Icariin rescues Reproductive damage caused by Manganese: Network pharmacology and Experimental validation

Icariin rescues Reproductive damage caused by Manganese: Network pharmacology and Experimental validation | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 8 July 2025 V1 Latest version Share on Icariin rescues Reproductive damage caused by Manganese: Network pharmacology and Experimental validation Authors : Jiaqi He , Tongci Li , Yan Liang , Ying Tian , Xiang Lu , Jidong Zhang , and Jun Tan 0009-0002-8566-0448 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175195452.27644103/v1 214 views 137 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The global escalation of male infertility, a critical reproductive health crisis affecting approximately 15% of couples worldwide, is increasingly attributed to environmental exposures to toxic heavy metals, with manganese (Mn) emerging as a significant occupational and environmental contaminant. Despite its association with impaired spermatogenesis and testicular dysfunction, no effective clinical interventions exist to counteract heavy metal-induced reproductive toxicity. This study investigates the therapeutic potential of icariin (ICA) in mitigating Mn-exposure male reproductive damage through a multi-omics strategy combining network pharmacology, molecular docking, and in vitro validation. Disease-drug-target network analysis prioritized sphingolipid metabolism and NF-κB signaling as key pathways modulated by ICA, with molecular docking confirming high-affinity interactions between ICA and core targets. Experimental validation using Mn-exposure mouse spermatogonial GC-1 cells demonstrated that ICA significantly rescued cytotoxicity (MTT assay) and reversed dysregulated gene expression patterns. Specifically, the qPCR revealed significant upregulation the expression of TRIAP1, HSP90AA1 and NF-κB in the Mn-exposure group compared to the control group. Compared with the Mn-exposure group, the ICA intervention group exhibited significant downregulation the expression these genes, thereby highlighting dual regulatory effects on apoptotic resistance and inflammatory homeostasis. These findings establish ICA as a promising candidate for addressing heavy metal-associated male reproductive disorders, offering mechanistic insights into its cytoprotective and anti-inflammatory actions. Icariin rescues Reproductive damage caused by Manganese: Network pharmacology and Experimental validation Jiaqi He a , Tongci Li a , Yan Liang a , Ying Tian b , Xiang Lu a , Jidong Zhang c,d* , Jun Tan a* a Department of Histology and Embryology, Zunyi Medical University, Zunyi 563000, China. b Laboratory of Morphology, Zunyi Medical University, Zunyi 563000, China. c Department of Immunology, Zunyi Medical University, Zunyi 563000, China. d Special Key Laboratory of Gene Detection & Therapy of Guizhou Province, Zunyi Medical University, Zunyi 563000, China. * Corresponding to: Department of Histology and Embryology, Zunyi Medical University, Zunyi, Guizhou 563000, China. E-mail addresses: [email protected] (J. He), [email protected] (T. Li), [email protected] (Y, Liang), [email protected] (Y. Tian), [email protected] (X, Lu), [email protected] (J. Zhang), [email protected] (J. Tan). Abstract The global escalation of male infertility, a critical reproductive health crisis affecting approximately 15% of couples worldwide, is increasingly attributed to environmental exposures to toxic heavy metals, with manganese (Mn) emerging as a significant occupational and environmental contaminant. Despite its association with impaired spermatogenesis and testicular dysfunction, no effective clinical interventions exist to counteract heavy metal-induced reproductive toxicity. This study investigates the therapeutic potential of icariin (ICA) in mitigating Mn-exposure male reproductive damage through a multi-omics strategy combining network pharmacology, molecular docking, and in vitro validation. Disease-drug-target network analysis prioritized sphingolipid metabolism and NF-κB signaling as key pathways modulated by ICA, with molecular docking confirming high-affinity interactions between ICA and core targets. Experimental validation using Mn-exposure mouse spermatogonial GC-1 cells demonstrated that ICA significantly rescued cytotoxicity (MTT assay) and reversed dysregulated gene expression patterns. Specifically, the qPCR revealed significant upregulation the expression of TRIAP1, HSP90AA1 and NF-κB in the Mn-exposure group compared to the control group. Compared with the Mn-exposure group, the ICA intervention group exhibited significant downregulation the expression these genes, thereby highlighting dual regulatory effects on apoptotic resistance and inflammatory homeostasis. These findings establish ICA as a promising candidate for addressing heavy metal-associated male reproductive disorders, offering mechanistic insights into its cytoprotective and anti-inflammatory actions. KEYWORDS Icariin, network pharmacology, molecular docking, manganese, reproductive damage 1 INTRODUCTION With the acceleration of industrialization, heavy metal exposure has emerged as a pervasive global health issue, with its gravity continually escalating. 