Effects Of Icariin Research Articles

Icariin protects human nucleus pulposus cells from hydrogen peroxide-induced mitochondria-mediated apoptosis by activating nuclear factor erythroid 2-related factor 2

Intervertebral disc degeneration (IVDD) is a well-known cause of lower back pain. Icariin has been shown to exert a protective effect on human nucleus pulposus (NP) cells and accordingly has implications for the prevention and treatment of IVDD; however, the molecular mechanisms underlying its action are not fully established. In this study, the mechanisms underlying its protection against hydrogen peroxide (H2O2)-induced oxidative stress injury were investigated. In vitro, we demonstrated that icariin inhibits H2O2-induced mitochondria-mediated apoptosis. It upregulates oxidative stress mediators, i.e., reactive oxygen species, and downregulates mitochondrial membrane potential. Nuclear factor erythroid 2-related factor 2 (Nrf-2) is a key factor involved in the regulation of the endogenous defense system. It was proved that icariin could activate the Nrf-2 signaling pathway, upregulate the protein expression of nuclear respiratory factor-1 and the mitochondrial transcription factor, promoting mitochondrial biogenesis in human NP cells. An Nrf-2 agonist and inhibitor promoted or partly abolished the protective effects of icariin on mitochondrial homeostasis. Moreover, it was demonstrated that the Nrf-2 signaling pathway could be inhibited by the phosphatidylinositol 3-Kinase/AKT pathway. In vivo, icariin ameliorated IVDD in a rat model by promoting Nrf-2 activity, and preserving extracellular matrix in NP cells. These data suggest that icariin could ameliorate IVDD in rat models in vivo. In summary, the protective effects of icariin on human NP cells may suppress the pathogenesis of IVDD via the Nrf-2 signaling pathway. Our findings suggest that the Nrf-2 signaling pathway is a novel therapeutic target for the treatment of IVDD.

Icariin reduces high glucose-induced endothelial progenitor cell dysfunction via inhibiting the p38/CREB pathway and activating the Akt/eNOS/NO pathway.

High glucose (HG) impairs endothelial progenitor cell (EPC) function. The activation of p38 mitogen-activated protein kinase and the inhibition of the Akt/eNOS/NO pathway serve central roles in this process. Icariin has protective effects in endothelial cells. The aim of the present study was to investigate the effects of icariin on HG-induced EPC dysfunction, including proliferation, migration and tube formation. Experiments were performed with EPCs isolated from the femurs and tibias of Sprague-Dawley rats in vitro. In a dose-dependent manner, icariin reversed the inhibition of EPC proliferation induced by HG treatment, and the maximal effective concentration of icariin was 1 µM [Fold change (FC):0.90±0.07, P=0.0124 vs. HG group]. The impaired EPC migration and tube formation induced by glucose was partially restored by 1 µM icariin treatment (FC: 0.81±0.08, P=0.0148 vs. HG group for migration; 0.82±0.03, P=0.0214 vs. HG group for tube formation). Furthermore, icariin significantly suppressed HG-induced p38 and cAMP response element binding protein (CREB) phosphorylation in EPCs (FC: 1.84±0.21, P=0.0238 vs. HG group for p38; FC: 2.24±0.15, P=0.0068 vs. HG group for CREB). Increased Akt and endothelial nitric oxide (NO) synthase (eNOS) activation was also observed after icariin treatment (FC: 0.64±0.08, P=0.0047 vs. HG group for Akt; FC:0.53±0.05, P=0.0019 vs. HG group for eNOS), which was followed by increased NO production (FC: 0.69±0.06, P=0.0064 vs. HG group). In conclusion, icariin attenuated HG-induced EPC dysfunction, which may be partially attributed to the inhibition of the p38/CREB pathway and the activation of the Akt/eNOS/NO pathway. Icariin may be a therapeutic candidate for improving the function of EPC.

Icariin promotes early and late stages of fracture healing in rats.

