Long-term CP chemotherapy is a cause of cytotoxicity which may affect healthy cells, including male reproductive cells (Leydig and sertoli cells), leading to sexual dysfunctions (Elangovan et al. 2006, Fusco et al. 2021, Park et al. 2021). To prevent the adverse effects of CP and its metabolites, the use of nanoparticles synthesized using plant materials such as AgNPs is attractive, due to their pharmacological properties, their stability, and their effectiveness and small size (Grace et al. 2007, Yin et al. 2020). P. zenkeri is a tropical plant widely used in the treatment of sexual disorders, but there is no study on the green synthesis of AgNPs using this plant. In the present work, we fabricated AgNPs using P. zenkeri extract, and determined its antioxidant potentials in vitro using DPPH radicals scavenging assay. Further, we investigated the effects of AgNPs on cell viability, cell morphology, apoptosis level, oxidative stress related markers as well as testosterone level and mRNA level of stAR in TM3 cells. This study confirms the ability of P. zenkeri for the fabrication of AgNPs for the first time. AgNPs exhibit potent antioxidant activity in vitro, modulate cell viability, apoptosis and oxidative stress, and improve testosterone production by up regulating stAR genes in TM3 cells.
During the fabrication of AgNPs using P. zenkeri, the reduction of Ag+ ion was confirmed by the color transformation of the solution from light brown to dark brown. Indeed, silver nanoparticle surface plasmon excitation induces color change, which is the evidence for the formation of AgNPs (Banerjee et al. 2014, Pirtarighat et al. 2019). AFM is a reliable method to characterize nanoparticles as it can be used to image and evaluate their surface morphology and property (Jayaseelan et al. 2012). AgNPs characterization using AFM revealed a regular morphology with high degree of uniformity and smooth topography with uniform grains distribution, which corroborated the work of Iqtedar et al. (2019) and Ullah et al. (2021). The DPPH scavenging activity of AgNPs and ascorbic acid was increased in a dose-dependent manner, ascorbic acid being the most effective at all doses. The in vitro antioxidant activity of ascorbic acid is evident as it has been already shown by previous researchers (Rajeshkumar et al. 2021, Kładna et al. 2021). The potent in vitro antioxidant potential of AgNPs could be due to its ability to quench free radicals. Previous works also reported the in vitro antioxidant potential of AgNPs synthesised using Fucus vesiculosus (Kładna et al. 2021) and Azadirachta indica (Chinnasamy et al. 2021) extracts. In the current study, this radical scavenging ability of AgNPs could be responsible of its beneficial effects against CP-induced oxidative stress in TM3 cells.
In the current work, AgNPs, decreased the cell viability of TM3 cells in a time and dose-dependent manner, compared with control. This is the first study to report the effect of AgNPs synthesized using P. zenkeri extract on the viability of TM3 cells. This result corroborated that of Zhang et al. (2015) who investigated the effects of AgNPs in Leydig (TM3), Sertoli (TM4) and spermatogonial stem cells (SSCs). In parallel, high concentrations of AgNPs (40–100 µg/ml) synthesized using the root extracts of Beta vulgaris reduced the viability of human hepatic normal (CHANG) and cancer (HUH-7) cells (Bin-Jumah et al. 2020). The effects of the treatments on cell morphology were investigated and the results showed that AgNPs, vitamin E and CP did not affect the morphology of TM3 cells after 24 hours of treatment. In all groups, the cells were normally attached with healthy appearances, but CP seems to inhibit the cell growth, when compared to the control (cells cultured without CP) (Fig. 4). Studies have shown that the antineoplastic potential of CP is mediated via its inhibitory action on the Notch signalling pathway (Jin et al. 2021) and it capacity to promote oxidative stress in pathogenic cells by triggering the production of excessive amounts of ROS (Yang et al. 2020).
It has been reported that ROS overproduction induces apoptosis by up-regulating caspase genes (Simon et al. 2000), leading to DNA defects and cell death (Görlach et al. 2015). Our results indicated that, CP application induced cytotoxicity in TM3 cells, characterised by the significantly increase in apoptosis level, mitochondrial membrane potential, ROS production, and caspase 3/9 activities, compared with control. Indeed, ROS overproduction induces apoptosis by increasing the mitochondrial membrane potential (Che et al. 2021) and up-regulating caspase genes (Mu et al. 2020), leading to DNA damage (Srinivas et al. 2019). Although no previous work has described the cytotoxic of CP in TM3 cells, CP was found to be cytotoxic for the murine 4T1 mammary carcinoma cell by increasing the ROS production and apoptosis levels via Bax/Bcl-2 pathway (Kurokawa et al. 2021). Moreover, CP induced cytotoxic in HuH-7 cells, characterized by increased intracellular ROS generation and suppression of antioxidant machineries (Al-Johani et al. 2022). Of great interest, treatment with AgNPs reversed the adverse effects of CP, probably due to their powerful antioxidant potentials. Similarly, low percentages of ROS production and apoptosis level have been reported in CHANG human hepatic normal cells (compared to HUH-7 cancer cells) after treatment with green AgNPs (Bin-Jumah et al. 2020).
Oxidative stress is the key provider causing CP-induced cytotoxicity (Caglayan 2019). We found in this study that CP induced oxidative stress in the TM3 cells by elevating MDA level and decreasing the activities of SOD, catalase and glutathione peroxidase. The cytotoxicity of CP has been deported in various cells like breast cancer cells (Engin et al. 2021), human hepatoma cells (HuH-7) (Al-Johani et al. 2022), and monocyte macrophage cells (Raw 264.7) (Yadav et al. 2020). Interestingly, oxidative stress associated with CP toxicity was attenuated after AgNPs treatment, which may be due to their inhibitory action on free radicals activators such as xanthine oxidase. More mechanistic studies are required to clarify this action. In parallel, AgNPs was reported to prevent oxidative stress in normal cells such as L-929 fibroblast cells, but had an opposite effect in cancer cells like human neuroblastoma cancer cells (SH-SY5Y) (Zhai et al. 2022), human cervical cancer cells (HeLa) (Yuan et al. 2018) and human epidermoid larynx carcinoma cells (HEp-2) (Muthukrishnan et al. 2019). Moreover, Khorrami et al. (2018) reported that AgNPs are nontoxic toward normal cell line, but are cytotoxic to cancer cells. Thus, the properties of AgNPs on testicular cancer cells are highly needed.
Leydig cell are the main source of production of testosterone in male. StAR acts in the steroidogenesis by controlling cholesterol transfer from the external to internal mitochondrial membrane, thus down regulation of StAR decrease testosterone synthesis (Walsh et al. 2000). CP impaired testicular function by decreasing sex hormones, including testosterone production (Jin et al. 2021, Rezaei et al. 2021). We noticed in the present study that CP significantly decreased testosterone and mRNA levels of StAR in the TM3 cells compared to control. Our findings are consistent with those of Fan et al. (2022) who reported that CP down regulated gene expression of genes related to spermatogenesis and lowered testosterone production by altering STAR, CYP11A1, and PRKACB genes. Of great interest, AgNPs elevated testosterone level and up regulated StAR genes after application. The significant increase in the mRNA levels of StAR in the TM3 cells incubated with AgNPs, indicated that their androgenic activities are due to the activation of StAR machineries. However more mechanistic mechanisms are needed to clarify this action. These results indicate that AgNPs synthesized using P. zenkeri could be a potential alternative drug in the management of androgen deficit associated to CP chemotherapy.