Icariside II in NSCLC and COVID-19: Network pharmacology and molecular docking study.

BACKGROUND: Patients with non-small cell lung cancer (NSCLC) are susceptible to coronavirus disease-2019 (COVID-19), but current treatments are limited. Icariside II (IS), a flavonoid compound derived from the plant epimedin, showed anti-cancer,anti-inflammation and immunoregulation effects. The present study aimed to evaluate the possible effect and underlying mechanisms of IS on NSCLC patients with COVID-19 (NSCLC/COVID-19). METHODS: NSCLC/COVID-19 targets were defined as the common targets of NSCLC (collected from The Cancer Genome Atlas database) and COVID-19 targets (collected from disease database of Genecards, OMIM, and NCBI). The correlations of NSCLC/COVID-19 targets and survival rates in patients with NSCLC were analyzed using the survival R package. Prognostic analyses were performed using univariate and multivariate Cox proportional hazards regression models. Furthermore, the targets in IS treatment of NSCLC/COVID-19 were defined as the overlapping targets of IS (predicted from drug database of TMSCP, HERBs, SwissTarget Prediction) and NSCLC/COVID-19 targets. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of these treatment targets were performed aiming to understand the biological process, cellular component, molecular function and signaling pathway. The hub targets were analyzed by a protein-protein interaction network and the binding capacity with IS was characterized by molecular docking. RESULTS: The hub targets for IS in the treatment of NSCLC/COVID-19 includes F2, SELE, MMP1, MMP2, AGTR1 and AGTR2, and the molecular docking results showed that the above target proteins had a good binding degree to IS. Network pharmacology showed that IS might affect the leucocytes migration, inflammation response and active oxygen species metabolic process, as well as regulate the interleukin-17, tumor necrosus factor and hypoxia-inducible factor-1 signaling pathway in NSCLC/COVID-19. CONCLUSIONS: IS may enhance the therapeutic efficacy of current clinical anti-inflammatory and anti-cancer therapy to benefit patients with NSCLC combined with COVID-19.

Baohuoside I suppresses the NLRP3 inflammasome activation via targeting GPER to fight against Parkinson's disease.

BACKGROUND: Accumulating evidence indicates the crucial role of microglia-mediated inflammation and the NLR family pyrin domain containing 3 (NLRP3) inflammasome-mediated pyroptosis in the pathogenesis of Parkinson's disease (PD). Baohuoside I, a natural flavonoid extracted from Herba Epimedii, has been shown to possess anti-inflammatory effects, but its potential neuroprotective effects and mechanism against PD have not been documented. STUDY DESIGN AND METHODS: The anti-inflammatory effects of Baohuoside I were evaluated by LPS-induced BV2 cells or primary microglia isolated from wide type or G protein-coupled estrogen receptor (GPER) gene knockout mice. The underlying mechanism related to GPER-mediated NLRP3 inflammasome inhibition was further explored using LPS-induced GPER+/+ or GPER-/- mouse models of PD. The neuroprotective effects of Baohuoside I were detected through western blot analysis, real-time PCR, molecular docking, mouse behavioral tests, immunofluorescence, and immunohistochemistry. RESULTS: Baohuoside I significantly alleviated LPS-induced neuroinflammation by inhibiting the activation of NF-κB signal and the increase of pyroptosis levels as evidenced by the downregulated expression of pyroptosis-related proteins (NLRP3, ASC, pro-Caspase-1, IL-1ß) in microglia cells. Intragastric administration of Baohuoside I protected against LPS-induced motor dysfunction and loss of dopaminergic neurons, reduced pro-inflammatory cytokines expressions, and inhibited microglial (Iba-1) and astrocyte (GFAP) activation in the nigrostriatal pathway in LPS-induced mouse model of PD. Pretreatment with GPER antagonist G15 in microglia cells or GPER gene deletion in mice significantly blocked the inhibitory effects of Baohuoside I on LPS-induced neuroinflammation and activation of the NLRP3/ASC/Caspase-1 pathway. Molecular docking further indicated that Baohuoside I might bind to GPER directly with a binding energy of -10.4 kcal/mol. CONCLUSION: Baohuoside I provides neuroprotective effects against PD by inhibiting the activation of the NF-κB signal and NLRP3/ASC/Caspase-1 pathway. The molecular target for its anti-inflammatory effects is proved to be GPER in the PD mouse model. Baohuoside I may be a valuable anti-neuroinflammatory agent and a drug with well-defined target for the treatment of PD.

