Testicular torsion is a urological emergency affecting 3.8 in 100 000 males under 18 annually in the United States [1]. The twisting of the spermatic cord impairs blood flow to the testicle, causing ischemia and subsequent oxygen and nutrient deprivation. This leads to energy depletion, electrolyte imbalance, and mitochondrial dysfunction, resulting in progressive tissue damage [2]. Without timely intervention, irreversible damage may occur, potentially necessitating orchiectomy [1]. Despite extensive research, there is currently no approved pharmacological option to protect against testicular torsion and mitigate damage. The rat model of testicular torsion-detorsion (T/D) provides a clinically relevant paradigm that closely mimics the pathophysiology of human cases, making it suitable for exploring potential interventions [3].

Ischemia triggers complex signaling cascades, with inflammatory pathways playing a critical role. Energy depletion and mitochondrial dysfunction activate inflammatory responses within affected cells. Additionally, the release of damage-associated molecular patterns recruits both local and systemic immune cells, intensifying the inflammatory response [4]. Given these mechanisms, agents with anti-inflammatory properties present promising therapeutic potential for mitigating ischemia-related damage in testicular torsion. The central role of mitogen-activated protein kinases (MAPKs) in inflammation further highlights them as a plausible target for anti-inflammatory therapies [5]. Elevated MAPK levels in testicular T/D injury [6] and the efficacy of MAPK-modulating drugs in testicular torsion [7-9] reinforce this pathway as a key therapeutic focus. Additionally, Toll-like receptor 4 (TLR-4) modulation, as a key upstream regulator of the MAPK pathway, has emerged as a potential therapeutic target in inflammatory conditions [10].

Linezolid, an oxazolidinone antibiotic, has exhibited notable anti-inflammatory properties in both infectious and non-infectious conditions [11-15], making it a potential therapeutic candidate for testicular torsion. While its primary mechanism involves inhibiting bacterial protein synthesis by targeting 23S ribosomal RNA, evidence suggests its effects extend to mammalian cells [12-14,16,17]. The structural similarity between mitochondrial and bacterial ribosomes may partially explain its influence on mitochondrial function and related anti-inflammatory actions. Additionally, studies have implicated pathways such as MAPK in mediating these effects [16]. Consistent with MAPK modulation, oxazolidinones have also been reported to influence the TLR-4 pathway [18,19].

Given the complex and not fully understood mechanism of linezolid’s anti-inflammatory action, we employed molecular docking techniques to predict potential pathways involved in its protective effects against testicular torsion. We hypothesized that linezolid exerts protection against T/D injury by modulating the TLR-4 pathway. Using a rat model of testicular T/D, we evaluated the efficacy of linezolid by assessing histopathological changes, measuring pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α), evaluating oxidative stress markers (malondialdehyde [MDA] and superoxide dismutase [SOD]), and quantifying nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and TLR-4 levels as key components of the hypothesized pathway.

To the best of our knowledge, this study is the first to demonstrate the protective effects of linezolid against ischemia-reperfusion injury in a rat model of testicular T/D. Recognizing inflammation as a key factor in testicular T/D, our findings reveal the anti-inflammatory properties of linezolid, independent of its effects in infectious conditions. This aligns with prior research on the anti-inflammatory actions of linezolid in both in vivo and in vitro models. Linezolid attenuated T/D injury by modulating the TLR-4 and MAPK pathways, reducing oxidative stress and inflammatory biomarkers. Although the clinically equivalent dosage for rats is approximately 100 mg/kg, we observed promising protective effects at a lower dose of 50 mg/kg, thereby minimizing concerns regarding toxicity.

Although the anti-inflammatory effects of linezolid in infectious diseases have been widely reported and are often attributed to inhibition of prokaryotic metabolism, previous research has demonstrated its anti-inflammatory actions across various conditions independent of infectious pathogens. For instance, treatment with linezolid in a carrageenan-induced paw edema model in rats reduced the edema rate, whereas other antibiotics failed to produce similar effects [11]. In human peripheral blood mononuclear cells activated with lipopolysaccharide (LPS), linezolid treatment reduced interleukin-6 (IL-6) expression [13]. An in vitro study using phorbol-12-myristate-13-acetate-stimulated neutrophils revealed decreased IL-8 secretion following linezolid treatment [15]. Furthermore, cytokine expression studies in LPS-treated human whole blood cells showed decreased levels of IL-1β, IL-6, TNF-α, and IL-8 mRNA following linezolid administration [14]. In a study examining neutrophils, linezolid did not impact methicillin-resistant Staphylococcus aureus (MRSA) viability in the presence of healthy neutrophils but reduced viability in impaired neutrophils treated with C5a, and it also inhibited IL-8-induced neutrophil transmigration [12]. Conversely, some studies reported increased IL-1β expression after linezolid incubation in immune cells under physiological conditions in the absence of bacteria or endotoxins [22], as well as reactive oxygen species (ROS)-independent activation of the NOD-like receptor family pyrin domain-containing 3 (NLRP-3) inflammasome by linezolid [23]. While these findings may appear contradictory, they are consistent with the mechanisms underlying linezolid's actions.