1 These pollutants, derived from a multitude of sources, exhibit remarkable persistence within the human body once introduced through the food chain, air, or water due to their recalcitrance to biodegradation. 2 Consequently, the elimination of heavy metals becomes exceptionally challenging for the body. Over an extended period, the accumulation of these substances poses a significant and ominous threat to human reproductive systems. 3 Beyond the reproductive toxicity associated with Mn exposure, the adverse effects of other heavy metals on reproductive health cannot be ignored. 4,5 These metals interfere with the endocrine system, disrupt DNA replication and repair mechanisms, or exert direct actions on reproductive cells, leading to a cascade of complex consequences, ranging from reproductive hormone imbalances to structural alterations in reproductive organs. 6,7 Heavy metals such as lead, cadmium, and mercury have been scientifically validated to cause varying degrees of reproductive damage. Specifically, lead exposure is correlated with decreased sperm quality in men, infertility in women, and abnormal fetal development. Cadmium disrupts hormonal balance, adversely affects ovarian function in women, and impairs sperm production in men. Mercury exposure may result in fetal neurological damage and genetic mutations in reproductive cells. 5,6,8,9 At present, therapeutic interventions for reproductive damage predominantly involve surgical procedures aimed at rectifying structural abnormalities, alongside pharmacological approaches designed to modulate hormonal levels and facilitate the restoration of reproductive cells. 10 Nevertheless, the progression of reproductive medicine has spurred a growing body of research investigating alternative treatment modalities, including stem cell technology and regenerative medicine. These emerging approaches hold the potential to offer novel insights into the repair and restoration of the reproductive system. 11-15 In the context of male reproductive impairment, research indicates that stem cell transplantation and reproductive tissue engineering hold potential as prospective therapeutic interventions. These approaches have the capability not only to repair damaged reproductive tissues but also to restore fertility. 11 Moreover, the application of antioxidants has demonstrated a protective effect against chemotherapy-induced damage to reproductive cells, thereby presenting novel opportunities for enhancing the survival rate of these cells. 13,15 While existing surgical and drug treatments can ameliorate symptoms of reproductive damage to some extent, the exploration of safer, more effective, and less side-effect-laden treatments remains a focal area of ongoing research. Traditional medicine, particularly Chinese medicine, has demonstrated unique efficacy in treating reproductive system diseases. Many Chinese medicines, including goji berries and cistanche have been proven to enhance reproductive function and fertility. 16-17 Notably, Chinese medicines with demonstrable reproductive protective effects, such as Epimedium, are increasingly recognized as adjunctive or alternative treatment options due to their gentle actions and minimal side effects. 4 The discovery of icariin (ICA) has provided novel insights into the treatment of reproductive damage caused by heavy metal exposure. 18 Preliminary studies have confirmed the role of ICA in scavenging free radicals, exhibiting antioxidant properties, and improving reproductive function. These Chinese medicines often exert their effects through a multi-target, multi-pathway mechanism, fostering the self-repair of the reproductive system. However, the specific mechanisms of action and their application effects in repairing reproductive damage caused by Mn exposure necessitate further in-depth research for verification and optimization. 