Bioactive Compounds Protect Mammalian Reproductive Cells from Xenobiotics and Heat Stress-Induced Oxidative Distress via Nrf2 Signaling Activation: A Narrative Review

Oxidative stress occurs when there is an imbalance between the production of reactive oxygen species (ROS) and the body’s antioxidant defenses. It poses a significant threat to the physiological function of reproductive cells. Factors such as xenobiotics and heat can worsen this stress, leading to cellular damage and apoptosis, ultimately decreasing reproductive efficiency. The nuclear factor erythroid 2–related factor 2 (Nrf2) signaling pathway plays a crucial role in defending against oxidative stress and protecting reproductive cells via enhancing antioxidant responses. Dysregulation of Nrf2 signaling has been associated with infertility and suboptimal reproductive performance in mammals. Recent advancements in therapeutic interventions have underscored the critical role of Nrf2 in mitigating oxidative damage and restoring the functional integrity of reproductive cells. In this narrative review, we delineate the harmful effects of heat and xenobiotic-induced oxidative stress on reproductive cells and explain how Nrf2 signaling provides protection against these challenges. Recent studies have shown that activating the Nrf2 signaling pathway using various bioactive compounds can ameliorate heat stress and xenobiotic-induced oxidative distress and apoptosis in mammalian reproductive cells. By comprehensively analyzing the existing literature, we propose Nrf2 as a key therapeutic target for mitigating oxidative damage and apoptosis in reproductive cells caused by exposure to xenobiotic exposure and heat stress. Additionally, based on the synthesis of these findings, we discuss the potential of therapies focused on the Nrf2 signaling pathway to improve mammalian reproductive efficiency.


