Icariin attenuates vascular endothelial dysfunction by inhibiting inflammation through GPER/Sirt1/HMGB1 signaling pathway in type 1 diabetic rats.

Icariin, a flavonoid glycoside, is extracted from Epimedium. This study aimed to investigate the vascular protective effects of icariin in type 1 diabetic rats by inhibiting high-mobility group box 1 (HMGB1)-related inflammation and exploring its potential mechanisms. The impact of icariin on vascular dysfunction was assessed in streptozotocin (STZ)-induced diabetic rats through vascular reactivity studies. Western blotting and immunofluorescence assays were performed to measure the expressions of target proteins. The release of HMGB1 and pro-inflammation cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The results revealed that icariin administration enhanced acetylcholine-induced vasodilation in the aortas of diabetic rats. It also notably reduced the release of pro-inflammatory cytokines, including interleukin-8 (IL-8), IL-6, IL-1ß, and tumor necrosis factor-alpha (TNF-α) in diabetic rats and high glucose (HG)-induced human umbilical vein endothelial cells (HUVECs). The results also unveiled that the pro-inflammatory cytokines in the culture medium of HUVECs could be increased by rHMGB1. The increased release of HMGB1 and upregulated expressions of HMGB1-related inflammatory factors, including advanced glycation end products (RAGE), Toll-like receptor 4 (TLR4), and phosphorylated p65 (p-p65) in diabetic rats and HG-induced HUVECs, were remarkably suppressed by icariin. Notably, HMGB1 translocation from the nucleus to the cytoplasm in HUVECs under HG was inhibited by icariin. Meanwhile, icariin could activate G protein-coupled estrogen receptor (GPER) and sirt1. To explore the role of GPER and Sirt1 in the inhibitory effect of icariin on HMGB1 release and HMGB-induced inflammation, GPER inhibitor and Sirt1 inhibitor were used in this study. These inhibitors diminished the effects of icariin on HMGB1 release and HMGB1-induced inflammation. Specifically, the GPER inhibitor also negated the activation of Sirt1 by icariin. These findings suggest that icariin activates GPER and increases the expression of Sirt1, which in turn reduces HMGB1 translocation and release, thereby improving vascular endothelial function in type 1 diabetic rats by inhibiting inflammation.

[Determination of 13 chemical components of Epimedii Folium in pharmacopoeia by UPLC method combined with quantitative analysis of multicomponents by single-marker].

The quantitative analysis of multicomponents by single-marker(QAMS) was established for 13 chemical components of Epimedii Folium, including neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuoside Ⅰ, so as to investigate the feasibility and accuracy of this method in evaluating the quality of Epimedii Folium materials from different origins and different varieties. Through the scientific and accurate investigation of the experimental method, the external standard method was used to determine the content of 13 chemical components in epimedium brevieornu. At the same time, icariin was used as the internal standard, and the relative correction factors of icariin with neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuoside Ⅰ were established, respectively. The contens of neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuosideⅠ in Epimedii Folium were calculated by QAMS. Finally, the difference between the measured value and the calculated value was compared to verify the accuracy and scientific nature of QAMS in the determination. The relative correction factor of each component had better repeatability, and there was no significant difference between the results of the external standard method and those of QAMS. With icariin as the internal standard, QAMS simultaneously determining neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuoside Ⅰ can be used for quantitative analysis of Epimedii Folium.

[Study on mechanism of icariin-induced ferroptosis in HepG2 hepatoma carcinoma cells through PPARG/FABP4/GPX4 pathway].

The aim of this study was to explore the mechanism of icaritin-induced ferroptosis in hepatoma HepG2 cells. By bioinformatics screening, the target of icariin's intervention in liver cancer ferroptosis was selected, the protein-protein interaction(PPI) network was constructed, the related pathways were focused, the binding ability of icariin and target protein was evaluated by molecular docking, and the impact on patients' survival prognosis was predicted and the clinical prediction model was built. CCK-8, EdU, and clonal formation assays were used to detect cell viability and cell proliferation; colorimetric method and BODIPY 581/591 C1 fluorescent probe were used to detect the levels of Fe~(2+), MDA and GSH in cells, and the ability of icariin to induce HCC cell ferroptosis was evaluated; RT-qPCR and Western blot detection were used to verify the mRNA and protein levels of GPX4, xCT, PPARG, and FABP4 to determine the expression changes of these ferroptosis-related genes in response to icariin. Six intervention targets(AR, AURKA, PPARG, AKR1C3, ALB, NQO1) identified through bioinformatic analysis were used to establish a risk scoring system that aids in estimating the survival prognosis of HCC patients. In conjunction with patient age and TNM staging, a comprehensive Nomogram clinical prediction model was developed to forecast the 1-, 3-, and 5-year survival of HCC patients. Experimental results revealed that icariin effectively inhibited the activity and proliferation of HCC cells HepG2, significantly modulating levels of Fe~(2+), MDA, and lipid peroxidation ROS while reducing GSH levels, hence revealing its potential to induce ferroptosis in HCC cells. Icariin was found to diminish the expression of GPX4 and xCT(P<0.01), inducing ferroptosis in HCC cells, potentially in relation to inhibition of PPARG and FABP4(P<0.01). In summary, icariin induces ferroptosis in HCC cells via the PPARG/FABP4/GPX4 pathway, providing an experimental foundation for utilizing the traditional Chinese medicine icariin in the prevention or treatment of HCC.

