Unraveling Icariin's Dual Mechanisms: From Antitumor to Reproductive Repair

Abstract

Icariin (ICA), a principal pharmacologically active constituent of the Chinese herbal medicine Epimedium, exhibits numerous therapeutic advantages. Current research has explored ICA's anti-tumor capabilities, including its inhibition of cell proliferation and promotion of cell death, as well as its potential therapeutic effects in repairing reproductive damage by promoting cell proliferation. However, the precise molecular mechanisms underlying ICA's ability to switch between promoting cell growth and inducing cell death remain inadequately understood. This review aims to elucidate the dual mechanisms of ICA in inhibiting cell proliferation and promoting cell proliferation.

Keywords

icariin reproductive system tumors proliferation apoptosis

Introduction

Epimedium brevicornu Maxim (EbM), is an extensively employed traditional Chinese medicine in clinical practice. EbM predominantly comprises flavonoids, polysaccharides, volatile oils, alkaloids, potassium, calcium, magnesium, and diverse trace elements.¹ The pharmacological effects of EbM are noteworthy and include free radical scavenging, antioxidant properties, inhibition of DNA damage, and modulation of male reproductive function.² Icariin (ICA) represents the primary bioactive constituent present in EBM.³ Evidence from studies indicates that ICA can enhance testosterone secretion in the testis and improve spermatogenic function.⁴ Additionally, the activation of the ERK1/2 signaling pathway has been demonstrated to promote the proliferation of Sertoli cells and to safeguard reproductive capacity.⁵ Furthermore, it has been demonstrated that this compound can protect matrix metalloproteinases and impede germ cell apoptosis, thereby improving the condition of the testes that has been caused by the consumption of nicotine and alcohol.⁶ The additional effects of this compound include the concurrent regulation of the hypothalamic-pituitary-gonadal axis, the initiation of the PI3K/Akt/eNOS/NO signaling pathway, the promotion of testosterone synthesis, the enhancement of sperm viability, and the subsequent enhancement of sexual function.⁷ Furthermore, the antitumor characteristics of ICA, including its mechanisms involving cell cycle arrest, angiogenesis and metastasis inhibition, facilitation of cell apoptosis, enhancement of the inflammatory microenvironment, regulation of the immune system, and augmentation of the body's immune response against tumors has been elucidated.⁸

Consequently, ICA has garnered recognition as a promising pharmaceutical agent for the therapeutic intervention of various human malignancies. However, the molecular mechanism of ICA toggles between promoting cell growth and inducing cell death is not yet understood. This paper summarizes the role of ICA in promoting cell death and inhibiting cell death, which can be applied in the fields of tumor treatment and reproductive injury repair respectively, aiming to identify the factors affecting these effects and provide insights for the future development and application of ICA.

The Metabolic Process of ICA

In the human body, ICA undergoes a series of metabolic processes, including absorption, distribution, metabolism, and excretion. Following oral administration, ICA is absorbed in the gastrointestinal tract and can enter the bloodstream through passive diffusion or active transport.⁹ After being assimilated into the bloodstream, the ICA swifts to diverse bodily tissues and organs, including the liver, kidney, and heart. The liver serves as the principal site for ICA metabolism, predominantly through enzymatic hydrolysis, resulting in the formation of metabolites that may possess comparable or distinct activities in comparison to ICA. Consequently, these metabolites may also influence the pharmacological effects of ICA.

Metabolites are eliminated from the body via urine and feces, primarily through the renal system, where a fraction undergoes glomerular filtration and subsequent reabsorption by the renal tubules. It is important to acknowledge that additional metabolic conversions of ICA may occur in vivo, including glucuronidation or sulfation, which can vary depending on individual variations and the method of drug administration, such as oral ingestion or intravenous injection.¹⁰ The diverse metabolic pathways and individual physiological traits can potentially influence the pharmacological activity and effectiveness of ICA. Currently, subcutaneous injection is the prevailing method for conducting antitumor experiments in vivo. Extensive research has demonstrated that ICA has in vivo inhibitory effects on tumor weight and volume in various mouse tumor models without any discernible adverse effects. Consequently, ICA exhibits promising potential as a cancer treatment.¹¹

ICA, an 8-isopentenyl flavonoid glycoside compound, undergoes primarily I-phase metabolic reaction in the metabolic pathway of rats, with the possibility of an II phase metabolic reaction as well.¹⁰ Icariside I and icariside II were identified in rat plasma following the administration of ICA via gavage, intravenous injection, or intraperitoneal injection.¹⁰ The absolute bioavailabilities of ICA were determined to be 72.0% and 14.1% after intraperitoneal injection and gavage, respectively.¹² These findings indicate that variations in dosing regimens influence the bioavailability of ICA, with intraperitoneal injection demonstrating an enhancement in bioavailability (Figure 1).¹³ Variations in bioavailability significantly modulate systemic drug exposure by altering pharmacokinetic parameters (absorption, distribution, metabolism, and excretion), which consequently determine the magnitude and duration of pharmacodynamic responses.

Figure 1.

Metabolic Pathways and Molecular Forms of ICA in Vivo.

The Molecular Mechanisms of ICA Promote the Death of Cancer Cells

Mitochondrial Pathway

ICA was found to induce apoptosis in lung adenocarcinoma A549 cells primarily through the PI3K-Akt pathway (Figure 2). The concentrations used for the ICA treatment groups ranged from 0 to 400 μM, with increases of 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 20, 50, 100, 200, 300, and 400 μM. The findings of this study indicate that the application of ICA at a concentration of 100 μM or higher resulted in notable cytotoxic effects on A549 cells. Moreover, the apoptotic percentage of A549 cells increased in a dose-dependent manner upon exposure to ICA. Furthermore, ICA downregulated the phosphorylation of PI3K and Akt, enhanced the expression of Bad, induced cellular apoptosis, and impeded the invasion and migration of A549 cells. Additionally, the anticancer properties of ICA were also evident in its ability to reduce the adhesion and migration capabilities of cancer cells. This was achieved through the modulation of N-cadherin, E-cadherin, and vimentin expression, as well as the reduction of intracellular GSH levels and the increase in the apoptosis index.¹⁴

Figure 2.