This study aims to evaluate the effects of locally applied icariin on bone fracture healing in femur fractured rat model. The study included 64 male Sprague-Dawley rats (mean age 6 months; weighing, 280-490 g) in eight main study groups. Fracture healing process and level were evaluated with radiography, histopathology and dual energy X-ray absorptiometry to investigate the effects of local administration of icariin at varying doses, which is an exogenous osteo-inductive substance. Activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured in the peripheral blood in addition to glutathione (GSH) and myeloperoxidase (MPO) levels to investigate the effects of icariin on the oxidant-antioxidant systems. Radiological bone mineral density measurements and histopathological findings revealed that icariin improved all these parameters in the two healing periods tested. Superoxide dismutase activity decreased in association with local icariin application to the fractured side whereas GPx and GSH increased and MPO remained unchanged. Icariin increased the GPx and GSH levels which are responsible from scavenging hydroxyl radical and hydrogen peroxide. Locally administered icariin to the fracture accelerated bone healing by reducing the oxidative stress.

Protective Effect of Icariin on Oxidative Damage of HUVECs Induced by Hydrogen Peroxide

Protective Effect of Icariin on Oxidative Damage of HUVECs Induced by Hydrogen Peroxide

PI3K-AKT Signaling Activation and Icariin: The Potential Effects on the Perimenopausal Depression-Like Rat Model.

Icariin is a prenylated flavonol glycoside isolated from Epimedium herb, and has been shown to be its main bioactive component. Recently, the antidepressant-like mechanism of icariin has been increasingly evaluated and demonstrated. However, there are few studies that have focused on the involvement of the phosphatidylinositol 3-kinase (PI3K)/serine-threonine protein kinase (AKT) signaling in mediating the perimenopausal depression effects of icariin. Perimenopausal depression is a chronic recurrent disease that leads to an increased risk of suicide, and poses a significant risk to public health. The aim of the present study was to explore the effect of icariin on the expression of the PI3K–AKT pathway related to proteins in a rat model of perimenopausal depression. Eighty percent of the left ovary and the entire right ovary were removed from the model rats. A perimenopausal depression model was created through 18 days of chronic unpredictable stimulation, followed by the gavage administration of target drugs for 30 consecutive days. We found that icariin administered at various doses significantly improved the apparent symptoms in the model rats, increased the organ indices of the uterus, spleen, and thymus, and improved the pathological changes in the ovaries. Moreover, icariin administration elevated the serum levels of female hormone estradiol (E2), testosterone (T), and interleukin (IL)-2, decreased those of follicle stimulating hormone (FSH) and luteotropic hormone (LH), promoted the expression levels of estrogen receptor (ER) and ERα in the hypothalamus, and increased those of serotonin (5-HT), dopamine (DA), and noradrenaline (NA) in the brain homogenate. Furthermore, icariin elevated the expression levels of AKT, phosphorylation-akt (p-AKT), PI3K (110 kDa), PI3K (85 kDa), and B-cell lymphoma 2 (Bcl-2) in the ovaries, and inhibited those of Bax. These results show that icariin administration rebalanced the disordered sex hormones in perimenopausal depression rats, regulated the secretion of neurotransmitters in the brain, boosted immune function, and improved the perimenopausal syndrome. The mechanism of action may be related to the regulation of the expression of PI3K–AKT pathway-related proteins.

Bioactive molecule Icariin inhibited proliferation while enhanced apoptosis and autophagy of rat airway smooth muscle cells in vitro.

