Pharmacological actions of the bioactive compounds of Epimedium on the male reproductive system: current status and future perspective

Abstract

Compounds isolated from Epimedium include the total flavonoids of Epimedium, icariin, and its metabolites (icaritin, icariside I, and icariside II), which have similar molecular structures. Modern pharmacological research and clinical practice have proved that Epimedium and its active components have a wide range of pharmacological effects, especially in improving sexual function, hormone regulation, anti-osteoporosis, immune function regulation, anti-oxidation, and anti-tumor activity. To date, we still need a comprehensive source of knowledge about the pharmacological effects of Epimedium and its bioactive compounds on the male reproductive system. However, their actions in other tissues have been reviewed in recent years. This review critically focuses on the Epimedium, its bioactive compounds, and the biochemical and molecular mechanisms that modulate vital pathways associated with the male reproductive system. Such intrinsic knowledge will significantly further studies on the Epimedium and its bioactive compounds that protect the male reproductive system and provide some guidances for clinical treatment of related male reproductive disorders.

INTRODUCTION

With the growing concern of an aging population, there has been an increasing emphasis on preserving reproductive capacity.1 But pessimistically, global fertility shows a yearly decline, which may be closely related to damage to the male reproductive system caused by viral infections, chemical disruptors, and systemic diseases.2 Currently, infertility affects about 8%–12% of couples worldwide, and 40%–50% of these cases are due to male factors.3 Consequently, how to protect the male reproductive system and maintain fertility is becoming a growing concern. To date, therapy for male infertility can be divided into noninvasive and invasive modes, which include lifestyle modifications, anti-oxidants, intrauterine insemination, and in vitro fertilization (IVF).4 Although these treatments have achieved some therapeutic success, the results are unsatisfactory and associated with adverse effects.5 Therefore, reasonably successful traditional therapies based on medicinal plants remain the leading choice for most male infertility patients.6

Epimedium, also known as Xian Ling spleen, was first recorded in Shennong Ben Cao Jing as a genus of 52 species of plants in the family Berberidaceae with a history of more than 2000 years (Supplementary Figure 1a ^{(137.3KB, tif)} and 1b ^{(137.3KB, tif)} ). The primary function of Epimedium in traditional Chinese applications is to tonify the kidneys and strengthen the “yang”, thereby restoring erectile function in males.7 In modern drug applications, many marketed proprietary Chinese medicines and health products contain Epimedium, of which Epimedium is the primary drug used to improve symptoms of impotence, premature ejaculation, and kidney yang deficiency.8 Currently, up to 80 species of Epimedium have been identified, most endemic to China, mainly in southwestern and central areas.7 In addition, several species are native to East Asia, South Asia, Central Asia, and Europe. In the Chinese Pharmacopoeia, Epimedium brevicornum Maxim., Epimedium sagittatum (Sieb. et Zucc.) Maxim., Epimedium pubescens Maxim., and Epimedium koreanum Nakai are considered major medicinal species.9 The main active compound of Epimedium is the total flavonoids of Epimedium (TFE); four of them, epimedin A, epimedin B, epimedin C, and icariin (ICA), are specified in the Chinese Pharmacopoeia as quality control standards for crude and processed products of Epimedium.9 Over the past two decades, various techniques such as heat extraction, percolation extraction, Soxhlet extraction, microwave-assisted extraction, and supercritical fluid extraction have been employed to extract flavonoids from Epimedium. In recent years, ultrasonic extraction has received more and more attention in the extraction of flavonoids from Epimedium because of its advantages of high extraction efficiency, short extraction time, and low extraction temperature. The TFE were separated and quantified by high-performance liquid chromatography (HPLC).10,11 Metabolic studies showed that epimedin A–C and ICA could converted into icaritin (ICT), icariside I (ICA I), and icariside II (ICA II), which have similar molecular structures with ICA in plasma (Supplementary Figure 1c ^{(137.3KB, tif)} –1f ^{(137.3KB, tif)} ). The quality and stability are controlled by various methods such as genetic identification, chemical evaluation, HPLC, and liquid chromatography-tandem mass spectroscopy (LC-MS/MS).12,13 Modern pharmacological studies and clinical practice have demonstrated a wide range of pharmacological effects of Epimedium and its active compounds,14 which are widely used in the clinical treatment of osteoporosis,15 Alzheimer’s disease,16 diabetes,17 rheumatoid arthritis,18 atherosclerosis,19 and cancer.20 Studies have shown that Epimedium is beneficial for reproduction. Its mechanism of action includes stimulating testosterone production, reducing oxidative stress, activating testicular signaling pathways, and regulating regulatory behavior.21 In this paper, we review the protective effects of Epimedium and its main active compounds on the male reproductive system and discuss their mechanisms of action from the perspective shown in vivo and in vitro models summarized in Table 1, providing a clear and comprehensive knowledge of how it exerts its protective effects on the male reproductive system.

Table 1.