The Molecular Mechanisms of ICA Inhibited the Proliferation of Cancer Cells.

According to recent academic research, the application of various concentrations of ICA (1, 5, 10, 15, 20, 25, 30, and 40 μM) to SMMC-7721 liver cancer cells results in apoptosis primarily through the ROS/INK-dependent mitochondrial pathway.¹⁵ In their study, Song et al reported that various concentrations of ICA (5, 10, and 20 μM) inhibited the proliferation of MDA-MB-231 and 4T1 breast cancer cells, treatment with 50 μg/mL ICA resulted in upregulating the expression of Bax and Cyto C in MLTC-1 mouse Leyden cell tumor cells and ultimately induced cell apoptosis.¹⁶ This phenomenon was primarily attributed to the activation of the SIRT6/NF-κB signaling pathway by ICA, which effectively triggered redox-induced apoptosis. Furthermore, an increase in the Bax/Bcl-2 ratio and ROS content was observed, leading to the inhibition of NF-κB/EMT pathway activation, inhibition of metastasis, and regulation of the immunosuppressive microenvironment in TNBC. The concentration-dependent pro-apoptotic effects of ICA in lung adenocarcinoma A549 cells demonstrate pharmacological potential, yet several critical issues warrant further investigation. Notably, the extreme concentration range (0-400 μM) employed in A549 studies contrasts sharply with hepatocarcinoma (1-40 μM) and breast cancer (5-20 μM) models, highlighting a lack of standardized dosing paradigms across cell lines, this difference makes clinical application difficult.^14–16

Death Receptor Pathway

While KIM et al demonstrated that ICA sensitizes HCT-116 and HT-29 colon cancer cells to TRAIL via the ROS-ERK-CHOP axis, upregulating DR4/DR5 and activating caspase-8.^17,18 However, the target of ICA on HCT-116 and HT-29 remains unclear. Notably, the study underscores NF-κB inhibition as a key mechanism for ICA-mediated suppression of antiapoptotic proteins (XIAP, Bcl-2, Bcl-xl, c-IAP). However, reliance on two cell lines may oversimplify tumor heterogeneity, as colon cancers exhibit diverse genetic backgrounds (eg, KRAS, BRAF, or TP53 mutations) that could differentially modulate NF-κB signaling and ICA responsiveness.^19,20

Endoplasmic Reticulum Stress (ER Stress) Pathway

The regulation of various precancerous characteristics and the functioning of dynamically reprogrammed immune cells have been demonstrated to be influenced by the state of endoplasmic reticulum stress.²¹ Consequently, the receptors and downstream signaling pathways associated with endoplasmic reticulum stress are regarded as pivotal regulators of tumor growth, metastasis, and the response to chemotherapy, targeted therapy, and immunotherapy. Apoptosis was observed in lung cancer A549 cells upon treatment with ICA and involved activation of both the mitochondrial and endoplasmic reticulum (ER) pathways. Furthermore, ICA treatment resulted in the upregulation of ERS-related molecules, including p-PERK, ATF6, GRP78, p-eIF2α, and CHOP. Conversely, downregulation of the ERS signaling pathway using PERK siRNA rendered lung adenocarcinoma cells less responsive to ICA treatment, suggesting that apoptosis primarily occurs via the PERK pathway within the ER pathway.²²

Likewise, Wang et al discovered that ICA at concentrations of 80 and 100 μM can activate the endoplasmic reticulum pathway, resulting in increased expression of the p-perk, p-eif2α, and Bip proteins.²³ This activation ultimately promotes apoptosis in the human colon cancer cell lines HCT116 and SW620. Similarly, the expression of ERS-related proteins was upregulated in EC109 and TE1 cells treated with 20, 40, and 80 μM ICA. NADPH oxidase activation results in heightened reactive oxygen species (ROS) generation and diminished glutathione (GSH) levels, thereby initiating the apoptotic pathway through the coupling of endoplasmic reticulum stress (ERS) and mitochondria.²⁴

Mitochondria-associated endoplasmic reticulum membranes (MAMs) are subcellular entities that facilitate the functional interplay between these vital organelles.²⁵ Furthermore, MAMs have the potential to impact or regulate the functioning and structure of other organelles.²⁶ When the intensity and duration of ERS surpass the regulatory threshold of the pathways above, the cellular quality control mechanism initiates the endoplasmic reticulum-mediated apoptosis pathway.¹⁸

ICA Promoted Autophagy of Cancer Cells

Autophagy has dual effects, as it can contribute to the elimination of tumor cells while also promoting the activity of cancer cells under stressful conditions.²⁷ Consequently, the investigation of autophagy occurrence in tumor cells has significant research implications for antitumor therapy. Jiang et al examined the impact of ICA on autophagy induction in cells and revealed that treatment with 19.5 μg/mL and 48.4 μg/mL ICA reduced cell proliferation and autophagy in drug-resistant ovarian cancer SKVCR cells.²⁸ The administration of the mTOR inhibitor rapamycin can counteract the inhibitory effects on autophagy and the promotion of apoptosis in cells. This finding suggested that ICA can impede autophagy and facilitate apoptosis in tumor cells via activation of the mTOR signaling pathway.²⁹ Finally, the authors showed that ICA enhanced the sensitivity of SKVCR cells to chemotherapy drugs by activating the AKT/mTOR signaling pathway and inhibiting autophagy. In addition, Xu et al reported that following ICA treatment of prostate cancer cell lines with DU145 and PC-3, cell proliferation was notably inhibited, ROS levels were increased, miR-7 expression was upregulated, and mTOR was targeted by miR-7, which subsequently regulated the expression of the mTOR/SREBP1 pathway.³⁰ Notably, ICA inhibited the proliferation of prostate cancer cells but had no substantial impact on the proliferation of normal prostate epithelial RWPE-1 cells. It is suggested that ICA may be selective to cancer cells, but the mechanism is not clear, which limits its further development and utilization.