Dietary supplementation of osthole and icariin improves the production performance of laying hens by promoting follicular development.

Osthole (Ost) and icariin (Ica) are extracted from traditional Chinese medicine Cnidium monnieri and Epimedii Folium, respectively, and both exhibit estrogen-like biological activity. This study aimed to determine the efficacy and safety of combining Ost with Ica on the production performance of laying hens and to explore their possible mechanisms. The production performance, egg quality, residues of Ost and Ica in eggs, serum reproductive hormone levels, expression of ovarian reproductive hormone receptor, proliferation of granulosa cells in small yellow follicles (SYF), and progesterone secretion in large yellow follicles (LYF) related genes and proteins expression were detected. The results showed that adding 2 mg/kg Ost + 2 mg/kg Ica to the feed increased the laying rate, average egg weight, Haugh unit, and protein height of laying hens. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and progesterone (P4) levels increased, and the expression of ovarian estrogen receptor (ER), follicle-stimulating hormone receptor (FSHR), and progesterone receptor (PGR) mRNA was up-regulated. Additionally, the mRNA and protein levels of steroidogenesis acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450scc), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) increased in LYF. Furthermore, mRNA and protein levels of proliferating cell nuclear antigen (PCNA), cyclin E1, and cyclin A2 were up-regulated in SYF. The residues of Ost and Ica in egg samples were not detected by high-performance liquid chromatography (HPLC). In conclusion, dietary supplementation of Ost and Ica increased granulosa cells proliferation in SYF and increased P4 secretion in granulosa cells of LYF, ultimately improving the production performance of laying hens.

Icariin promotes bone marrow mesenchymal stem cells osteogenic differentiation via the mTOR/autophagy pathway to improve ketogenic diet-associated osteoporosis.

BACKGROUND: Icariin, a traditional Chinese medicine, has demonstrated anti-osteoporotic properties in ovariectomized mice. However, its effectiveness in preventing bone loss induced by ketogenic diet (KD), which mimics osteoporosis in human, remains unexplored. This study aims to investigate icariin's impact on KD-induced bone loss in mice. METHODS: Thirty mice were divided into: sham, KD, and KD + icariin groups. Post a 12-week intervention, evaluation including bone microstructures, serum concentrations of tartrate-resistant acid phosphatase (TRAP) and bone-specific alkaline phosphatase (ALP), and femoral tissue expression levels of osteocalcin (OCN) and TRAP. The expression levels of mammalian target of rapamycin (mTOR), ALP, peroxisome proliferator-activated receptor gamma (PPAR-γ), phosphorylated mTOR (p-mTOR), and the autophagy adaptor protein (p62) were also analyzed. Alizarin granule deposition and cellular ALP levels were measured following the induction of bone marrow mesenchymal stem cells (BMSCs) into osteogenesis. RESULTS: The study found that KD significantly impaired BMSCs' osteogenic differentiation, leading to bone loss. Icariin notably increased bone mass, stimulated osteogenesis, and reduced cancellous bone loss. In the KD + icariin group, measures such as bone tissue density (TMD), bone volume fraction (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were significantly higher than in the KD group. Additionally, bone trabecular separation (Tb.Sp) was markedly lower in the KD + icariin group. Moreover, icariin increased OCN and ALP levels while suppressing PPAR-γ, TRAP, p62, and p-mTOR. In cellular studies, icariin encouraged osteogenic development in BMSCs under KD conditions. CONCLUSIONS: Icariin effectively counteracts bone thinning and improves bone microstructure. Its mechanism likely involves stimulating BMSCs osteogenic differentiation and inhibiting bone resorption, potentially through mTOR downregulation. These findings suggest icariin's potential as an alternative treatment for KD-induced bone loss.

