Alpha-Lipoic Acid and Ginkgo Biloba Ameliorate Testicular Dysfunctions Induced by Silver Nanoparticles in Rats

Silver nanoparticles (AgNPs) are more commonly utilised in medicine, however they have negative effects on the majority of organs, including the reproductive system. AgNPs were reported to be able to reach the testes due to their small size, which allows them to pass through blood testicular barriers. The goal of this study was to see if LA (alpha lipoic acid) or GB (ginkgo biloba) might protect adult rat testes after intraperitoneal injection of AgNPs. Forty male healthy adult Wister albino rats were randomly assigned to four groups (10 rats each); control, AgNPs-intoxicated group intraperitoneally injected AgNPs 50 mg/kg b.w, 3 times a week, LA + AgNPs group intoxicated with AgNPs and orally gavaged with 100 mg LA/kg b.w, and GB + AgNPs group injected with AgNPs and orally given GB extract 120 mg/kg b.w were continued for 30 consecutive days. Biochemical changes in testicular tissue (testosterone, ACP, and Prostatic acid phosphatase), oxidative indices in testicles tissues, mRNA expression of pro-apoptotic (BAX) and anti-apoptotic (BCL-2) biomarkers, histological, and immunohistochemical changes were studied. Signicant decrease in serum testosterone level and elevation in ACP and PACP enzyme activity in AgNPs treated group than in the control. In addition, lowering in tGSH, GSH GR, GPx and elevation MDA and GSSG were observed in AgNPs treated group than control. Decreasing in mRNA expression thirodexin-1 (Txn-1), transforming growth factor-1β (TGF-1β), anti-apoptic (BCL-2) and elevation the expression of proapoptic biomarkers (BAX) in the testis homogenates of rats exposed to silver nanoparticles. Strong positive action to BAX and lowering the action of Ki-67antibody were observed. Because of their antioxidant, anti-inammatory, and anti-apoptotic properties, co-treatment with LA or GB


Introduction
Surface cleansing agents, washing machines, toys and textiles, air and water lters, food canning, and antimicrobial coatings all contain silver nanoparticles (AgNPs) (Tolaymat et al., 2010). AgNPs' antibacterial activity has led to their use as a sterilising agent in medical devices such as contraceptives, wound dressings, surgical equipment, and medical catheters (Wijnhoven et al., 2009). AgNPs were also used in the administration of medications for tumour and retinal treatment (Kalishwaralal et al., 2010).
Because of the vast range of AgNPs uses, the amount of AgNPs released into the environment is increasing, as is the vulnerability of living species to AgNPs exposure via various routes such as inhalation, ingestion, cutaneous, and injection (Marambio- Jones and Hoek, 2010). AgNPs can pass through the blood-testicular barrier and the cell membrane (Schrand et al., 2010). The negative effects of AgNPs on the male reproductive system at various doses, include a decrease in spermatocytes, spermatids, and spermatozoa counts in rats (Miresmaeili et al., 2013). Moreover, (Baki et al., 2014a) reported the disruption of male sexual hormones, with deleterious consequences for sexual organ maturation and sperm characteristics in rats exposed to varied dosages of AgNPs. The endocrine; follicle stimulating hormone (FSH) and luteinizing hormone (LH) and paracrine hormonal pathways; testosterone, which affect germ cell proliferation and effective spermatogenesis and are implicated in apoptosis when their levels are low, are required for optimal testicular activities (So kitis et al., 2008).
Despite the fact that AgNPs caused testicular toxicity, the putative molecular processes underpinning this remained unknown. The proposed hypothesis includes three levels: disruption in endocrine hormones, decreased spermatogonia and primary spermatocyte proliferation, and direct in uence on germ and leydig cells via induction of oxidative stress and apoptosis, all of which are intertwined in AgNPs-induced testicular dysfunctions. Many attempts have been made to mitigate the negative effects of AgNPs on testicular function, including the use of natural compounds.
The natural product generated by mitochondria, alpha lipoic acid (ALA), had a role in enzymatic mitochondrial bioenergetics as a cofactor and possessed substantial antioxidative action via many pathways (Sadek et al., 2018). Biewenga et al. (1997) reported that ALA and its reduced counterpart dihydrolipoic acid have antioxidant properties through scavenging activity, antioxidant regeneration, metal chelation, and repair mechanisms. Furthermore, ALA showed an anti-in ammatory effect, lowering pro-in ammatory cytokines such as IL-6 and IL-1 and modulating NF-B (Dinicola et al., 2017). In rats with surgical varicocele, ALA was found to improve sperm parameters and minimise lipid peroxidation and DNA damage (Shaygannia et al., 2018). Consequently, (Lebda et al., 2014) revealed ALA protects rats from acrylamide-induced testicular damage by increasing blood testosterone, testicular antioxidative enzymes, and decreased glutathione levels while lowering MDA levels.
Flavonoids and terpenoids such as quercetin, kaempferide, rutin, and ginkgolides are the primary ingredients of Ginkgo biloba tree leaves extract (GBE) (Watanabe et al., 2001), showing free radical scavenging activity (Li et al., 2003). GBE's antioxidative and antiapoptotic properties make it a good candidate for treating and preventing oxidative damage in many tissues (Akgül et al., 2008). The goal of this study was to see if ALA and GBE could protect rats from AgNPs-induced testicular injury by looking at endocrine hormonal balance, cell proliferative activity, oxidative stress, and apoptotic pathways.