Icariin is the main active compound extracted from epimedium Flavonoids (EFs) and involved in regulation of cell behaviors (proliferation, apoptosis, and autophagy etc.) for many cell types, but the effect of Icariin on airway smooth muscle cells (ASMCs) is still unknown. The aim of the present study is to examine the role of Icariin on rat ASMCs proliferation, apoptosis and autophagy. CKK8 assay showed that Icariin inhibited rat ASMCs growth in dose-time-dependent manner, and the flow cytometry assay showed that the Icariin interfered with ASMCs cell cycle, when treated with Icariin, S phase shortened while G2 phase extended, cyclin E1 and cyclinA1 gene and protein expression decreased. Next apoptosis was detected, Flow cytometry and TdTmediated dUTP Nick-End Labeling (TUNEL) assay showed that Icariin promoted ASMCs apoptosis, and enhanced apoptosis protein cleaved-caspase-3 expression. Finally, it was found Icariin induced rat ASMCs autophagy, with enhancement expression of autophagy marker LC3 II. In conclusion, Icariin inhibited ASMCs proliferation while promoted apoptosis and autophagy, revealing its potential role in treatment of airway remodeling in asthma.

Icariin as a Preservative to Maintain the Fruit Quality of Banana During Postharvest Storage

Banana is a tropical fruit with good palatability and multiple health benefits. However, the fruit quality is readily deteriorated during postharvest storage. In this work, icariin was used to treat banana to maintain the fruit quality. NMR spectroscopy was applied to analyze the effect of icariin on the metabolite profiles of banana. It was found that the fruit quality of banana was well maintained after icariin treatment. The data of 1D and 2D NMR spectra revealed the composition of metabolites, whose contents greatly varied during storage. The first principal components included ethanol, acetic acid, saponin b, salsolinol, dopamine, glucose, and linoleic acid. After icariin treatment, the conversion of starch to α-d-glucose and β-d-glucose was delayed, and the production of γ-aminobutyrate, glutamine, and alanine increased. These results indicated that icariin could effectively maintain the fruit quality and delay the senescence of banana.

Protective effects of icariin on dorsal random skin flap survival: An experimental study

This study was performed to examine the protective effects of icariin (ICA) on ischemic random skin flaps. A rat random-pattern skin flap model was established, and animals in the low-dose and high-dose experimental groups were administered ICA intraperitoneally at doses of 40 and 80 mg/kg, respectively, once daily for 7 days after the initial surgery. Control rats received vehicle according to the same schedule. Survival rates were observed and recorded using transparent graph paper, and flaps were obtained and stained with hematoxylin and eosin (H&E). The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in the flap tissue were assessed. The blood flow volume was determined by the laser Doppler method, and tissue expression levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), interleukin-1β, and phosphodiesterase 5 (PDE5) were scored immunohistochemically. The levels of proinflammatory cytokines, including tumor necrosis factor-α and IL-6, were determined by enzyme-linked immunosorbent assay. The main flap survival area was significantly larger in rats treated with ICA than in vehicle-treated controls. H&E staining showed an inhibitory effect of ICA on inflammation, especially at the high dose. In addition, ICA treatment was associated with decreases in the tissue MDA level, proinflammatory cytokine production, and the level of PDE5, but increases in SOD activity, blood flow volume, and the level of VEGF expression. The findings of the present study suggest that ICA is a potential therapeutic agent for random-pattern skin flap necrosis in the clinical setting.

The protective effect of icariin and phosphorylated icariin against LPS-induced intestinal goblet cell dysfunction

In this study, we used LS174T cells as a model to investigate the protective effects of icariin and phosphorylated icariin on LPS-induced goblet cell dysfunction. Our results indicated that icariin and phosphorylated icariin increased the cell viability and decreased lactate dehydrogenase activity in LPS-treated LS174T cells. Icariin and phosphorylated icariin attenuated LPS-induced changes in mucin 2 synthesis and secretion. Besides, Icariin and phosphorylated icariin reduced the levels of ROS, MDA, and H2O2 and increased the activity of SOD, GPx, CAT, and T-AOC in LPS-treated LS174T cells. Moreover, the levels of IL-1β, IL-6, IL-8, and TNF-α were reduced in the Icariin and phosphorylated icariin group. Furthermore, Icariin and phosphorylated icariin decreased gene abundance or enzyme activity of Bip, XBP1, GRP78, CHOP, caspase-3, and caspase-4 in LPS-treated LS174T cells. Our data suggest that Icariin and phosphorylated icariin effectively attenuate LPS-induced intestinal goblet cell function damage through regulating oxidative stress, inflammation, apoptosis, and mucin expression.