Effect of icariin on depressive behaviour in rat pups. Evidences for its mechanism of action by integrating network pharmacology, metabolomics and gut microbiota composition.

BACKGROUND: Prenatal stress (PS) can cause cognitive disorder and a range of psychological illnesses, including anxiety and depression. Icariin (ICA) has shown promising effects in improving PS-induced depressive behaviour. However, its mechanism of action remains unclear. PURPOSE: This study was performed to reveal the key targets, metabolites and gut microbiota for ICA in improving depressive behaviour in PS rat pups. METHODS: A prenatal restraint stress animal model was established for Sprague-Dawley (SD) rats in late pregnancy. Male pups were randomly divided into six groups: no stress group (NS), PS group, PS + saline group (PS_S), PS + high-dose ICA group (ICAH, 80 mg/kg*day), PS + low-dose ICA group (ICAL, 40 mg/kg*day) and PS + fluoxetine group (FLU, 10 mg/kg*day). The depressive behaviour of each group of rat pups was evaluated using open field test, forced swimming test and sucrose preference test. Different metabolites were identified using untargeted metabolomics of serum and faeces, and metabolic pathways were analyzed through MetaboAnalyst. Targets for ICA acting on depression were determined after network pharmacology was applied. An integrated network of network pharmacology and metabolomics were constructed using Cytoscape software, and molecular docking were performed to verify the interactions between ICA and key targets. Finally, gut microbiota of rat pups in each group were analyzed after 16S rDNA sequencing. RESULTS: PS could cause rat pups to exhibit depressive behaviour, and ICA could significantly improve this depressive behaviour. A total of 49 differential metabolites were found in serum and 23 differential metabolites were found in faeces, and 24 metabolites in serum and 6 metabolites in faeces could be reversed following ICA administration. Integrated analysis focused on five key targets (i.e. adenosyl homocysteinase; medium-chain specific acyl-CoA dehydrogenase, mitochondrial; thymidine phosphorylase; cGMP-specific 3',5'-cyclic phosphodiesterase and xanthine dehydrogenase/oxidase) and three metabolites (i.e. palmitoylcarnitine, methionine and hypoxanthine). Molecular docking indicated that ICA combined well with key targets. Gut microbiota analysis showed that g_Bacteroides, f_Bacteroidaceae and s_Lactobacillus reuteri were required for ICA to improve depressive behaviour. CONCLUSION: In this study, the antidepressant mechanism of ICA was clarified with a strategy of integrating metabolomics, network pharmacology and gut microbiota. ICA has a good effect on improving metabolism and increasing the abundance of probiotics in the intestine. The present research provided new insights into the anti-depressant mechanism of ICA.

Icariin Regulates EMT and Stem Cell-Like Character in Breast Cancer through Modulating lncRNA NEAT1/TGFß/SMAD2 Signaling Pathway.

Metastases and drug resistance are the major risk factors associated with breast cancer (BC), which is the most common type of tumor affecting females. Icariin (ICA) is a traditional Chinese medicine compound that possesses significant anticancer properties. Long non-coding RNAs (lncRNAs) are involved in a wide variety of biological and pathological processes and have been shown to modulate the effectiveness of certain drugs in cancer. The purpose of this study was to examine the potential effect of ICA on epithelial mesenchymal transition (EMT) and stemness articulation in BC cells, as well as the possible relationship between its inhibitory action on EMT and stemness with the NEAT1/transforming growth factor ß (TGFß)/SMAD2 pathway. The effect of ICA on the proliferation (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony assays), EMT (Western blotting, immunofluorescence, and wound healing), and stemness (mammosphere formation assays, Western blotting) of BC cells were examined. According to the findings, ICA suppressed the proliferation, EMT, and stem cell-like in MDA-MB-231 cells, and exerted its inhibitory impact by downregulating the TGFß/SMAD2 signaling pathway. ICA could significantly downregulate the expression of lncRNA NEAT1, and silencing NEAT1 enhanced the effect of ICA in suppressing EMT and expression of different stem cell markers. In addition, silencing NEAT1 was found to attenuate the TGFß/SMAD2 signaling pathway, thereby improving the inhibitory impact of ICA on stemness and EMT in BC cells. In conclusion, ICA can potentially inhibit the metastasis of BC via affecting the NEAT1/TGFß/SMAD2 pathway, which provides a theoretical foundation for understanding the mechanisms involved in potential application of ICA for BC therapy.

The protective effects of icariin against testicular dysfunction in type 1 diabetic mice Via AMPK-mediated Nrf2 activation and NF-κB p65 inhibition.