Chemicals and reagents
AgNPs ne powder was purchased from Sigma Aldrich (St. Louis, MO, USA). The particles were suspended in deionized water by vigorous vortexing and sonication prior to use to ensure the prevention of particle aggregation and characterized previously for their size and shape (Lebda et al., 2018b). LA was obtained from EVA Pharma Co. (Cairo, Egypt). Ginkgo biloba L. extract code# GK501 standardized to 24% Ginkgo avonoids was purchased from Pharmaton SA (Lugano, Switzerland). ELISA testosterone kit was purchased from Cayman Chemical Co, USA. Other bio-diagnostic kits were obtained from Biodiagnostic Co, Giza, Egypt.

Animals, housing conditions, and experimental protocol
Forty male Wistar Albino rats weighing 180-200 g were purchased from Animal Breeding Unit, Medical Research Institute, Alexandria University. The animals were kept in metal cages under environmentalcontrolled conditions with optimum temperature, humidity, and dark/light cycle and free access to rat chow and drinking water. The international ethical guidelines for the care and use of laboratory animals were performed to handle the animals and the experimental procedures were approved by the Experimental Animal Use and Ethics Committee at the Faculty of Veterinary Medicine, Alexandria University, Egypt. The rats were randomly assigned to four groups (10 rats each); control, AgNPsintoxicated group intraperitoneally injected AgNPs 50 mg/kg b.w, 3 times a week, LA + AgNPs group intoxicated with AgNPs and orally gavaged with 100 mg LA/kg b.w (Pari and Murugavel, 2004), and GB + AgNPs group injected with AgNPs and orally given GB extract 120 mg/kg b.w (Huang et al., 2017). All treatments were continued for 30 consecutive days. The dose of AgNPs was selected based on the study of Tiwari et al., (2011). Twenty-four hours after the last doses, the rats were anesthetized using ketamine/xylazine (7.5-10 mg/kg, 1 mg/kg i.p). The blood was collected from the inner canthus, and the sera were separated for estimation of testosterone, total acid phosphatase (ACP) and prostatic acid phosphatase (pACP) activities according to the manufacturers' guidelines. The rats were then euthanized and the whole testicles were immediately dissected, rinsed with chilled normal saline 0.9% and divided longitudinal into two halves; one was used for histopathological and immunohistochemical analyses and the other half was used for estimation of oxidative indices, and transcriptome analyses.

Lipid peroxidation and antioxidant pro le
The testicular tissue (about 500 mg) was homogenized using Te on and pestle homogenizer in ice-cold 0.1 M phosphate buffer saline pH 7.4. The supernatant was separated after centrifuging the crude homogenate at 14,000 rpm for 10 min at 4°C. Lipid peroxide was measured after the reaction with thiobarbituric acid and expressed as nmol malondialdehyde (MDA) per tissue weight (Ohkawa et al., 1979). Glutathione peroxidase (GPX) activity was analysed according to Paglia and Valentine (1967), which based on the reaction of hydrogen peroxide (H 2 O 2 ) in the presence of NADPH, GSH, and glutathione reductase. The absorbance measured at 340 nm and the result was expressed as IU per tissue weight. The enzymatic method described by (Gri th, 1980) was used to measure the total glutathione content where it depends on the oxidation of GSH by 5, 5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to yield GSSG and 5-thio-2-nitrobenzoic acid (TNB). Oxidized GSSG is reduced enzymatically by the action of glutathione reductase and NADPH to regenerate GSH which reacts again. The rate of TNB formation is monitored at 412 nm and is proportional to the sum of GSH and GSSG present in the sample The GSSG content is determined by the same assay as total glutathione, but where the reduced glutathione is bound by 2-vinylpyridine.

