Up to 10%–15% of couples worldwide struggle with infertility, and 50% of those cases are linked to a male component (Leslie et al., 2024). Men become infertile for three main reasons: hypogonadism caused by hypothalamic-pituitary abnormity, seminal outflow impairment, and dysfunctional testicles (Shen et al., 2022). Orchitis is a common inflammatory reproductive disorder that results in male infertility and a deterioration in sperm quality (Guo et al., 2024), and impairs spermatogenesis resulting in significant harm to both human and animal reproduction, as well as substantial economic losses in animal breeding (Theas et al., 2018). Numerous factors contribute to orchitis, including bacterial and viral infections as well as other conditions such as autoimmune diseases (Klein et al., 2016) and obesity (Leisegang et al., 2019). A male reproductive system infection or inflammation accounts for 13%–15% of occurrences of male infertility (Balló et al., 2023). Orchitis is characterized by intercellular lymphocytic infiltrations, damaging the seminiferous tubules. In extreme cases, this may completely arrest the process of sperm development (Fijak et al., 2011).

It is known that the pathophysiology of orchitis involves oxidative stress, apoptosis, and an imbalance in inflammatory cytokines (Petrella et al., 2022). Orchitis may be induced by discrepancies in the inflammatory and anti-inflammatory cytokine levels in testicular cells, including TNF-α, interleukin (IL)-1β, (IL-6), and IL-10 (Inoue et al., 2020; Chen et al., 2017). In the same context, the production of reactive oxygen species (ROS) by macrophages is a crucial factor contributing to inflammation and cell death in testicular tissue (Inoue et al., 2020). Thus, eliminating testicular oxidative stress and apoptotic damage and balancing inflammatory cytokines has gained global attention for the management of orchitis (Rival et al., 2006). Lipopolysaccharides (LPS) is a key element of the external membrane found in Gram-negative bacteria and can cause orchitis by promoting the production of ROS and inflammatory mediators such as TNF-α, IL-6, and IL-1β in testicular tissue (Zhu et al., 2022). Consequently, these events result in diminished sperm production, interruption of hormone synthesis in the testes, and compromise of the blood testis barrier in Sertoli cells (Shen et al., 2022; Zhu et al., 2022).

OXY is a hormone produced in the supraoptic and paraventricular hypothalamic nuclei and is referred to as a neuro-pituitary hormone (Matsuura et al., 2015). It facilitates the expulsion of milk during breastfeeding and the contraction of the uterus during childbirth (Uvnäs-Moberg et al., 2019). It has a fundamental role in adjusting a broad spectrum of physiological activities, such as appetite, pain perception, emotion, and social behavior (Jurek and Neumann, 2018). OXY enhances testosterone synthesis via Leydig cells and promotes spermiation (Frayne et al., 1996; Thackare et al., 2006). Ghasemnezhad et al. (2015) established a testicular damage model induced by ischemia-reperfusion in rats; thereby, OXY has shown a therapeutic potential where it could improve spermatogenesis, raise the Johnson score, and lower the apoptotic index.

OXY has been shown to exhibit antioxidative, antiapoptotic, and anti-inflammatory characteristics in sepsis-associated organ damage (Mehdi et al., 2022). OXY could suppress the synthesis of pro-inflammatory mediators in microglia triggered by lipopolysaccharides (LPS) in mouse brain via interaction with mitogen-activated protein kinases (MAPKs) pathway and inhibition of the LPS-stimulated secretion of TNF-α and IL-1β in these microglial cells (Yuan et al., 2016). OXY diminished macrophage sensitivity to LPS stimulation, resulting in reduced expression of inflammatory cytokines such as IL-1β, IL-6, and TNF-α, while simultaneously augmenting sensitivity to IL-4 stimulation, leading to enhanced expression of M2-type genes accompanied by suppression of NF-κB signaling (Tang et al., 2019).

Since orchitis significantly contributes to male animal reproductive problems, developing medicines that may prevent and cure it promptly is imperative. The present research aimed to investigate the potential therapeutic benefits of OXY against LPS-induced orchitis in a rat model.