BACKGROUND: Owing to the early suffering age and the rising incidence of type 1 diabetes (T1D), the resulting male reproductive dysfunction and fertility decline have become a disturbing reality worldwide, with no effective strategy being available. Icariin (ICA), a flavonoid extracted from Herba Epimedium, has been proved its promising application in improving diabetes-related complications including diabetic nephropathy, endothelial dysfunction and erectile dysfunction. Ensuring the future reproductive health of children and adolescents with T1D is crucial to improve global fertility. However, its roles in the treatment of T1D-induced testicular dysfunction and the potential mechanisms remain elusive. PURPOSE: The purpose of this present study was to investigate whether ICA ameliorates T1D-induced testicular dysfunction as well as its potential mechanisms. METHODS: T1D murine model was established by intraperitoneal injection of STZ with or without treated with ICA for eleven weeks. Morphological, pathological and serological experiments were used to determine the efficacy of ICA on male reproductive function of T1D mice. Western blotting, Immunohistochemistry analysis, qRT-PCR and kit determination were performed to investigated the underlying mechanisms. RESULTS: We found that replenishment of ICA alleviated testicular damage, promoted testosterone production and spermatogenesis, ameliorated apoptosis and blood testis barrier impairment in streptozotocin-induced T1D mice. Functionally, ICA treatment triggered adenosine monophosphate protein kinase (AMPK) activation, which in turn inhibited the nuclear translocation of nuclear factor kappa B p65 (NF-κB p65) to reduce inflammatory responses in the testis and activated nuclear factor erythroid 2-related factor 2(Nrf2), thereby enhancing testicular antioxidant capacity. Further studies revealed that supplementation with the AMPK antagonist Compound C or depletion of Nrf2 weakened the beneficial effects of ICA on testicular dysfunction of T1D mice. CONCLUSION: Collectively, these results demonstrate the feasibility of ICA in the treatment of T1D-induced testicular dysfunction, and reveal the important role of AMPK-mediated Nrf2 activation and NF-κB p65 inhibition in ICA-associated testicular protection during T1D.

Icariin regulates acidic microenvironment to alleviate pain caused by postmenopausal osteoporosis in the elderly / 中国组织工程研究

BACKGROUND:Previous studies have demonstrated that icariin has important roles in promoting bone formation and inhibiting bone resorption,but its effects on osteoporosis-mediated bone pain have not been reported. OBJECTIVE:To investigate the possible mechanism of icariin alleviating bone pain in postmenopausal senile osteoporosis. METHODS:(1)Animal experiment:200 C57BL/6 mice were randomly divided into 4 groups:sham group(n=50),model group(n=50),icariin treatment group(n=50),and carbonic anhydrase Ⅱ inhibitor(Brinzolamide)treatment group(n=50).Ovariectomy was performed on C57BL/6 mice to establish a postmenopausal osteoporosis model in all groups except the sham group.The icariin group was given icariin on the second day after modeling,and pain behavior tests(Von Frey,Hot Plate,and Tail Flick tests)were performed every 2 weeks for 20 weeks.After sampling,bone mass was detected by microCT,osteoclast activity was detected by hematoxylin-eosin and tartrate-resistant acid phosphatase staining,and neuronal morphology and related ion channel expression were detected by tissue immunofluorescence staining.(2)Cell experiment:Osteoclast precursor cells derived from mouse bone marrow were extracted and induced to differentiate into osteoclasts using the RANKL/M-CSF system in vitro and supplemented with icariin of different concentrations(1 and 10 μmol/L).Tartrate-resistant acid phosphatase staining was used to detect osteoclast differentiation,ghost pen cyclic peptide staining was used to detect osteoclast actin ring,bone plate absorption assay was used to detect osteoclast osteophagy function,pH value of the system was detected by pH meter,and expression of osteoclast differentiation-related proteins was detected by western blot.In addition,mouse dorsal root ganglion-derived nerve cells were extracted and treated with icariin.Cell counting kit-8 was used to detect neuronal activity and CGRP staining was used to detect neuronal morphology. RESULTS AND CONCLUSION:Compared with the model group,the icariin treatment group had higher bone mineral density,fewer tartrate-resistant acid phosphatase-positive osteoclasts in bone tissue,decreased neuronal activity,and decreased neuronal transient receptor potential vanilloid subtype 1 channel and carbonic anhydrase Ⅱ expression.Behavioral results showed that the icariin treatment group was less sensitive to pain than the model group.Icariin inhibited osteoclast differentiation and bone phagocytosis in vitro.Icariin enhanced the activity of dorsal root ganglion neurons and inhibited the expression of calcitonin gene-related peptide and transient receptor potential vanilloid subtype 1 channels in dorsal root ganglia in a relatively non-toxic pH range.To conclude,icariin alleviates bone pain caused by postmenopausal osteoporosis by regulating the acidic microenvironment through its effect on osteoclasts.