9 The potential of these traditional remedies to revolutionize the treatment landscape for heavy metal-induced reproductive damage warrants rigorous scientific investigation and exploration. 19-20 2 MATERIALS AND METHODS 2.1 Study Design The flowchart of the study is shown in Figure 1. 2.2 Chemicals and reagents MnCl 2 •4H 2 O (batch number: R000534, purity: 99%) was purchased from Shanghai Yi En Chemical Technology Co., Ltd. Prepared a 0.1 M stock solution of MnCl₂, filtering it through a 0.22 μm filter. Followed this by preparing serial dilutions of MnCl₂ to achieve concentrations of 1, 10, 100, 1000, and 10000 μM. ICA (Batch number: 160920; purity: 98.61%) was purchased from Chengdu Plant Standard Pure Biotechnology Co., Ltd. ICA solutions of 0, 0.05, 0.5, 5, 50 μM were prepared and then filtered with a 0.22 μm filter. The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies. 21 Cell lines and experimental groups: Mouse spermatogonial GC-1 cells were obtained from the Cell Bank of the Chinese Academy of Sciences. The culture medium consisted of 89% DMEM supplemented with l0% FBS and 1% penicillin–streptomycin. The cells were divided into the control, Mn-exposure, and ICA (0, 0.05, 0.5, 5 and 50 μM) treatment groups, Cell viability was assessed by MTT assay. 2.3 Screening of ICA-related targets Information regarding ICA was retrieved from the Database and Analysis Platform for Systematic Pharmacology of Traditional Chinese Medicine (TCMSP, https://www.tcmsp-e.com/). Subsequently, the Swiss Target Prediction information database (http://www.swisstargetprediction.ch/) was utilized to obtain the corresponding component targets based on the PubChem database SMILES, with parameters set as ” Homo sapiens ” and probability≥0. Duplicate targets were subsequently eliminated. 2.4 Screening of targets related to male reproductive damage induced by Mn Subsequently, the GeneCards database (https://www.genecards.org/) was utilized with a relevance score threshold set at 5 or higher for target selection. Disease targets and duplication targets were then screened in the OMIM database (http://www.omim.org/). The Venny online analysis tool (http://bioinfogp.cnb.csic.es/tools/venny) was used to identify overlapping targets and diseases associated with ICA components, resulting in a Venn diagram that facilitated the identification of potential ICA targets for mitigating Mn-exposure reproductive damage. 2.5 Construction of the protein‒protein interaction (PPI) network The selected intersection targets were input into the STRING database (https://www.string-db.org/, Version 11.5), with the parameters set as ”multiple proteins” and species specified as ” Homo sapiens ”. Subsequently, relevant data were extracted, and these targets’ degree, betweenness centrality, and closeness centrality were computed using the CytoNCA tool integrated within Cytoscape 3.8 software. Consequently, pivotal targets were identified, and a core target map was generated. 2.6 Gene-set enrichment analysis: GO terms, KEGG pathways Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the common targets were performed using Metascape (https://Metascape.org/). GO enrichment analysis included three components: biological process (BP), molecular function (MF), and cellular component (CC). The results were visualized using the Microscopic Letter platform (http://www.bioinformatics.com.cn/). 2.7 Construction of the ”Disease-drug-target-pathway” network Cytoscape 3.8 software was used to construct a comprehensive ”disease-drug-target-pathway” network for investigating the therapeutic potential of ICA against Mn-exposure reproductive damage. This network integrated the chemical constituents of ICA, intersecting target genes, and their corresponding pathways. Furthermore, the CytoNCA tool within Cytoscape 3.8 software facilitated the calculation of crucial topological properties such as degree, betweenness centrality, and closeness centrality for each component. 2.8 Molecular docking and result visualization The three-dimensional structures of the aforementioned components were obtained from the TCMSP database. Subsequently, target information (species selection: human) was screened from the UniProt database (https://www.uniprot.org/) to acquire the 3D structure of the protein targets. The molecular docking software provided by HOME for Researchers (http://www.home-for-researchers.com/static/index.html#/) was used to perform docking analysis on targets with known three-dimensional structures and record their optimal affinity. The docking results were visualized using PyMOL software. 