Infections and inflammation can negatively impact male fertility in the body and testes, which can result in a decrease in sperm production that may not recover even with antibiotic therapy (Li et al., 2023). Inflammatory cytokine imbalance influences spermatogenesis by altering cell-to-cell interactions, producing reactive oxygen species (ROS), and activating Toll-like receptors, potentially leading to male infertility (Syriou et al., 2018). In response to LPS insult, severe inflammatory reactions are induced, liberating pro-inflammatory cytokines from testicular cells, including Sertoli and Leydig cells, leading to testicular dysfunction (Reddy et al., 2006; Takeda and Akira, 2015).

In the current study, serum testosterone levels and anti-inflammatory IL-10 were considerably lowered by LPS injection. Conversely, LPS injection elevated the TNF-α, IL-1β, and IL-6 levels. LPS also increased aberrant sperm morphology, decreased sperm motility, and decreased sperm count. The disproportion of pro- and anti-inflammatory mediators, including TNF-α, IL1β, IL-6, and IL-10, in testicular cells promotes the induction of acute orchitis (Chen et al., 2017; O’Bryan et al., 2000). Intraperitoneal LPS injection prompts acute inflammatory responses, inhibiting testicular steroidogenesis and producing male germ cell apoptosis (Reddy et al., 2006; Metukuri et al., 2010; Wang et al., 2019a). LPS-induced inflammation alters testicular physiology (Hedger and Meinhardt, 2003). Moreover, it stimulates the synthesis of TNF-α, IL-1β, and IL-6 (Reddy et al., 2006; Metukuri et al., 2010; Wang et al., 2019a). Elevated TNF-α levels might impede steroidogenesis under inflammatory conditions and induce germ-cell apoptosis in experimental autoimmune orchitis (Hong et al., 2004; Li et al., 2013; Theas et al., 2008). Furthermore, IL-6 and IL-1β inhibit testosterone production by suppressing the expression of steroidogenic enzymes (Wang et al., 2019a; Abarikwu et al., 2024; Huang et al., 2020).

The current results demonstrated that LPS-induced oxidative stress, which increased MDA, reduced the antioxidant defense by decreasing GSH. Oxidative stress and ROS overproduction significantly influence testicular impairment, which primarily involves damage to the germ and Leydig cells, along with reduced sperm vitality and testosterone levels (Metukuri et al., 2010; Abd-Allah et al., 2009; Al-Olayan et al., 2014). Previous studies reported that LPS caused excessive ROS generation, which set off a chain reaction of oxidative damage manifested as a marked drop in GSH concentration and a sharp rise in MDA levels (Reddy et al., 2006; Li et al., 2013; Allen et al., 2004).

There have been suggestions that inflammatory reactions may be related to oxidative damage. An essential function of the TLR4-MyD88 pathway is to produce inflammatory mediators (Kawai and Akira, 2010). The current investigation showed that LPS increased TLR4, MyD88, and NF-κB expression, enhancing the production of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) which was in paralel with the previous reports (Tsubaki et al., 2015; Wang et al., 2019b; Mulero et al., 2019; Shen et al., 2020). In the present investigation, the rats injected with LPS exhibited upregulated PK2 and PKR1 expression levels. Consistent with earlier findings, exposure to LPS has resulted in elevated expressions of pro-inflammatory mediators, PK2, and PKR1 (Chen et al., 2016; Huang et al., 2020; Martucci et al., 2006). Testicular inflammation has been associated with PK2, which is mostly expressed in the testes, via the PK2/PKR1 cascade, which is implicated in the pathophysiology of the LPS-induced orchitis model (Chen et al., 2016; LeCouter and Ferrara, 2003). PK2 induces macrophage recruitment and the release of pro-inflammatory mediators (Martucci et al., 2006). Furthermore, the present results showed that the rats in the LPS group exhibited decreased IL-10. Previous research has shown that overexpression of PK2 weakens the immune system’s ability to fight inflammation by drastically lowering the production of IL-10 (Martucci et al., 2006; Franchi et al., 2008; Cook et al., 2010). These findings point to the significance of the PK2/PKR1 signal cascade in controlling the inflammatory response during infection. Histopathological analysis demonstrated the adverse effects of LPS on the testis, which included vacuolation, a decrease in the number of spermatids, and a focal loss of the tubular germinal epithelium. Other studies corroborated these findings (Chen et al., 2016; Wang et al., 2019a; Shen et al., 2020).