Icariin improves learning and memory function in Aß1-42-induced AD mice through regulation of the BDNF-TrκB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Epimedium brevicornu Maxim. is a traditional medicinal Chinese herb that is enriched with flavonoids, which have remarkably high medicinal value. Icariin (ICA) is a marker compound isolated from the total flavonoids of Epimedium brevicornu Maxim. It has been shown to improve Neurodegenerative disease, therefore, ICA is probably a potential drug for treating AD. MATERIALS AND METHODS: The 6-8-week-old SPF-class male ICR mice were randomly divided into 8 groups for modeling, and then the mice were administered orally with ICA for 21 days. The behavioral experiments were conducted to evaluate if learning and memory behavior were absent in mice, confirming that infusion of Amyloid ß-protein (Aß)1-42 caused significant memory impairment. The morphological changes and damage of neurons in the mice's brains were observed by HE and Nissl staining. The spinous protrusions (dendritic spines) on neuronal dendrites were investigated by Golgi-Cox staining. The molecular mechanism of ICA was examined by Western Blot. The protein docking of ICA and Donepezil with BDNF were analyzed to determine their interaction. RESULTS: The behavioral experimental results showed that in Aß1-42-induced AD mice, the learning and memory abilities were improved after using ICA. At the same time, the low, medium, and high doses of ICA could reduce the content of Aß1-42 in the hippocampus of AD mice, repair neuronal damage, enhance synaptic plasticity, as well as increase the expression of BDNF, TrκB, CREB, Akt, GAP43, PSD95, and SYN proteins in the hippocampus of mice. However, the effect with high doses of ICA is more pronounced. The high-dose administration of ICA has the best therapeutic effect on AD mice. After administering the inhibitor k252a, the therapeutic effect of ICA was reversed. The macromolecular docking results of ICA and BDNF protein demonstrated a strong interaction of -7.8 kcal/mol, which indicates that ICA plays a therapeutic role in AD mice by regulating the BDNF-TrκB signaling pathway. CONCLUSIONS: The results confirm that ICA can repair neuronal damage, enhance synaptic plasticity, as well as ultimately improve learning and memory impairment through the regulation of the BDNF-TrκB signaling pathway.

Icariin induces developmental toxicity via thyroid hormone disruption in zebrafish larvae.

Icariin (ICA) is a natural flavonoid isolated from the traditional Chinese medicinal herb, Epimedium brevicornu Maxim. Although previous studies have reported that ICA exhibits various pharmacological activities, little is known about its toxicology. Herein, zebrafish embryos were exposed to ICA at 0, 2.5, 10, and 40 µM. In developmental analysis, reduced hatching rates, decreased body length, and abnormal swim bladder were found after treatment with 10 and 40 µM ICA. In addition, the ability of locomotor behavior was impaired by ICA. Two important thyroid hormones (THs), triiodothyronine (T3) and thyroxine (T4), were tested. The exposure resulted in a remarkable alteration of T4 level and a significant decrease of the T3/T4 ratio in the 40 µM, indicating thyroid endocrine disruption. Furthermore, gene transcription analysis showed that genes involved in thyroid development (nkx2.1) and THs synthesis (tg) were up-regulated after ICA exposure. Significant down-regulation of iodothyronine deiodinase (dio1) was also observed in the 10 and 40 µM groups compared to the control. Taken together, our study first demonstrated that ICA caused developmental toxicity possibly through disrupting thyroid development and hormone synthesis. These results show that it is necessary to perform risk assessments of ICA in clinical practice.

Icariin inhibits hypertrophy by regulation of GPER1 and CaMKII/HDAC4/MEF2C signaling crosstalk in ovariectomized mice.