Effect and mechanism of Epimedium and its bioactive compounds on male reproductive system diseases

Disease	Brief description of effects	Mechanisms	Drugs	In vitro/in vivo	Cells or animals	Route of administration and dose	Reference
Male infertility	Promote testosterone synthesis and secretion	Activated Creb and activated Drp1-dependent steroidogenesis by Creb-mediated signaling pathway	Epimedium	In vitro In vivo	Primary LCs ICR mice	1 µg ml−1 100 mg kg−1 oral	30
		Upregulated the activity of Creb and Sf-1 via the Esr1/Akt/Creb Sf-1 signaling pathway indirectly	ICA	In vitro	LC-540	100 µg ml−1	31
	Promote the proliferation of Sertoli cells	Promoted the proliferation of Sertoli cells by activating the ERK1/2 signal pathway	ICA	In vitro	Primary SCs	10 µmol l−1, 15 µmol l−1, 20 µmol l−1	40
	Protect against destruction of BTB	Prevented the degradation of GJIC and impairment of Cx43 via suppressing the Akt pathway	ICA	In vitro	TM4	100 µmol l−1	42
		Reduced PFOS-induced testicular toxicity by downregulating the p38/MAPK/MMP9 pathway	ICA	In vitro In vivo	TM4 C57BL/6 mice	1 µmol l−1, 2 µmol l−1, 4 µmol l−1 5 mg kg−1, 20 mg kg−1 oral	43
	Anti-oxidant	Unclear	Epimedium	In vivo	SD rat	0.81 g kg−1, 2.43 g kg−1 per day	47
		Reduced ROS and DNA damage through anti-oxidant activity by mediating ROS-DNA damage-P16-CDK6 pathway signaling	Epimedium	In vivo	C57BL/6 mice	-	49
		Reduced DNA damage caused by aging by inhibiting p53 phosphorylation	TFE	In vivo	SD rat	10 mg kg−1, 20 mg kg−1 oral	50
		Reduced testicular oxidative damage by increasing SOD activity and reducing MDA levels	TFE	In vivo	SD rat	80 mg kg−1 i.p	51
		Enhanced the activity of anti-oxidant enzymes	TFE ICA ICA	In vivo In vivo In vivo	Balb/c mice Kunming mice SD rat	200 mg kg−1, 400 mg kg−1 oral 75 mg kg−1 per day oral 80 mg kg−1 per day oral	52 53 54
Erectile dysfunction	As an endothelial function-protective agent	Increased eNOs levels through the Akt-eNOs signaling pathway	ICA II	In vitro In vivo	HCECs Wistar rats	0.01 µmol l−1, 0.1 µmol l−1, 1 µmol l−1 10 mg kg-1 per day oral	60 61
		Regulated the MAPK pathway in human cavernous endothelial cells through miR-126/SPRED1	ICA II	In vitro	HCECs	0.1 µmol l−1, 1 µmol l−1, 10 µmol l−1	62
		Increased eNOs in penile endothelial cells and NO levels in serum through PI3K/AKT signaling pathway	ICA	In vivo	ICR mice	50 mg kg−1, 100 mg kg−1, 200 mg kg−1 per day oral	25
		Increased p-eNOs/eNOs levels through inhibition of IP3R1, PACs2, and FACL4 expression	ICA	In vivo	SD rat	10 mg kg−1 per day oral	67
	Neurotrophic effect	Increased nNOs levels in penile tissue	ICA ICA II	In vivo In vivo	SD rat SD rat	1 mg kg−1, 5 mg kg−1, 10 mg kg−1 oral 2.5 mg kg−1 per day oral	69 70
	As an inhibitor of PDE5	Increased the concentration of cGMP in penile smooth muscle cells via binds to the cGMP catalytic site	ICA	In vitro	SH-SY5Y	1–6 µmol l−1	68
	Enhance the therapeutic effect of ADSCs	Protected ADSCs against oxidative stress by mediating the PI3K/Akt-STAT3 signaling pathway	ICA	In vivo	SD rat	5 mg kg−1 oral	63
	Promote the differentiation of ADSCs	Promoted the differentiation of ADSCs to SCs via miR-33/GDNF axis	ICA II	In vivo	SD rat	4.5 mg kg−1 per day oral	64
Tumor	Inhibit cell proliferation	Induced G1 phase arrest by the elevated expression level of p16, p21, and p27, and inhibit the expression of cell cycle proteins D1 and CDK4	ICT	In vitro	PC-3 cells	30–100 µmol l−1	74
		Inhibited the proliferation and invasion of prostate cancer cells by modulating miR-381-3p and target gene UBE2C	ICT	In vitro In vivo	PC-3 cells TRAMP mice	32 µg ml−1 30 mg kg−1 oral	79
	Promote apoptosis, inhibits migration and invasion	Regulated the expression and release of cytochrome c and the activities of caspase-9 and caspase-3	ICA	In vitro	MLTC-1 cells	50 µg ml−1	81
		Induced apoptosis and inhibits cell invasion by regulating PEA3/HER2/AR signaling	ICT	In vitro	LNCaP cells	35 µg ml−1	82
	Enhance autophagy	Enhanced autophagy by modulating the PI3K-Akt-mTOR signaling pathway	ICA II	In vitro	DU145 cells	20 µmol l−1, 40 µmol l−1	88
Other diseases	Anti-inflammatory	Reduced ROS levels and decrease inflammatory cell infiltration to alleviate the inflammatory response	ICA II	In vivo	C57BL/6 mice	5 mg kg−1 per day oral	90
	Exerted an anti-inflammatory response by downregulating NF-κB and upregulating Nrf-2/HO-1 signaling pathway	ICA	In vitro In vivo	PC-12 cells Albino mice	1 µmol l−1 25 mg kg−1 i.p	91

