Icariin upregulates methyltransferase-like 14-mediated prolyl 4-hydroxylase beta subunit m6A modification to promote osteogenic differentiation of bone marrow stem cells.

Prolyl 4-hydroxylase beta subunit (P4HB) plays a vital role in bone formation. This study intends to clarify the role of P4HB in the therapeutic effect of Icariin (ICA) on osteoporosis. Herein, in vivo and in vitro models were constructed by performing ovariectomy (OVX) in rats and inducing osteogenic differentiation in bone marrow stem cells (BMSCs), respectively. Hematoxylin and eosin staining and micro-computed tomography analysis were performed to evaluate osteoporosis in OVX rats. Alizarin Red staining, alkaline phosphatase staining, and the ALP activity test were employed to assess osteogenesis. m6A dot blotting and methylated RNA immunoprecipitation were used to determine m6A modification. We found that P4HB was downregulated in bone tissues of patients with osteoporosis and OVX rats. P4HB facilitated osteogenic differentiation of BMSCs. What's more, ICA upregulated P4HB expression, promoted osteogenic differentiation of BMSCs, and alleviated osteoporosis in OVX rats, which were reversed by knocking down P4HB. ICA enhanced the stability and m6A modification of P4HB. METTL14 mediated m6A modification of P4HB mRNA. In addition, METTL14 knockdown overturned the promotive effects of ICA on P4HB m6A level and BMSC osteogenic differentiation. To sum up, ICA elevated the METTL14-mediated m6A modification of P4HB to facilitate BMSC osteogenic differentiation.

ICA/SDF-1α/PBMSCs loaded onto alginate and gelatin cross-linked scaffolds promote damaged cartilage repair.

A three-dimensional alginate-coated scaffold (GAIS) was constructed in the present study to showcase the multidifferentiation potential of peripheral blood mesenchymal stem cells (PBMSCs) and to investigate the role and mechanism by which Icariin (ICA)/stromal cell-derived factor (SDF-1α)/PBMSCs promote damaged articular repair. In addition, the ability of ICA, in combination with SDF-1α, to promote the migration and proliferation of stem cells was validated through the utilization of CCK-8 and migration experiments. The combination of ICA and SDF-1α inhibited the differentiation of PBMSCs into cartilage, as demonstrated by in vivo experiments and histological staining. Both PCR and western blot experiments showed that GAIS could upregulate the expression of particular genes in chondrocytes. In comparison to scaffolds devoid of alginate (G0), PBMSCs seeded into GAIS scaffolds exhibited a greater rate of proliferation, and the conditioned medium derived from scaffolds containing SDF-1α enhanced the capacity for cell migration. Moreover, after a 12-week treatment period, GAIS, when successfully transplanted into osteochondral defects of mice, was found to promote cartilage regeneration and repair. The findings, therefore, demonstrate that GAIS enhanced the in vitro capabilities of PBMSCs, including proliferation, migration, homing and chondrogenic differentiation. In addition, ICA and SDF-1α effectively collaborated to support cartilage formation in vivo. Thus, the ICA/SDF-1α/PBMSC-loaded biodegradable alginate-gelatin scaffolds showcase considerable potential for use in cartilage repair.

Icariin alleviates oxygen-induced retinopathy by targeting microglia hexokinase 2.

Retinopathy of prematurity (ROP) is a retinal disease-causing retinal neovascularization that can lead to blindness. Oxygen-induced retinopathy (OIR) is a widely used ROP animal model. Icariin (ICA) has anti-oxidative and anti-inflammation properties; however, whether ICA has a regulatory effect on OIR remains unclear. In this study, ICA alleviated pathological neovascularization, microglial activation and blood-retina barrier (BRB) damage in vivo. Further results indicated that endothelial cell tube formation, migration and proliferation were restored by ICA treatment in vitro. Proteomic microarrays and molecular mimicry revealed that ICA can directly bind to hexokinase 2 (HK2) and decrease HK2 protein expression in vivo and in vitro. In addition, ICA inhibited the AKT/mTOR/HIF1α pathway activation. The effects of ICA on pathological neovascularization, microglial activation and BRB damage disappeared after HK2 overexpression in vivo. Similarly, the endothelial cell function was revised after HK2 overexpression. HK2 overexpression reversed ICA-induced AKT/mTOR/HIF1α pathway inhibition in vivo and in vitro. Therefore, ICA prevented pathological angiogenesis in OIR in an HK2-dependent manner, implicating ICA as a potential therapeutic agent for ROP.

