sperm abnormalities [35]. In the metabolic process of 6MP, xanthine oxidase converts 6MP to thiouric acid, a reaction capable of producing ROS, almost H2O2 [36]. We demonstrated that 6MP alters testicular antioxidant/ oxidative redox by decreasing testicular antioxidants; GSH concentration, GR, catalase and SOD activities with a concurrent increase of testicular lipid peroxidation by increasing MDA level. Protein concentration of Nrf2 and HO1 were significantly decreased in 6MP treated rats. Türk et al., (2010) proved the correlation between oxidative stress and sperm abnormalities [37]. 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 [40–42].
To insight into 6MP-induced testicular apoptosis, we evaluated PI3K and AKT protein expression, pro-apoptotic 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 [51–53]. 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 combination with 6MP to reduce or mitigate 6MP toxicity on testicular tissues through up-regulation of PI3K/AKT pathway, Nrf2/HO-1 pathway and down regulation of caspase-3, P53 gene expression and TNF-α. In confirm to our results Ahmed, W.M., et al., report that RJ prevent hepatotoxicity induced by azathioprine [13]. RJ acts as scavenger to ROS therefore decrease DNA fragmentation percent, increase antioxidants activity and decrease peroxidation levels in testicular tissues. The efficacy of RJ as anti-inflammatory, antioxidant and anti-apoptotic agent qualify it to attenuate chemotherapy induced toxicity and prevent testicular dysfunction caused by 6MP