BTB: blood–testis barrier; PDE5: phosphodiesterase 5; ADSCs: adipose tissue-derived mesenchymal stem cells; NO: nitric oxide; NOs: nitric oxide synthase; eNOs: endothelial NOs; nNOs: neuronal NOs; AR: androgen receptor; cGMP: cyclic guanosine monophosphate; TFE: total flavonoids of Epimedium; ICA: icariin; ICT: icartin; ICA II icariside II; Creb: cAMP response element binding proteins; Sf-1: steroidogenic factor 1; Esr1: estrogen receptor 1; Akt: serine/threonine protein kinase B; ERK1/2: extracellular signal-regulated kinases 1 and 2; GJIC: gap junctional intercellular communication; Cx43: connexin 43; PFOS: perfluorooctane sulfonate; MAPK: mitogen-activated protein kinase; MMP9: matrix metallopeptidase 9; ROS: reactive oxygen species; SOD: superoxide dismutase; MDA: malondialdehyde; CDK: cyclin-dependent kinase; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; Nrf-2: nuclear factor-erythroid 2-related factor 2; NF-κB: nuclear factor kappa-B; i.p: intraperitoneal; SCs: Schwann cells; LC: liquid chromatography; SPRED1: sprouty-related, EVH1 domain containing 1; IP3R1: inositol 1,4,5-trisphosphate receptor type I; PAcS2: phosphofurin acidic cluster sorting protein 2; FACL4: long-chain-fatty-acid-CoA ligase 4; UBE2C: ubiquitin-conjugating enzyme E2C

MALE INFERTILITY

Male infertility can be broadly categorized into three types: hypothalamic–pituitary disorders that result in secondary hypogonadism; alterations in sperm quantity and quality, commonly referred to as obstructive azoospermia (OA) and nonobstructive azoospermia (NOA); and testicular dysfunction.21 For different types of male infertility problems, Epimedium and its compounds may play a therapeutic role.

Testosterone synthesis and secretion

Testosterone, a steroid hormone, is a significant component of androgens. Testosterone in the testis binds to the androgen-binding protein (ABP). It is secreted into the seminiferous tubules and attaches to the Sertoli cells’ androgen receptor (AR). It activates signaling pathways that initiate and sustain spermatogenesis to ensure proper spermatogenesis.22 Testosterone is mainly synthesized and secreted by interstitial cells and regulated by the hypothalamic–pituitary–testicular axis. The hypothalamus secretes gonadotropin-releasing hormone (GnRH), which governs the follicle-stimulating hormone (FSH) and luteinizing hormone (LH). LH binds to receptors on the surface of Leydig cells and activates adenylate cyclase. This promotes the production of cyclic adenosine monophosphate (cAMP) and activates protein kinase A (PKA). PKA, in turn, encourages the expression of steroid hormone synthase and ultimately leads to testosterone production.23

Experiments in mice and rats have shown that Epimedium has androgen-like effects that promote the synthesis and secretion of testosterone and increase the mass of the epididymis and seminal vesicle glands. This effect is achieved directly or indirectly by upregulating the activity of the relevant enzymes in the testosterone synthesis pathway.24,25 These enzymes and proteins include steroidogenic enzymes such as cytochrome P450, family 11, subfamily A, polypeptide 1 (Cyp11a1), three beta-hydroxysteroid dehydrogenases (3β-HSD), steroidogenic factor 1 (Sf-1), steroidogenic acute regulatory proteins (StAR), peripheral benzodiazepine receptors (PBR), and cAMP response element binding proteins (Creb). When rats were treated with appropriate doses of ICA, testosterone production increased by regulating the expression of genes such as StAR and PBR, and the effect of promoting testosterone synthesis was more pronounced when ICA was combined with zinc.24,26 In addition, although ICA has been shown to boost testosterone synthesis, a recent study has shown that several new compounds isolated from some biotransformation products of Epimedium promote testosterone production.27

With the rapid development of industrial society, many endocrine-disrupting chemicals are spreading in the environment, which may be found in pesticides, food contaminants, cosmetics, water, and air, leading to effects on male fertility.28 It has been shown that ICA protects mouse testes from damage caused by chemicals such as di(2-ethylhexyl) phthalate (DEHP) and endosulfan (ES) by preventing reactive oxygen species (ROS) and promoting the synthesis and secretion of testosterone.29 The specific mechanism of promoting testosterone synthesis and secretion is mediated indirectly by regulating Creb and Sf-1 expression through the estrogen signaling pathway estrogen receptor 1/sarcoma/the serine/threonine kinase Akt (Esr1/Src/Akt) and the direct activation of Creb expression.30,31 These results indicated that Epimedium and its bioactive compounds protected the reproductive system by promoting the synthesis and secretion of testosterone and protecting the Leydig cells from injury caused by chemicals (Figure 1). However, among all the studies on the effects of Epimedium and its bioactive compounds on the synthesis and secretion of testosterone, only ICA has a precise mechanism of action involving Creb.