Introduction
External environmental stressors such as high temperatures and exposure to xenobiotics significantly contribute to the initiation of oxidative stress and apoptosis processes, which have a negative impact on the functionality of reproductive cells [1][2][3][4].Normally, an organism's intrinsic antioxidant mechanisms are able to counteract the harmful effects of reactive oxygen species (ROS) overproduction, thus maintaining cellular integrity [5].However, when there is chronic and excessive ROS generation, oxidative stress occurs, resulting in cellular damage and disruption of normal physiological processes [6].To mitigate these effects, the use of external antioxidants is recommended to improve cellular antioxidant capacity and influence important biochemical pathways, including the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway.This intervention aims to protect mammalian reproductive cells from oxidative damage and apoptosis [2,[7][8][9].
The Nrf2 protein serves as a vital transcription factor essential for preserving the integrity of redox signaling when cells face oxidative stress [10,11].As a member of the cap'n'collar basic leucine zipper transcription factor family, Nrf2 plays a vital role in coordinating antioxidant and detoxification responses by upregulating downstream genes [12][13][14].Under normal conditions, Nrf2 predominantly resides in the cytoplasm, forming a complex with its inhibitory partner, Kelch-like ECH-associated protein 1 (Keap1).However, in the presence of elevated levels of ROS, this complex dissociates, allowing Nrf2 to translocate from the cytoplasm into the nucleus [2,15,16].Once activated, Nrf2 binds to the antioxidant response element (ARE) sequence, starting the transcription of genes involved in antioxidant defenses to counteract ROS-induced damage [9,[17][18][19].Recent research suggests that p62 competes with Keap1 for binding to the Nrf2 site, disrupting their association, releasing ubiquitinated Nrf2, and subsequently activating the Nrf2-antioxidant systems [20,21].
The role of Nrf2 signaling in safeguarding the reproductive cells/organs against oxidative stress has been extensively studied [22][23][24].It has been well documented that supplementation of bioactive compounds protects reproductive cells from oxidative stress induced by heat stress and environmental toxicants, via regulation of Nrf2 signaling [25].Nrf2 has demonstrated protective effects on bovine granulosa cells against H 2 O 2 -induced oxidative stress [26].Additionally, research by Sun et al. [27] illustrated that supplementation of melatonin safeguarded cryopreserved ovarian tissues from oxidative stress and apoptosis through the Nrf2/HO-1 signaling pathway.They observed an elevation in Nrf2 levels following melatonin administration, leading to the regulation of antioxidant genes [glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD), and heme oxygenase 1 (HO-1)] and a reduction in malondialdehyde (MDA) content [27].Antioxidant responses, including autophagy and Nrf2 activation, are triggered in response to heat stress-induced apoptosis [9,21].Alterations in autophagy dynamics play a crucial role in regulating the protective function of the Nrf2 signaling pathway in the testes.This protection involves the suppression of MDA levels and the promotion of an antioxidant status that shields the testes from the detrimental effects of heat stress [28][29][30].Notably, inhibition of Nrf2 leads to decreased cell viability, increased MDA levels, and Sertoli cell death [11].Consistently, studies have shown that exposure to heavy metals such as aluminum results in downregulated Nrf2 expression, increased oxidative stress, and toxicity, negatively impacting male reproductive function [31].
Nrf2 regulates several critical antioxidant genes, such as CAT, heme oxygenase 1 (HMOX1), peroxiredoxin 1 (PRDX1), SOD1, and thioredoxin 1 (TXN1).These genes collectively enhance antioxidant activity, thereby mitigating oxidative stress in mouse testis cells and safeguarding germ cells and Leydig cells from oxidative damage [30,32].Recent research has revealed that heat stress-induced ROS overproduction suppresses the expression of antioxidant genes (SOD, CAT, quinone oxidoreductase 1 (NQO1), and GSH-Px) in uterine tissue [33].In Sertoli cells, heightened ROS levels due to heat stress elevate MDA levels and reduce antioxidant enzyme levels [34].Additionally, heat stress increases the expression of apoptotic markers such as Fas, FasL, caspase 3, and caspase 9 in mouse Sertoli cells [34].Consequently, the Keap1/Nrf2 signaling pathway is significantly associated with the protective effects observed in mouse uterine tissue, characterized by increased levels of antioxidant genes [33].
Moreover, oxidative stress affects various crucial signaling pathways, including the Nrf2/Keap1 signaling axis in the testes [24].Recent studies emphasize Nrf2's protective role in shielding mouse Sertoli cells from heat-induced oxidative stress through the Nrf2/Keap1 signaling pathway [11].Similarly, another investigation demonstrated that Nrf2 significantly reduces caspase 3 levels, consequently decreasing cell death induced by heat stress treatment in Sertoli cells [29].Under conditions of severe heat stress, heightened expression of Keap1 and Nrf2 facilitates the regulation of genes associated with antioxidants through forming complexes with antioxidant regulated elements (ARE), thus establishing a defensive mechanism against heat stress within bovine endometrial epithelial cells [35].These findings collectively underscore the critical role of Nrf2 in alleviating oxidative stress and apoptosis in various cellular contexts, particularly under heat stress conditions.
Overall, Nrf2 plays a significant role in regulating the physiology and pathology of reproductive cells via modulating cellular resistance to oxidative stress and apoptosis induced through various factors such as chemicals, environmental toxicants, and heat stress [20].However, it is notable that several compounds act as both activators and inhibitors of testicular Nrf2.Nrf2 activators potentially hold therapeutic promise in preventing and treating testicular dysfunction, while Nrf2 inhibitors may contribute to dysfunction within testicular components.Activators of Nrf2 confer cellular protection against oxidative damage by stimulating Nrf2-related signaling pathways, facilitating its translocation into the nucleus, and enhancing Nrf2 function and expression, thereby upregulating downstream antioxidant gene expression.Conversely, Nrf2 inhibitors exacerbate oxidative stress by interfering with the Nrf2 signaling pathway.Therefore, this narrative review aims to investigate the impact of xenobiotics and heat stress-induced oxidative distress and apoptosis on the physiology of reproductive cells, while also addressing the protective role of activating the Nrf2 signaling pathway against oxidative stress and apoptosis in mammalian reproductive cells via supplementation of bioactive compounds.