RNA extraction and qRT-PCR
About 100 mg testicular tissues were rinsed in sterilized phosphate buffer saline and homogenized in liquid nitrogen using Te on and pestle homogenizer then the homogenates were stored at − 80°C till RNA isolation. Total RNA was isolated using the RNeasy Mini Kit (Qiagen GmbH, Germany) according to the manufacturer instructions. cDNA was synthesized from the puri ed RNA using QuantiTect Reverse Transcription Kit (Qiagen). The reaction mixture included RNA and master mix were placed at 42°C then inactivated at 95°C. The qRT-PCR for the target genes were performed using QuantiTect SYBR Green PCR Master Mix (Qiagen Rotor-Gene Q). The primer sequences of all target and reference genes and the PCR conditions were recorded in Table 1. The fold change of mRNA expression was calculated after recording the Ct values for reference and target genes using the 2 −∆∆Ct method.

Immunohistochemical assessment
The standard horseradish-peroxidase immunohistochemistry technique was applied to positive charged slides of para n testicular tissue sections. Several 4-µm thick sections of the testicular tissues were depara nized in xylene, rehydrated in descending grades of ethanol, and pre-treated with 3% H 2 O 2 to block endogenous peroxidase activity. Antigen retrieval was accomplished by placing slides in a microwave for 10 min in 10 mM sodium citrate buffer (pH 6.0). Slides were incubated with the speci c primary antibody; monoclonal rat anti-ki67 (Abcam, Cat: ab156956), and polyclonal rabbit anti-Bax (Abcam, Cat: ab53154) diluted in 1% BSA/PBS pH 7.4 at 1:100 then rinsed with PBS and the sections were incubated with biotin-conjugated goat anti-rat IgG antiserum (Abcam, Cat: 182018) for 60 min and then rinsed with PBS followed by Streptavidin-peroxidase conjugate (Histo ne Kit, Nichirei Corp) incubation for 30 min. The sections were visualized using 3,3'-diaminobenzidine tetra-hydrochloride substrate chromogen solution then counterstained with hematoxylin stain and examined under light microscope.

Histopathologic examination
Testicular samples of 5 rats per group were collected and immediately xed in 10% buffered formalin for at least 24 h. Tissue specimens were washed, dehydrated by serial dilutions of alcohol, cleared in xylene, and embedded in para n at 60°C in a hot air oven. Para n sections of 4-5 microns in thickness were prepared and stained with hematoxylin and eosin (HE) and examined under the light microscope.

Statistical analyses
The obtained values are expressed as the mean ± standard error (SE). Using the SPSS statistical package v22.0 for Windows (IBM, Armonk, NY, USA), one-way ANOVA followed by post hoc multiple comparisons Duncan's test were used to analyze obtained data. The signi cance level was set at p ≤ 0.05.

Reproductive hormonal changes
The adverse impacts of AgNPs on the reproductive hormonal status of male rats and the protective potency of LA and GB are shown in Table (2). Comparatively with controls, serum concentrations of testosterone showed a signi cant (p ≤ 0.05) reduction in AgNPs-treated rats, which had been associated with a signi cant (p ≤ 0.05) enhancement of total and prostatic ACP levels. However, these hormonal disturbances were signi cantly (p ≤ 0.05) attenuated following LA or GB co-treatment, compared with AgNPs-treated groups. The attenuation was more noticeable with LA, particularly in relation to testesterone (2.81 ± 0.12 IU/L) and total ACP (13.82 ± 1.28 IU/L) levels, (Group LA + AgNPs) vs GB + AgNPs-treated rats.

