فایل ورد کامل تاثیر سمیت سلولی نانوذرات نقره بر بافت بیضه: شواهد بیوشیمیایی استرس و بیان پروتئین Hsp70-2

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تعداد صفحات این فایل: ۲۰ صفحه

بخشی از مقاله انگلیسیعنوان انگلیسی:Cytotoxic Effect of Nanosilver Particles on Testicular Tissue; Evidence for biochemical Stress and Hsp70-2 Protein Expression~~en~~

Abstract

Lastly, there are growing evidences that nanosilver (NS) particles highly induce cytotoxic impacts in vitro and in vivo. Here, we analyzed the dose dependent effect of NS on histological changes, biochemical alterations and endocrine statuses, sperm parameters as well as chaperone Hsp70-2 expression. NS particles (50–۶۰ nm) were administrated in 3 doses of 0.5, 1 and 5 mg/kg, intraperitoneally, for 35 days. The 0.3 mL normal saline was administrated in control-sham group. Histological alterations, sperm parameters, serum levels of LH, FSH and testosterone were evaluated. Germinal and Leydig cells RNA damage, Leydig cells steroidogenic foci, the testicular and sperm total antioxidant capacity (TAC), malondialdehyde (MDA), nitric oxide (NO) levels, immunohistochemical (IHC) expression and mRNA level of Hsp70-2 were analyzed. The NS, dose dependently, resulted in enhanced germinal cells degeneration, necrosis, seminiferous tubules atrophy and decreased serum levels of LH, FSH and testosterone. Elevated germinal and Leydig cells RNA damage associated with increased sperm abnormalities were observed in NS-treated groups. Expression of Hsp70-2 was up-regulated in 0.5 mg/kg, while its expression was decreased in 1 and 5 mg/kg NS-treated groups. Testicular and sperm TAC levels reduced. However, the MDA and NO levels significantly (P < 0.05) increased in all NS-treated groups. No histological and biochemical changes were detected in control-sham group. In conclusion, the NS particles exert their pathological impact via affecting testicular antioxidant and endocrine statuses, which in turn lead to diminished expression of Hsp70-2. Ultimately, by this mechanism NS particles adversely impact the cellular RNA, DNA and protein contents.