Methodology
This study's methodology entailed a comprehensive literature review, primarily focusing on scholarly articles published between 2018 and April 2024.Additionally, select publications dating back to 2013 were also incorporated, specifically those addressing the role of Nrf2 signaling in mitigating oxidative stress and apoptosis induced by heat stress in mammalian reproductive cells.The literature search was conducted using esteemed academic databases, including Google Scholar, Web of Science, X-MOL, and PubMed.The selection of literature was guided by a set of predetermined keywords: "Oxidative Stress", "Apoptosis", "Mammalian Reproductive Cells", "Nrf2 Signaling", "Xenobiotics", "Heat Stress", and "Bioactive Compounds Regulating Nrf2 Signaling".To ensure the credibility and relevance of the sourced information, only articles published in English and indexed in Science Citation Index (SCI) Journals were considered for this review.In addition, book chapters and articles published in non-English languages were excluded from this review to maintain a focused and high-quality dataset for analysis.

Administration of Bioactive Compounds Protects Mammalian Reproductive Cells against Xenobiotic and Heat Stress-Induced Oxidative Stress through Nrf2 Signaling Activation
The regulation of Nrf2 is intricately managed through its interaction with Keap1.In a state of equilibrium, Keap1 confines Nrf2 within the cytoplasm, maintaining it at minimal levels.This confinement is achieved through the binding of Keap1 to Nrf2 at its C-terminal region, which triggers the ubiquitination of Nrf2.The ubiquitination process, facilitated by the Keap1-Cullin3-RING box protein complex, leads to the subsequent degradation of Nrf2 by the 26S proteasome [29].During episodes of oxidative distress caused by heat stress or xenobiotics, the increased expression of Keap1 inhibits the translocation of Nrf2 to the nucleus [3,27,33], consequently reducing the antioxidant response (Figure 1B).Conversely, supplementation with bioactive compounds has been observed to downregulate Keap1 expression, resulting in increased Nrf2 levels and subsequent elevation of downstream antioxidant response genes, such as NAD(P)H quinone dehydrogenase 1 (NQO1), HO-1, SOD1, CAT, and GPx (Figure 1A) [30,36].Consistently, the pivotal role of Nrf2 in antioxidant defense has been well documented, highlighting its importance in combating oxidative stress and mitigating cellular damage [37,38].In addition, the therapeutic potential of modulating Nrf2 signaling via administration of bioactive compounds to alleviate oxidative stress has garnered considerable attention in the recent literature [11,14,[39][40][41][42][43][44].Furthermore, the Nrf2 signaling cascade, in conjunction with other protective mechanisms, is crucial in preserving the integrity of mammalian reproductive cells against oxidative stress.This safeguarding is essential for maintaining reproductive health and function.
subsequent elevation of downstream antioxidant response genes, such as NAD(P)H quinone dehydrogenase 1 (NQO1), HO-1, SOD1, CAT, and GPx (Figure 1A) [30,36].Consistently, the pivotal role of Nrf2 in antioxidant defense has been well documented, highlighting its importance in combating oxidative stress and mitigating cellular damage [37,38].In addition, the therapeutic potential of modulating Nrf2 signaling via administration of bioactive compounds to alleviate oxidative stress has garnered considerable attention in the recent literature [11,14,[39][40][41][42][43][44].Furthermore, the Nrf2 signaling cascade, in conjunction with other protective mechanisms, is crucial in preserving the integrity of mammalian reproductive cells against oxidative stress.This safeguarding is essential for maintaining reproductive health and function.considerable attention in the recent literature [11,14,[39][40][41][42][43][44].Furthermore, the Nrf2 signaling cascade, in conjunction with other protective mechanisms, is crucial in preserving the integrity of mammalian reproductive cells against oxidative stress.This safeguarding is essential for maintaining reproductive health and function.