Icariin improves the cognitive function of APP/PS1 mice via suppressing endoplasmic reticulum stress

AimThis study aimed to investigate the effect of icariin (referred as ICA) on Alzheimer's disease (AD) model through endoplasmic reticulum (ER) stress pathway. Main methodsNine months male APP/PS1 and wild-type (WT) mice were randomly divided into four groups: APP/PS1 control, APP/PS1 + ICA, WT control and WT + ICA groups. The treated mice were given ICA 60 mg/kg/d and control mice were received the same volume distilled water for consecutive 3 months. The Morris water maze and Novel object recognition were used to detect animals' behavior. Nissl staining was used to observe the neuronal morphology in hippocampus area. Aβ deposition in hippocampal region was observed by immunofluorescence staining. TUNEL staining was used to observe apoptosis. Detection of expression of ER stress related factors by Western blot and real time RT-PCR. Key findingsChronically administrated with ICA compared with APP/PS1 control mice significantly improved the behavior performance, reduced neuronal apoptosis, as well as suppressing the ER stress signaling pathway, including that decreased the level of glucose-regulated protein 78, phosphorylated ER-regulated kinase and phosphorylated eukaryotic initiation factor α, as well activating transcription factor-4, C/EBP homologous protein, DNA damage inducible protein 34 and tribbles homologous protein 3. SignificanceOur data indicated that ICA suppressed the ER stress signaling to protect against AD animal model, these findings suggest that a potential point for researching the effect of ICA on neurodegeneration.

The protective effect of icariin and phosphorylated icariin against LPS-induced intestinal epithelial cells injury

Icariin (ICA) and phosphorylated icariin (pICA) have excellent antiviral and antioxidant effects. However, whether ICA and pICA cause anti-LPS-induced intestinal damage remains unclear. In this study, we used Caco-2 cells as a model to investigate the protective effects of ICA and pICA on human colonic epithelial cells and explore their potential mechanisms. Our results indicated that ICA and pICA increased cell viability and decreased lactate dehydrogenase activity in Caco-2 cells. ICA and pICA also attenuated LPS-induced changes in intestinal epithelial cell permeability and reduced the levels of oxidative stress indicators, such as reactive oxygen species, malondialdehyde, and hydrogen peroxide, in Caco-2 cells. Antioxidant indicators, such as superoxide dismutase, glutathione peroxidase, catalase and total antioxidant capacity, were increased, while the levels of IL-1β, IL-6, IL-8 and TNF-α were reduced in the ICA and pICA groups. Furthermore, ICA and pICA decreased the gene abundance and enzyme activities of caspase-3, -8, -9 and -10 in Caco-2 cells. Our data suggest that ICA and pICA effectively attenuated LPS-induced changes in the oxidative stress, inflammation, apoptosis and intestinal permeability of intestinal epithelial cells. These findings provide new insight for treating LPS-induced intestinal injury.

Optimization of the process for purifying icariin from Herba Epimedii by macroporous resin and the regulatory role of icariin in the tumor immune microenvironment

Pancreatic cancer is a digestive tract malignancy that poses a serious threat to human health. Compounds derived from traditional Chinese medicines have been an important source of anticancer drugs and adjuvant agents to regulate the tumor immune microenvironment in patients with pancreatic cancer. In this study, icariin was purified from Herba Epimedii using macropores, and its bioactivity against pancreatic cancer was also investigated. We found that icariin has direct inhibitory and immunomodulatory effects on tumor cells. In vitro experiments showed that icariin can inhibit the migration and proliferation of Panc02 pancreatic cancer cells and induce apoptosis. Our in vivo experiments show that icariin inhibits the development of mouse pancreatic cancer by inhibiting tumor-infiltrating M2 macrophages and polymorphonuclear myeloid-derived suppressor cells (MDSCs) (PMN-MDSCs). In addition, icariin inhibits the polarization of RAW 264.7 cells into M2 macrophages by inhibiting the expression of ARG1 and MRC1 and downregulating the IL4-STAT6 signaling pathway. In conclusion, the inhibitory effect of icariin on pancreatic cancer can not only directly affect tumor cells but also inhibit tumor development by regulating the tumor immune microenvironment.