Figure 1.

Diagram of the mechanism of Epimedium and icariin regulation of steroid synthesis. Cholesterol, the starting molecule for testosterone synthesis (cholesterol–pregnenolone–progesterone–testosterone), is transported to the mitochondria by the StAR. Steroidogenic enzymes, such as Cyp11a1, Hsd3b, and Hsd17b, play important roles in testosterone synthesis. Creb binds to DNA response elements to regulate the transcription of genes encoding Cyp11a1, Hsd3b, and StAR. Epimedium interacts with Creb directly, and ICA interacts with estrogen receptors on the cell membrane, which increases the phosphorylation levels of the Akt and Creb proteins, thereby promoting testosterone synthesis. ICA: icariin; Esr1: estrogen receptor 1; Src: sarcoma; PI3K: phosphatidylinositol 3-kinase; Akt: the serine/threonine kinase Akt; p: phosphorylation; Creb: cAMP response element binding proteins; Sf-1: steroidogenic factor 1; SR-B1: scavenger receptor, class B type 1; StAR: steroidogenic acute regulatory proteins; Cyp11a1: cytochrome P450, family 11, subfamily A, polypeptide 1; 3β-HSD: three beta-hydroxysteroid dehydrogenases; Cyp17: cytochrome P450, family 17.

In contrast, most other studies have not explored the specific mechanism of how to activate Creb in depth. However, the precise mechanism of action shows that the activation pathway of Creb is still relatively singular because the activation of Creb can only occur through the Esr1/Src/Akt pathway. Are there other activation pathways that have not yet been confirmed? This is the direction we need to explore in the future.

The microenvironment of spermatogenesis

Spermatogenesis is a process that occurs in the seminiferous tubules and is complexly regulated to achieve continuous germ cell proliferation and differentiation.32 Furthermore, a suitable microenvironment is necessary for a constant spermatogenesis process to guarantee lifelong fertility in males.33 Sertoli cells are the only somatic cells in the microenvironment that provide protection and guidance to germ cells during spermatogenesis.34 In the whole process of spermatogenesis, Sertoli cells embrace various types of germ cells, play the roles of structural support35 and nutritional support,36 participate in intercellular material transport and signaling,37 and build an immune-protective environment through the blood–testis barrier (BTB),38 which creates a suitable microenvironment for spermatogenesis.

Sertoli cells can suffer attrition due to age, damage caused by exogenous endocrine disruptors, or disruption of the BTB. This can lead to spermatogenic dysfunction and ultimately result in male infertility.39 Experiments showed that ICA promotes the proliferation of Sertoli cells through activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway in vitro. This effect is dose-dependent, which explains the protective effect of ICA on male fertility.40 In a study of rat testis in an age-related model, ICA significantly increased the level of estrogen receptor α (Erα) in vivo and alleviated the damage to Sertoli cells by activating the Erα/nuclear factor-erythroid 2-related factor 2 (Nrf2) signaling pathway, which improved the age-related degenerative lesions in rat testes.41 Apart from aging factors, some exogenous factors can also lead to Sertoli cell damage. In vivo and in vitro experiments showed that microcystin-leucine-arginine (MC-LR) disrupted BTB, reduced gap junctional intercellular communication (GJIC), and impaired connexin 43 (Cx43) expression, which was associated with MC-LR activation of the PI3K/Akt cascade. At the same time, ICA prevented MC-LR-induced GJIC degradation and Cx43 damage by inhibiting the Akt pathway, thus attenuating the toxic effects of MC-LR.42 It has also been shown that ICA effectively reduces perfluorooctane sulfonate (PFOS)-induced BTB disruption through downregulation of the p38/ mitogen-activated protein kinase (MAPK)/matrix metalloproteinase 9 (MMP9) pathway.43 Collectively, Epimedium and its main bioactive compounds promote the proliferation of Sertoli cells and rescue the destruction of the BTB caused by multiple factors, thus protecting the microenvironment required for spermatogenesis (Figure 2). Interestingly, only ICA seems to be reported to have a protective effect on Sertoli cell proliferation and BTB destruction among Epimedium and its main bioactive compounds. Its specific mechanism of action is also clearly described. Still, there is a critical point that is not clearly defined in these studies: how ICA enters into the cell and acts, which is worth investigating. In addition, it is also worthwhile to explore whether the bioactive compounds in Epimedium, other than ICA, have the same effect on Sertoli cells as ICA.

Figure 2.

