Protective effect of Jinkui Shenqi pills against polystyrene-induced reproductive injury
Article information
Abstract
Objective
Polystyrene exposure poses an increasing threat to reproductive health, and effective interventions remain limited. Jinkui Shenqi pills (JSP), a traditional Chinese medicine used to treat reproductive disorders, may protect against such damage. This study integrated network pharmacology with experimental validation to explore the mechanisms by which JSP counteracts polystyrene-induced reproductive toxicity.
Methods
Potential targets related to polystyrene toxicity and bioactive components of JSP were retrieved from relevant databases. Overlapping targets were identified using VENNY 2.1. Protein-protein interaction networks were constructed using STRING, and core targets were identified with Cytoscape. Enrichment analyses were performed using DAVID. Molecular docking was used to evaluate ligand-target binding. A polystyrene-exposed mouse model was established, and tissue injury and recovery were assessed by hematoxylin-eosin (HE) staining and Western blotting (WB).
Results
In-depth screening identified 111 overlapping targets, and three key molecules—AKT serine/threonine kinase 1 (AKT1), caspase-3 (CASP3), and B-cell lymphoma 2 (BCL2)—were selected for further investigation. Gene Ontology analysis highlighted phosphorylation-related processes and responses to xenobiotics, while Kyoto Encyclopedia of Genes and Genomes analysis indicated enrichment in pathways related to lipid metabolism, atherosclerosis, cancer, and phosphatidylinositol-3-kinase (PI3K)-AKT signaling. Molecular docking confirmed effective binding of sitosterol, alisol B monoacetate, acetate, and alisol to these targets. HE staining revealed polystyrene-induced damage in testicular and ovarian tissues, which was effectively alleviated by JSP administration. WB results further demonstrated that JSP upregulated the expression of core target proteins, supporting its protective role against polystyrene-induced reproductive toxicity.
Conclusion
JSP mitigates polystyrene-induced reproductive toxicity by modulating AKT1, CASP3, and BCL2, supporting its potential as a protective agent.
Introduction
Polystyrene consists of small plastic particles less than 5 mm in diameter [1]. This versatile material has widespread applications across sectors including plastic manufacturing, packaging, and building insulation. However, recent environmental studies have raised concerns that these small particles have entered ecosystems, contaminating oceans, soils, and even the atmosphere [2]. This infiltration has contributed to substantial environmental pollution and warrants urgent attention. Research has demonstrated that, under certain environmental conditions, polystyrene can release toxic chemicals [3]. These substances may enter the human body through multiple routes, including inhalation via the respiratory tract, ingestion through contaminated food and water, and absorption through contact with clothing and cosmetics [4]. These released substances are particularly concerning due to their potential harm to the human reproductive system [5,6]. Traces of these chemicals have been detected in key reproductive tissues, including the testes, semen, and placenta [7,8]. Moreover, experimental studies have confirmed polystyrene-associated reproductive toxicity, demonstrating that exposure can directly damage germ cells, disrupt hormonal balance, and impair reproductive organ function [9]. Such disruptions can have serious health consequences, including infertility [10]. Exposure to polystyrene nanoplastics during pregnancy and lactation can adversely influence neural stem cell function and hormone (e.g., luteinizing hormone [LH])-driven neuronal composition in offspring, potentially increasing the risk of neurodevelopmental disorders [11]. In addition, oral exposure to polystyrene microplastics (PS-MPs) can disrupt glycolipid metabolism, thereby increasing the likelihood of insulin resistance [12]. Elevated insulin levels can stimulate the ovaries to produce excess androgens, potentially resulting in polycystic ovary syndrome [13]. Experiments in mice have further shown that oral microplastic exposure may activate apoptosis in spermatogenic cells via the p53 signaling pathway, ultimately leading to reproductive toxicity [14]. Exposure to PS-MPs can also induce histological changes in mouse testes, impair normal spermatogenesis, and disrupt serum hormone secretion. Prolonged exposure to PS-MPs may result in reproductive system toxicity, characterized by decreased testosterone levels mediated through the LH-driven LH receptor/cyclic adenosine monophosphate/protein kinase A/steroidogenic acute regulatory protein signaling pathway [15]. Given the widespread contamination by polystyrene and the serious reproductive health risks it poses, prioritizing the exploration of effective detoxification strategies and protective measures is essential. Such approaches will be crucial for addressing environmental threats and safeguarding human health against the potential dangers associated with polystyrene exposure.