Neuroprotective role of icariin in experimental spinal cord injury via its antioxidant, anti‑neuroinflammatory and anti‑apoptotic properties.

Icariin is a type of flavonoid derived from the Chinese herbal plant Epimedium sagittatum Maxim. Mounting evidence has confirmed the beneficial effects of icariin in neurological diseases, including spinal cord injury (SCI). The aim of the present study was to investigate the neuroprotective effects of icariin in SCI and the precise underlying mechanism. The weight‑drop injury technique was applied to construct an SCI model in Sprague‑Dawley rats. Icariin (35µmol/kg) was administered orally once daily for 7 consecutive days to examine its neuroprotective effects. The Basso, Beattie and Bresnahan scoring system was used for neurobehavioral evaluation. The water content of the injured spinal cord was measured via the dry‑wet weight method. Biochemical indices were examined by colorimetric assay using commercially available kits. Western blot analysis was used to detect protein expression. Icariin significantly accelerated the recovery of the locomotor function of SCI rats and decreased spinal cord water content. Icariin also attenuated SCI‑induced pro‑apoptotic protein expression and activity, while it increased anti‑apoptotic protein levels. In addition, icariin alleviated oxidative stress in SCI rats and decreased the levels of inflammatory molecules, including interleukin (IL)‑1β, IL‑6, tumor necrosis factor‑α, nitric oxide, nuclear factor‑κB and inducible nitric oxide synthase, and increased the expression of anti‑inflammatory proteins, including NADPH‑quinone oxidoreductase‑1, heme oxygenase‑1 and nuclear factor erythroid 2‑related factor 2 in the injured spinal cord. Therefore, icariin treatment accelerated locomotor function recovery in SCI, and its protective effects may be mediated via its antioxidant, anti‑inflammatory and anti‑apoptotic bioactivity.

Effects of icariin on long noncoding RNA and mRNA expression profile in the aortas of apoE-deficient mice.

Objective: The beneficial effects of icariin (ICA) in ameliorating atherosclerosis (AS) are well known, but the underlying protective mechanism has not been fully elucidated. The present study aimed to investigate altered long noncosing RNA (lncRNA) and mRNA expression profiles in ApoE−/− mice after ICA treatment.Method: The atherosclerotic plaque area was evaluated on high-fat diet (HFD)-induced ApoE−/− mice treated with either ICA or vehicle. LncRNA and mRNA integrated microarrays was performed on aortic tissues. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were utilized to explore the significant function and pathway of the differentially expressed (DE) mRNAs, global signal transduction network were constructed to select key mRNAs, and lncRNA–mRNA co-expression network was built to find out the interactions between lncRNA and mRNA. Quantitative real-time PCR (qPCR) was used to further validate the expressions of selected lncRNAs and mRNAs.Results: Administration of ICA significantly reduced plaque size after 12 weeks (P<0.05). A total of 1512 DE lncRNAs and 2059 DE mRNAs were identified. The mRNAs: protein kinase C, β (Prkcb), Cyp2c65, Mapk10, Calmodulin 5 (Calm5), Calmodulin-like 3 (Calml3) and Camk4 were selected as hub mRNAs, the correlated lncRNAs in co-expression network were identified as important regulatory lncRNAs. The identified target pairs such as lncRNA-NONMMUT000659/Prkcb may play critical roles in AS development mediated by ICA.Conclusion: Taken together, our study highlights a panel of DE lncRNAs and mRNAs that could explain the molecular mechanism of ICA’s anti-atherosclerotic effects. The work lays a foundation for subsequent genes functional researches, which could contribute to provide new therapeutic targets for AS.