Testicular oxidant/antioxidant status
The effect of AgNPs on the testicular oxidant/antioxidant capacity of rats and the ameliorative effect of LA and GB are shown in Table (3). The testicular oxidants -MDA and GSSG concentrations were found to be signi cantly (p ≤ 0.05) elevated, and antioxidants -total GSH, reduced GSH, GR and GPx activities were signi cantly (p ≤ 0.05) lowered in the AgNPs-intoxicated rats. Meanwhile, compared to AgNPs group, LA and GB cotreatment were signi cantly (p ≤ 0.05) reduced MDA and GSSG levels, and increased total GSH, reduced GSH content and GR and GPx activities in the testicular tissues. LA and GB administration reduced AgNPs-induced testicular oxidative stress in rat, without ensuring complete protection in relation to control ones. Moreover, LA cotreatment was generally offered better antioxidant properties than GB do.

Histopathological analysis
Testicular tissue of the control healthy rats had normal histoarchitecture that composed of uniform, wellorganized seminiferous tubules with complete spermatogenesis and normal interstitial connective tissue (Fig. 1a).Testicular sections of silver-nanoparticles-treated group showed degenerative changes of the seminiferous tubules as shrunken, disorganized seminiferous tubules with irregular basement membrane and vacuolar degeneration of spermatogonia cells (Fig. 1b). Some seminiferous tubules showed coagulative necrosis and depletion of germinal epithelium with hyalinization of the luminal contents, exfoliating of degenerated germinal epithelial cells and interstitial edema that was represented by faint eosinophilic material (Fig. 1c). The lumina of some seminiferous tubules contained giant cell formations (Fig. 1d). The microscopic pictures in silver-nanoparticles + lipoic acid treated group were interstitial edema and few tubules contained sloughed germinal epithelium (Fig. 1e) while the noticeable lesions in silver-nanoparticles + gink trated group the majority of seminiferous tubules had normal germinal epithelium and marked improvementof spermatogenesis with elongated spermatids and spermatozoa. Some tubules showed few vacuolated germinal epitheliums (Fig. 1f).
Hematoxiline and Eosin stained sections of the control Caput epididymis revealednormal histologicalarchitecture with normal sperm density (Fig. 2a).epididymalsections of silver-nanoparticlestreated group showedvacuolationof some caput epididymal epithelium (Fig. 2b)beside sloughed germ cells in its lumina (Fig. 2c) and congestion of interstitial blood vessel with perivascular in ammatory cell in ltrations (Fig. 2d). Silver-nanoparticles + lipoic acid treated group and silver-nanoparticles + gink trated group showed normal histological structure with normal sperm density ( Fig. 2e and f).
The control Couda epididymis sections had normal histological architecture with normal sperm density (Fig. 3a). The detectable histopathological pictures in silver-nanoparticles-treated groupwere vacuolation of some Coudaepididymal epithelium (Fig. 3b), sloughed germ cells in its luminawith hyalinized of the luminal contents of some epididymalductules (Fig. 3c) and low sperm density in the most of epididymalductulesbeside congestion of interstitial blood vessel with perivascular in ammatory cell in ltrations (Fig. 3d).Silver-nanoparticles treated withlipoic acid and Gink showed normal histological structure with normal sperm density ( Fig. 3e and f).