Icariin improves oxidative stress injury during ischemic stroke via inhibiting mPTP opening.

BACKGROUND: Ischemic stroke presents a significant threat to human health due to its high disability rate and mortality. Currently, the clinical treatment drug, rt-PA, has a narrow therapeutic window and carries a high risk of bleeding. There is an urgent need to find new effective therapeutic drugs for ischemic stroke. Icariin (ICA), a key ingredient in the traditional Chinese medicine Epimedium, undergoes metabolism in vivo to produce Icaritin (ICT). While ICA has been reported to inhibit neuronal apoptosis after cerebral ischemia-reperfusion (I/R), yet its underlying mechanism remains unclear. METHODS: PC-12 cells were treated with 200 µM H2O2 for 8 h to establish a vitro model of oxidative damage. After administration of ICT, cell viability was detected by Thiazolyl blue tetrazolium Bromide (MTT) assay, reactive oxygen species (ROS) and apoptosis level, mPTP status and mitochondrial membrane potential (MMP) were detected by flow cytometry and immunofluorescence. Apoptosis and mitochondrial permeability transition pore (mPTP) related proteins were assessed by Western blotting. Middle cerebral artery occlusion (MCAO) model was used to establish I/R injury in vivo. After the treatment of ICA, the neurological function was scored by ZeaLonga socres; the infarct volume was observed by 2,3,5-Triphenyltetrazolium chloride (TTC) staining; HE and Nissl staining were used to detect the pathological state of the ischemic cortex; the expression changes of mPTP and apoptosis related proteins were detected by Western blotting. RESULTS: In vitro: ICT effectively improved H2O2-induced oxidative injury through decreasing the ROS level, inhibiting mPTP opening and apoptosis. In addition, the protective effects of ICT were not enhanced when it was co-treated with mPTP inhibitor Cyclosporin A (CsA), but reversed when combined with mPTP activator Lonidamine (LND). In vivo: Rats after MCAO shown cortical infarct volume of 32-40%, severe neurological impairment, while mPTP opening and apoptosis were obviously increased. Those damage caused was improved by the administration of ICA and CsA. CONCLUSIONS: ICA improves cerebral ischemia-reperfusion injury by inhibiting mPTP opening, making it a potential candidate drug for the treatment of ischemic stroke.

Icariin attenuates asthmatic airway inflammation via modulating alveolar macrophage activation based on network pharmacology and in vivo experiments.

BACKGROUND: Icariin (ICA) inhibits inflammatory response in various diseases, but the mechanism underlying ICA treating airway inflammation in asthma needs further understood. We aimed to predict and validate the potential targets of ICA against asthma-associated airway inflammation using network pharmacology and experiments. METHODS: The ovalbumin-induced asthma-associated airway inflammation mice model was established. The effects of ICA were evaluated by behavioral, airway hyperresponsiveness, lung pathological changes, inflammatory cell and cytokines counts. Next, the corresponding targets of ICA were mined via the SEA, CTD, HERB, PharmMapper, Symmap database and the literature. Pubmed-Gene and GeneCards databases were used to screen asthma and airway inflammation-related targets. The overlapping targets were used to build an interaction network, analyze gene ontology and enrich pathways. Subsequently, flow cytometry, quantitative real-time PCR and western blotting were employed for validation. RESULTS: ICA alleviated the airway inflammation of asthma; 402 targets of ICA, 5136 targets of asthma and 4531 targets of airway inflammation were screened; 216 overlapping targets were matched and predicted ICA possesses the potential to modulate asthmatic airway inflammation by macrophage activation/polarization. Additionally, ICA decreased M1 but elevated M2. Potential targets that were disrupted by asthma inflammation were restored by ICA treatment. CONCLUSIONS: ICA alleviates airway inflammation in asthma by inhibiting the M1 polarization of alveolar macrophages, which is related to metabolic reprogramming. Jun, Jak2, Syk, Tnf, Aldh2, Aldh9a1, Nos1, Nos2 and Nos3 represent potential targets of therapeutic intervention. The present study enhances understanding of the anti-airway inflammation effects of ICA, especially in asthma.

