Antiapoptotic and antioxidative effects of cerium oxide nanoparticles on the testicular tissues of streptozotocin-induced diabetic rats: An experimental study

Abstract Background Cerium dioxide nanoparticles (CNPs) due to the antidiabetic and antioxidant activities are proposed for the treatment of oxidative stress-associated diseases. Objective To examine the impact of CNPs on hyperglycemia-induced apoptosis and oxidative stress in the testis of diabetic rats. Materials and Methods Twenty-four male rats were divided into four groups (n = 6/each) as diabetic rats, CNPs group, diabetic + CNPs rats, and controls. The control group was fed only mouse food and water. Rats became diabetic through receiving streptozotocin (STZ) 60 mg/kg. CNPs were given to the rats at a dose of 30 mg/kg daily for 2 wk. Malondialdehyde and total thiol group (TTG) levels were measured using spectrofluorometer. Expression of b-cell lymphoma protein 2-associated X protein (BAX) and b-cell lymphoma protein 2 (Bcl-2) were investigated using quantitative real-time polymerase chain reaction. Western blot analysis was used to examine caspase 3 protein levels. Results The content of malondialdehyde significantly increased in the STZ-diabetic rats, while TTG levels demonstrated a remarkable decrease. Caspase-3, BAX, and BAX/Bcl-2 mRNA ratio raised significantly in the STZ-diabetic rats. On the other hand, Bcl-2 mRNA levels reduced in the testis of diabetic rats (p = 0.006). Intervention with CNPs caused a substantial increase in the TTG levels, while the malondialdehyde contents, caspase-3, BAX levels, as well as BAX/Bcl-2 mRNA ratio were considerably decreased following CNPs treatment. Administration of CNPs increased mRNA levels of Bcl-2 (p < 0.0001). Conclusion CNPs treatment attenuates testicular apoptosis and oxidative stress induced by diabetes. This nanoparticle might be suggested for the treatment of diabetes-associated reproductive disorders.


Introduction
Diabetes is associated with the impairment of semen quality, erectile dysfunction, testicular damage, decreased impotence, and fertility potential in men (1). A previous study on diabetes has shown that hyperglycemia may influence male infertility and reproductive complications via activation of oxidative stress and apoptosis (2). Recent studies have introduced different natural products with remarkable antioxidant and antidiabetic activities (5)(6)(7)(8). Cerium dioxide nanoparticles (CNPs), identified as nanoceria, frequently exhibit powerful medicinal effects on diabetes because of their self-regenerative antioxidant activity with minimum toxicity (5). The antioxidant characteristics of CNPs attribute to the electronic configuration at the nanoscale. A high proportion of surface area to the volume as particle size decreases, accompanied with a unique ability of quick and reversible switching between Ce3+ and Ce4+ oxidation states, (6) provide suitable "reactive sites" or "hot spots" for scavenging radicals and ROS (7). CNPs present effective antidiabetic action via modulation of nuclear factor kappa-light-chain-enhancer of activated B cells/nuclear factor erythroid-2-related factor 2 (NF-B/Nrf2) signaling, and significant abrogation of the apoptosis (8,9). Nanoceria, as a sensor for ROS, can reduce apoptotic cell death and reveals a pro-survival effect (10). There is no report on the impact of CNPs on the apoptosis and the thiol levels in the testicular tissues of the diabetic rats. Therefore, the present study was designed to examine the antioxidative and antiapoptotic potential of the CNPs as a therapeutic agent on the testicular injuries of the diabetic rats.

Quantitative real-time polymerase chain reaction analysis
The total RNA was extracted with RNX-Plus TM (Cinnagen, Iran), and any genomic contamination was checked using DNase 1 (Fermentas, Vilnius, and Lithuania). First-strand complementary DNA (cDNA) was synthesized from isolated RNA using superscript II and reverse transcribed by oligo (dt) primers (Fermentas, Vilnius, and Lithuania).
The expression of b-cell lymphoma protein 2 (Bcl-2) and Bcl-2-associated X protein (BAX) genes were examined by quantitative real-time PCR using specific primers prepared by the Bioneer Corporation under the PCR condition as described previously. Table I shows the characteristics of the primers. Beta-actin was used as an internal

Western blot analysis
Western blot analysis was performed to measure the protein expression levels of

Statistical analysis
Data were presented as mean ± SEM and analyzed with Social Sciences statistical package v. 16 (SPSS, Inc., Chicago, IL). One-way ANOVA with Tukey's post hoc test was used to compare the differences between the multiple groups. P < 0.05 was considered statistically significant. When CNPs were administered to the diabetic + CNPs group, a considerable enhancement in the concentration of TTG was noticed (p = 0.034, Figure 1B).  Figure 2B).

Effect of CNPs on Bcl-2 and BAX gene expression and BAX/Bcl-2 mRNA ratio
Furthermore, the BAX/Bcl-2 mRNA ratio in the diabetic rat was remarkably higher than controls (p < 0.0001). CNPs treatment of the diabetic rats resulted in a noticeable decline in the BAX/Bcl-2 mRNA ratio (p < 0.0001, Figure 2C).

WB analysis of Caspase-3 expression
The WB analysis showed a specific reaction

Discussion
In this study, the impact of CNPs treatment was evaluated on the apoptosis and oxidative stress status in the testicular tissue of the STZ-  It was reported that CNPs can enhance mitochondrial membrane potential and inhibit mitochondrial dysfunction caused by proapoptotic factors (25). ROS cause releasing of Ca2 + from the endoplasmic reticulum and mitochondria, which activates the mitochondrial Ca2 + uptake (28).
It has been shown that nanoceria by blocking the Ca2 + channels attenuates mitochondrial depolarization and apoptosis (29). Nanoceria scavenges ROS through the cycling between Ce3 + and Ce4 + states and mimics superoxide dismutase and catalase activities (30). Reduction of DNA fraction and decline in the ratio of necrosis and apoptosis, as well as enhancement of mitochondrial membrane potential, indicate apoptosis alleviation induced by ROS. These findings demonstrate that nanoceria is a novel nanoparticle with a strong ability to prevent oxidative stress injuries to the testicular tissue, which can be used as a therapeutic agent to control diabetes complications associated with the reproductive disorders (25).

Conclusion
In the present study, STZ-induced diabetic rats exhibited elevation of oxidative stress identified by higher levels of MDA and lower TTG levels. Moreover