Immunohistochemistry
As appeared in gures (4 and 5)  The current study found that repeated intraperitoneal injections of AgNPs to rats result in changes in serum male sex hormone and enzymes. In comparison to the control and another treated group, the AgNPs-treated group saw a substantial reduction in serum testosterone levels. AgNPs block cholesterol transport into the inner mitochondrial membrane by lowering steroidogenic acute regulatory protein (STAR) expression, which eventually stops the conversion of cholesterol to pregnenolone levels (Baki et al., 2014b). Furthermore, decreased TGF-1 expression results in substantially insu cient male serum and intratesticular testosterone, as well as serum androstenedione. Because serum LH and serum FSH were lowered, and exogenous LH replacement with human chorionic gonadotropin (hCG) caused serum testosterone to decrease, the testosterone hormone shortage was secondary to disturbed pituitary gonadotropin release (Ingman and Robertson, 2007).
The concentration of serum prostatic acid phosphatase (Prostatic ACP) and the rise of serum acid phosphatase (ACP) produced in the liver, spleen, and prostate gland in the AgNPs group. This increase is a sign of benign prostatic hyperplasia (BPH) and the early stages of prostate cancer (Goto et al., 2009, Lee andFinn, 2012). Using of LA with AgNPs group improved the serum male sex hormones than AgNPs group. These result was correlated with Othman et al. (2012) who revealed that lipoic acid kept cholesterol and sex hormone-binding globulin (SHBG) levels the same as in control rats. These results could be attributable to LA's antioxidant activity, which improves the signal transduction pathways required for optimal hypothalamus-testicular axis function, resulting in normal testosterone release and sperm generation. Also, the treatment with GB can enhances testosterone synthesis and secretion of Leydig cells (Wu et al., 2008a). The prostatic acid phosphatase level is reduced when the general body condition and testicles improved in using GB or LA due to their antioxidant activity. It has anti-in ammatory, anti-neoplastic, and anti-proliferative properties as well. The antioxidant activity of GB was related to its components of terpenoids and avonoids, which operate as broadspectrum free radical scavengers and lower lipid peroxidation, which may explain why cotreatment with GB improved antioxidant indices (Yeh et al., 2009, Mohamed and Abd El-Moneim, 2017). By blocking and terminating radical chain reactions and suppressing ROS and lipid peroxidation reactions, avonoid glycosides can either eliminate free radicals to reduce the consumption of SOD and GSH-Px or promote the production of SOD and GSH-Px to scavenge free radicals such as superoxide anion and hydrogen peroxide as a result, the antioxidant testicular enzymes SOD and catalase were restored, and the concentration of testicular MDA was reduced, as described in our ndings (Wu et al., 2008a, Amin et al., 2012).
When ROS generation surpasses the capacity of the antioxidant defence, which is connected to lipid peroxidation, an imbalance in oxidative stress and antioxidant capacity ensues (Quinteros et al., 2018) and induction of apoptosis. . Thioredoxin is found in a variety of biological systems. It prolongs life and guards against oxidative stress in various organs and cell types (Mitsui et al., 2002).
To prevent stress and cytokine-induced apoptosis, the reduced/dithiol form of Trxs binds to apoptotic signal-regulating kinase 1 (ASK1) and suppresses its activity. Due to ROS, thioredoxin-1 expression was found to be lower in the silver nanoparticles treated group compared to the control and other groups, indicating that it dissociates from Ask1 and stimulates apoptosis. Trx interacting protein (TXNIP), which binds to Trx and removes Trx from ASK1, also contributes to the apoptotic process (Jun and Arne 2012).
AgNP-induced p53 activation has also been observed in mouse and human cells (Ahamed et al., 2008, Gopinath et al., 2010. P53 is activated by a variety of cell death events that can activate gene expression or permeabilize mitochondria, causing apoptosis. P53 builds up in the nucleus and regulates the production of the proapoptotic protein Bax (Wu et al., 2008b). P53 can enter mitochondria, interact with antiapoptotic Bcl-2 proteins, neutralise them, and trigger cell death (Li et al., 2015). The proapoptotic Bax and antiapoptotic Bcl-2 molecules are two key players in cell death, and the ratio of Bax/Bcl-2 is the numerator that determines whether or not cells will die. The AgNP-treated rats had higher Bax expression, as well as a strong positive immunological response and lower Bcl-2 expression, resulting in a higher Bax/Bcl-2 ratio, as seen in humans treated with AgNPs (Gopinath et al., 2010, Piao et al., 2011. By upregulating the expression of two genes that encode the anti-apoptotic proteins Bcl-2 and Xiap via a process that appears to include NF-B, cotreatment with LA substantially prevented apoptosis of testicular cells (Antonio et al., 2011). Ginkgo biloba also, has antiapoptotic effects through the protection of mitochondrial membrane integrity, possibly by its avonoid constituents (Takao, 2000) this agree with (Guan et al., 2014) who found that the apoptotic index was decreased with the antioxidant and antiin ammatory effects of the Ginkgo biloba treatment on the organs.