Icariin (ICA), a flavonoid phytoestrogen, was isolated from traditional Chinese medicine Yin Yang Huo (Epimedium brevicornu Maxim.). Previous studies reporting the cardioprotective effects of ICA are available; however, little is known about the impact of ICA on cardioprotection under conditions of reduced estrogen levels. This study aimed to provide detailed information regarding the antihypertrophic effects of ICA in ovariectomized female mice. Female mice were subjected to ovariectomy (OVX) and transverse aortic constriction and then orally treated with ICA at doses of 30, 60 or 120 mg/kg/day for 4 weeks. Morphological assessments, echocardiographic parameters, histological analyses, and immunofluorescence were performed to evaluate cardiac hypertrophy. Cardiomyocytes from mice or rats were stimulated using phenylephrine, and cell surface and hypertrophy markers were tested using immunofluorescence and qPCR. Western blotting, qPCR, and luciferase reporter gene assays were used to assess the expression of proteins and mRNA and further investigate the proteins related to the G-protein coupled estrogen receptor (GPER1) and CaMKII/HDAC4/MEF2C signaling pathways in vivo and in vitro. ICA blocks cardiac hypertrophy induced by pressure overload in OVX mice. Additionally, we demonstrated that ICA activated GPER1 and inhibited the nuclear export or promoted the nuclear import of histone deacetylase 4 (HDAC4) through regulation of phosphorylation of calmodulin-dependent protein kinase II (CaMKII) and further improved the repression of myocyte enhancer factor-2C (MEF2C). ICA ameliorated cardiac hypertrophy in OVX mice by activating GPER1 and inhibiting the CaMKII/HDAC4/MEF2 signaling pathway.

Liver protecting effects and molecular mechanisms of icariin and its metabolites.

As a detoxification and metabolism organ, the liver plays a vital role in human health. However, an excessive consumption of drugs and toxins, exposure to pathogenic viruses, and unhealthy living habits can lead to liver damage, which may even develop into liver cirrhosis and liver cancer. Epimedium brevicornum Maxim. is a traditional Chinese medicine and dietary supplement in which the flavonoid icariin is a main functional component. Although the protective mechanisms of icariin and its metabolites against liver injury are not yet comprehensively understood, an increasing number of studies have confirmed their liver-protective and anticancer effects. Indeed, icaritin, one of the metabolites of icariin, is currently utilized as an active component of an anti-cancer drug. This paper presents a review of the molecular mechanisms through which icariin and its metabolites actively protect against the occurrence and development of liver injury, and, thus, provides a comprehensive reference for further research and their application in liver protection.

Icariin Supplementation Suppresses the Markers of Ferroptosis and Attenuates the Progression of Nonalcoholic Steatohepatitis in Mice Fed a Methionine Choline-Deficient Diet.

Icariin, a flavonoid abundant in the herb Epimedium, exhibits anti-ferroptotic activity. However, its impact on nonalcoholic steatohepatitis (NASH) development remains unclear. This study aimed to investigate the potential role of icariin in mitigating methionine choline-deficient (MCD) diet-induced NASH in C57BL/6J mice. The results showed that icariin treatment significantly reduced serum alanine aminotrasferase and aspartate aminotransferase activities while improving steatosis, inflammation, ballooning, and fibrosis in the liver tissues of mice fed the MCD diet. These improvements were accompanied by a substantial reduction in the hepatic iron contents and levels of malondialdehyde and 4-hydroxynonenal, as well as an increase in the activities of catalase and superoxide dismutase. Notably, icariin treatment suppressed the hepatic protein levels of ferroptosis markers such as acyl-CoA synthetase long-chain family member 4 and arachidonate 12-lipoxygenase, which were induced by the MCD diet. Furthermore, transmission electron microscopy confirmed the restoration of morphological changes in the mitochondria, a hallmark characteristic of ferroptosis, by icariin. Additionally, icariin treatment significantly increased the protein levels of Nrf2, a cystine/glutamate transporter (xCT), and glutathione peroxidase 4 (GPX4). In conclusion, our study suggests that icariin has the potential to attenuate NASH, possibly by suppressing ferroptosis via the Nrf2-xCT/GPX4 pathway.

Icariin activates far upstream element binding protein 1 to regulate hypoxia-inducible factor-1α and hypoxia-inducible factor-2α signaling and benefits chondrocytes.