Apoptosis and autophagy are two pivotal mechanisms of cell death in the field of cell biology. Apoptosis, characterized by its passive and routine nature, is typically linked to pathological circumstances, DNA impairment, or acute stress induced by external agents.³¹ Conversely, autophagy is a meticulously regulated process of cellular self-degradation that upholds intracellular homeostasis while concurrently supplying energy and nutrients through the degradation of diverse cellular constituents via lysosomes.³² During the cell life cycle, apoptosis and autophagy, the two modes of cell death, can exert mutual influence and interaction, with various molecules serving as switches between these cellular processes.³³

ICA can Regulate the Cell Cycle and Inhibit the Growth of Cancer Cells

Wang et al observed that treatment with 50 μg/mL ICA upregulated p27 mRNA expression, indicating that ICA may induce differentiation and inhibit proliferation of HepG2.2.15 cells.³⁴ Furthermore, the cell cycle distribution of HepG2.2.15 cells was altered after ICA treatment, with a significant decrease in the proportion of cells in the S phase and an increase in the proportion of cells in the G1 phase. The findings of the present study indicated a decrease in the number of proliferative cells and a relative increase in the number of quiescent cells following the administration of ICA. This finding suggested that the impact of ICA on the distribution of the tumor cell cycle may serve as a mechanism for inducing tumor cell differentiation. Additionally, treatment with 100 μg/mL ICA decreased AFP (Alpha-fetoprotein) expression and increased Tf expression. High AFP expression and low Tf synthesis are recognized as indicators of a malignant phenotype in HCC cells. Therefore, these results suggest that ICA has the potential to reverse the malignant phenotype.³⁴

In a study conducted by Gu et al, treatment of the thyroid cancer cell line SW579 with varying concentrations of ICA (6.25, 12.5, 25, 50, 100, 200, and 400 mg/L) inhibited ID-L gene expression, while the expression of p21 mRNA was upregulated. These changes exhibited a dose-dependent relationship.³⁵ Considering the role of p21 as a cell cycle inhibitor, it is highly important for cell differentiation. ICA treatment promoted the transition of SW579 cells from the S phase to the G0/G1 phase. Notably, the number of S phase cells was significantly lower than that in the control group, whereas the number of G0/G1 phase cells was increased. These findings suggest that ICA can impede tumor cell proliferation and induce cell differentiation. Equally, Liu et al reported that ICA can induce the expression of AMPK, enhance the expression of PPARγ, induce G0/G1 cell cycle arrest, facilitate apoptosis in glioblastoma multiforme T98G and U87MG cells, and impede cell growth in a time- and dose-dependent manner.³⁶

While these studies highlight the therapeutic potential of ICA, there are still some shortcomings that warrant further exploration. First, the molecular link between ID-L inhibition and p21 induction is unclear. Whether ICA directly disrupts carcinogenic transcription or indirectly alters chromatin accessibility through epigenetic regulation remains an open question. Second, reliance on single-cell lines (SW579, T98G, U87MG) limits generality, as cancer heterogeneity may lead to different responses in different patient-derived models or genetic subtypes. For example, ID-L overexpression is prevalent in braf mutated papillary thyroid carcinoma, but the efficacy of ICA in this case has not been validated.³⁷

ICA can Regulate the Immune System

The initiation and progression of tumors are initially marked by cellular dysplasia, but the subsequent development and treatment of tumors are contingent upon the intricate interplay between tumor cells and the tumor microenvironment (TME). Variations in the TME can be observed among patients with the same tumor, potentially influenced by factors such as age, sex, lifestyle, Body Mass Index (BMI), and in vivo microbiota. Furthermore, distinct tumor types exhibit diverse TME characteristics.³⁸ Xu et al treated the prostate cancer cell lines DU145 and PC-3 with ICA, leading to the observation that ICA potentially influences lipid metabolism through its impact on the expression of SREBP1.³⁰

The existing body of literature has demonstrated that the activation of NF-κB is involved in the immune response of the host and is pivotal in various types of cancers, potentially leading to distant metastasis and evasion of the tumor immune system via epithelial-mesenchymal transition (EMT).³⁹ In this context, ICA intervention has been found to enhance the expression of SIRT6 while reducing the activation of the NF-κB/EMT pathway, thereby impeding the migration and invasion of breast cancer cells. Different concentrations of ICA (10, 20, and 40 mg/kg) were injected into tumor-bearing mice to systematically evaluate their effects on tumor progression and survival time.⁴⁰ They observed a dose-dependent enhancement of humoral immunity in the mice, as evidenced by increased spleen and thymus indices. Additionally, the secretion of immune cell-derived cytokines such as IFN-γ, IL-2, and TNF-α increased with increasing doses of ICA. Conversely, the levels of the inflammatory cytokines IL-6, IL-8, and IL-1β, which are associated with tumor proliferation, were reduced. These findings suggest that ICA significantly enhances immune function in the body.