Icariin exerts a protective effect against d-galactose induced premature ovarian failure via promoting DNA damage repair

Icariin is one of the most common active ingredients in traditional Chinese medicine, while its function against Premature ovarian failure (POF) has not been explored. POF animal model was induced by d-galactose, and icariin at different doses was administered. Ovarian structure and follicle counting were observed via hematoxylin and eosin staining. The levels of serum hormones were measured by ELISA. Primary ovarian granulosa cells were cultured to compare the protective effects of icariin on cell aging, and DNA damage markers including γH2AX and 53BP1 were assessed by Western Blot. Administration of icariin promoted ovary/body weight, follicles numbers and fertility outcomes. In addition, icariin downregulated the levels of follicle stimulating hormone and luteinizing hormone, and upregulated the levels of estradiol and anti-Müllerian hormone. Icariin protected ovarian granulosa cells from d-galactose induced aging, with increased cell viability and lower endogenous β-galactosidase activity. The alterations of expression level of γH2AX and 53BP1 by icariin indicated that the protection is via promoting DNA damage repair. In this study we tested the biological function of icariin against the d-galactose induced POF. Our results demonstrated that icariin effectively attenuated ovarian injury via promoting DNA damage repair, suggesting that icariin can be developed as a protective agent against POF.

The cardioprotective effects of icariin on the isoprenaline-induced takotsubo-like rat model: Involvement of reactive oxygen species and the TLR4/NF-κB signaling pathway

IntroductionTakotsubo syndrome (TS) is an acute cardiac syndrome that mimics acute coronary syndrome (ACS) but lacks coronary obstruction and is associated with sudden physical or psychiatric episodes. Several hypotheses have been proposed for the TS mechanism, but the precise cause of this syndrome remains poorly known. Recent studies noted TS patients with acute endogenous catecholamine discharge, which could trigger an oxidative stress response and inflammatory action. MethodsA single dose of the selective β-adrenergic agonist isoprenaline (ISO) was used to induce a takotsubo-like (TS-like) model. Different icariin or metoprolol doses were supplied as cardioprotective agents by intragastric administration (IG), and lipopolysaccharides (LPS) were assessed to investigate the possible mechanism of action of icariin. Transthoracic echocardiography was used to study cardiac function and morphology. The amounts of intracellular lipids and myocardial fibrosis, which represent the degree of cardiac impairment, were assessed by histological analysis. Real-time polymerase chain reaction (RT-PCR) was performed to analyze a variety of anti-oxidant elements and inflammatory factors, and Western blotting was conducted to analyze the expression of signaling pathway proteins involved in the development of TS. ResultsThe TS-like incidence in rats was lowest with icariin precondition at 2-h post-ISO administration, and both the left ventricular ejection fraction (LVEF) and ejection volume per minute were higher than those of the other groups. However, LPS administration increased the incidence of TS and aggravated cardiac impairment. Moreover, ISO significantly increased the levels of both reactive oxygen species (ROS) and TLR4/NF-κB signaling pathway proteins compared to those of the Sha-group, whereas icariin remarkably decreased the ROS levels and increased anti-oxidant element expression while reducing pro-inflammatory factor secretion and suppressing TLR4/NF-κB signaling pathway protein expression. However, the cardioprotective effect of icariin was significantly weakened by combining treatment with LPS. ConclusionIcariin prevented ISO-induced TS-like cardiac dysfunction in rats. The effects were induced mainly through maintenance of the dynamic balance of the ROS system, promotion of anti-oxidant element activity, and suppression of TLR4/NF-κB signaling pathway protein expression. Furthermore, the ability of icariin to increase anti-inflammatory and reduce pro-inflammatory factor secretion may be involved in the protective process.