ICA promotes the proliferation of Sertoli cells and rescues the destruction of BTB caused by multiple factors. ICA: icariin; PI3K: phosphatidylinositol 3-kinase; Akt: the serine/threonine kinase Akt; p: phosphorylation; Erk1/2: extracellular signal-regulated kinase 1/2; MEk1/2: mitogen-activated protein kinase kinase 1/2; BTB: blood–testis barrier; MAPK: mitogen-activated protein kinase; MMP9: matrix metalloproteinase 9; CX43: connexin 43; ZO-1: zonula occludens-1.

Anti-oxidative stress

Oxidative stress (OS) is considered to cause male infertility in approximately 40%–50% of cases. The endogenous testicular anti-oxidant defensed mechanisms can not inhibit the occurrence of oxygen species (OS), and high levels of reactive OS (ROS) can reduce cell viability and induce DNA damage.44 Therefore, anti-oxidant therapy has become an acceptable treatment method among international male infertility experts. A study has shown that oral anti-oxidant therapy can significantly improve sperm quality, resulting in higher clinical pregnancy and live birth rates.45 In contrast, most anti-oxidant therapy is based on the use of synthetic or naturally derived anti-oxidants.46 Consequently, an increasing number of traditional Chinese medicines (TCMs) with anti-oxidant properties, such as Epimedium, are being used to improve male infertility caused by high levels of OS.47

For practical clinical applications, Epimedium is used chiefly as an ingredient in multi-component mixed formulas to exert anti-oxidant effects. However, the active components or constituents with testicular-protective activity remain unclear.48,49 TFE and ICA are the main active components of Epimedium. Among them, TFE exerts anti-oxidative stress effects in animal models induced by aging, streptozotocin, and cyclophosphamide. The specific mechanism involves the reduction of senescence-induced DNA damage by inhibiting p53 phosphorylation,50 reducing testicular oxidative damage by increasing superoxide dismutase (SOD) activity and reducing malondialdehyde (MDA) levels to maintain anti-oxidant capacity,51 and directly scavenging ROS and upregulating anti-oxidant enzymes to repair damaged testes and epididymis, especially superoxide dismutase 3 (SOD3) and glutathione peroxidase 1 (GPx1).52 Another study found that ICA enhanced the activity of anti-oxidant enzymes in mice/rats with reproductive damage caused by nicotine and diabetes, which reduced the testicular tissue damage caused by OS.53,54

In conclusion, Epimedium and its bioactive compounds have strong anti-oxidant effects and can effectively improve oxidative damage of testicular tissue from various causes. Epimedium’s anti-oxidant effects have a closer relationship to clinical applications compared to other products that target the male reproductive system. This is because studies on the anti-oxidant effects of Epimedium have used formulations that are either already in clinical use or approved for marketing, but this is the biggest shortcoming of these studies. Although it has been clarified that the active compounds of Epimedium, such as TFE and ICA, have potent anti-oxidant effects, it is still unclear whether the active compounds of Epimedium play a significant role in the formula because they contain a variety of herbs. Therefore, further experimental studies are needed to identify the main components that play anti-oxidant roles in the procedure.

Erectile dysfunction (ED)

ED is an essential cause of male infertility. It usually presents as inadequate or absent erections during sexual intercourse, which can lead to impaired fertility for natural conception.55 Penile erection is due to the nitric oxide (NO)-mediated increase in intracellular cyclic guanosine monophosphate (cGMP) levels, causing diastole of penile cavernous trabeculae and vascular smooth muscle. At the same time, phosphodiesterase (PDE) hydrolyzes cGMP and reverses this process. ED may occur when any of these processes is interrupted.56

According to epidemiological surveys, ED has been identified as the most common chronic complication in individuals with diabetes mellitus (DM), prostate cancer, and cardiovascular disease, which seriously threaten both qualities of life and the health of patients.57 Among them, DM is highly correlated with the development of ED. DM can produce oxidative stress injuries in cavernous tissue, leading to loss of physiological properties of the endothelium and a shift toward vasoconstriction, prothrombotic, and pro-inflammatory state, which is regarded as a key to the early development of diabetes mellitus-associated erectile dysfunction (DMED).58 It suggests that ICA II plays a core role in regulating cavernous nerves, smooth muscle cells, and endothelial cells and facilitates the differentiation of endogenous stem cells to neuronal cells.59 Experiments in vitro and in vivo demonstrate that ICA II is a protective agent of endothelial function and can attenuate high glucose-induced injury in human cavernous endothelial cells in a dose-dependent manner by mechanisms including increasing endothelial nitric oxide synthase (eNOs) levels through the Akt-eNOs signaling pathway and regulating the MAPK pathway in human cavernous endothelial cells through sprouty-related, EVH1 domain containing 1 (SPRED1).60,61,62 In addition, when ICA II is combined with adipose-derived mesenchymal stem cells (ADSCs), ICA II can protect ADSCs against OS by mediating the phosphatidylinositol 3-kinase (PI3K)/Akt/STAT3 signaling pathway, which enhances the therapeutic effect of ADSCs on DMED.63 For ED caused by cavernous nerve injury, ICA II promotes the differentiation of ADSCs to Schwann cells (SCs) via the miR-33/glial cell-derived neurotrophic factor (GDNF) axis, which facilitates the recovery of erectile function.64