The ICA is associated with immunomodulatory pathways in tumor cells and can also modulate the function of immune cells. Research has demonstrated that ICA can enhance the expression of MHC-I in breast cancer cells, augment the tumor recognition capabilities of T cells and CTL cells, improve immune function, and prevent immune evasion by tumor cells.^40–42 Moreover, ICA also impacts peritoneal macrophages, thereby enhancing immune surveillance and suppressing tumor growth.⁴³ Macrophages play a crucial role in regulating the tumor microenvironment throughout tumor progression.²¹ M2 macrophages can secrete certain substances that aid in the evasion of immune killing by tumor cells, facilitate the formation of blood and lymphatic vessels within tumors (tumor angiogenesis and lymphangiogenesis), and enhance the invasion and spread of tumor cells (tumor cell invasion and metastasis). Research has demonstrated that ICA hampers the proliferation of polymorphonuclear MDSCs (PMN-MDSCs) in adenocarcinoma, suppresses the expression of ARG1 and MRC1, and impedes IL4-STAT6 signaling.⁴⁴ These effects subsequently impact M2 macrophages, which are advantageous for tumor progression. ICA exerts a direct inhibitory effect on pancreatic cancer growth and proliferation. The administration of ICA at varying concentrations (0, 5, 10, 20, and 40 M) resulted in the downregulation of TLR4, p-P65, and NF-κB expression; decreased cell viability; and impacted cell invasion in oral squamous cells.⁴⁵

Furthermore, ICA exhibited notable effects on immune organs within the immune system.⁴⁶ To investigate this further, SAMP8 mice were subjected to gavage administration of ICA at varying concentrations (20, 40, or 80 mg/kg). The findings indicated a decrease in β-galactosidase staining of blue particles in the liver, kidney, and thymus, along with a reduction in malondialdehyde (MDA) levels and an increase in the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). The potential protective mechanism may be attributed to in vivo antioxidation.⁴⁷

Chemotherapy, as a tumor treatment modality, exhibits comparable cytotoxicity toward tumor cells and normal cells, particularly in terms of its impact on the immune system. According to previous reports, the administration of cyclophosphamide plus ICA resulted in increased spleen and thymus indices in C57BL/6 mice compared to those in the positive control group. Additionally, there was an increase in the proportion of CD3+ T cells and NKT cells in vivo. These findings suggest that ICA has the potential to enhance T-cell maturation in the spleen and thymus of mice, thereby improving immune function following chemotherapy.⁴⁸

ICA Promoted Oxidative Stress

Oxidative stress occurs in all cells of the body, with tumor cells demonstrating elevated oxygen consumption and potentially higher intracellular levels of reactive oxygen species (ROS) compared to normal cells. Previous research has shown that subacute aging in mice results in a diminished proliferative response of T and B lymphocytes, which can be restored through the administration of ICA via gavage.⁴⁹ Furthermore, this intervention has been found to significantly enhance the activity of SOD in the spleens of aging mice. In contrast, the application of ICA to tumor cells may stimulate ROS production while inhibiting SOD activity, leading to irreversible cellular damage and subsequent apoptosis. Consequently, ICA administration exacerbates oxidative stress within cells, potentially pushing those already under oxidative stress beyond their tolerance threshold, thereby triggering apoptosis. The potential synergistic effects of ICA in combination with chemotherapy agents, such as doxorubicin, in patient-derived xenografts warrant further investigation.

The Molecular Mechanisms of ICA Promotes Proliferation of Cells

Mitochondrial Pathway

The balanced regulation of mitochondrial apoptosis by Bcl-2 family proteins is crucial for the normal development of germ cells and reproductive tissues.⁵⁰ The quantity of adult sperm is contingent upon the generation of Sertoli cells during the perinatal period. Sertoli cells play a significant role in governing the self-renewal and differentiation of spermatogonial stem cells.⁵¹

Nan et al reported that the proliferation of Sertoli cells can be enhanced by ICA at concentrations of 0, 5, 10, 15, 20, and 25 μM through the activation of the MEK/ERK signaling pathway in a dose-dependent manner.⁵ The underlying mechanism is associated with the upregulation of p-ERK. However, the limitation of this study lies in the exclusive reliance on cell experiments in vitro, without conducting any in vivo experiments in vivo, thereby diminishing the persuasiveness of the findings. Moreover, oral ICA (100 mg/kg) and testosterone (50 mg/kg) were administered via intramuscular injection to subacute aging male rats.⁵² The findings revealed that the ICA suppressed the expression of P16 protein, whereas no significant alteration was observed in the testosterone group. This discrepancy between the two groups may explain why ICA is more effective at inhibiting cell apoptosis than testosterone. Furthermore, Zhang et al elucidated the multi-faceted protective mechanisms of ICA against germ cell apoptosis, demonstrating its capacity to attenuate oxidative stress-induced damage, restore cell cycle dynamics, and preserve intratesticular androgen homeostasis through a coordinated modulation of redox signaling, cyclin-dependent kinase activity, and steroidogenic enzyme expression.⁵² In a separate study, Wang investigated the impact of a daily dosage of 6 mg/kg ICA on reproductive damage induced by diabetes.⁵³ Their findings revealed that diabetic rats induced by a high-fat diet (HFD) and streptozotocin (STZ) exhibited a significant decrease in PCNA expression, resulting in impaired DNA replication, dysfunction of replication factors, and disruption of protein assembly for cell cycle control and repair, ultimately leading to sperm dysfunction. Furthermore, SIRT1 is predominantly expressed in spermatids and spermatogonia, and a decrease in its expression may lead to impaired spermatogenesis, reproductive dysfunction, and disruption of spermatogonial stem cell differentiation. The administration of ICA can activate the SRIT1-HIF-1α signaling pathway. Moreover, the inhibition of the intrinsic mitochondria-dependent apoptosis pathway results in the upregulation of SIRT1 expression and an increase in the number of primary spermatids.