2.9 The impact of ICA on the proliferation of GC-1 cells Cells in the logarithmic growth phase were seeded at a density of 5×10 3 cells per well in a 96-well plate, with 5 replicate wells for each group. The experiment was independently repeated three times. After 24 hours of incubation, 10 μL of MTT reagent (5 mg/mL) was added, and the mixture was incubated for an additional 4 hours before the removal of the supernatant. Subsequently, 100 μL of DMSO was added to ensure complete dissolution of the crystals. The absorbance at a wavelength of 570 nm was measured using an enzyme-linked immunosorbent assay, and the cell viability was calculated as follows: cell viability (%) = (OD value of the experimental group - OD value of the blank group)/(OD value of the control well - OD value of the blank group) × 100%. 2.10 The impact of ICA on the expression of key regulatory genes in male reproductive damage induced by Mn Total RNA from mouse spermatogonial GC-1 cells was extracted using a TRIzol reagent. Following RNA quantification, 1μg of cDNA was synthesized using a reverse transcription kit and subsequently amplified by PCR according to the amplification kit’s instructions. The reaction conditions involved predenaturation at 95°C for 30 min, followed by 44 cycles of denaturation at 95°C for 5 s and annealing at 60°C for 30 s. The 2 -ΔΔCt method was employed to determine the relative expression of the target gene, with their corresponding sequences provided in Table 1. 2.11 Statistical analysis All experimental data were analyzed using SPSS 29.0 (IBM Corp., Armonk, NY, USA, Version 29.0) statistical software, and the results are presented as the mean ± standard error. The differences between groups were assessed through one-way ANOVA and pairwise comparisons using the least significant difference (LSD) method. A significance level of P <0.05 was considered to indicate statistical significance. Figure 6 was drawn by Figdraw. 3 RESULTS 3.1 Acquisition of relevant targets The 2D and 3D structures of the ICA from the TCMSP database are depicted in Figure 2. Table 2 presents the biologically relevant properties of ICA. A total of 305 targets associated with ICA were retrieved from the PubChem and Swiss Target Prediction databases. In comparison, the GeneCards and OMIM databases provided a collection of 1538 targets related to reproductive damage caused by Mn exposure. Figure 3 illustrates the presence of 49 targets associated with components and diseases. 3.2 Construction of the drug-component-disease-target visual network The drug-component-disease-target visual network was constructed using Cytoscape software, incorporating pathways associated with 305 drug targets, 1538 disease targets, and their overlapping targets. This network comprised 167 nodes and 188 edges, as illustrated in Figure 3. The depicted pathways included the sphingolipid signaling pathway, NF-κB signaling pathway, HIF-1 signaling pathway, C-type lectin receptor signaling pathway, hepatitis B infection pathway, lipids and atherosclerosis pathway, chemical oncogenicity-reactive oxygen species pathway, and chemical oncogenicity-receptor activation pathway. 3.3 Construction of the protein-protein interaction (PPI) network The 49 overlapping targets were subjected to analysis using the STRING database, and the resulting data were visualized using Cytoscape 8.3.0 software to construct a protein-protein interaction (PPI) network, as depicted in Figure 4. Subsequently, the CytoNCA tool was used to calculate the degree, betweenness centrality, and closeness centrality of these targets to identify key targets. Among them, the top 10 targets with high degrees may hold potential as therapeutic targets: HSP90AA1 (5WG8), TP53 (IG0), NF-κB (4MTZ), TNF (4ZCH), AKT1 (1UNQ), EGFR (2RGP), ESR1 (6VPF), RELA (4EWE), PTGS2 (5F19), and PRKCD (1YRK). 3.4 GO enrichment analysis of intersection targets The DAVID database was utilized for conducting GO functional enrichment analysis, with a significance threshold set at P <0.05. A total of 286 enriched terms were identified, including 209 BP terms, 32 CC terms, and 45 MF terms. Figure 5 presents the top ten enriched terms based on gene count. In the BP category, the predominant processes included negative regulation of apoptotic processes, response to xenogenic stimuli, replicative senescence, positive regulation of nitric oxide biosynthesis processes, positive regulation of cell growth, positive regulation of peptidyl serine phosphorylation, peptidyl serine phosphorylation itself and protein and peptidyl threonine phosphorylation. The CC category mainly encompasses macromolecular complexes, plasma membrane structures, perinuclear regions of cytoplasm and cytoplasmic components, extracellular exosomes, and nucleoplasmic entities. The MF category primarily involved enzyme binding activities alongside protein kinase activity and binding functions specifically related to protein serine/threonine/tyrosine kinases; it also included protein homodimerization activity as well as identical protein binding capabilities while encompassing nitric oxide synthase regulatory activity along with ubiquitin-protein ligase binding abilities. 