Icariin rescues developmental BPA exposure induced spatial memory deficits in rats.

Bisphenol A (BPA) has been implicated in cognitive impairment. Icariin is the main active ingredient extracted from Epimedium Herb with protective function of nervous system. However, the potential therapeutic effects of Icariin on spatial memory deficits induced by developmental BPA exposure in Sprague-Dawley rats have not been investigated. This study investigated the therapeutic effect of Icariin (10 mg/kg/day, from postnatal day (PND) 21 to PND 60 by gavage) on spatial memory deficits in rat induced by developmental BPA exposure (1 mg/kg/day, from embryonic to PND 60), demonstrating that Icariin can markedly improve spatial memory in BPA-exposed rat. Furthermore, intra-gastric administration of Icariin could attenuate abnormal hippocampal cell dispersion and loss, improved the dendritic spine density and Nissl bodies. Moreover, Icariin reversed BPA induced reduction of frequency of miniature excitatory postsynaptic currents(mEPSC) and decrease of Vesicular glutamate transporter 1(VGlut1). Collectively, Icariin could effectively rescue BPA-induced spatial memory impairment in male rats by preventing cell loss and reduction of dendritic spines in the hippocampus. In addition, we also found that VGlut1 is a critical target in the repair of BPA-induced spatial memory by Icariin. Thus, Icariin may be a promising therapeutic agent to attenuate BPA-induced spatial memory deficits.

Icariin exerts anti-tumor activity by inducing autophagy via AMPK/mTOR/ULK1 pathway in triple-negative breast cancer.

BACKGROUND: Breast cancer is the most prevalent female tumor, of which triple-negative breast cancer (TNBC) accounts for about 15%. Characterized by its aggressive nature and limited treatment options, TNBC currently stands as a significant clinical challenge. This study aimed to investigate the effects of icariin (ICA) on TNBC and explore the underlying molecular mechanism. METHODS: Cell viability was assessed using CCK-8 assay, whereas the impact of ICA on cell proliferation was determined using colony formation assay and detection of proliferating cell nuclear antigen protein. Wound healing and transwell assays were used to evaluate the effects of ICA on cell migration and invasion, respectively. Flow cytometry was used to analyze cell cycle distribution and apoptosis. Transmission electron microscopy and monodansylcaverine staining were performed to detect the induction of autophagy, whereas molecular docking was conducted to predict the potential targets associated with autophagy. The in vivo anti-tumor effects of ICA were evaluated using a TNBC 4T1 xenograft mouse model. Protein expression levels were examined using immunoblotting and immunohistochemistry. RESULTS: In vitro, ICA effectively suppressed the viability, proliferation, migration, and invasion of TNBC cells and induced G0/G1 phase cell cycle arrest, apoptosis, and autophagy in TNBC cells by regulating the adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) signaling pathway. The knockdown of AMPK and inhibition of autophagy with 3-methyladenine reversed the effects of ICA, highlighting the importance of AMPK and autophagy in the anti-cancer mechanism of ICA. In vivo, ICA significantly inhibited TNBC growth, promoted autophagy, and regulated AMPK/mTOR/ULK1 pathway. CONCLUSIONS: Our findings demonstrated that ICA exerts anti-cancer effects against TNBC and the associated molecular mechanisms. This study will help to facilitate further preclinical and clinical investigations for the treatment of TNBC.

Binary Nano-inhalant Formulation of Icariin Enhances Cognitive Function in Vascular Dementia via BDNF/TrkB Signaling and Anti-inflammatory Effects.