Furthermore, it has been discovered that the application of 1 μg•mL⁻¹ ICA for 3 h significantly decreases ROS levels and restores the mitochondrial membrane potential, consequently mitigating the apoptosis induced by DEHP in Leyden cells (Figure 3).⁵⁴

Figure 3.

The Molecular Mechanisms of ICA Promoted the Proliferation of Cells in Testicle.

ER Pathway

Previous studies demonstrated that the nuclear phosphoprotein c-fos disrupts blood-testis barrier (BTB) integrity by modulating occludin expression in Sertoli cells, ultimately impairing reproductive function.^55,56 Notably, ATF6 knockout suppresses c-fos expression, suggesting a regulatory axis between endoplasmic reticulum stress responses and BTB homeostasis. The findings reveal that ICA administration upregulates ATF6 and c-fos in Sertoli cells across dose groups, implying a potential protective mechanism against BTB dysfunction via ATF6/c-fos signaling.⁵⁷

While these results highlight ICA's role in modulating ATF6/c-fos-dependent BTB integrity, several critical gaps remain. First, the study assumes a linear relationship between ATF6 activation and c-fos expression, yet emerging evidence suggests that ATF6 may heterodimize with other transcription factors (eg, XBP1) under prolonged stress, potentially altering downstream targets.⁵⁸ Second, the functional dichotomy of c-fos in spermatogenesis—where it promotes germ cell adhesion but disrupts BTB—raises questions about context-dependent outcomes.⁵⁹ The lack of temporal analysis in ICA-treated models precludes determination of whether ATF6/c-fos activation represents an adaptive or maladaptive response.

ICA can Inhibit Autophagy of Cells in Testicle

Several studies have demonstrated the significant influence of autophagy on male reproductive capacity. The findings indicate that the protein expression of ATG5 and LC3 in Sertoli cells diminishes with advancing age, resulting in a reduction in the number of autophagic vacuoles within the cytoplasm, the emergence of vacuoles, and an increase in damage to mitochondria and the endoplasmic reticulum.⁶⁰ These observations highlight the need to mitigate cellular damage and enhance the decline in testicular reproductive function induced by aging in rats.

To further investigate the impact of ICA on reproductive injury repair, the Sertoli cells TM4 were treated with varying concentrations of chloroquine (0, 12.5, 25, and 50 μM).⁶¹ Chloroquine induced apoptosis in TM4 cells, while 0.5 μM ICA reduced the protein expression of LC3II and p62. Additionally, the accumulation of autophagosomes and autolysosomes in the cytoplasm decreased. Based on these findings, it can be inferred that the inhibition of autophagy may lead to detrimental effects on Sertoli cells, whereas the activation of autophagy through ICA administration may serve as a potential strategy to mitigate Sertoli cell damage. However, the authors discovered that chloroquine has the potential to enhance the apoptosis of Sertoli cells, as evidenced by the observed increase in LC3II and p62 protein expression in the chloroquine group. Consequently, ICA has been shown to impede the autophagic process in Sertoli cells by diminishing the protein expression of LC3II and p62, contradicting the authors’ initial conclusion that ICA can induce autophagy in Sertoli cells. The exact mechanism of ICA regulates autophagy in Sertoli cells requires further investigation and clarification.

ICA can Regulate the Cell Cycle and Promote the Growth of Germ Cells

Zhang et al Subacute aging male rats were treated with 100 mg/kg ICA or 50 mg/kg testosterone to investigate the impact on P16 protein levels. The findings revealed that ICA inhibited P16 protein expression, whereas no significant changes were observed in the testosterone group.⁶² This discrepancy suggests that compared with testosterone, ICA may have superior ability to suppress cell apoptosis. Notably, P16 protein functions as a cyclin-dependent kinase inhibitor and is produced by tumor suppressor genes.

ICA can Regulate the Immunity System to Promote Growth of Cells

The BTB is a crucial component of tight junctions between adjacent Sertoli cells. Its primary function is to prevent the entry of cytotoxic substances, thereby ensuring the survival of the microenvironment necessary for germ cells. Researchhas demonstrated that the impairment of BTB integrity frequently coincides with spermatogenic dysfunction during the aging process.⁶³ The occludin protein is capable of modulating the tight junction protein ZO-1, allowing preleptotene and leptotene spermatocytes to traverse the BTB while preserving its overall integrity throughout spermatogenesis. Zhu et al established an experimental design consisting of an aging model group, a young control group, and two ICA treatment groups (2 mg/kg and 6 mg/kg) to investigate the impact of Sertoli cells on the BTB. ICA administration resulted in the upregulation of ZO-1, occludin, and β-catenin expression in the testes of naturally aged rats, suggesting that ICA possesses the potential to ameliorate the structural deterioration of the BTB. Zhu et al conducted an investigation employing HE staining to determine the potential of ICA to ameliorate degenerative alterations in the testicular tissue of naturally aged rats.⁵⁷ However, the impact of ICA on enhancing the structure of the BTB through the upregulation of BTB-related proteins remains uncertain. Consequently, we propose that using transmission electron microscopy as an adjunctive experiment could enable the observation of structural modifications in the BTB.