3.5 KEGG enrichment analysis of intersection targets The intersection targets were subjected to KEGG enrichment analysis using the DAVID database, resulting in the identification of 129 significantly enriched pathways ( P <0.05). Figure 5 illustrates the top 30 pathways ranked by gene number. The ten most enriched pathways included the sphingolipid signaling pathway, chemical cancer-receptor activation, cancer pathway, human T-cell leukemia virus type 1 infection, neurodegenerative disease pathway, the role of the AGE-RAGE signaling pathway in diabetic complications, blood lipids and atherosclerosis, chemical cancer-reactive oxygen species, human cytomegalovirus infection, and the MAPK signaling pathway. 3.6 Molecular docking The components and top 10 key targets were subjected to molecular docking analysis using the researchers’ home website. A small molecule is considered to possess a strong binding ability to the docking protein if its affinity is less than -5. Table 3 presents the molecular binding energy between the components and targets, while Figure 6 visualizes the combination with the lowest affinity toward the target. 3.7 Confirmatory assessment of the repair effect of the ICA The repair effect of ICA on GC-1 cells was confirmed through a confirmatory assessment, wherein the cells were treated with a diluted solution of ICA at concentrations of 0.05, 0.5, 5 and 50 μM for 24 hours. The cell survival rate was determined using the MTT assay. The results demonstrated a significant decrease in the cell survival rate in the Mn-exposure group compared to that in the control group ( P <0.05). However, compared with the Mn-exposure group, the ICA-treated group exhibited a significantly increased cell survival rate ( P <0.05), as depicted in Figure 7. 3.8 The expression of the genes in male reproductive damage induced by Mn According to the results of molecular docking and pathway enrichment analysis, genes downstream of TP53 in the sphingolipid signaling pathway and NF-κB signaling pathway genes, including RELA , NF-κB , HSP90AA1 , TRIAP1 , and PTGS2 , were selected for assessing the impact of ICA on the expression of these genes in Mn-exposure spermatogonia. As depicted in Figure 8, the qPCR results demonstrated a significant increase in the mRNA expression levels of TRIAP1 , HSP90AA1 , and NF-κB in the Mn-exposure group compared to the control group ( P <0.05). Conversely, there was a significant decrease in the mRNA expression levels of PTGS2 and RELA ( P <0.05). In comparison to the Mn-exposure group, intervention with ICA led to a noteworthy reduction in the mRNA expression levels of TRIAP1 , HSP90AA1 , and NF-κB mRNA expression ( P <0.05). 4 DISCUSSION The health status of the reproductive system is crucial for human reproduction and survival. In recent years, male reproductive health has become a concerning issue, particularly due to the detrimental effects of chronic heavy metal poisoning on the reproductive system. 22 Traditional Chinese medicine (TCM) has gained acceptance among people due to its safety profile and minimal side effects. ICA, the main active component in Epimedium, a Chinese herbal medicine, has been reported to ameliorate spermatogenic disorders. 23 However, it remains unclear whether it can effectively repair Mn-exposure damage to the reproductive system and the specific underlying mechanism. Therefore, this study aimed to utilize network pharmacology and molecular docking techniques to identify potential targets of ICA in the Mn-exposure reproductive system. Furthermore, we will verify these hypotheses using GC-1 cells, a key cell involved in spermatogenesis, via in vitro experiments. The ultimate goal of this study was to explore the potential mechanisms by which ICA repairs male germ cell damage caused by Mn exposure and to provide valuable insights for treating male infertility. By constructing a PPI network and performing molecular docking, we identified the top 7 target proteins: HSP90AA1, TP53, RELA, TNF, AKT1, NF-κB, and PTGS2. Subsequently, HSP90AA1, TRIAP1, RELA, NF-κB, and PTGS2 were chosen for experimental validation. Our results demonstrated that the mRNA expression levels of HSP90AA1 , TRIAP1 , and NF-κB were significantly greater in the Mn-exposure group than in the control group ( P <0.05), while the mRNA expression of PTGS2 and RELA were substantially lower ( P <0.05). Notably, an independent study suggested a potential association between the rs11547523 variant in HSP90AA1 and idiopathic male infertility among Chinese individuals. 