Vascular dementia (VaD) has a serious impact on the patients' quality of life. Icariin (Ica) possesses neuroprotective potential for treating VaD, yet its oral bioavailability and blood-brain barrier (BBB) permeability remain challenges. This research introduced a PEG-PLGA-loaded chitosan hydrogel-based binary formulation tailored for intranasal delivery, enhancing the intracerebral delivery efficacy of neuroprotective agents. The formulation underwent optimization to facilitate BBB crossing, with examinations conducted on its particle size, morphology, drug-loading capacity, in vitro release, and biodistribution. Using the bilateral common carotid artery occlusion (BCCAO) rat model, the therapeutic efficacy of this binary formulation was assessed against chitosan hydrogel and PEG-PLGA nanoparticles loaded with Ica. Post-intranasal administration, enhanced cognitive function was evident in chronic cerebral hypoperfusion (CCH) rats. Further mechanistic evaluations, utilizing immunohistochemistry (IHC), RT-PCR, and ELISA, revealed augmented transcription of synaptic plasticity-associated proteins like SYP and PSD-95, and a marked reduction in hippocampal inflammatory markers such as IL-1ß and TNF-α, highlighting the formulation's promise in alleviating cognitive impairment. The brain-derived neurotrophic factor (BDNF)/tropomyosin related kinase B (TrkB) pathway was activated significantly in the binary formulation compared with the other two. Our study demonstrates that the intranasal application of chitosan hydrogel loaded with Ica-encapsulated PEG-PLGA could effectively deliver Ica into the brain and enhance its neuroprotective effect.

Icariin alleviates diabetic renal interstitial fibrosis aggravation by inhibiting miR-320a-3p targeting BMP6.

Diabetic nephropathy is a common complication of diabetes, accumulating evidence underscores the pivotal role of tubulointerstitial fibrosis in the progression of diabetic nephropathy. However, the underlying mechanisms remain incompletely understood. Although the mechanisms in diabetic nephropathy fibrosis have been the focus of many studies, only limited information is currently available concerning microRNA regulation in tubulointerstitial fibrosis. In this study, we aimed to investigate the roles of miR-320a-3p and bone morphogenetic protein-6 (BMP6) in tubulointerstitial fibrosis. After inducing fibrosis with high glucose in HK-2 cells, we found that miR-320a-3p is significantly up-regulated, whereas BMP6 is markedly down-regulated. These changes suggest close link between miR-320a-3p and BMP6 in tubulointerstitial fibrosis. To elucidate this phenomenon, miR-320a-3p mimic, inhibitor and siBMP6 were employed. We observed in miR-320a-3p mimic group the fibrosis marker include alpha smooth muscle actin and type I collagen was significantly up-regulated, whereas BMP6 exhibited the opposite trend. Additionally, we found icariin could alleviate tubulointerstitial fibrosis by downregulation the miR-320a-3p expression. In conclusion, miR-320a-3p promotes tubulointerstitial fibrosis during the development of DN by suppressing BMP signal pathway activity via inhibiting BMP6 expression. Suggesting that miR-320a-3p represents a potential therapeutic target for tubulointerstitial fibrosis induced by diabetic nephropathy.

Icariin alleviates high-fat diet-induced nonalcoholic fatty liver disease via up-regulating miR-206 to mediate NF-κB and MAPK pathways.

Nonalcoholic fatty liver disease (NAFLD) is an abnormal lipid accumulation disease in hepatocytes. The existing drugs for NAFLD have some side effects, so new therapeutic agents are required to be explored. In this study, the effect and mechanism of icariin (ICA) on high-fat diet-induced NAFLD were investigated. Firstly, a high-fat diet was used to construct a NAFLD rat model and HepG2 cells were treated with 1 mM free fatty acid (FFA). After ICA treatment, the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were measured; liver injury and lipid deposition were observed by H&E and Oil Red O staining; interleukin-1ß (IL-1ß), IL-12, and IL-6 were measured by enzyme-linked immunosorbent assay. Additionally, qRT-PCR and western blot were performed to detect miR-206 expression and NF-κB/MAPK pathway-related protein expression in liver tissues and cells. After a variety of trials, we discovered that compared with the NAFLD group, ICA significantly reduced ALT, AST, TBil, TG, TC, and LDL-C levels and increased HDL-C levels, and improved liver tissue injury and lipid deposition. Moreover, ICA reduced IL-1ß, IL-12, and IL-6 levels in liver tissues and cells as well as inhibited MAPK and NF-κB-related protein expression in the liver tissues. Notably, ICA could significantly increase miR-206 expression in liver tissues and cells. Further experiments confirmed that inhibition of miR-206 was able to reverse the effect of ICA on NAFLD. In conclusion, ICA can alleviate NAFLD by upregulating miR-206 to mediate NF-κB and MAPK pathways.