Jinkui Shenqi pills (JSP) are a traditional Chinese medicine formulation with a long history, reflecting the accumulated clinical insights of ancient medical practitioners. This herbal formulation, classified as a mildly warming therapy, combines several medicinal herbs, including Rehmannia glutinosa (Di Huang), Dioscorea opposita (Shan Yao), Cornus officinalis (Shan Zhu Yu), Poria cocos (Fu Ling), Mudan bark (Mu Dan Pi), Alisma orientalis (Ze Xie), Cinnamomum cassia (Gui Zhi), Aconitum carmichaelii (Fu Zi), Achyranthes bidentata (Niu Xi), and Plantago asiatica (Che Qian Zi), among others [16]. Pharmacological studies have shown that JSP contains multiple bioactive constituents, such as dicinnamic acid, paeonol, quercetin, and stigmasterol, which collectively exert antioxidant, anti-inflammatory, anti-apoptotic, and endocrine-modulatory effects. These compounds are known to regulate key signaling pathways, including phosphatidylinositol-3-kinase (PI3K)-AKT, mitogen-activated protein kinase (MAPK), and hypoxia-inducible factor 1 (HIF1), thereby enhancing cellular resistance to exogenous stressors [17,18]. Each constituent herb contributes distinct therapeutic properties that collectively enhance the overall efficacy of JSP. This versatile formulation primarily targets kidney deficiencies, which may manifest as conditions such as kidney-yang deficiency, characterized by edema, weakness of the lower back and knees, urinary dysfunction, and sensitivity to cold. JSP is used in various clinical contexts in which renal insufficiency is either a primary disorder or a contributing factor. In a rat model of diabetes, JSP has demonstrated early intervention effects by activating protective mechanisms in the early stages of the disease. This intervention slows disease progression, reduces the risk of complications, and provides a basis for the development of new therapeutic strategies for diabetes and its associated conditions [19]. From a pharmacological perspective, JSP acts as a warming agent that helps restore internal homeostasis by reinforcing kidney yang and promoting balanced qi circulation and water metabolism.
Recent research suggests that JSP may exert beneficial effects on reproductive health. In particular, it may aid individuals with asthenozoospermia by improving sperm motility, increasing sperm quality, and ultimately promoting fertility [20,21], reflecting its multifaceted therapeutic profile. JSP is also used to support female reproductive health by warming yang and nurturing the kidneys, thereby improving reproductive function. In addition, it may help regulate endocrine balance, alleviate irregular menstrual cycles, and enhance overall physical well-being, contributing to improved fertility potential. By modulating complex hormonal interactions and improving overall organ function, this herbal formulation may support favorable reproductive outcomes, especially in contexts where reproductive toxicity from external agents, such as polystyrene, poses a risk.
Methods
1. Screening of polystyrene-induced reproductive toxicity targets
To identify reproductive toxicity targets associated with polystyrene, searches were conducted of the Comparative Toxicogenomics Database (CTD; https://ctdbase.org/) and the GeneCards database (www.genecards.org) using the keyword ‘polystyrene.’ The term ‘reproductive toxicity’ was then entered into the GeneCards database to obtain targets specifically related to reproductive toxicity. Targets were screened with the category set to ‘protein coding’ and a relevance score >1 to identify reproductive toxicity-related targets. Using the VENNY 2.2 online tool (https://bioinfogp.cnb.csic.es/tools/venny/index.html), overlapping targets associated with polystyrene-induced biological toxicity were identified.
2. Active ingredients and targets of JSP
The primary ingredients of JSP include Poria, Aconite, Guizhi, Mudan bark, yam, dogwood, R. glutinosa, and Alisma. We searched for these eight components in the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database (https://tcmsp-e.com/index.php) with the following screening criteria: oral bioavailability (OB) ≥30% and drug likeness (DL) ≥0.18. Subsequently, we extracted the corresponding chemical structure data from the PubChem database and predicted potential targets using the SwissTargetPrediction database (http://swisstargetprediction.ch/).