The concentration gradient of low and high doses of ICA is insufficient to establish a definitive lack of dose-dependent effects on BTB function. Furthermore, the underlying causes for the negligible disparity in effects between the low- and high-dose groups remain unexplored. Consequently, to further substantiate the effective concentration range of ICA, it is imperative to augment the concentration gradient, expand the number of experimental groups, and conduct protein expression analysis. Consequently, the optimal dosage of ICA can be investigated for clinical guidance.

Chen's study demonstrated that varicocele can lead to oxidative stress, damage to tissue structure and function, and the induction of cell apoptosis in the epididymis of SD rats.⁶⁴ However, the administration of 100 mg/kg ICA was found to enhance the activity of SOD and GSH-Px and decrease the content of MDA, thereby improving the antioxidant capacity. Chen previously established a mouse Sertoli cell line, TM4, cell senescence model induced by D-galactose, and the success of the model was determined by evaluating apoptosis. The findings from the cell cycle analysis indicated that the proliferation and division of TM4 cells were influenced by D-galactose stimulation. The authors specifically investigated the impact of ICA on oxidative stress-induced senescence. ICA at concentrations of 0.5 and 1 μM upregulated the expression of GDNF, BMP4, SCF, PLZF, Nrf2, HO-1, NQO-1, ERα, and ERβ while simultaneously reducing ROS levels and enhancing the antioxidant capacity of senescent TM4 cells.⁶⁵ Enhancement of senescent TM4 cell function, specifically through the ERα-Nrf2 signaling pathway, can potentially ameliorate cell senescence. The authors evaluated the success of their model based on apoptosis outcomes, yet no conclusive evidence was found to establish a direct link between apoptosis and oxidative stress. Notably, apoptosis can be triggered by various mechanisms.⁶⁵ Consequently, the absence of complete evidence implicating oxidative stress as the sole cause of apoptosis in this study prevents definitive confirmation that D-gal-induced cell senescence is solely attributable to oxidative stress. Furthermore, the author posits that there is a strong correlation between cell senescence and the cell cycle, suggesting that ICA may impede cell senescence through its impact on the cell cycle. Consequently, it is imperative to investigate the influence of ICA on the cell cycle. If the anticipated outcomes are achieved, it would substantiate the notion that ICA can decelerate cell senescence by modulating the proliferation and division of TM4 cells.

Furthermore, You et al observed significant upregulation of Nrf2 and its downstream target molecules HO-1 and NQO1 in the testes of naturally aged rats treated with 2 or 6 mg/kg ICA.⁶⁶ This finding suggested that ICA has the potential to mitigate oxidative damage via activation of the Nrf2/HO-1 pathway. In their study, Chen et al conducted a study to investigate the impact of varying doses (50, 100, and 200 mg/kg) of ICA on reproductive function.⁶⁷ This was achieved by assessing SOD activity and MDA levels in the testes of the rats. The results revealed that in the experimental groups receiving 50 and 100 mg/kg ICA, SOD activity increased, and MDA levels decreased. However, in the group receiving 200 mg/kg ICA, SOD activity did not significantly change, while the MDA level increased. The aforementioned experimental observations suggest that a suitable dosage of ICA can enhance the antioxidant capacity of the testis and decrease lipid peroxidation levels. Conversely, an excessive concentration of ICA may increase oxidative stress levels and exacerbate reproductive damage in rats. The authors elucidated the dose-dependent nature of ICA treatment by noting that a dose of 200 mg/kg did not yield significant alterations in StAR or PBR mRNA, both of which play crucial roles in steroidogenic production.⁶⁷ The findings of this experiment demonstrated that low and medium doses of ICA significantly ameliorated male reproductive damage. In contrast, high doses did not exhibit a significant difference in effect, suggesting a dose-dependent relationship in ICA's capacity to promote cell growth.

ICA can Regulate Hormone Levels and Promote Cell Growth

ICA, a flavonoid drug, has a discernible sex hormone-like impact by directly stimulating the secretion of estradiol by follicular granulosa cells and enhancing the secretion of corticosterone by adrenal cortical cells.⁶⁸ Within an appropriate dosage range, ICA can directly facilitate the synthesis and secretion of gonadotropin (GTH) by the pituitary gland. ICA has the potential to enhance the synthesis and secretion of LH and FSH by GTH cells.⁶⁹

In a study conducted by Li Bo et al, it was observed that the administration of 0.6 mL/100 g ICA in an animal model of kidney Yang deficiency resulted in the regulation of the hypothalamic corticotropin-releasing hormone (CRH) gene and pituitary promelanocortin (POMC) gene expression, bringing them closer to normal levels.⁷⁰ In their study, Zhang et al conducted a study using a rat model of D-gal injury in which the rats were treated with 100 mg/kg ICA and 50 mg/kg testosterone.⁵² The results indicated that the administration of ICA and testosterone led to a significant increase in the serum SOD activity and testosterone concentration, while the E2 content remained relatively unchanged. Additionally, the protein expression level of P16 decreased in the ICA treatment group. Nevertheless, the testosterone group did not exhibit any noteworthy disparity, as the testosterone level gradually reverted to its baseline in the ICA treatment group. The superior efficacy of ICA treatment to that of testosterone might be attributed to the sex hormone-like properties of ICA and its ability to regulate testosterone secretion via the hypothalamic-pituitary-gonadal axis. Furthermore, experimental evidence has demonstrated that prolonged administration of hormones at supraphysiological doses can impact spermatogenesis.⁷¹ According to our findings, it is hypothesized that ICA plays a regulatory role in testosterone secretion, preventing excessive accumulation within the body. This regulation may be attributed to the feedback mechanism of the hypothalamic-pituitary-gonadal axis.