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.

Icariin alleviates renal inflammation and tubulointerstitial fibrosis via Nrf2-mediated attenuation of mitochondrial damage.

Tubulointerstitial fibrosis is an inevitable consequence of all progressive chronic kidney disease (CKD) and contributes to a substantial health burden worldwide. Icariin, an active flavonoid glycoside obtained from Epimedium species, exerts potential antifibrotic effect. The study aimed to explore the protective effects of icariin against tubulointerstitial fibrosis in unilateral ureteral obstruction (UUO)-induced CKD mice and TGF-ß1-treated HK-2 cells, and furthermore, to elucidate the underlying mechanisms. The results demonstrated that icariin significantly improved renal function, alleviated tubular injuries, and reduced fibrotic lesions in UUO mice. Furthermore, icariin suppressed renal inflammation, reduced oxidative stress as evidenced by elevated superoxide dismutase activity and decreased malondialdehyde level. Additionally, TOMM20 immunofluorescence staining and transmission electron microscope revealed that mitochondrial mass and morphology of tubular epithelial cells in UUO mice was restored by icariin. In HK-2 cells treated with TGF-ß1, icariin markedly decreased profibrotic proteins expression, inhibited inflammatory factors, and protected mitochondria along with preserving mitochondrial morphology, reducing reactive oxygen species (ROS) and mitochondrial ROS (mtROS) overproduction, and preserving membrane potential. Further investigations demonstrated that icariin could activate nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway both in vivo and in vitro, whereas inhibition of Nrf2 by ML385 counteracted the protective effects of icariin on TGF-ß1-induced HK-2 cells. In conclusion, icariin protects against renal inflammation and tubulointerstitial fibrosis at least partly through Nrf2-mediated attenuation of mitochondrial dysfunction, which suggests that icariin could be developed as a promising therapeutic candidate for the treatment of CKD.

Adipose-derived stem cell exosomes loaded with icariin alleviates rheumatoid arthritis by modulating macrophage polarization in rats.

Rheumatoid arthritis (RA) is a chronic autoimmune disease marked by synovitis and cartilage destruction. The active compound, icariin (ICA), derived from the herb Epimedium, exhibits potent anti-inflammatory properties. However, its clinical utility is limited by its water insolubility, poor permeability, and low bioavailability. To address these challenges, we developed a multifunctional drug delivery system-adipose-derived stem cells-exosomes (ADSCs-EXO)-ICA to target active macrophages in synovial tissue and modulate macrophage polarization from M1 to M2. High-performance liquid chromatography analysis confirmed a 92.4 ± 0.008% loading efficiency for ADSCs-EXO-ICA. In vitro studies utilizing cellular immunofluorescence (IF) and flow cytometry demonstrated significant inhibition of M1 macrophage proliferation by ADSCs-EXO-ICA. Enzyme-linked immunosorbent assay, cellular transcriptomics, and real-time quantitative PCR indicated that ADSCs-EXO-ICA promotes an M1-to-M2 phenotypic transition by reducing glycolysis through the inhibition of the ERK/HIF-1α/GLUT1 pathway. In vivo, ADSCs-EXO-ICA effectively accumulated in the joints. Pharmacodynamic assessments revealed that ADSCs-EXO-ICA decreased cytokine levels and mitigated arthritis symptoms in collagen-induced arthritis (CIA) rats. Histological analysis and micro computed tomography confirmed that ADSCs-EXO-ICA markedly ameliorated synovitis and preserved cartilage. Further in vivo studies indicated that ADSCs-EXO-ICA suppresses arthritis by promoting an M1-to-M2 switch and suppressing glycolysis. Western blotting supported the therapeutic efficacy of ADSCs-EXO-ICA in RA, confirming its role in modulating macrophage function through energy metabolism regulation. Thus, this study not only introduces a drug delivery system that significantly enhances the anti-RA efficacy of ADSCs-EXO-ICA but also elucidates its mechanism of action in macrophage function inhibition.

Enhanced cartilage regeneration by icariin and mesenchymal stem cell-derived extracellular vesicles combined in alginate-hyaluronic acid hydrogel.