3. Identification of overlapping targets
Targets corresponding to the effective active ingredients of JSP and the reproductive toxicity targets of polystyrene were entered into the VENNY 2.1 tool to identify overlapping targets. Venn diagrams were generated to visually illustrate the relationships among these overlapping targets.
4. Network pharmacology analysis
Relevant target data were imported into Cytoscape v3.9.1 (https://cytoscape.org/) to construct a network illustrating the relationships among polystyrene-induced reproductive toxicity, JSP, its active ingredients, and the corresponding targets. The NetworkAnalyzer tool in Cytoscape was used to extract network topology and node attribute information, which was then visually represented. Subsequently, the overlapping targets were imported into the STRING database (https://cn.string-db.org/) to construct a protein-protein interaction (PPI) network.
5. Enrichment analysis
The DAVID database (https://david.ncifcrf.gov/) was used to analyze the overlapping targets associated with the action targets of JSP and polystyrene-induced reproductive toxicity. By selecting ‘Official Gene Symbol,’ ‘Human (Homo sapiens),’ and ‘Gene List,’ Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. The enrichment results were subsequently imported into Wei Sheng Xin, an online bioinformatics visualization platform (https://www.bioinformatics.com.cn/), for graphical presentation.
6. Molecular docking
AutoDock Tools v1.5.7 (https://autodocksuite.scripps.edu/) was used to perform molecular docking between the core components of JSP and target proteins associated with polystyrene-induced reproductive toxicity, thereby validating interactions between active compounds and key targets. The docking results were then imported into PyMOL 2.6 (https://pymol.org/) for visualization, generating molecular docking binding mode diagrams to illustrate spatial conformations.
7. Main reagents
Polystyrene microspheres were obtained from the Besler Chromatography Technology Development Center (2.5% w/v; particle size, 5.0 μm; Item No. 6-1-0500). JSP (water honey pills) were purchased from Tong Ren Tang. A hematoxylin-eosin (HE) staining kit (G1076) was obtained from Servicebio. The following antibodies were used: anti-AKT serine/threonine kinase 1 (AKT1) rabbit polyclonal antibody (P900012; Epizyme); anti-B-cell lymphoma 2 (Bcl-2) rabbit polyclonal antibody (P012161; Epizyme); anti-cleaved caspase-3 (CASP3; p17) rabbit polyclonal antibody (P011406; Epizyme); glyceraldehyde 3-phosphate dehydrogenase rabbit polyclonal antibody (AC001; ABclonal); and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (BA1054; Boster).
8. Experimental animals and groups
A total of 60 (4-week-old) specific pathogen-free (SPF) Institute of Cancer Research (ICR) mice (30 males and 30 females) were obtained from SPF (Beijing) Biotechnology Co. Ltd. and acclimatized for 1 week. The mice were randomly divided into three groups (n=20 per group; 10 males and 10 females per group): control, model, and JSP treatment. During the initial 2-week exposure phase, both the model and JSP treatment groups received 50 mg/L PS-MPs in their drinking water to induce reproductive toxicity. This concentration was selected based on previous studies demonstrating reproductive impairment at environmentally relevant exposure levels [9,22]. The control group received standard drinking water throughout the entire experimental period.
From weeks 3 to 4, the JSP treatment group received JSP by daily oral gavage. The administered dose was calculated based on the human equivalent dose of 8 g/day for a 60-kg adult and converted to a mouse dose using body surface area normalization according to the formula: mouse dose (g/kg)=human dose (g/kg)×(human Km/mouse Km). This dose, which has been validated in previous rodent models of reproductive dysfunction [23], was selected for the present study based on pre-experimental confirmation that it significantly improved reproductive function indicators in model animals without observable toxicity. During this phase, the control group received equivalent volumes of saline by gavage to control for procedural stress. The model group received no intervention during weeks 3–4, allowing assessment of the persistence and stability of polystyrene-induced reproductive injury.