Research has demonstrated that the hypothalamic-pituitary-gonadal axis plays a significant role in the reproductive function of animals, and any drug that affects any component of this axis may have an impact on reproductive function. In light of the potential of ICA to enhance sexual desire in animals, Zhang Sen conducted a study to investigate the effects of ICA on the gonadal axis. This study involved measuring FSH, LH, and E2 (estradiol) levels in the serum of female rats during proestrus and oestrus.⁷² The results revealed that 10 mg/kg, 20 mg/kg, and 30 mg/kg ICA could increase the levels of FSH, LH, and E2 in the serum of female rats during proestrus. These findings indicate that ICA can potentially augment the expression of FSH and LH transcription factors, stimulate the secretion of FSH and LH by the rat pituitary, and modulate the basal secretion of the gonadal axis as a cohesive unit.⁷³ In contrast to the conclusions of Zhang et al, their use of 100 mg/kg ICA had no noteworthy impact on E2 levels. Conversely, Zhang Sen employed a lower concentration of ICA and observed an increase in E2 levels. It was demonstrated that the influence of ICA on E2 levels is contingent upon the dosage administered.

Chen et al conducted a study wherein they observed a substantial increase in testosterone levels when the concentration of ICA was 50 mg/kg or 100 mg/kg.⁶⁷ However, no significant change in testosterone levels was observed when the ICA concentration was 200 mg/kg. These findings indicate that the regulation of testosterone production by ICA is contingent upon its dosage, although the authors did not explain this phenomenon.

Furthermore, real-time PCR revealed that the administration of ICA resulted in dose-dependent upregulation of StAR and PBR mRNA in Sertoli cells. Additionally, ICA influenced the expression of FSHR and claudin-11 mRNA, thereby facilitating testosterone production. This effect was accompanied by enhanced transport of cholesterol across the cellular membrane, ultimately promoting testosterone synthesis. Chen M's earlier investigation proposed a potential association between these findings and the expression of StAR.⁶⁷ Andric SA conducted a study that revealed the regulatory role of the NO/cGMP signaling pathway in steroid activity.⁷⁴ Additionally, they discovered that PDE5-IS can effectively inhibit PDE5, an enzyme responsible for the degradation of cGMP guanylate cyclase. Furthermore, this study suggested that PDE5-Is may have a significant impact on the steroidogenesis pathway and testosterone secretion. In a separate study, Zhang et al identified ICA as a PDE5-I that can enhance NOS in the cavernous tissue of rats with diabetic ED, thereby increasing cGMP levels.⁷⁵ The mechanism of action of PDE5 inhibitors involves binding to the catalytic site of cGMP on PDE5, thereby preventing its degradation. The author posited, based on the aforementioned experimental investigations, that ICA can elicit an effect akin to that of PDE5 inhibitors. Binding to the catalytic site of cGMP on PDE5 diminishes the degradation of cGMP, thereby preserving the functionality of the NO/cGMP signaling pathway and ensuring the maintenance of normal steroid activity.

Wang reported a decrease in the levels of FSH, LH, and testosterone in diabetic animals.⁵³ However, they found that an intervention of 80 mg•kg⁻¹ ICA for a duration of 6 weeks increased serum testosterone, LH, and FSH levels, thereby promoting the recovery of reproductive function in diabetic male rats. Similarly, Chen et al discovered that ICA doses of 50 mg/kg and 100 mg/kg not only can increase testosterone production by modulating the expression of the steroidogenic acute regulatory protein (StAR) and peripheral benzodiazepine receptor (PBR) but also can enhance spermatogenesis by regulating the expression of follicle-stimulating hormone receptor (FSHR) and claudin-11.⁶⁷ The in vitro and in vivo biochemical effects of ICA through different administration routes and dosages are summarized in Table 1.

Table 1.

Summary of the Biochemical Effects of ICA Through Different Administration Routes and at Different Dosages in Vitro and in Vivo.