Discussion 

Many previous studies showed that NS particles exert degenerative impacts on liver, kidney, nervous tissue (Reddy et al., 2008; Limbach et al., 2007) and even respiratory system (Aaseth et al., 1981). In present study, we extended the evidences to show NS-treated adverse impact at testicles and sperm levels. The NS-treated animals exhibited reduced percentage of TDI, RI, SPI, elevated germinal cells RNA damage and lowered sperm quality. To understand involved mechanism(s), we analyzed the testicular endocrine activity by assessing the Leydig cells histological features, steroidogenic activity as well as RNA damage. On the other hand, we focused on biosynthesis of chaperone Hsp70-2 at different layers of the germinal epithelium as well as Leydig cells. Moreover, the mRNA level of Hsp70-2 was assessed in all groups. Observations revealed that NS, dose dependently, increased RNA damage both at Leydig and germinal cell levels and reduced Leydig cells distribution as well as steroidogenic activity. The NS-treated animals exhibited significantly lowered Hsp70-2 expression at higher doses. Finally, our data showed that administrated NS down-regulated antioxidant status and elevated MDA and NO contents both at testicular and sperm levels. As a primordial finding, our analyses showed that the NS directly impacted the testicular tissue. Accordingly, the total body weight of the NS-treated animals did not significantly differ with those in control-sham group. Meanwhile, the testicular weight relative to body weight was remarkably decreased versus control-sham animals. These alterations may be attributed to degenerative effect of NS particles on testicular tissue. It is well known that the gonadotropins LH and FSH are the major endocrine regulators of spermatogenesis. The LH stimulates the Leydig cells in order to synthesis androgens (mainly testosterone) and FSH targets the Sertoli cells to regulate the spermatogenesis (Shan et al., 1995). Significant reduction at serum levels of LH and FSH suggests that NS affects the testicular endocrine potential via systemically influencing the pituitary-gonadal axis. This hypothesis supported by diminished steroid foci in Leydig cells and reduced testosterone level. On the other hand, our histological analyses showed that NS, in a dose dependent manner, decreased Leydig cells number per one mm2 of the connective tissue, enhanced hypertrophied Leydig cells distribution and resulted in severe RNA damage in these cells. Therefore, we can conclude that further to NS-induced derangement in pituitary-gonadal axis, it was able to reduce the testicular endocrine status by directly influencing the Leydig cells. In order to understand the mechanism governing the NS impact on testicular endocrine status, one should note that there is an appositional correlation between androgen evacuation and increasing oxidative stress. As a result for androgen deficiency-induced oxidative stress, the apoptosis rate increases in germinal cells (Lue et al., 1990; Agarwal et al., 1994; Dierich et al., 1998). On the other hand, the pathologically produced oxidative stress affects all damaged and intact cellular DNA, RNA, and protein contents and eventually results in protein and lipids peroxidation (Agarwal et al., 1994; Agarwal et al., 2008). In order to show these impairments, we analyzed testicular and sperm TAC levels. Our biochemical analyses showed that, NS downregulated the TAC both at testicular and sperm levels. Previous researched have shown that pathologically increased reactive oxygen and nitrogen species (ROS/RNS) including; superoxides, peroxides and nitric oxide (NO), result in cellular apoptosis/necrosis via inducing structural and biochemical changes (Calabrese et al., 2000; Forlenza and Miller, 2006). To confirm these findings, the NO content of the testicular tissue assessed and observations demonstrated that NS, dose dependently, enhanced the NO level. Therefore, we can come close to this fact that, the elevated NO content and reduced TAC level coordinated with endocrine deficiency-induced damages, and provoked cellular degeneration in NS-treated groups. Increased percentage of tubules with reduced TDI, RI and SPI indices confirmed this hypothesis. Although in physiological condition the Hsp70-2 involves in intacytoplasmic proteins assembling, folding and refolding processes (Matsumoto M, Fujimoto, 1990; Shonhai et al., 2007), under different stress conditions this chaperone plays an essential role in homeostasis (Shonhai et al., 2007). Accordingly, biosynthesis of the Hsp70-2 protein in testicular tissue alters depending on free radicals generation ratio (Zhu et al., 1997) and indirectly changes depending on androgens withdrawal (Ricci et al., 2004; Khosravanian et al., 2014). Our IHC and semiquantitative RT-PCR analyses showed that in low dose NS-exposed animals (0.5mg/kg) the expression of Hsp70-2 increased versus control-sham group. However, medium (1mg/kg) and high (5mg/kg) dose NS-treated animals exhibited significantly reduced expression of the Hsp70- 2 both at IHC and mRNA levels. Considering these finding, we can suggest that under lower stress condition (low dose NS-induced NOS/ROS stress and androgen evacuation), the Hs70-2 overexpression was occurred in testicular tissue in order to control the stress-induced derangements. In contrast, at higher dose levels, the NS acted via different mechanism. Indeed, different stimulant agents, including superoxide, free radicals and NO adversely impact the cellular protein structures even the Hsp70-2 (Kaur and Bansal, 2003). Beside this fact, it should be noted that the NS particles are capable to highly bind to proteins, lipids and DNA and consequently result in remarkable peroxidation at these levels (Lamb et al., 2010). Thus, we can hypothesis that, in high dose NS-treated animals the NS alone-induced damages in association with ROS/NOS-induced impairments leaded to remarkable damage at protein and RNA levels of Hsp70-2. Decreased biosynthesis and mRNA levels of Hsp70-2 associated with significant reduction at total RNA and protein levels confirmed this hypothesis. It has been reported that the spermatocytes, with lack of Hsp70-2 cannot complete the meiosis and are subsequently deleted by apoptosis (Tesarik, 1998). Therefore we can suggest that, the NS resulted in severe damages at spermatocyte cell levels (Marked with diminished TDI and SPI indices) via affecting the expression and/or biosynthesis of Hsp70-2. Moreover, function and expression of the Hsp70-2 changes during late stages of spermiogenesis process and becomes specifically associated with spermatid-specific-DNA-packing proteins (Govin et al., 2006). In other words, the synthesis of DNA-packing transition proteins 1 and 2, and protamines 1 and 2 largely depend on Hsp70-2 chaperones expression (Zhu et al., 1997; Govin et al., 2006). Our analyses for sperm nuclear maturation showed that the percentage of sperms with chromatin condensation decreased depending on administrated doses of NS. Thus, it can be concluded that, the NS reduced protamination partly by down-regulating the Hsp70-2 expression. In this regard, the sperms with impaired packing of DNA are highly susceptible against oxidative and nitrosative stresses (Malekinejad et al., 2012; Razi et al., 2011). In the line with this issue, the percentage of sperms with DNA damage was increased in NS-treated animals. Intensive arrangement of unsaturated fatty acids, plasmalogenes and sphingomyelins in cell membrane of the sperms are responsible to make these cells susceptible against free radicalsinduced lipid peroxidation (Sanocka and Kurpisz, 2004). In order to identify the effect of NSinduced oxidative stress on lipid peroxidation at sperm level, we analyzed the epididymal sperm viability and the MDA content. Observations revealed that the sperm viability decreased in NStreated animals and the MDA level increased depending on administrated doses. More analyses for sperm TAC showed a remarkable decrease in TAC level of sperm samples from NS-treated groups. Therefore, it would be more logic to hypothesis that, the NS-induced lipid peroxidation associated with NS-caused oxidative stress resulted in loss of the sperm viability that consequently leaded to loss of sperm motility (note Fig 12). Current hypothesis has been illustrated with other studies about the effect of free radicals on sperm viability and motility (Sanocka and Kurpisz, 2004; Aticken and Sawyer, 2003).

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