All gavage procedures were performed under consistent conditions with respect to timing and volume. Fresh PS-MP solutions were prepared daily at 7:00 AM, and drinking bottles were shaken every 7 hours to maintain suspension homogeneity. At the end of the 4-week experimental period, all mice were anesthetized with isoflurane, and reproductive tissues (testes from males and ovaries from females) were collected for subsequent analyses.
9. Histomorphological analysis by HE staining
To evaluate testicular and ovarian tissue damage induced by PS-MP exposure and the effects of JSP treatment, fresh testicular and ovarian tissues from mice in the model and JSP treatment groups were collected for HE staining. Tissues were fixed in 4% paraformaldehyde for 24 hours, followed by graded alcohol dehydration, paraffin embedding, and sectioning. Sections were stained with hematoxylin for 3–5 minutes, rinsed with tap water, differentiated, rinsed again, and blued using a bluing solution, followed by further rinsing with running water. The sections were then sequentially treated with 85% and 95% ethanol for 5 minutes each, stained with eosin for 5 minutes, dehydrated, and mounted. Histological changes were examined under a light microscope, and representative images were captured and analyzed.
10. Western blotting analysis of protein expression
Mouse testicular and ovarian tissues (20 mg) were homogenized in lysis buffer, incubated on ice for 10 minutes, and sonicated to ensure complete cell disruption. The lysates were centrifuged at 12,000 r/min for 10 minutes at 4 °C, and the supernatants were collected. Protein concentrations were determined, and samples were prepared by adding protein loading buffer and heating at 100 °C for 10 minutes. Equal amounts of protein (20 µg) were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto 0.45 µm nitrocellulose membranes. After blocking, membranes were incubated overnight at 4 °C with primary antibodies under gentle agitation. Following washing with Tris-buffered saline with Tween 20, membranes were incubated with secondary antibodies at room temperature. Protein bands were visualized using a colorimetric detection method, and band intensities were quantified using ImageJ ver. 1.8.0 (National Institutes of Health). Details of the primary and secondary antibodies are provided in the Supplementary Table 1.
11. Ethical review
This study was reviewed and approved by the Ethics Committee of Shanxi Provincial People’s Hospital under the ethics approval number 2022(267). All animal experiments were conducted in accordance with the Animal Research: Reporting of In Vivo Experiments guidelines.
Results
1. Core targets of JSP against PS-induced reproductive toxicity
To elucidate the potential mechanisms underlying polystyrene-induced reproductive toxicity, a comprehensive set of polystyrene-related targets was first compiled. Searches of the CTD and GeneCards databases identified 814 unique targets associated with polystyrene. In parallel, a search for ‘reproductive toxicity’ in the GeneCards database (relevance score >1) yielded 3,689 potential reproductive toxicity-related targets. Intersection analysis of these two target sets using VENNY 2.1 revealed 578 overlapping targets (Figure 1, right set). This substantial overlap suggests a strong molecular association between polystyrene exposure and reproductive dysfunction, providing a focused target pool for subsequent mechanistic investigations. To predict how JSP might counteract PS-induced reproductive damage, its potential active components and corresponding targets were identified. Based on the OB and DL screening criteria, active ingredients from the eight constituent herbs of JSP—Poria, Aconite, Guizhi, Mudan bark, yam, dogwood, R. glutinosa, and Alisma—were selected. Target prediction using the SwissTargetPrediction database yielded 864 potential protein targets for these active components. Importantly, intersection analysis between these 864 putative drug targets and the 578 disease-related targets identified above resulted in 111 shared targets (Figure 1, central overlap). These 111 overlapping targets represent the core predicted molecular targets through which JSP may exert therapeutic effects against polystyrene-induced reproductive toxicity.
Venn diagram illustrating target overlap between Jinkui Shenqi pills (JSP) and polystyrene-induced reproductive toxicity. The left circle (864 targets) represents JSP-related targets, whereas the right circle (578 targets) represents targets common to polystyrene exposure and reproductive toxicity. The overlapping region (111 targets) indicates shared targets that may mediate the protective effects of JSP.