Icariin (ICA) is a typical flavonoid glycoside derived from epimedium plants. It has both anabolic and anti-catabolic effects to improve bone mineral density and reduce bone microstructural degradation. However, the effect and underlying mechanism of ICA on the proliferation and metabolism of chondrocyte and synthesis of extracellular matrix are still unclear. This study aimed to investigate the role and regulation of far upstream element binding protein 1 (FUBP1) in chondrocytes treated with ICA to maintain homeostasis and suppress inflammatory responses. In the study, the effect of ICA on chondrocytes with overexpressed or silenced FUBP1 was detected by the MTS and single-cell cloning methods. The expression of hypoxia-inducible factor-1/2α (HIF-1/2α), FUBP1, matrix metalloproteinase (MMP)9, SRY-box transcription factor 9 (SOX9), and type II collagen (Col2α) in ATDC5 cells, a mouse chondrogenic cell line, treated with ICA was evaluated by immunoblotting. Western blotting revealed 1 µM ICA to have the most significant effect on chondrocytes. Alcian blue staining and colony formation assays showed that the promoting effect of ICA was insignificant in FUBP1-knockdown cells (P > 0.05) but significantly enhanced in FUBP1-overexpressed cells (P < 0.05). Western blot results from FUBP1-knockdown cells treated with or without ICA showed no significant difference in the expression of FUBP1, HIF-1/2α, MMP9, SOX9, and Col2α proteins, whereas the same proteins showed increased expression in FUBP1-overexpressed chondrocytes; moreover, HIF-2α and MMP9 expression was significantly inhibited in FUBP1-knockdown chondrocytes (P < 0.05). In conclusion, as a bioactive monomer of traditional Chinese medicine, ICA is beneficial to chondrocytes.

Icariin promotes the proliferation and osteogenic differentiation of bone-derived mesenchymal stem cells in patients with osteoporosis and T2DM by upregulating GLI-1.

BACKGROUND: The function of mesenchymal stem cells (MSCs) from patients with osteoporosis (OP) is impaired and worsens in patients with type 2 diabetes mellitus (T2DM). Icariin (ICA) is the major active flavonoid glucoside isolated from traditional Chinese herbal Epimedium pubescens, and confirmed able to improve bone mass of OP patients. OBJECTIVE: To investigate the effect of ICA on the proliferation and osteogenic differentiation of bone-derived MSCs (BMSCs) from patients with OP and T2DM and uncover the potential mechanism. METHODS: BMSCs were treated with ICA, and proliferation and osteogenic potency were evaluated using the 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and detection of osteogenic markers (ALP, RUNX2, SPP1, COL1A1, and mineralized nodules) was performed. RNA sequencing and bioinformatic analysis were performed to identify differentially expressed genes (DEGs) after ICA treatment and screen proliferation- and osteogenic differentiation-related processes. Gene gain and loss were performed to confirm the role of the key candidate gene. RESULTS: ICA significantly promoted the proliferation and osteogenic differentiation of BMSCs. A total of 173 DEGs were identified after ICA treatment. Six DEGs (GLI-1, IGF2, BMP6, WNT5A, PTHLH, and MAPK14) enriched in both proliferation- and osteogenic differentiation-related processes were screened; GLI-1 had the highest validated |log2FC| value. Overexpression of GLI-1 enhanced the proliferation and osteogenic differentiation of BMSCs, and knockdown of GLI-1 weakened the positive effect of ICA on BMSCs. CONCLUSION: ICA promoted the proliferation and osteogenic differentiation of impaired BMSCs by upregulating GLI-1.

Icariin, a phytoestrogen, exerts rapid estrogenic actions through crosstalk of estrogen receptors in osteoblasts.

Icariin, a flavonoid glycoside derived from Epimedium brevicornum Maxim, exerts bone protective effects via estrogen receptors (ERs). This study aimed to investigate the role of ER-α66, ER-α36, and GPER in bone metabolism in osteoblasts following treatment with icariin. Human osteoblastic MG-63 cells and osteoblast-specific ER-α66 knockout mice were employed. The ERs crosstalk in the estrogenic action of icariin was evaluated in ER-α66-negative human embryonic kidney HEK293 cells. Icariin, like E2, regulated ER-α36 and GPER protein expression in osteoblasts by downregulating them and upregulating ER-α66. ER-α36 and GPER suppressed the actions of icariin and E2 in bone metabolism. However, the in vivo administration of E2 (2 mg/kg/day) or icariin (300 mg/kg/day) restored bone conditions in KO osteoblasts. ER-α36 and GPER expression increased significantly and rapidly activated and translocated in KO osteoblasts after treatment with E2 or icariin. ER-α36 overexpression in KO osteoblasts further promoted the OPG/RANKL ratio induced by E2 or icariin treatment. This study showed icariin and E2 elicit rapid estrogenic responses in bone through recruiting ER-α66, ER-α36, and GPER. Notably, in osteoblasts lacking ER-α66, ER-α36, and GPER mediate the estrogenic effects of icariin and E2, while in intact osteoblasts, ER-α36 and GPER act as negative regulators of ER-α66.

Icariin alleviates ferroptosis-related atherosclerosis by promoting autophagy in xo-LDL-induced vascular endothelial cell injury and atherosclerotic mice.