NO is an essential neuromodulator during penile erection. A study has shown that the downregulation of nitric oxide synthase (NOs) and cGMP-phosphodiesterase activity is a significant event in the development of ED.65 The nitric oxide synthase family includes three isoforms, composed of neuronal nitric oxide synthase (nNOs), eNOs, and inducible nitric oxide synthase (iNOs); and ICA was found to be a potential drug to treat ED by enhancing NOs activity.66 These include modulating the central and peripheral nervous system, stimulating the PI3K/Akt signaling pathway, increasing eNOs in penile endothelial cells and NO levels in serum, and promoting penile erection in mice.25 It also improves penile erection by increasing phosphorylation-eNOs (p-eNOs)/eNOs levels by inhibiting inositol 1,4,5-trisphosphate receptor type I (IP3R1), phosphofurin acidic cluster sorting protein 2 (PACs2), and long-chain-fatty-acid-CoA ligase 4 (FACL4) expression in rats after prostate radiation treatment.67 It has also been shown that ICA, an inhibitor of PDE5, specifically binds to the cGMP catalytic site, inhibits the hydrolysis of cGMP by PDE5, increases the concentration of cGMP in penile smooth muscle cells, and significantly enhances the erectile effect with increasing action concentrations.68 ICA has a neurotrophic possible impact and its known type 5 phosphodiesterase inhibitory effect. It could improve neurological function in radiation-injured rats by increasing nNOs levels in penile tissue.69,70

In summary, Epimedium and its main bioactive compound improve penile ED by treating oxidative damage to penile cavernous endothelial cells caused by high sugar and radiation (Figure 3). Epimedium has a long history as a traditional aphrodisiac; thus, the effect of Epimedium and its main bioactive compounds on penile ED has become a hot topic of research on the male reproductive system. Compared with sildenafil, the active components of Epimedium have the same therapeutic effect and are already on the market. They are more effective than sildenafil in improving erectile function, and when they are used in combination, the therapeutic effect is even more significant,59 suggesting that it could be a potential treatment for penile ED. However, these are only studies based on animal models, and we still need to use this as a basis for the early clinical application of this treatment method.

Figure 3.

Possible mechanism by which ICA and ICA II ameliorate erectile dysfunction. ROS: reactive oxygen species; L-Arg: L-arginine; L-Cit: L-citrulline; NO: nitric oxide; HCEC: human cavernous endothelial cells; HCSMC: human cavernous smooth muscle cells; GC: guanylate cyclase; GTP: guanosine-5’-triphosphate; cGMP: cyclic guanosine monophosphate; PKG: cGMP-dependent protein kinase; PDE5: phosphodiesterase type 5; 5’-GMP: guanosine 5’-monophosphate; eNOs: endothelial nitric oxide synthase; Erk: extracellular signal-regulated kinase; PI3K: phosphatidylinositol 3-kinase; Akt: the serine/threonine kinase Akt; ICA II: icariside II; ICA: icariin; O₂: oxygen.

ANTI-TUMOR EFFECTS ON THE MALE REPRODUCTIVE SYSTEM

Cancer is a rising health problem and one of the primary causes of death worldwide. With increasing incidence and decreasing age of onset, tumors are an increasing threat to humans.71 Common treatments for cancer include chemotherapy, radiotherapy, immunotherapy, and surgery, but there are still unsatisfactory side effects, easily resistant to drugs, and even fatal. TCM is an ideal choice for anti-tumor drug development because of its multipotency, strong targeting, and few side effects. Epimedium has received wide attention for its highly effective and extensive anti-tumor results. However, the targets and potential mechanisms of its anticancer effects seem diverse, depending on the different types of tumors.72

Proliferation

The cell cycle consists of G1, S, G2, and M phases, resulting in DNA replication and cell division to produce two daughter cells. The cell cycle process is influenced by the precise regulation of cell cycle protein machinery, metabolic enzymes, and cellular redox status, and aberrant changes can lead to tumorigenesis. Targeting the cyclin machinery to induce cell cycle arrest has been shown to be a potential therapeutic approach.73

Epimedium and its main bioactive compounds can regulate the expression of various cell cycle-related proteins. In human prostate cancer PC-3 cells, ICT can induce G1 phase arrest by upregulating the expression of p16, p21, and p27 and further inhibit the expression of cell cycle proteins D1 and CDK4.74 Except for inhibiting the proliferation of tumor cells by affecting the cell cycle, abnormal expression of microRNAs (miRNAs) has been associated with various diseases, including tumorigenesis, in recent years. Depending on the target mRNAs of different tumor types, miRNAs can act as tumor suppressor genes and oncogenes.75 Therefore, functional regulation of miRNAs is an effective strategy for tumor therapy.76 Natural products and other small molecules have been reported to exhibit anti-tumor effects by regulating miRNA expression.77 ICA has been shown to affect several physiological and pathological processes by regulating specific miRNAs.78 In a study on reproductive tumors, ICT has been shown to inhibit the proliferation and invasion of prostate cancer cells by modulating miR-381-3p and its target gene, ubiquitin-conjugating enzyme E2C (UBE2C).79 These findings revealed that Epimedium and its main bioactive compounds could exert anti-tumor effects by inducing cell cycle arrest and regulating miRNAs and their downstream targets to inhibit the proliferation of tumor cells (Figure 4).