The present work demonstrated that OXY could mitigate the testicular damage in the LPS-induced acute orchitis model. OXY demonstrated these actions by reducing the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and simultaneously boosting the anti-inflammatory response by elevating the IL-10 level. Additionally, OXY could improve the antioxidant ability of the testicles by raising the concentration of GSH and diminishing the level of MDA. Furthermore, our study has demonstrated OXY’s antioxidant and anti-inflammatory characteristics through its capacity to restore histological architecture and improve semen qualities. Herein, the administration of OXY improved sperm count and motility and reduced sperm morphological abnormalities. Besides, OXY markedly decreased the expression of immune-related proteins such as MyD88, TLR4, NF-κB, PK2, and PKR1. It's interesting to note that Leydig cells produce OXY, which may have a localized effect on testosterone synthesis. Additionally, it controls how the epididymis ducts and seminiferous tubules contract (Thackare et al., 2006; Nicholson et al., 1994).

It has been shown that exogenous OXY treatment decreases tissue pathology in several animal models (Houshmand et al., 2009; Akdemir et al., 2014; An et al., 2019). OXY has reportedly been proven to prevent inflammation induced by LPS in microglial cells (Yuan et al., 2016), and effectively guard against experimental colitis through the AKT/Pi3K pathway (Dou et al., 2021). The OXY receptor gene has response components for interleukins and acute-phase reactants; meanwhile, this receptor is upregulated in the LPS-activated macrophages (Szeto et al., 2017). OXY can decrease the release of pro-inflammatory cytokines like IL-1β and IL-6 and enhance anti-inflammatory cytokines like IL-4 and IL-10 (An et al., 2019). Surprisingly, in another study, OXY could be utilized to counteract COVID-19-induced inflammation due to its anti-inflammatory properties (Buemann et al., 2021). It was also reported that OXY attenuated acute lung injury induced by LPS in mice by inhibiting the TLR4/NLRP3/NF-κB inflammatory signaling cascade along with upregulation of OXY receptors in the alveolar macrophages (An et al., 2019). In addition, OXY could counteract the sepsis-induced flow in the pro-inflammatory cytokine TNF-α and block the shift of macrophages from a neutral to a pro-inflammatory state (Tang et al., 2019). When OXY binds to its receptor, it inhibits the NF-κB signaling pathway (Szeto et al., 2017) and, therefore diminishes the production of TNF-α and IL-6 (Tang et al., 2019; Buemann et al., 2021). According to earlier research, the OXY-treated torsion group had higher Johnsen scores than the testicular torsion group (Ghasemnezhad et al., 2015; Fırat et al., 2018). Moreover, OXY has a significant testicular vasodilator effect on the testicular vascular tone and steroid levels in rams (El-Shalofy and Hedia, 2021).

Interestingly, in light of all the previously described findings, the PCA data demonstrate the extent of testicular damage caused by exposure to LPS, with the toxic group being able to be distinguished from the control or LPS + OXY groups. Similarly, since the LPS + OXY group is situated near the control group along component 1, the overall statistics show that OXY has a large preventative impact. In addition, the VIP scores affirmed that inflammation and oxidative stress were the main modulators in the LPS-induced testicular damage; thereby, the OXY could counteract such toxic actions of LPS. Meanwhile, the correlation heatmap attested to a significant correct correlation between all studied parameters, which supported all the above-obtained data. In the same data frame, the difference in each variable’s concentration levels across the various treatment groups was indicated by the clustering heatmap. According to the heatmap, rats exposed to LPS were more likely to show greater testicular damage. Alternatively, rats responded well to the protective effect of OXY therapy when it was co-administered with LPS. The proposed mechanisms behind the pharmacological potential of OXY toward LPS-induced testicular damage are summarized in Figure 8.

This study found that OXY effectively alleviates testicular dysfunction induced by LPS. This preferential action was achieved by mitigating the oxidative stress caused by LPS by enhancing GSH and reducing MDA tissue contents. OXY could also decrease pro-inflammatory cytokines levels and boost anti-inflammatory by downregulating TLR4, MyD88, NF-κB, and PK2/PKR1 pathways, which are linked to LPS-induced orchitis. In addition, OXY improves sperm count, motility, and morphology and maintains normal testicular histology. These results imply that OXY might be a viable curative option for testicular dysfunction and male infertility resulting from acute inflammatory disorders.