Bioactive Compound Supplementation to Combat Xenobiotic-Induced Oxidative Stress and Apoptosis in Reproductive Cells via Activation of the Nrf2 Signaling Pathway
Xenobiotic agents have been identified as initiators of ROS generation, subsequently inducing oxidative stress [45].This oxidative milieu has been implicated in impairing the integrity of key reproductive cells, including Sertoli cells, spermatogonial cells, and granulosa cells, potentially underpinning reduced reproductive efficiency and health [46][47][48][49].The perturbation is manifested through mechanisms such as increased DNA fragmentation in spermatozoa, disruption of mitochondrial membrane lipids in sperm, and compromised functionality of granulosa cells.In response, a spectrum of therapeutic interventions has been explored to fortify reproductive cells against xenobiotic-induced oxidative insult, specifically through the modulation of the Nrf2 signaling cascade [50].To combat xenobiotic-induced oxidative stress and apoptosis in reproductive cells, several exogenous bioactive compounds with antioxidant properties have been given to animals.These operate via regulating Nrf2 signaling in reproductive cells and consequently ameliorate oxidative damage.Notably, Ji et al. [51] elucidated the ameliorative effects of salidroside on oxidative stress and apoptosis in dihydrotestosterone-challenged human granulosa cells via the AMP-activated protein kinase (AMPK)/Nrf2 pathway, marked by upregulation of Nrf2, HO-1, and NQO1.In a similar way, sulforaphane has been shown to confer protection to bovine granulosa cells against H 2 O 2 -induced oxidative stress through Nrf2 pathway activation, enhancing antioxidant defenses including SOD, CAT, NQO1, and HO-1, thereby mitigating oxidative stress and apoptosis [26,52].
Further investigations have revealed that lycopene counteracts dihydrotestosteroneinduced oxidative stress in human granulosa cells by activating the Nrf2 signaling pathway [53], while anthocyanins have been reported to safeguard testicular tissue from cadmium-induced oxidative harm through Nrf2 signaling mediation, also revitalizing the activity of key antioxidant enzymes [3].Targeting the Nrf2/HO-1 axis, carvacrol administration in rats has shown promise in alleviating oxidative stress and apoptosis, evidenced by modulated expression of Bcl-2, Nrf2, CAT, GPx, and HO-1 and reduced MDA and Bax levels in testicular tissue [54].Another study highlighted sitagliptin's efficacy in attenuating cadmium-induced oxidative stress and toxicity in rats via the Nrf2/HO-1 pathway, resulting in improved testicular health markers [55].Complementary to these findings, treatments with Artemisia judaica extract, ellagic acid, and cardamonin significantly curtailed oxidative stress and apoptosis in diabetic rat testes, underscoring the therapeutic potential of these agents in modulating oxidative balance [56][57][58].Vitamin D3 has also been recognized for its capacity to mitigate lead-induced oxidative stress and toxicity in rat testes through Nrf2 signaling pathway regulation [59].