Angiogenic and MMPs modulatory effects of icariin improved cutaneous wound healing in rats

Icariin is a flavonoid from plant belonging to the genus Epimedium, commonly known as Horny goat weed or Yin Yang Huo. The compound possesses multiple biological activities which are associated with the modulation of many signalling pathways, like NF-κB, Erk-p38-JNK, and release of various cytokines and growth factors. The present study determined wound healing potential of icariin in male Wistar rats. Icariin ointment (0%, 0.004%, 0.02%, 0.1% and 0.5%), was applied daily (b.i.d.) for 14 days on ≈ 400 mm2 cutaneous wound in different groups of rats. On day 14 post-wounding, 0.1% and 0.5% icariin treatment significantly (P < 0.01 and P < 0.001, respectively) increased wound contraction, as compared to control. Western blots revealed upregulation of IL-10 and downregulation of NF-κB and TNF-α. Increased expression of CD-31 showed abundance of microvessels in healing tissues after treatment with icariin. The MMP-2 and MMP-9 activities were reduced in icariin treated groups. Masson's trichrome staining revealed relatively better completion of re-epithelisation as well as increased deposition of well organised collagen fibres in the healing tissues compared to control. It is concluded that icariin has potential to accelerate cutaneous wound healing in rats.

Icariin induces apoptosis by suppressing autophagy in tamoxifen-resistant breast cancer cell line MCF-7/TAM

BackgroundIcariin is a major component isolated from Epimedium brevicornum Maxim and has been reported to exhibit anti-tumor activity. However, whether icariin could reverse the acquired drug resistance in breast cancer remains largely unclear. Therefore, this study was designed to explore the antitumor effects of icariin and its underlying mechanisms in a tamoxifen-resistant breast cancer cell line MCF-7/TAM.Methods3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Lactate dehydrogenase (LDH) assay were performed to determine the effects of icariin on cell viability and cell death. Cell cycle progression and apoptosis were detected by flow cytometry analysis. Transmission electron microscopy (TEM) assay was utilized to observe cell autophagy. The downstream protein levels were measured using western blotting.ResultsHere, we observed that icariin treatment not only inhibited the growth of MCF-7 but also has a potential function to overcome tamoxifen resistance in MCF-7/TAM. Moreover, icariin significantly induced cell cycle G0/G1 phase arrest and apoptosis, as well as suppressed autophagy. At molecular levels, icariin treatment remarkably down-regulated the expression levels of CDK2, CDK4, Cyclin D1, Bcl-2, LC3-1, LC3-II, AGT5, Beclin-1, but upregulated the expression levels of caspase-3, PARP and p62. Most importantly, we found inhibition of autophagy via 3-MA treatment could significantly enhance the effects of icariin on cell viability and apoptosis. Enhanced autophagy via autophagy related 5 (ATG5) overexpression could partially reverse the effects of icariin on cell viability and apoptosis.ConclusionThese results revealed that icariin might potentially be useful as an adjuvant agent in cancer chemotherapy to enhance the effect of tamoxifen through suppression of autophagy in vitro and provide insight into the therapeutic potential of icariin for the treatment of chemo-resistant breast cancer.

Effects of icariin on autophagy and exosome production of bone microvascular endothelial cells