24 This finding is supported by our study, as increased levels of HSP90AA1 may impact the secretion of reproductive hormones, such as anti-Mullerian hormone and estradiol, by Sertoli cells. 10,25 Research indicates that the mechanism underlying male reproductive toxicity resulting from cadmium exposure may involve the regulation of p53 by piRNA-DQ717867, which subsequently leads to disruptions in the cell cycle of mouse spermatogonia GC-2 cells. 26 Furthermore, p53 has been implicated in abnormal pregnancies in which it inhibits embryonic cell proliferation and trophoblast cell growth while inducing apoptosis, ultimately leading to embryonic disorders resulting in implantation failure and recurrent miscarriages. 27 In our experiment, we utilized the expression level of TRIAP1 to represent the transcriptional activity status of TP53 . As TP53 functions as a transcription factor, its functional assessment should focus on ”transcriptional activity” rather than merely measuring the expression level of the TP53 gene. Direct measurement of TP53 mRNA or total protein levels may not accurately reflect its actual transcriptional function. The role of TP53 in regulating cell apoptosis is primarily mediated through the modulation of its downstream target genes. TRIAP1, a key anti-apoptotic factor, is directly suppressed by TP53. Therefore, monitoring changes in TRIAP1 expression provides an effective means to evaluate the transcriptional activity of TP53. In this study, the Mn-exposure group exhibited significant upregulation of TRIAP1 gene expression compared to the control group ( P <0.05), leading to inhibited cell proliferation, which is consistent with previous findings. It has been reported that estrogen can suppress NF-κB signaling pathway activation by promoting estrogen receptor (ERα) and that ERα concentration-dependently inhibits NF-κB activity. Spermatogenesis relies on abundant androgens, while elevated levels of estrogen are risk factors for male infertility and sexual dysfunction, which is consistent with our results. Notably, during pathway enrichment analysis, the sphingolipid signaling pathway emerged as an enriched KEGG pathway, aligning with the direction of sphingolipid signaling supported by a previous ”drug-disease-target-pathway” intersection target network and GO enrichment analysis. Sphingolipid signaling is widely recognized as one of the most crucial intracellular secondary signaling systems in the mammalian reproductive system. By activating specific sphingolipid kinases to generate sphingosine-1-phosphate (S1P), S1P binds to G protein-coupled receptors, initiating diverse signaling pathways related to cell proliferation, apoptosis, differentiation, migration, the inflammatory response, and the immune response involving NF-κB and P53. 28,29,30,31,32,33 In response to DNA damage, neutral sphingomyelinase 2 can upregulate p53 mRNA expression. In this study, ICA treatment significantly attenuated Mn-exposure upregulation of TP53 and NF-κB expression in testicular tissues compared to the Mn-exposure group. Accumulating evidence suggests that sphingolipid metabolites play pivotal roles in male reproductive physiology, with studies demonstrating their involvement in spermatogenesis regulation and stress-induced germ cell apoptosis. These findings collectively suggest a mechanistic link between aberrant sphingolipid metabolism, impaired spermatogenic potential, and male infertility. 34,35,36 Suomalainen et al. investigated alterations in intracellular sphingolipid levels and apoptosis markers and revealed increased ceramide content in apoptotic cells during the early stage of Caspase 3 activation. 37 Exposure to Mn in the nervous system also affects the sphingolipid signaling pathway, leading to the activation of Caspase 3, 7, 8, and 9; upregulation of FasL; cleavage of Bid; disruption of Δφm; release of cytochrome c; and induction of oxidative stress in astrocytoma C6 cells, resulting in cellular apoptosis. 