Model	Route of Administration	Function and Dosage	References
Lung adenocarcinoma A549 cells	In vitro	Inhibition proliferation (10, 20, 50 μM)	¹⁴
Hepatocellular carcinoma SMMC-7721 cells	In vitro	Inhibition proliferation（5, 10, 20 μM）	¹⁵
Breast cancer cells MDA-MB-231 and 4T1	In vitro	Inhibition proliferation (5, 10, 20 μM)	¹⁶
Leydig tumor cells MLTC-1	In vitro	Inhibition proliferation (50 μg/mL)	¹⁸
Ovarian cancer cell SKVCR	In vitro	Inhibition proliferation (19.5, 48.4 μg/mL)	²⁹
Esophageal squamous cells EC109 and TE1	In vitro	Inhibition proliferation (20, 40, 80 μM)	²⁴
Thyroid cancer cell SW579	In vitro	Inhibition proliferation (6.25(48, 72 h), 12.5(48, 72 h), 25(48, 72 h), 50, 100, 200, 400 mg/L)	³⁵
Colon cancer cells HCT116	In vitro	Inhibition proliferation (20, 40, 80 μM)	²³
Colon cancer cells SW620	In vitro	Inhibition proliferation (25, 50, 100 μM)	²³
GBM cell lines U87MG	In vitro	Inhibition proliferation (10, 20 μM)	³⁶
GBM cell lines T98G	In vitro	Inhibition proliferation (10, 20 μM)	³⁶
Prostate cancer DU145 and PC3 cell	In vitro	Inhibition proliferation (10, 20, 30, 40, 80 μM)	³⁰
Hepatocellular carcinoma cells HepG2.2.15	In vitro	Inhibition proliferation (50 mg/L)	³⁴
cervical cancer cell SiHa	In vitro	Inhibition proliferation (30, 90, 120 uM)	⁴⁰
pancreatic cancer cell Panc02	In vitro	Inhibition proliferation (100, 150, 200 μM)	⁴⁵
Oral squamous cell carcinoma cells SCC-9 and SCC-15	In vitro	Inhibition proliferation (0, 5, 10, 20, and 40 M)	⁴⁵
HCC cells	In vitro	Inhibition proliferation (100 μg/mL)	³⁴
Sertoli cells TM4	In vitro	Promotion proliferation (0.5, 1, 5, 10, 15, 20, 25 μM)	⁵
Kunming mice	In vivo (Intragastric administration)	Inhibition proliferation (10, 20, 40 mg/kg/d)	⁴⁰
C57BL/6 mice	In vivo (Intragastric administration)	Inhibition proliferation (40, 80, 150 mg/kg/d)	⁴⁸
SD rats	In vivo (Intragastric administration)	Promotion proliferation(100 mg/kg/d)	⁵²
SD rats	In vivo (Intraperitoneal injection)	Promotion proliferation (80 mg/kg/d)	⁴⁵
SD rats	In vivo (Gastric gavage)	Promotion proliferation (2, 6 mg/kg)	⁵³
SD rats	In vivo (Oral administration)	Promotion proliferation (50, 100 mg/kg)	⁶⁷
SD rats	In vivo (Gastric gavage)	Promotion proliferation (200 mg/kg/d)	⁶⁹
SD rats	In vivo (Intragastric administration)	Promotion proliferation (0.6 mL/100 g/d)	⁷⁰
Wistar rats	In vivo (Subcutaneous injection)	Promotion proliferation (10, 20, 30 mg/kg)	⁷²
SAMP8 rats	In vivo (Intragastric administration)	Promotion proliferation (20, 40, 80 mg/kg)	⁴⁷
SD rats	In vivo (Intragastric administration)	Promotion proliferation (100 mg/kg)	⁶⁴
SD rats	In vivo (Intraperitoneal injection)	Promotion proliferation (2, 6 mg/kg)	⁵⁹

Discussion and Future Perspective

ICA, an active compound derived from Epimedium, has garnered significant attention in the field of traditional Chinese medicine for the management of reproductive system disorders. ICA can promote the synthesis and secretion of testosterone, increase sperm count and motility, and thus improve male reproductive ability. In addition, ICA can inhibit the proliferation of tumor cells, induce cell apoptosis, and inhibit tumor-related angiogenesis, thereby impeding the nutrient supply and growth of tumors.^76–81 Overall, these findings highlight that the biological effects of ICA are environment-dependent and governed by multi-dimensional interactions of cell type specificity, the unique microenvironmental niche in which the cells are located, and pharmacokinetic parameters, including the pathway and regimen of administration.

In addition, the effect of ICA is also related to its concentration, with low concentrations inhibiting apoptosis, possibly due to the abundant blood supply in the tumor cell microenvironment.⁸² After entering the blood through the gastrointestinal tract, ICA has a prolonged residence time and is widely distributed in tumor cells. However, male reproductive system BTB selectively allows passive diffusion of small molecules while limiting larger macromolecules, and the molecular size of ICA may limit effective diffusion through tight junctions.¹⁰ Critically, quantitative data on icariin's BTB permeability remains scarce. Advanced in vitro models incorporating primary Sertoli cells and in vivo imaging techniques are needed to clarify transport mechanisms. Such studies could inform the design of targeted delivery systems (eg, lipid nanoparticles, cell-penetrating peptides) to enhance testicular drug deposition.^83,84

Conclusion

Icariin (ICA), a bioactive flavonoid from Epimedium, exhibits paradoxical roles in cancer suppression and reproductive repair. In the field of anti-tumor, ICA induces apoptosis via mitochondrial pathways (eg, PI3K/Akt inhibition, ROS elevation), death receptor activation (eg, DR4/5 upregulation), and endoplasmic reticulum (ER) stress, while also modulating autophagy and cell cycle arrest (G1/S phase blockade). These effects vary across cancer types, with concentration-dependent cytotoxicity observed in lung, liver, and breast cancers. Conversely, in reproductive systems, ICA promotes cell proliferation by activating ERK1/2 and SIRT1-HIF-1α pathways, enhancing antioxidant defenses (SOD/GSH-Px), and regulating hormone synthesis (Testosterone, FSH/LH). Notably, ICA's duality hinges on micro-environment specific factors: high concentrations in tumors trigger oxidative stress and apoptosis, whereas lower doses in testes restore mitochondrial function and blood-testis barrier integrity. However, discrepancies in dosing regimens and unclear molecular switches between pro-survival and pro-death pathways limit clinical translation. Further studies are needed to standardize dosing, elucidate tissue-specific pharmacokinetics, and validate dual mechanisms in human models. ICA's multifunctionality highlights its potential as a therapeutic agent but underscores the necessity for precision in application.

Footnotes

Abbreviations

ORCID iDs

Jun Tan

Jidong Zhang

Author Contributions

Jiaqi He drafted the manuscript and generated the figures. Tongci Li collected the information and constructed the table. Yue Su and Keyu Wu collected the information and references. Jun Tan, Ying Tian, and Jidong Zhang revised the manuscript. All authors approved the final version of the manuscript. Informed consent was obtained from all individual participants included in the study. The Figure 1 was drawn by Biorender. The Figures 2 and were both drawn by Figdraw (UWYTI344da, SOITAc00d0).

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Science and Technology Support Program of Guizhou Province (QKH-MS[2025]373); Zunyi City Science and Technology and Big Data Bureau & Zunyi Medical University joint project (HZ[2023]175, HZ[2023]189, HZ[2023]235, [2021]1350-011, [2021]1350-025).

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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