OBJECTIVE: Osteoarthritis (OA) is characterized by progressive cartilage degeneration and absence of curative therapies. Therefore, more efficient therapies are compellingly needed. Both mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) and Icariin (ICA) are promising for repair of cartilage defect. This study proposes that ICA may be combined to potentiate the cartilage repair capacity of MSC-EVs. MATERIALS AND METHODS: MSC-EVs were isolated from sodium alginate (SA) and hyaluronic acid (HA) composite hydrogel (SA-HA) cell spheroid culture. EVs and ICA were combined in SA-HA hydrogel to test therapeutic efficacy on cartilage defect in vivo. RESULTS: EVs and ICA were synergistic for promoting both proliferation and migration of MSCs and inflammatory chondrocytes. The combination therapy led to strikingly enhanced repair on cartilage defect in rats, with mechanisms involved in the concomitant modulation of both cartilage degradation and synthesis makers. CONCLUSION: The MSC-EVs-ICA/SA-HA hydrogel potentially constitutes a novel therapy for cartilage defect in OA.

Icariin decreases cell proliferation and inflammation of rheumatoid arthritis-fibroblast like synoviocytes via GAREM1/MAPK signaling pathway.

OBJECTIVE: Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic inflammation and joint damage, leading to pain and reduced joint function. Icariin, a flavonoid compound, has been studied for its potential therapeutic role in RA due to its anti-inflammatory and anti-proliferative effects. Here, we aimed to investigate the action mechanism of icariin in regulating RA. MATERIALS AND METHODS: Fibroblast-like synoviocytes (FLS) were obtained from RA and trauma patients, generating RA-FLS and normal FLS. The cells were treated with varying concentrations of icariin (0, 10, 20, 40, 80 µM). We assessed the effects of icariin on cell proliferation, apoptosis, and levels of inflammatory factors using the CCK-8 assay, flow cytometry, and enzyme-linked immunosorbent assay, qRT-PCR, and western blotting. RESULTS: Icariin treatment had no significant impact on the cell proliferation of normal FLS. However, it dose-dependently repressed cell proliferation, reduced TNF-α, IL-6, and IL-1ß levels, and increased apoptosis in RA-FLS. The expression of GAREM1, p-p38, and p-ERK1/2 was upregulated in RA-FLS, which was reversed by icariin treatment. Overexpression of GAREM1 reversed the inhibitory effects of icariin on cell proliferation and inflammatory factor levels in RA-FLS. CONCLUSION: Our findings suggest that icariin treatment can alleviate the development of RA by reducing cell proliferation and inflammation in RA-FLS through the regulation of the GAREM1/MAPK signaling pathway. These results support the potential of icariin as a therapeutic agent for RA treatment. As icariin is safe and well-tolerated in previous studies, further research is warranted to explore its efficacy in clinical settings.

New insights into the anticancer therapeutic potential of icaritin and its synthetic derivatives.

Icaritin is a natural prenylated flavonoid derived from the Chinese herb Epimedium. The compound has shown antitumor effects in various cancers, especially hepatocellular carcinoma (HCC). Icaritin exerts its anticancer activity by modulating multiple signaling pathways, such as IL-6/JAK/STAT3, ER-α36, and NF-κB, affecting the tumor microenvironment and immune system. Several clinical trials have evaluated the safety and efficacy of icaritin in advanced HCC patients with poor prognoses, who are unsuitable for conventional therapies. The results have demonstrated that icaritin can improve survival, delay progression, and produce clinical benefits in these patients, with a favorable safety profile and minimal adverse events. Moreover, icaritin can enhance the antitumor immune response by regulating the function and phenotype of various immune cells, such as CD8+ T cells, MDSCs, neutrophils, and macrophages. These findings suggest that icaritin is a promising candidate for immunotherapy in HCC and other cancers. However, further studies are needed to elucidate the molecular mechanisms and optimal dosing regimens of icaritin and its potential synergistic effects with other agents. Therefore, this comprehensive review of the scientific literature aims to summarize advances in the knowledge of icaritin in preclinical and clinical studies as well as the pharmacokinetic, metabolism, toxicity, and mechanisms action to recognize the main challenge, gaps, and opportunities to develop a medication that cancer patients can use. Thus, our main objective was to clarify the current state of icaritin for use as an anticancer drug.