Bioactive Compound Supplementation to Combat Heat Stress Induced Oxidative Stress and Apoptosis in Mammalian Reproductive Cells via Activation of the Nrf2 Signaling Pathway
Exposure to high temperature has been linked to the activation of cell death and oxidative stress responses within reproductive cells.This phenomenon is supported by a study conducted by Sammad et al. [137], wherein bovine granulosa cells exposed to a thermal stress of 43 • C for 2 h showed a decrease in Nrf2 signaling, resulting in increased apoptosis and oxidative stress markers.Consistently, another study found that heat treatment increased ROS production in granulosa cells with silenced HO-1 and Nrf2 genes [138].However, granulosa cells with overexpressed HO-1 and Nrf2 genes demonstrated significant resistance, including increased antioxidant response and anti-apoptotic activities [138].Sertoli cells, which play a vital role in supporting the development of germ cells, rely on normal glucose metabolism for effective spermatogenesis.Melatonin has emerged as a potential therapeutic agent for mitigating the negative effects of heat stress on spermatogenesis.Research by Deng et al. [139] revealed that melatonin reduced heat-induced oxidative stress and apoptotic pathways by activating the KEAP1/Nrf2 signaling axis, thereby enhancing antioxidant defenses.In a parallel finding, He et al. [11] demonstrated the protective role of the Nrf2 signaling pathway against heat stress-induced oxidative challenges in Sertoli cells.Inhibition of the Nrf2 signaling pathway was associated with increased cellular apoptosis, reduced viability, and higher levels of intracellular ROS production.Additionally, melatonin, as reported by Sun et al. [27], upregulated the expression of heat-shock protein 90 (HSP90) through the melatonin receptor 1B (MTNR1B), which stabilized hypoxia-inducible factor-1α (HIF-1α).This activation of HIF-1α signaling promoted glycolysis, enhanced the pentose phosphate pathway, and improved cell viability.
In the domain of uterine physiology, Li et al. [33] observed that heat stress compromised normal uterine function by downregulating Nrf2 expression and its downstream antioxidant genes while upregulating the MDA level.The administration of baicalin significantly improved antioxidant responses and restored normal uterine function.Similarly, Alemu et al. [32] reported downregulated expression of Nrf2 and its target antioxidant genes (SOD, CAT) in bovine granulosa cells exposed to heat stress, resulting in reduced cell proliferation and increased cell death.Conversely, Li et al. [29] reported an increase in Nrf2 expression after scrotal heat treatment in mouse testes, suggesting a time-dependent response of the Nrf2-antioxidant system to heat stress.Moreover, Li et al. [21] demonstrated that heat stress induced autophagy in mice, activating the Nrf2 signaling pathway as a protective response to oxidative stress, safeguarding testicular tissue from damage.Furthermore, comprehensive research indicates that bioactive compounds given to animals can mitigate oxidative stress and cell death caused by heat stress, while also enhancing the antioxidant response through the regulation of Nrf2 signaling pathways [32,[139][140][141][142][143].
The collective body of evidence highlights the crucial role of Nrf2 signaling in counteracting oxidative stress caused by heat exposure in mammalian reproductive cells, as summarized in Table 2.These findings emphasize the importance of Nrf2 signaling pathways in the cellular defense mechanism against heat stress-induced reproductive dysfunction.

Limitations and Future Recommendations
Based on the existing literature, it has been established that most of the evidence presented to date has been derived from in vitro studies and animal models.The absence of clinical trials limits the direct applicability of these findings to reproductive health and, therefore, the therapeutic use of bioactive compounds in clinical settings.Thus, to validate the therapeutic potential of Nrf2 signaling pathway activation in improving reproductive health, clinical trials are needed.These studies should assess the safety, efficacy, and optimal dosing of bioactive compounds in human populations.In addition, the existing review has presented only the protective roles of Nrf2 signaling pathway activation, and it may underrepresent the potential negative effects of prolonged or excessive Nrf2 signaling pathway stimulation, such as possible interference with normal cellular functions or promotion of tumorigenesis in certain contexts.Investigating the long-term effects of chronic Nrf2 signaling pathway mediation on reproductive health is crucial.More detailed mechanistic studies are necessary to better understand how Nrf2 interacts with other cellular pathways under stress conditions.Such studies could lead to the development of more targeted therapies that minimize side effects while enhancing therapeutic efficacy.

Conclusions
Overall, this review provides compelling evidence that the Nrf2 signaling pathway plays a crucial role in safeguarding mammalian reproductive cells from oxidative stress and apoptosis induced by heat stress and xenobiotic exposure.Through the activation of antioxidant defense genes, the Nrf2 signaling pathway mitigates the harmful effects of heat stress and xenobiotic-induced oxidative stress and apoptosis, thereby preserving the functionality and viability of reproductive cells.Furthermore, the review highlights the pivotal bioactive compounds capable of alleviating oxidative distress caused by heat stress and xenobiotics, safeguarding mammalian reproductive cells from oxidative damage through the activation of the Nrf2 signaling pathway.These findings underscore the importance of Nrf2 in maintaining cellular homeostasis under environmental stressors, highlighting its potential as a therapeutic target for enhancing reproductive health.The intricate regulation of Nrf2 through its interaction with Keap1 and subsequent activation in response to oxidative stress illustrates a sophisticated cellular mechanism for combating cellular damage and maintaining reproductive integrity.Future studies should explore the development of targeted therapies that enhance the Nrf2 signaling pathway, offering new avenues for protecting reproductive health against environmental stressors.Additionally, investigating the long-term effects of Nrf2 modulation on reproductive function could provide deeper insights into its therapeutic potential.