Figure 4.

Possible mechanisms of the active compounds of Epimedium affecting the biological functions of reproductive system tumors. ICT: icaritin; CDK4: cyclin-dependent kinase 4; pRb: phosphorylation retinoblastoma protein; E2F: early 2 factor; TNF-α: tumor necrosis factor; FasL: factor-related apoptosis ligand; Bcl-xL: B-cell leukemia/lymphoma 2 like 1; BaX: bcl-2 associated x; ROS: reactive oxygen species; G1: gap 1 phase; S: synthesis phase; ICA II: icariside II; Cyc C: cytochrome C; Apaf-1: apoptotic protease activating factor-1; CDK6: cyclin-dependent kinase 6; Erk: extracellular signal-regulated kinase.

Apoptosis

Apoptosis is the primary form of cell-programmed death through which damaged and unneeded cells can be eliminated. Dysregulation of this dying process is related to irrepressible cell proliferation, tumor development, and resistance to tumor therapy. Producing apoptosis is the foremost treatment strategy for cancer.80 Studies have demonstrated that Epimedium and its main biologically active compounds have well-induced apoptosis in cancer cells, among which ICA can regulate the expression and release of cytochrome c and the activities of caspase-9 and caspase-3, which have proapoptotic training in the mouse Leydig tumor cell line MLTC-1.81 ICT could induce apoptosis in prostate cells by regulating polyomavirus enhancer activator 3/human epidermal growth factor receptor type 2/androgen receptor (PEA3/HER2/AR) signaling.82

Interestingly, ICT induces explicit apoptosis in chemoresistant tumor cells. When treated by androgen deprivation, prostate cancer prefers progression to chemoresistant neuroendocrine prostate cancer (NEPC).83 Sequential treatment even stimulates tumor cells to respond to autocrine interleukin-6 (IL-6), accelerating the formation of highly aggressive NEPC with poor prognoses. Optimistically, ICT induces apoptosis in prostate cancer cells and inhibits the development of NEPC.84 In addition, ICT also has a practical proapoptotic effect on cisplatin-resistant ovarian cancer cells by activating p53 and inhibiting the Akt/mTOR pathway.85 In conclusion, Epimedium and its main bioactive compounds have shown apoptosis-inducing substantial effects on tumor cells and significant efficacy against treatment-resistant tumors by chemotherapy and radiotherapy.

Autophagy

Autophagy occurs when cells wrap specific proteins and organelles for degradation into lysosomes to repurpose small molecules and generate energy. The sequential occurrence of autophagy blocks the accumulation of impaired proteins and organelles, thereby protecting cells from apoptosis or carcinogenesis.86 Mutual interactions between autophagy and apoptosis are complex, as autophagy can induce apoptosis or necrosis and promote cell death itself, which means that regulating the occurrence of autophagy affects apoptosis.87 Epimedium and its major bioactive compounds have complex roles in regulating autophagy in tumor cells. In human prostate cancer cells, ICA II treatment can enhance the autophagic process by controlling the PI3K-AKT-mTOR signaling pathway. Enhanced autophagy further induces the onset of apoptosis in prostate cancer cells.88

Interestingly, when combined with anti-tumor drugs, Epimedium and its major bioactive compounds could increase the drug sensitivity of tumor cells by modulating autophagy. ICT exhibits synergistic effects with epirubicin to inhibit the growth of bladder cancer by suppressing autophagy.89 To summarize, Epimedium and its main biologically active compounds can induce autophagy as a promoter or blocker of apoptosis by inhibiting autophagy from increasing apoptosis in combination with chemotherapeutic agents. These conflicting results emphasize the complex role of Epimedium and its main biologically active compounds in regulating autophagy in tumor cells.

Other effects on tumors

The capacity for extensive infiltration and spread is closely related to migration and invasion in tumor cells, contributing to the difficulty of tumor eradication. Epimedium and its main bioactive substances for various male reproductive system tumors have sound inhibitory effects on tumor cell migration and invasion.76,82,88 These studies suggest that Epimedium and its main active compounds can inhibit the migration and invasion process of tumor cells and exert their sound anti-tumor effects.

OTHER EFFECTS ON THE REPRODUCTIVE SYSTEM

Anti-inflammatory

Torsional testicular injury is a typical ischemia-reperfusion injury that can lead to ROS production, activation of the inflammatory response, and attendant apoptosis. ICA II treatment can reduce ROS levels and decrease inflammatory cell infiltration to alleviate the inflammatory response.90 In contrast, in cyclophosphamide-induced cystitis in mice, ICA could exert an anti-inflammatory response by downregulating nuclear factor kappa-B (NF-кB) and upregulating the Nrf-2/heme oxygenase-1 (HO-1) signaling pathway.91 Taken together, Epimedium and its main bioactive compounds exhibited sound anti-inflammatory effects in the male reproductive system.