Linezolid binds to the bacterial 23S ribosomal RNA of the 50S subunit, thereby preventing the formation of a functional 70S initiation complex and inhibiting bacterial protein synthesis by disrupting translation. Due to structural similarities between mitochondrial and bacterial ribosomes, linezolid administration can lead to mitochondrial dysfunction, which may explain its adverse effects during prolonged clinical use, such as thrombocytopenia, optic neuropathy, peripheral neuropathy, and lactic acidosis [24]. Additionally, linezolid has been shown to inhibit mitochondrial complex IV, impairing oxidative phosphorylation [25]. This impairment leads to oxidative and nitrosative stress, resulting in increased production of ROS [26]. While elevated ROS levels can activate the NLRP-3 inflammasome and enhance IL-1β maturation, linezolid-induced mitochondrial dysfunction can also activate the NLRP-3 pathway in a ROS-independent manner through cardiolipin-mediated mechanisms [23]. These oxidative and proinflammatory effects are typically observed under physiological conditions. However, in the context of a pathological inflammatory state, linezolid exhibits an opposing, protective role.

During the ischemic phase of T/D, oxygen and glucose deprivation inhibits cellular respiration at mitochondrial complex IV. So, the additional inhibition of complex IV by linezolid may not significantly exacerbate the existing mitochondrial dysfunction. Also, linezolid's anti-inflammatory effects may outweigh its pro-oxidant actions. In some studies, linezolid treatment has been associated with reduced neutrophil infiltration in inflamed tissues [27]. Given that neutrophils contribute significantly to ROS production during reperfusion, reduction in neutrophil infiltration could help minimize oxidative damage during the reperfusion phase, thus indirectly protecting the testicular tissue in the T/D model [28]. Also, in a T/D model, this limited exposure could mean that any mitochondrial dysfunction induced by linezolid does not have enough time to significantly affect testicular cells or exacerbate I/R injury. It’s possible that linezolid’s effect on mitochondria only becomes problematic at higher doses or with prolonged treatment, so in a short-term or moderate-dose context, the effects might not be substantial enough to worsen T/D injury.

Overactivity of tissue-specific and circulating immune cells is a well-documented phenomenon during ischemia-reperfusion, including in testicular T/D. Mitigating this immune overactivity can provide protective effects against tissue damage. In fact, various mitochondrial inhibitors have demonstrated protective effects against ischemia [29-31]. Linezolid has been shown to confer protection in experimental autoimmune encephalomyelitis through the inhibition of T helper-17 cell effector function [32]. Additionally, linezolid has been found to dampen neutrophil-mediated inflammation in MRSA-induced pneumonia, thereby protecting the lungs from associated damage [27]. These observations are consistent with our findings of protection against testicular T/D, characterized by a reduction in TNF-α levels. Interestingly, linezolid treatment of human peripheral blood mononuclear cells stimulated with LPS did not reduce TNF-α levels [13], suggesting that the observed TNF-α reduction in our study likely results from indirect modulation via anti-inflammatory mechanisms rather than direct suppression of TNF-α production.

Despite the oxidative effects of linezolid, our study demonstrated protective effects against testicular T/D, reflected by reduced MDA levels and increased SOD activity. Oxidative stress during ischemia-reperfusion occurs in three phases: oxygen and glucose deprivation inhibit cellular respiration at mitochondrial complex IV; intracellular ATP depletion activates xanthine oxidase, resulting in ROS production; and rapid blood flow restoration causes a mismatch in mitochondrial complexes, leading to additional ROS generation [33]. Linezolid's inhibition of complex IV suggests that it does not exacerbate oxidative stress, as this complex is already inhibited. However, during the reperfusion phase, slowing electron transport may help reduce ROS production, a phenomenon supported by the protective role of carbon monoxide-known to inhibit complex IV—against ischemia-reperfusion injury [34].