Figure 1 .
Figure 1.The role of Nrf2 signaling in mitigating heat stress and xenobiotic-induced oxidative distress and apoptosis.(A) Exogenous supplementation of bioactive compounds with antioxidant ability activates the Nrf2/KEAP1 signaling pathway.This activation leads to NRF2 translocation to the nucleus and heterodimerization with sMaf proteins.Subsequently, there is binding with ARE to activate antioxidant genes (SOD, CAT, NQO1, HO1, and GPx).The activation of these antioxidant genes enhances the antioxidant response by suppressing oxidative stress and apoptosis induced via heat stress and xenobiotics.(B) Oxidative stress induced by xenobiotics/heat stress increases the level of KEAP1 and inhibits the translocation of NRF2 to the nucleus, leading to oxidative damage and apoptosis of reproductive cells.Note: Blunt arrows ( ) indicate inhibition and sharp arrows (→) indicate stimulation.

Figure 1 .
Figure 1.The role of Nrf2 signaling in mitigating heat stress and xenobiotic-induced oxidative distress and apoptosis.(A) Exogenous supplementation of bioactive compounds with antioxidant ability activates the Nrf2/KEAP1 signaling pathway.This activation leads to NRF2 translocation to the nucleus and heterodimerization with sMaf proteins.Subsequently, there is binding with ARE to activate antioxidant genes (SOD, CAT, NQO1, HO1, and GPx).The activation of these antioxidant genes enhances the antioxidant response by suppressing oxidative stress and apoptosis induced via heat stress and xenobiotics.(B) Oxidative stress induced by xenobiotics/heat stress increases the level of KEAP1 and inhibits the translocation of NRF2 to the nucleus, leading to oxidative damage and apoptosis of reproductive cells.Note: Blunt arrows (

Figure 1 .
Figure 1.The role of Nrf2 signaling in mitigating heat stress and xenobiotic-induced oxidative distress and apoptosis.(A) Exogenous supplementation of bioactive compounds with antioxidant ability activates the Nrf2/KEAP1 signaling pathway.This activation leads to NRF2 translocation to the nucleus and heterodimerization with sMaf proteins.Subsequently, there is binding with ARE to activate antioxidant genes (SOD, CAT, NQO1, HO1, and GPx).The activation of these antioxidant genes enhances the antioxidant response by suppressing oxidative stress and apoptosis induced via heat stress and xenobiotics.(B) Oxidative stress induced by xenobiotics/heat stress increases the level of KEAP1 and inhibits the translocation of NRF2 to the nucleus, leading to oxidative damage

Funding:
The author(s) declare financial support was received for the research, authorship, and/or publication of this article.This research was funded by the National Key R&D Program of China (grant number 2022YFD1600103), The Shandong Province Modern Agricultural Technology System Donkey Industrial Innovation Team (grant no.SDAIT-27), Livestock and Poultry Breeding Industry Project of the Ministry of Agriculture and Rural Affairs (grant number 19211162), The National Natural Science Foundation of China (grant no.31671287), The Open Project of Liaocheng University Animal Husbandry Discipline (grant no.319312101-14), The Open Project of Shandong Collaborative Innovation Center for Donkey Industry Technology (grant no.3193308), Research on Donkey Pregnancy Improvement (grant no.K20LC0901) and Liaocheng University scientific research fund (grant no.318052025).

Table 1 .
Summary of studies targeting bioactive compound supplementation to combat xenobiotic-induced oxidative stress and apoptosis in reproductive cells via activation of the Nrf2 signaling pathway.

Table 2 .
Summary of studies investigation supplementation with bioactive compounds to combat heat stress-induced oxidative distress and apoptosis in mammalian reproductive cells via activation of the Nrf2 signaling pathway.