CONCLUSION AND OUTLOOK

Growing evidence suggests that the primary active components of Epimedium, especially ICA and its metabolite ICT, ICA II, play a crucial role in male reproductive protection; the main molecular mechanisms of the regulation of the primary active components of Epimedium are fully displayed in Figure 5, which involve multiple protective effects of the primary functional components of Epimedium on the male reproductive system.

Figure 5.

Effects of Epimedium and its main active compounds on major signaling pathways on the male reproductive system. Esr1: estrogen receptor 1; PI3K: phosphatidylinositol 3-kinase; Akt: the serine/threonine kinase Akt; p: phosphorylation; Creb: cAMP response element binding proteins; Sf-1: steroidogenic factor 1; StAR: steroidogenic acute regulatory proteins; PBR: peripheral benzodiazepine receptors; SOD: superoxide dismutase; GPx: glutathione peroxidase; CAT: catalase; Sirt6: sirtuin 6; NF-кB: nuclear factor kappa-B; CDK6: cyclin-dependent kinase 6; p-p53: phosphorylation-p53; Erk1/2: extracellular signal-regulated kinase 1/2; Erα: estrogen receptor α; Nrf2: nuclear factor-erythroid 2-related factor 2; MAPK: mitogen-activated protein kinase; HO-1: heme oxygenase-1; CDK2: cyclin-dependent kinase 2; mTOR: mechanistic target of rapamycin; CDK4: cyclin-dependent kinase 4; PAE3: polyomavirus enhancer activator 3; HER2: human epidermal growth factor receptor type 2; AR: androgen receptor; UBE2C: ubiquitin-conjugating enzyme E2C; ↑: rising expression; ↓: decreasing expression.

The role of Epimedium and its active components in the male reproductive system involves many aspects, the majority of which focus on male infertility. Furthermore, the specific mechanisms are mainly related to promoting testosterone secretion, improving the microenvironment of spermatogenesis, alleviating oxidative stress, and improving ED.

For the treatment of reproductive tumors, Epimedium and its active components have great potential to act through various mechanisms, not limited to single targets and pathways but involving targets such as UBE2C and insulin-like growth factor 2 receptor (IGF2R) and routes such as the PI3K/Akt and Akt/mTOR signaling pathways. Furthermore, some biological processes, such as oxidative stress, combined with the multi-target and multi-pathway involved in the related network pharmacology results, indicate the promising mechanism of Epimedium and its active components in male reproductive tumors.

Although great progress has been made in the study of the mechanism of the improvement of male reproductive function by Epimedium and its active substances, it still needs to be improved in the following aspects. (1) The main components of the current research are focused on ICA and its metabolites. However, the active ingredients of Epimedium are complex and need to be further explored for the components with therapeutic effects in the male reproductive system. (2) The research needs more work to reveal the related proteins and genes to lay the foundation for developing new drugs with Epimedium as the main active ingredient and its clinical application. (3) The current research on Epimedium is mainly based on experimental animal studies and lacks clinical research data.

In addition, by summarizing the relevant studies, we found several questions that deserve in-depth study. (1) Epimedium and its main bioactive compounds can protect the male reproductive system in many ways, such as regulating the synthesis and secretion of related hormones, anti-oxidative stress, anti-inflammatory, etc. However, in practical application, which aspect of Epimedium and its main bioactive compounds exert protective effects? Or is it a combination of multiple elements? (2) Although most studies have discussed in detail the mechanism of action of Epimedium and its main bioactive compounds on the male reproductive system, the description of how these active compounds act intracellularly needs to be more specific.27,42,52,63 This is a crucial point to understanding the specific mechanism of their protective effect and deserves to be studied in depth. (3) Currently, Epimedium is mainly used as an herbal medicine in clinical applications, and it is a long process to extract, process, and modify the active compounds of Epimedium that play a role in male reproductive protection and apply them to clinical applications. (4) Although most of the studies reported the protective effects of Epimedium and its main bioactive compounds on the male reproductive system, some of them also mentioned that they may cause some damage to the male reproductive system and it depends on their specific dosage, and this is an urgent question whether Epimedium and its main bioactive compounds can be successfully used in clinical applications.

AUTHOR CONTRIBUTIONS

SPL and YFL were responsible for review design and manuscript writing. DZ was responsible for data collection and form creation. JC, XFD, CYL, HZ, and JT were responsible for revising the manuscript. TS, DFL, and YYZ were involved in the design of the images. ZXH, JDZ, and JT were engaged in the review design, funding application, and project supervision and management. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declared no competing interests.

Supplementary Figure 1

Epimedium and its main bioactive components. (a) Image of the medicinal plant of Epimedium. (b) Image of the central therapeutic part (leaves) of Epimedium. (c) Icariin chemical structure formula. (d) Icariside I chemical structure formula. (e) Icaritin chemical structure formula. (f) Icariside II chemical structure formula.