Vascular endothelial cells (VECs) are located between the blood plasma and the vascular tissue, and the ferroptosis (iron-dependent programmed cell death) of VECs can lead to a range of cardiovascular diseases. Icariin is the main active ingredient of Epimedium brevicornum Maxim., which can improve endothelial cell dysfunction. In the present study, the protective effects of icariin on oxidised low-density lipoprotein (ox-LDL)-treated VECs and high-fat diet-fed Apolipoprotein E-deficient mice were investigated. Inflammatory fibrosis in tissues and inflammatory factors in serum and cell supernatants were detected, and mitochondrial membrane potential and the expression levels of ferroptosis-associated proteins were also detected. The results revealed that icariin reduced the endothelial atherosclerotic plaque area and collagen fibres in aortic sinus tissue, and increased the viability and mitochondrial membrane potential, whereas it reduced the reactive oxygen species levels of VECs. The nucleation of transcription factor EB (TFEB) and subsequent autophagy were negatively associated with ferroptosis in endothelial cells, and the more prominent the autophagy, the lower the levels of ferroptosis. Furthermore, by co-treating the cells with icariin and the two autophagy inhibitors, Bafilomycin A1 (blocking autophagosome and lysosome fusion) and 3-methyladenine (blocking autophagosome formation), respectively, the promoting effects of icariin on autophagy were found to be mediated through the process of autophagosome-lysosome fusion. In in vivo experiments, icariin reduced ferroptosis, alleviated atherosclerotic lesions and increased the rate of TFEB nucleation. Additionally, it was found that ARG304, THR308 and GLN311 were the optimal binding sites for the interaction between icariin and TFEB. Taken together, these results suggest that the fusion of autophagosomes and lysosomes promoted by icarrin enhances autophagy and thus reduces ferroptosis. Therefore, icariin may be a potential candidate for the prevention of ferroptosis of VECs and, thus, for the treatment of cardiovascular diseases.

Molecular mechanisms and therapeutic potential of icariin in the treatment of Alzheimer's disease.

BACKGROUND: Icariin (ICA) is the main active component of Epimedium, a traditional Chinese medicine (TCM), known to enhance cognitive function in Alzheimer's disease (AD). This study aims to investigate and summarize the mechanisms through which ICA treats AD. METHODS: The PubMed and CNKI databases were utilized to review the advancements in ICA's role in AD prevention and treatment by analyzing literature published between January 2005 and April 2023. To further illustrate ICA's impact on AD development, tables, and images are included to summarize the relationships between various mechanisms. RESULTS: The study reveals that ICA ameliorates cognitive deficits in AD model mice by modulating Aß via multiple pathways, including BACE-1, NO/cGMP, Wnt/Ca2+, and PI3K/Akt signaling. ICA exhibits neuroprotective properties by inhibiting neuronal apoptosis through the suppression of ER stress in AD mice, potentially linked to NF-κB, MAPK, ERK, and PERK/Eif2α signaling pathways. Moreover, ICA may safeguard neurons by attenuating mitochondrial oxidative stress injury. ICA can also enhance learning, memory, and cognition by improving synaptic structure via regulation of the PSD-95 protein. Furthermore, ICA can mitigate neuroinflammation by inactivating microglial activity through the upregulation of PPARγ, TAK1/IKK/NF-κB, and JNK/p38 MAPK signaling pathways. CONCLUSION: This study indicates that ICA possesses multiple beneficial effects in AD treatment. Through the integration of pharmacological and molecular biological research, ICA may emerge as a promising candidate to expedite the advancement of TCM in the clinical management of AD.

Icariin promotes osteogenic differentiation by upregulating alpha-enolase expression.

Osteogenic differentiation is a crucial biological process for maintaining bone remodelling. Aerobic glycolysis is the main source of energy for osteogenic differentiation. Alpha-enolase (Eno1), a glycolytic enzyme, is a therapeutic target for numerous diseases. Icariin, a principal active component of the traditional Chinese medicine Epimedium grandiflorum, can stimulate osteogenic differentiation. Here, we aimed to determine if icariin promotes osteogenic differentiation via Eno1. Icariin (1 µM) significantly promoted osteogenic differentiation of MC3T3-E1 cells. Icariin upregulated Eno1 protein and gene expressions during osteogenic differentiation. Moreover, ENOblock, a specific inhibitor of Eno1, markedly inhibited icariin-induced osteogenic differentiation. Futhermore, western blot assay showed that Eno1 might mediate osteogenic differentiation through the BMP/Smad4 signalling pathway. Collectively, Eno1 could be a promising drug target for icariin to regulate osteogenic differentiation.