2. Construction of the drug-disease-target network
To elucidate the potential mechanisms by which JSP alleviates polystyrene-induced reproductive toxicity, a systematic network pharmacology approach was applied. First, the active components of JSP and their corresponding targets were integrated with known targets associated with polystyrene-induced reproductive toxicity to construct a comprehensive ‘drug-component-disease-target’ interaction network using Cytoscape. This heterogeneous network consisted of 108 nodes and 1,392 edges, visually depicting the complex interactions between the botanical formulation and the pathological condition (Figure 2A). Network topology analysis of this integrated model identified four key active components of JSP with the highest connectivity, suggesting their central roles in the therapeutic effects of JSP (Table 1). Comparative analysis of the targets derived from these JSP components and those associated with reproductive toxicity yielded 111 overlapping targets, representing putative key mediators of the intervention (Figure 2A).
Network pharmacology analysis of Jinkui Shenqi pills (JSP) against polystyrene-induced reproductive toxicity. (A) Drug-component-target-disease interaction network. The outer ring represents JSP components, the middle ring denotes disease-related targets, and the inner ring indicates overlapping targets between JSP and polystyrene-induced reproductive toxicity. (B) Protein-protein interaction (PPI) network constructed using the STRING database (confidence score >0.4). (C) PPI network visualized and analyzed in Cytoscape v3.9.1. Node size and color intensity reflect degree centrality. (D) Top hub targets identified by the CytoHubba plugin using the Degree algorithm.
Drug active ingredient information
To further explore the functional relationships among these common targets, the 111 overlapping genes were imported into the STRING database to construct a PPI network. Using a confidence score threshold of >0.4, a PPI network was generated. Three targets were excluded as isolated nodes due to the absence of documented interactions, resulting in a final high-confidence network comprising 108 nodes and 1,392 edges (Figure 2B). This network was subsequently visualized and analyzed using Cytoscape (v3.9.1). In the network visualization (Figure 2C), node size and color intensity were scaled according to degree values, allowing an initial visual assessment of node importance.
To objectively identify the most central targets within the PPI network, topological analysis was performed using the CytoHubba plugin. The ‘Degree’ algorithm, which ranks nodes based on the number of direct connections, was applied. Based on this analysis, eight targets with the highest degree values were identified as core hubs: AKT1, CASP3, BCL2, tumor necrosis factor (TNF), estrogen receptor 1 (ESR1), hypoxia-inducible factor 1 alpha (HIF1A), epidermal growth factor receptor (EGFR), and C-X-C motif chemokine ligand 8 (CXCL8) (Figure 2D). The high connectivity of these hub targets underscores their pivotal regulatory roles within the network, suggesting that they are key signaling molecules through which JSP may exert protective effects against polystyrene-induced reproductive damage.
3. Enrichment analysis results
Enrichment analysis yielded a total of 595 GO terms, including 522 biological processes (BPs), 69 cellular components (CCs), and 104 molecular functions (MFs). The top 10 terms in each category (BP, CC, and MF) were selected for visualization in a bubble chart (Figure 3A). The most prominently enriched terms included phosphorylation, protein phosphorylation, and responses to xenobiotic stimuli. In addition, KEGG pathway analysis identified 165 pathways, of which the top 20 were selected for visualization in a bubble chart (Figure 3B). The major enriched pathways included lipids and atherosclerosis, pathways in cancer, and the PI3K-AKT signaling pathway.
Enrichment analysis. (A) Gene Ontology (GO) enrichment bubble chart. Bubble color indicates −log10(p-value), while bubble size corresponds to gene count. The top 10 terms from each GO category (biological processes [BP], cellular components [CC], and molecular functions [MF]) are shown. (B) Kyoto Encyclopedia of Genes and Genomes pathway enrichment bubble chart. The top 20 enriched pathways are displayed, with bubble size reflecting gene count and color indicating significance level. ATP, adenosine triphosphate; AGE-RAGE, advanced glycation end products-receptor for advanced glycation end products; IL, interleukin; TNF, tumor necrosis factor; HIF1, hypoxia-inducible factor 1; PI3K, phosphatidylinositol-3-kinase.