Although linezolid is primarily recognized as an inhibitor of the formation of a functional 70S initiation complex, evidence suggests it may also target other proteins. For instance, Enterococcus faecium cultured with subinhibitory concentrations of linezolid exhibited altered protein expression patterns, with some proteins showing decreased levels and others showing increased levels—a phenomenon not solely attributable to inhibition of protein synthesis [35]. Furthermore, linezolid reduced MRSA supernatant-induced MUC5AC overexpression in human airway epithelial cells, accompanied by a decrease in ERK phosphorylation [16]. Additionally, 2,3,4-Triaryl-1,2,4-oxadiazol-5, a synthetic oxazolidinone structurally similar to linezolid, demonstrated potent inhibitory effects on p38 MAPK [36]. Similarly, 2-pentadecyl-2-oxazoline inhibited LPS-induced microglia activation by interfering with TLR-4 signaling [18], while 4-oxo-4-(2-oxo-oxazolidin-3-yl)-but-2-enoic acid ethyl ester, another oxazolidinone, inhibited TLR-4 homodimerization [19]. These findings prompted us to focus on the TLR-4 pathway, which acts through MAPKs, as potential targets for linezolid’s mechanism of action. Consistent with previous studies and molecular docking results, our data indicate decreased TLR-4 levels and a reduction in NF-κB-p65 as a downstream mediator.

Testicular torsion induces an elevation in p38, ERK, and JNK levels [6]. Modulating p38 and JNK activity by Acetyl-11-keto-β-boswellic acid has been shown to prevent testicular T/D injury in rats [7]. Dysregulation of p38 is also implicated in male infertility, highlighting it as a potentially effective therapeutic target [37]. Activation of peroxisome proliferator-activated receptors protects the testes from ischemia-reperfusion injury by reducing ERK phosphorylation [8]. Additionally, exposure to acrylamide inhibits testosterone production in mice testes and Leydig cells through ERK1/2 phosphorylation activation [38]. SP600125, a JNK inhibitor, alleviates oxidative DNA damage, germ cell apoptosis, and mitochondrial dysfunction during testicular torsion [9]. IL-1β has been shown to induce JNK phosphorylation, leading to neutrophil recruitment to the testes through increased E-selectin expression [39]. Collectively, these findings underscore the critical role of MAPK activation in testicular T/D, suggesting that modulating this pathway could yield protective effects. Consistent with these observations, our docking results indicate that MAPKs are probable targets for linezolid's mechanism of action, which is supported by decreased NF-κB levels.

The present study uncovered several key findings with potential implications for future research. Protective anti-inflammatory effects of linezolid observed at subinhibitory concentrations, along with the lower doses used in our study compared to equivalent therapeutic doses, minimize concerns regarding safety in clinical settings. Furthermore, adverse effects associated with linezolid treatment typically arise during prolonged administration, whereas pharmacological intervention for testicular torsion is inherently acute [24]. Given that testicular T/D serves as a model for ischemia-reperfusion injury, linezolid's protective effects may extend to other ischemia-reperfusion conditions, such as myocardial infarction, stroke, and renal or hepatic ischemia. Additionally, structural modifications of linezolid could potentially yield more potent and selective oxazolidinones with reduced side effects.

Despite the novel and promising findings, our study faces several limitations. First, while the testicular T/D rat model closely replicates the pathophysiology of human testicular torsion, further clinical trials are necessary to validate the efficacy and safety of linezolid in human subjects. Second, although our data demonstrating decreased levels of TNF-α and NF-κB align with our docking results, further assessment of MAPKs activity would strengthen our hypothesis. Third, the use of a four-hour ischemic period caused extensive damage, potentially obscuring the protective effects of lower doses of linezolid under milder ischemic conditions. Some studies have suggested using shorter ischemic periods to better evaluate therapeutic interventions [40]. Finally, while administering linezolid during the ischemic phase aligns with typical clinical scenarios, where the median delay from diagnosis to surgery is approximately 1.8 hours [41], evaluating administration during different phases of T/D injury could more thoroughly elucidate its therapeutic role.

In conclusion, this study uniquely demonstrates the protective effects of linezolid against testicular T/D injury, primarily through its anti-inflammatory properties independent of infectious states. Linezolid’s modulation of the TLR-4 and MAPK pathways, alongside reduced oxidative stress and inflammatory biomarkers, underscores its therapeutic potential. Effective at doses significantly below conventional clinical levels, it mitigates concerns about toxicity. Despite recognized mitochondrial dysfunction-related proinflammatory effects in physiological settings, linezolid’s targeted inhibition of immune overactivity during ischemia-reperfusion highlights its potential for acute therapeutic applications. Future clinical trials and mechanistic studies are warranted to further elucidate its efficacy, safety, and dosing strategies in ischemic contexts.