38 5 CONCLUSION In this study, we innovatively applied network pharmacology and molecular docking methods to investigate the mechanism and target prediction of ICA in the repair of Mn-exposure reproductive damage. The results demonstrated that the sphingomyelin pathway may be involved in the reproductive damage caused by ICA and Mn exposure, which could be restored through the regulation of TRIAP1 , HSP90AA1 , and NF-κB mRNA expression. However, there are certain limitations in our study. First, the mRNA expression levels of some related genes were not detected during the gene selection process, possibly leading to missed targets such as TNF, ATK1, and INF-α. Second, since only gene-level verification was conducted without protein-level detection in this study, further research is warranted. Moreover, it should be noted that our experiments were performed solely in vitro , as spermatogenesis relies on intricate processes; thus, additional investigations are required to determine whether ICA can effectively reverse the testicular effects induced by Mn exposure. AUTHOR CONTRIBUTIONS J.Q.H. drafted the original draft. T.C.L. and Y.L. performed the experiments. X.L. supervised the research activity planning. J.T., Y.T., and J.D.Z revised the manuscript. All authors approved the final version of the manuscript. CONFLICT OF INTEREST STATEMENT All authors disclosed no relevant relationships. DATA AVAILABILITY STATEMENT The dataset used and/or analyzed during the current study is available from the corresponding author on reasonable request. FUNDING This work was supported by the Science and Technology Support Program of Guizhou Province (QKH-ZK[2023]506; QKH-ms[2025]373); Zunyi City Science and Technology and Big Data Bureau & Zunyi Medical University joint project (HZ[2023]175, HZ[2023]189, [2021]1350-011, [2021]1350-025). ORCID Jun Tan , https://orcid.org/0009-0002-8566-0448 Jidong Zhang, https://orcid.org/0000-0002-7934-7079 REFERENCES 1. Rehman K, Fatima F, Waheed I, et al. Prevalence of exposure of heavy metals and their impact on health consequences. J Cell Biochem . 2018;119(1):157-184. doi:10.1002/jcb.26234 2. 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Note: (A) ICA target map, (B) target map of reproductive damage induced by Mn, and (C) Venn diagram of ICA-Mn-exposure reproductive damage targets. Figure 4. PPI network of common targets. Figure 5. GO and KEGG enrichment analysis of common targets. Note: (A) GO chord diagram and correlation analysis. (B) GO enrichment analysis of common targets. (C) KEGG enrichment analysis of common targets. Figure 6. Visualization of combinations of components and targets. Note: (A) HSP90AA1; (B) TP53; (C) RELA; (D) TNF; (E) ATK1; (F) NF-κB; (G) PTGS2 Figure 7. Anti-proliferation Effects of different concentrations of Mn on mouse spermatogonial GC-1 cells for 24h in vitro . Note: P <0.05 between group a and group b. Figure 8. The expression of the genes in Mn-exposure spermatogonia. Note: Different lowercase letters above columns indicate significant differences at P <0.05. Supplementary Material File (fig 1.tif) Download 2.12 MB File (fig 2.tif) Download 690.07 KB File (fig 3.tif) Download 1.76 MB File (fig 4.tif) Download 891.20 KB File (fig 5.tif) Download 1.80 MB File (fig 6.tif) Download 3.13 MB File (fig 7.tif) Download 311.80 KB File (fig 8.tif) Download 6.98 MB File (tables.docx) Download 32.68 KB Information & Authors Information Version history V1 Version 1 08 July 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords icariin manganese molecular docking network pharmacology reproductive damage Authors Affiliations Jiaqi He Zunyi Medical University View all articles by this author Tongci Li Zunyi Medical University View all articles by this author Yan Liang Zunyi Medical University View all articles by this author Ying Tian Zunyi Medical University View all articles by this author Xiang Lu Zunyi Medical University View all articles by this author Jidong Zhang Zunyi Medical University View all articles by this author Jun Tan 0009-0002-8566-0448 [email protected] Zunyi Medical University View all articles by this author Metrics & Citations Metrics Article Usage 214 views 137 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Jiaqi He, Tongci Li, Yan Liang, et al. Icariin rescues Reproductive damage caused by Manganese: Network pharmacology and Experimental validation. Authorea . 08 July 2025. 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