4. Molecular docking results and visualization
Within the network linking JSP active ingredients to targets involved in mitigating polystyrene-induced reproductive toxicity, four key components were selected as ligands, while core targets identified from the PPI network—AKT1, CASP3, and BCL2—were chosen as receptors. Molecular docking was subsequently performed, and the results were visualized and analyzed using PyMOL (Table 2, Figure 4).
Binding energies of major components of JSP
Molecular docking models and binding interactions between Jinkui Shenqi pills (JSP) components and core targets. Predicted binding conformations of sitosterol, alisol B monoacetate, acetate, and alisol within the binding pockets of AKT serine/threonine kinase 1 (AKT1), caspase-3 (CASP3), and B-cell lymphoma 2 (BCL2) are shown.
5. HE staining results
HE staining revealed that, in female mice, ovarian tissues in the model group exhibited a substantial degree of granulosa cell detachment within follicles compared with the control group. A small number of granulosa cells showed necrosis and nuclear fragmentation, and mild granulocyte infiltration was observed around the follicles. After 2 weeks of JSP treatment, granulosa cells were restored to a regular and orderly arrangement, with no obvious necrosis, detachment, or inflammatory infiltration (Figure 5A). In male mice, compared with the normal control group, testicular tissues from the model group exhibited disorganization of the seminiferous epithelium, with loosely arranged and sparsely distributed spermatogenic cells and widened intercellular spaces. Shedding of spermatogenic cells at various developmental stages was observed, resulting in a marked reduction in mature spermatozoa within the lumen. Following 2 weeks of JSP treatment, histopathological damage was markedly improved: the seminiferous epithelium was restored, spermatogenic cells were arranged in a compact and orderly manner, and the sperm count increased (Figure 5B). Overall, these preliminary HE staining results indicate that JSP treatment can ameliorate PS-MP–induced histopathological abnormalities in both ovarian and testicular tissues. Although quantitative histological assessments of necrotic cell proportions or immune cell infiltration were not performed in this study, subsequent Western blotting (WB) analyses provided quantitative molecular-level evidence, demonstrating that JSP significantly regulates the expression of key proteins involved in cell survival and apoptosis.
Histomorphological changes in reproductive tissues assessed by hematoxylin-eosin (HE) staining. (A) Ovarian sections. (B) Testicular sections. Arrows indicate key pathological features, including granulosa cell detachment in the ovary, disorganized seminiferous epithelium in the testis, and inflammatory cell infiltration. Groups include control, model (polystyrene microplastics)(arrows), and Jinkui Shenqi pills (JSP)-treated. Scale bar: 50 µm.
6. WB analysis of target proteins
WB analysis provided molecular-level evidence for the protective effects of JSP. In murine testicular and ovarian tissues, exposure to PS-MPs resulted in significant downregulation of AKT1 and BCL2 expression, accompanied by upregulation of CASP3 expression compared with the control group. Notably, JSP administration markedly attenuated these PS-MP–induced alterations, normalizing the protein expression profiles. The absence of statistically significant differences between the JSP-treated and control groups further supports the capacity of JSP to reestablish molecular homeostasis disrupted by polystyrene toxicity (Figure 6).
Western blot quantification of target protein expression. (A) Ovarian tissue. (B) Testicular tissue. Data are presented as relative expression levels. AKT1, AKT serine/threonine kinase 1; BCL2, B-cell lymphoma 2; CASP3, caspase-3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ns, not significant; JSP, Jinkui Shenqi pills. a)p<0.05; b)p<0.01.
Discussion
This study represents the first integrated investigation combining network pharmacology with experimental validation to systematically elucidate the multi-target and synergistic mechanisms by which JSP counteracts reproductive damage induced by PS-MPs. The central finding is that JSP effectively reverses PS-MP–induced histopathological damage and functional dysregulation by modulating a cell survival-apoptosis regulatory network centered on AKT1, CASP3, and BCL2.
To experimentally validate the predictions derived from network pharmacology, three core targets—AKT1, CASP3, and BCL2—were prioritized for detailed analysis based on their central roles in regulating cell survival and apoptosis in the context of reproductive toxicity. Other predicted targets, such as TNF, ESR1, HIF1A, EGFR, and CXCL8, are involved in inflammatory, hormonal, and hypoxic responses and may also contribute to the multi-target synergistic effects of JSP. However, their functional validation remains a focus for future investigations aimed at further clarifying the comprehensive protective network of JSP.