In fact, histological and immunohistochemical examinations revealed that AgNPs have a harmful effect. Damage to testicular tissue was observed in the AgNPs-treated group, which could be attributed to silver nanoparticles crossing the blood-testis barrier (BTB) and accumulating in the testicles, as previously reported in multiple studies (Asare et al., 2012) due to particle size of it ( (Amin et al., 2015). The toxicity is related to the nano-surface. silver's In the environment and biological systems, it is easily oxidised by O2 and other molecules, resulting in the release of Ag, a recognised hazardous ion. Nano-silver has been demonstrated to in ltrate and internalise cells. As a result, nano-silver is frequently used as an Ag source within cells (McShan et al., 2014). In a concentration-and time-dependent way, AgNPs are more cytotoxic, producing apoptosis, necrosis, and reduced proliferation (Asare et al., 2012). In comparison to the control group, AgNPs treated groups had lower Ki-67 antibody expression. The reduction in KI67 expression as a result of reduced cell proliferation is owing to AgNPs' inhibitory action in cell proliferation, which explains the damaged seminiferous tubules. AgNPs can bind to membrane proteins and activate signalling pathways, resulting in cell proliferation inhibition (Asharani et al., 2008). AgNPs can also enter the cell via diffusion or endocytosis, causing mitochondrial malfunction and the production of reactive oxygen species (ROS), which causes damage to proteins and nucleic acids inside the cell, as well as cell proliferation inhibition (Lim et al., 2012).
Histopathological analysis of testicular sections from the silver nanoparticles-treated group revealed degenerative alterations in the seminiferous tubules, such as smaller, disordered seminiferous tubules with uneven basement membranes, which could lead to signi cant testis functional impairment (Liu et al., 2013). The loss of germinal epithelium could also be attributed to a signi cant disruption of the Sertoli-germ cell connection. The sloughing of the germ cells from the seminiferous epithelium could have been caused by a breakdown in this physical contact (Erkanlı Şentürk et al., 2012). Hyalinization of luminal contents, exfoliation of degenerated germinal epithelial cells, and interstitial edoema in seminiferous tubules with giant cell formations were also seen. The earliest physical symptom of testicular injury is vacuolation degeneration of spermatogonia cells, also known as coagulative necrosis. It is thought that vacuolation causes spermatogenic cells to detach from Sertoli cells, which is the rst step toward cell death or apoptosis (Asare et al., 2012).
The epididymal sections of the silver-nanoparticles-treated group showed vacuolation of some caput epididymal epithelium alongside sloughed germ cells in its lumina and congestion of interstitial blood vessel with perivascular in ammatory cell in ltrations, indicating that continued exposure to toxic chemicals may lead to some histologic changes. The vacuolation of some Couda epididymal epithelium, sloughed germ cells in its lumina, hyalinization of the luminal contents of some epididymal ductulus, and low sperm density in the majority of epididymal ductulus were all observed in this study, along with congestion of interstitial blood vessels and perivascular in ammatory cell in ltrations. The blood-testis barrier must remain intact in order to maintain reproductive potential. Exposure to environmental toxins can compromise this barrier, causing the production of reactive oxygen species (ROS) in the testes, resulting in oxidative DNA damage and infertility. Because LA is a fat-and water-soluble antioxidant, it is found in cellular membranes (Prahalathan et al., 2006). LA protects against nanoparticle-induced oxidative disruption of the blood-testis barrier and testicular histological alterations, according to our ndings.

Conclusion
AgNPs had a deleterious impact on male reproductive processes in rat testis, according to recent ndings (biochemical, oxidative stress, apoptosis, immunohistochemical and pathological examination). As a result, it's possible to conclude that AgNPs are very hazardous to reproductive function and may affect animal fertility. Hormonal disturbances, apoptosis, mRNA expression abnormalities, and pathological alterations could all be caused by mechanisms linked to oxidative stress after rats were exposed to AgNPs. Furthermore, LA and GB are reported to be a recent and effective preventative treatment for AgNPinduced reproductive damage in male rats.   Table 2 Effect of α-lipoic acid and Ginkgo biloba L extract on serum testicular biomarkers (testosterone, prostatic ACP, and total ACP) and in rats exposed to silver nanoparticles   Table 4 :Effect of α-lipoic acid and Ginkgo biloba L extract on thirodexin-1 (Txn-1), transforming growth factor-1β (TGF-1β) proapoptic biomarkers (BAX) and anti-apoptic (BCL-2) in the testis homogenates of rats exposed to silver nanoparticles.