To evaluate the effects of icariin on autophagy induced by low-concentration of glucocorticoid and exosome production in bone microvascular endothelial cells (BMECs). BMECs were isolated from femoral heads resected in total hip arthroplasty and then intervened with hydrocortisone of low concentration (0, 0.03, 0.06, 0.10 mg/mL), which were set as groups A, B, C, and D, respectively. On the basis of hydrocortisone intervention, 5×10 -5 mol/L of icariin was added to each group (set as groups A1, B1, C1 and D1, respectively). Western blot was used to detect the expressions of microtubule-associated protein 1 light chain 3B (LC3B) and dead bone slice 1 (p62) after 24 hours. Exosomes were extracted from BMECs treated with icariin (intervention group) and without icariin (non-intervention group), and the diameter and concentration of exosomes were evaluated by nanoparticle tracking analysis technique. The total protein content of exosomes was detected by BCA method, and the expressions of proteins carried by exosomes including CD9, CD81, transforming growth factor β 1 (TGF-β 1), and vascular endothelial growth factor A (VEGFA) were assessed by Western blot. The BMECs were further divided into three groups: BMECs in the experimental group and the control group were co-cultured with exosomes secreted by BMECs treated with or without icariin, respectively; the blank control group was BMECs without exosome intervention. The three groups were treated with hydrocortisone and Western blot was used to detect the expressions of LC3B and p62. The scratching assay was used to detect cell migration ability; angiogenic ability of BMECs was also assessed. With the increase of hydrocortisone concentration, the protein expression of LC3B-Ⅱ increased gradually, and the protein expression of p62 decreased gradually ( P<0.01). Compared with group with same concentration of hydrocortisone, the protein expression of LC3B-Ⅱ decreased and the protein expression of p62 increased after the administration of icariin ( P<0.01). The concentration of exosomes in the intervention group was significantly higher than that in the non-intervention group ( t=-10.191, P=0.001); and there was no significant difference in exosome diameter and total protein content between the two groups ( P>0.05). CD9 and CD81 proteins were highly expressed in the non-intervention group and the intervention group, and the relative expression ratios of VEGFA/CD9 and TGF-β 1/CD9 proteins in the intervention group were significantly higher than those in the non-intervention group ( P<0.01). After co-culture of exosomes, the protein expression of p62 increased in blank control group, control group, and experimental group, while the protein expression of LC3B-Ⅱ decreased. There were significant differences among groups ( P<0.05). When treated with hydrocortisone for 12 and 24 hours, the scratch closure rate of the control group and experimental group was significantly higher than that of the blank control group ( P<0.05), and the scratch closure rate of the experimental group was significantly higher than that of the control group ( P<0.05). When treated with hydrocortisone for 4 and 8 hours, the number of lumens, number of sprouting vessels, and length of tubule branches in the experimental group and the control group were significantly greater than those in the blank control group ( P<0.05); the length of tubule branches and the number of lumens in the experimental group were significantly greater than those in the control group ( P<0.05). Icariin and BMECs-derived exosomes can improve the autophagy of BMECs induced by low concentration of glucocorticoid.

Icariin induces irisin/FNDC5 expression in C2C12 cells via the AMPK pathway

Backgroud Icariin, a major bioactive pharmaceutical component of the Chinese herbal medicine Epimedii Herba, has demonstrated lipid-lowering and anti-obesity effects. Irisin/ fibronectin type III domain-containing 5 (FNDC5) protects against obesity by inducing browning in white adipose tissue. Objectives This study investigated the effects of icariin on irisin/FNDC5 expression in C2C12 myotubes. Method Cultured murine C2C12 myocytes were used to study the effects of icariin on irisin/FNDC5 expressions by Western-blot, qPCR, Elisa and Immunofluorescence. We also investigated FNDC5 expression in icariin-treated intact mice. Results Icariin increased irisin/FNDC5 protein levels. mRNA levels of irisin/FNDC5 were also increased in C2C12 myocytes after treatment with icariin. Icariin increased peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1α) protein and mRNA levels. Additionally, icariin exposure resulted in phosphorylation of AMP-activated protein kinase (AMPK) in a dose-dependent manner. The regulatory effect of icariin on FNDC5 protein expression was blocked by the AMPK antagonist compound C or silencing of AMPK, suggesting that icariin increased FNDC5 protein expression via the AMPK pathway. In vivo, icariin decreased body weight gain in C57BL/6 mice and increased FNDC5, PGC-1α, and p-AMPK expression levels in skeletal muscle. Conclusions Taken together, our results indicated that icariin induces irisin/FNDC5 expression via the AMPK pathway, indicating that icariin may be promising as an anti-obesity drug.