AKT1, a central hub of the PI3K-AKT signaling pathway, plays an essential role in maintaining cell survival, proliferation, and metabolism [24,25]. Our PPI network analysis identified AKT1 as a key target, a finding further corroborated by WB results demonstrating that PS-MP exposure significantly downregulated AKT1 protein expression in both mouse testicular and ovarian tissues. This downregulation directly weakens intrinsic germ cell survival signaling. This observation is consistent with previous reports describing inhibition of the PI3K-AKT pathway by environmental toxins [26,27]. Importantly, JSP intervention restored AKT1 expression levels, indicating that its primary protective mechanism involves reactivation of the PI3K-AKT signaling pathway. By enhancing this pathway, JSP likely promotes germ cell survival under PS-MP–induced toxic stress, thereby laying the foundation for subsequent cellular repair and functional recovery.
Apoptosis represents the primary execution mechanism of PS-MP–induced reproductive toxicity [28]. The present study indicates that PS-MP exposure activates the canonical mitochondrial apoptotic pathway. On the one hand, inhibition of AKT1 attenuates phosphorylation-dependent inactivation of downstream pro-apoptotic proteins, such as Bad [29]. On the other hand, PS-MPs may directly induce oxidative stress, leading to mitochondrial membrane potential loss, cytochrome c release, and subsequent activation of the caspase cascade [30]. Our experimental data provide direct evidence for this mechanism: in the model group, expression of the anti-apoptotic protein BCL2 was significantly reduced, whereas levels of the activated executioner protein cleaved CASP3 were markedly increased. This molecular profile indicates large-scale initiation of the apoptotic program.
The therapeutic effects of JSP were particularly pronounced at this stage. Following treatment, BCL2 expression was significantly upregulated, whereas CASP3 activation was effectively suppressed. These findings indicate that JSP inhibits apoptosis through a dual mechanism: first, by directly stabilizing the outer mitochondrial membrane via upregulation of BCL2, thereby preventing cytochrome c release; and second, by indirectly suppressing apoptotic initiation through activation of AKT1. This precise regulation of the BCL2/CASP3 axis provides direct protection to key germ cells, such as spermatogonia and oocytes, against programmed cell death [31,32]. Consistent with these molecular effects, histological analyses demonstrated marked improvements in seminiferous tubule architecture and the orderly arrangement of follicular granulosa cells.
The primary strength of this study is the demonstration that JSP exerts its therapeutic effects not through a single molecular target, but via network-based regulation involving multiple components and targets. The four core active compounds identified by network pharmacology showed strong binding affinities to AKT1, CASP3, and BCL2. Notably, the robust interaction between sitosterol and BCL2 was particularly pronounced, providing a molecular-level explanation for the anti-apoptotic effects of JSP. Moreover, GO and KEGG enrichment analyses highlighted key BPs—including protein phosphorylation, responses to xenobiotic stimuli, and the PI3K-AKT signaling pathway—as central to the mechanism of action of JSP.
Pharmacological evidence further supports the multi-target therapeutic profile of JSP. Network-based analyses indicate that JSP modulates targets involved in oxidative stress, inflammation, apoptosis, and hormonal regulation, consistent with the present focus on the AKT1/BCL2/CASP3 axis. Integrated serum pharmacochemistry and pharmacokinetics further identify key bioactive components of JSP that contribute to its efficacy in conditions associated with reproductive dysfunction. Collectively, these findings indicate that JSP exerts its effects through synergistic regulation of multiple signaling pathways—such as PI3K-AKT, MARK, and HIF1—rather than through a single mechanism [17,18]. These pathways are consistent with the documented pharmacological properties of JSP, including antioxidant, anti-inflammatory, and stress-adaptive activities, thereby linking its traditional kidney-warming and qi-regulating applications with a contemporary mechanistic understanding of its protective effects against environmental toxicants. In line with this interpretation, our experimental results demonstrate that JSP establishes a synergistic protective network by upregulating AKT1 to enhance cell survival signaling and BCL2 to reinforce anti-apoptotic defenses, while simultaneously inhibiting CASP3 to block apoptotic execution. Restoration of this survival-apoptosis balance provides a clear molecular basis for the capacity of JSP to preserve reproductive tissue structure and function. Furthermore, although KEGG-enriched pathways related to lipid metabolism and cancer are not directly associated with reproductive toxicity, their enrichment suggests that JSP may also exert metabolic and anti-inflammatory effects that indirectly contribute to its protective role, opening new avenues for future investigation. Thus, the present study elucidates a central, albeit partial, component of JSP’s broader regulatory network.