Icariin ameliorates the cuprizone-induced demyelination associated with antioxidation and anti-inflammation.

The treatment of immunomodulation in multiple sclerosis (MS) can alleviate the severity and relapses. However, it cannot improve the neurological disability of patients due to a lack of myelin protection and regeneration. Therefore, remyelinating therapies may be one of the feasible strategies that can prevent axonal degeneration and restore neurological disability. Natural product icariin (ICA) is a flavonol compound extracted from epimedium flavonoids, which has neuroprotective effects in several models of neurological diseases. Here, we attempt to explore whether ICA has the potential to treat demyelination and its possible mechanisms of action using lipopolysaccharide-treated BV2 microglia, primary microglia, bone marrow-derived macrophages, and cuprizone-induced demyelination model. The indicators of oxidative stress and inflammatory response were evaluated using commercial kits. The results showed that ICA significantly reduced the levels of oxidative intermediates nitric oxide, hydrogen peroxide, malondialdehyde, and inflammatory cytokines TNF-α, IL-1ß, and increased the levels of antioxidants superoxide dismutase, catalase, glutathione peroxidase, and anti-inflammatory cytokines IL-10 and TGF-ß in vitro cell experiments. In vivo demyelination model, ICA significantly alleviated the behavioral abnormalities and enhanced the integrated optical density/mm2 of Black Gold II and myelin basic protein myelin staining, accompanied by the inhibition of oxidative stress/inflammatory response. Immunohistochemical staining showed that ICA significantly induced the expression of nuclear factor erythroid derived 2/heme oxygenase-1 (Nrf2/HO-1) and inhibited the expression of toll-like receptor 4/ nuclear factor kappa B (TLR4/NF-κB), which are two key signaling pathways in antioxidant and anti-inflammatory processes. Our results strongly suggest that ICA may be used as a potential agent to treat demyelination via regulating Nrf2/HO-1-mediated antioxidative stress and TLR4/NF-κB-mediated inflammatory responses.

[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.

[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.

Inhibition of ferroptosis by icariin treatment attenuates excessive ethanol consumption-induced atrial remodeling and susceptibility to atrial fibrillation, role of SIRT1.

Ferroptosis contributes to the pathogenesis of atrial fibrillation (AF), although the mechanisms are still largely uncovered. The current study was designed to explore the pharmacological effects of icariin against ethanol-induced atrial remodeling, if any, and the mechanisms involved with a focus on SIRT1 signaling. Excessive ethanol-treated animals were administered with Ferrostatin-1, Erastin or icariin to evaluate the potential effects of icariin or ferroptosis. Then, the underling mechanisms was further explored in the in vitro experiments using HL-1 atrial myocytes. Excessive ethanol administration caused significant atrial damage as evidenced by increased susceptibility to AF, altered atrial conduction pattern, atrial enlargement, and enhanced fibrotic markers. These detrimental effects were reversed by Ferrostatin-1 or icariin treatment, while Erastin co-administration markedly abolished the beneficial actions conferred by icariin. Mechanistically, ethanol-treated atria exhibited markedly up-regulated pro-ferroptotic protein (PTGS2, ACSL4, P53) and suppressed anti-ferroptotic molecules (GPX4, FTH1). Icariin treatment inhibited ethanol-induced atrial ferroptosis by reducing atrial mitochondrial damage, ROS accumulation and iron overload. Interestingly, the in vivo and in vitro data showed that icariin activated atrial SIRT1-Nrf-2-HO-1 signaling pathway, while EX527 not only reversed these effects, but also abolished the therapeutic effects of icariin. Moreover, the stimulatory effects on GPX4, SLC7A11 and the suppressive effects on ACSL4, P53 conferred by icariin were blunted by EX527 treatment. These data demonstrate that ferroptosis plays a causative role in the pathogenesis of ethanol-induced atrial remodeling and susceptibility to AF. Icariin protects against atrial damage by inhibiting ferroptosis via SIRT1 signaling. Its role as a prophylactic/therapeutic drug deserves further clinical study.