In this investigation, initial observations from HE staining revealed that JSP exerted a restorative influence on reproductive tissue damage inflicted by PS-MPs. This was evident in the reestablishment of the orderly arrangement of ovarian granulosa cells and the improvement of structural integrity in testicular spermatogenic cells. Although HE staining did not yield detailed phenotypic data, such as precise counts of necrotic cells or quantitative measurements of immune cell infiltration, the observed histological changes were consistent with trends identified in subsequent WB analyses. Specifically, the upregulation of AKT1 and BCL2, together with the downregulation of CASP3, further substantiated the protective role of JSP, in terms of both tissue morphology and molecular expression patterns. Looking forward, future research could incorporate more precise quantitative assessments of apoptosis, inflammatory infiltration, and other relevant phenotypes. Such analyses may include techniques such as terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, immunohistochemical quantification, and flow cytometry to further clarify the mechanisms underlying the protective effects of JSP.
Although this study provides mechanistic insight into how JSP alleviates PS-MP–induced reproductive injury via the AKT1/BCL2/CASP3 axis, several limitations must be acknowledged. First, experimental validation was confined to this signaling axis, while other network-predicted core targets—such as TNF, ESR1, HIF1A, EGFR, and CXCL8, which are implicated in inflammatory, hormonal, and hypoxic responses—were not examined and may also contribute to the multi-target synergistic effects of JSP. Second, changes in AKT1 and CASP3 activity were inferred primarily from total protein expression; future studies should assess phosphorylated AKT levels and CASP3 enzymatic activity to confirm functional modulation. Third, although JSP upregulated BCL2 and downregulated CASP3 expression, direct evidence of anti-apoptotic effects requires further validation using approaches such as TUNEL assays. Additionally, only a single dose of JSP was evaluated; dose–response studies are necessary to determine optimal therapeutic ranges for clinical translation. Finally, while JSP demonstrated efficacy in a murine model, its safety, bioavailability, and potential interactions with existing therapies in humans warrant rigorous clinical evaluation.
In conclusion, by integrating network pharmacology, molecular docking, and both in silico and in vivo experimental approaches, this study demonstrates that JSP can effectively mitigate PS-MP-induced reproductive toxicity through a multi-target, synergistic mechanism. The core mechanism involves the modulation of the AKT1/BCL2/CASP3 signaling axis, thereby restoring the critical balance between cell survival and apoptosis. These findings not only provide a robust scientific rationale for the use of JSP in addressing reproductive dysfunction caused by environmental pollutants but also open new avenues for the development of reproductive protective strategies based on traditional Chinese medicine.
Notes
Conflict of interest
No potential conflict of interest relevant to this article was reported.
Acknowledgments
We gratefully acknowledge financial support from the Technology Bureau of Lvliang City, the National Natural Science Foundation of China Youth Project, and Fenyang Hospital of Shanxi Province. We are sincerely grateful to Dr. Xiaofang Han for her invaluable guidance throughout the research design, data analysis, and manuscript preparation. We also thank our colleagues for their dedicated contributions and the staff of the institutional animal facility for their professional animal care.
Author contributions
Conceptualization: HL, LY. Methodology: HL, LY. Formal analysis: HL. Data curation: HL, LY. Funding acquisition: XH. Project administration: XH. Visualization: HL, LY, AA. Writing – original draft: HL. Writing – review & editing: AA, XH. Approval of final manuscript: HL, LY, AA, XH.
Supplementary materials
Supplementary material can be found via https://doi.org/10.5653/cerm.2025.08914.
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