Production of Rhamnosyl Icariside II by snailase hydrolysis of Epimedium wushanense extracts.

Rhamnosyl Icariside II is a rare secondary flavonoid glycoside isolated from Epimedium L. plants. It has better stability and physiological activity than the primary flavonoid glycosides of Epimedium L., therefore, conversion of the primary flavonoid glycoside into Rhamnosyl Icariside II would be desirable. In this study, a method for the enzymatic production of Rhamnosyl Icariside II from the total flavonoids of Epimedium wushanense was established, and the conditions were optimized. Six commercial enzymes were screened, and the reaction conditions for the best enzyme were optimized. Snailase was the most effective hydrolase, and the highest yield was obtained under the optimized conditions. To facilitate industrial production of Rhamnosyl Icariside II, a scaled-up pilot test was performed. The reaction solution was extracted with n-butanol to obtain the Rhamnosyl Icariside II crude product, which was then subjected to silica gel column chromatography and preparative chromatography. Finally, a product of Rhamnosyl Icariside II with purity of 99.1 % was achieved, in a total yield of 46.8 %. Compared to direct extraction and acid hydrolysis, this method improves the product yield and purity, which is of great significance for the large-scale production of Rhamnosyl Icariside II. This study provides a basis for the physiological activity study of Rhamnosyl Icariside II, and offers possibilities for future applications in the healthcare sector.

Icariside II prevents kidney fibrosis development in chronic kidney disease by promoting fatty acid oxidation.

Renal interstitial fibrosis (RIF) is the main pathological basis for the progression of chronic kidney disease (CKD), however, effective interventions are limited. Here, we investigated the effect of Icariside II (ICA-II) on RIF and explored the underlying mechanisms. Rats receiving 5/6 ablation and infarction (A/I) surgery were gavaged with ICA-II (5 or 10 mg/kg) for 8 weeks. In vitro, TGF-ß1-stimulated NRK-52E cells were treated with ICA-II and (or) oleic acid, etomoxir, ranolazine, fenofibrate, and GW6471. The effects of ICA-II on RIF, fatty acid oxidation, lipid deposition, and mitochondrial function were determined by immunoblotting, Oil red O staining, colorimetric, and fluorometric assays. Using adeno-associated virus injection and co-culture methods, we further determined mechanisms of ICA-II anti-RIF. ICA-II ameliorated the fibrotic responses in vivo and in vitro. RNA-seq analysis indicated that ICA-II regulated fatty acid degradation and PPAR pathway in 5/6 (A/I) kidneys. ICA-II attenuated lipid accumulation and up-regulated expression of PPARα, CPT-1α, Acaa2, and Acadsb proteins in vivo and in vitro. Compared to ICA-II treatment, ICA-II combined with Etomoxir exacerbated mitochondrial dysfunction and fibrotic responses in TGF-ß-treated NRK-52E cells. Importantly, we determined that ICA-II improved lipid metabolism, fatty acid oxidation, mitochondrial function, and RIF by restoring PPARα. Co-culture revealed that ICA-II decreased the expression of Fibronectin, Collagen-I, α-SMA, and PCNA proteins in NRK-49F cells by restoring PPARα of renal tubular cells. ICA-II may serve as a promising therapeutic agent for RIF in 5/6 (A/I) rats, which may be important for the prevention and treatment of CKD.

Icariside II protects dopaminergic neurons from 1­methyl­4­phenylpyridinium­induced neurotoxicity by downregulating HDAC2 to restore mitochondrial function.

Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease (AD). Icariside II (ICS II) is known to confer notable therapeutic effects against a variety of neurodegenerative diseases, such as AD. Therefore, the present study aimed to evaluate the possible effects of ICS II on 1-methyl-4-phenylpyridinium (MPP+)-induced SK-N-SH cell injury, in addition to understanding the underlying mechanism of action. The MPP+-induced SK-N-SH cell model was used to simulate PD in vitro. The viability and mitochondrial membrane potential of SK-N-SH cells were detected by MTT assay and JC-1 staining, respectively. Lactate dehydrogenase (LDH) release, ATP levels and complex I activity in treated SK-N-SH cells were measured using LDH activity, ATP and Complex I assay kits, respectively. The protein expression levels of histone deacetylase 2 (HDAC2) and γ-H2A histone family member X and the copy number of mitochondrial DNA were measured by western blotting or reverse transcription-quantitative PCR, respectively. Autodock 4.2 was used to predict the molecular docking site of ICS II on HDAC2. The results of the present study demonstrated that ICS II mitigated SK-N-SH cytotoxicity induced by MPP+. Specifically, ICS II alleviated DNA damage and restored mitochondrial function in SK-N-SH cells treated with MPP+. In addition, ICS II reduced the HDAC2 protein expression levels in MPP+-induced SK-N-SH cells. However, overexpression of HDAC2 reversed the protective effects of ICS II on DNA damage and mitochondrial dysfunction in MPP+-induced SK-N-SH cells. In conclusion, the results of the present study suggest that ICS II can protect dopaminergic neurons from MPP+-induced neurotoxicity by downregulating HDAC2 expression to restore mitochondrial function.

Icariside II suppresses ferroptosis to protect against MPP+-Induced Parkinson's disease through Keap1/Nrf2/GPX4 signaling.

Parkinson's disease (PD) is recognized as a degenerative and debilitating neurodegenerative disorder. The novel protective role of icariside II (ICS II) as a plant-derived flavonoid compound in neurodegenerative diseases has aroused much attention. Herein, the definite impacts of ICS II on the process of PD and the relevant action mechanism were studied. Human neuroblastoma SK-N-SH cells were challenged with 1-methyl-4-phenylpyridinium ion (MPP+) to construct the PD cell model. MTT assay and flow cytometry analysis, respectively, appraised cell viability and apoptosis. Caspase 3 Activity Assay examined caspase 3 activity. Corresponding kits examined oxidative stress levels. BODIPY 581/591 C11 assay evaluated lipid reactive oxygen species. Iron Assay Kit assessed iron content. Western blot tested the expression of apoptosis-, ferroptosis- and Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPX4) signaling-associated proteins. Molecular docking verified the binding of ICS II with Keap1. The existing experimental results unveiled that ICS II elevated the viability whereas reduced the apoptosis, oxidative stress, and ferroptosis in MPP+-treated SK-N-SH cells in a concentration-dependent manner. Furthermore, ICS II declined Keap1 expression while raised Nrf2, heme oxygenase 1, and GPX4 expression. In addition, ICS II had a strong binding with Keap1 and Nrf2 inhibitor ML385 partially abolished the suppressive role of ICS II in MPP+-triggered apoptosis, oxidative stress, and ferroptosis in SK-N-SH cells. To summarize, ICS II might inhibit apoptosis, oxidative stress, and ferroptosis in the MPP+-stimulated PD cell model, which might be due to the activation of Keap1/Nrf2/GPX4 signaling.

Effects of Icariin and Its Metabolites on GPCR Regulation and MK-801-Induced Schizophrenia-Like Behaviors in Mice.

Icariin, a major bioactive compound found in the Epimedium genus, has been reported to exert protective effects against neurodegenerative disorders. In the current study, we aimed to investigate the regulatory effect of icariin and its active metabolites (icariside II and icaritin) against prime G-protein-coupled receptor targets, considering their association with neuronal disorders. Icariside II exhibited selective agonist activity towards the dopamine D3 receptor (D3R), with half-maximal effective concentrations of 13.29 µM. Additionally, they effectively inhibited the specific binding of radioligands to D3R. Molecular docking analysis revealed that icariside II potentially exerts its agonistic effect through hydrogen-bonding interaction with Asp110 of the D3R, accompanied by negative binding energy. Conversely, icaritin demonstrated selective antagonist effects on the muscarinic acetylcholine M2 receptor (M2R). Radioligand binding assay and molecular docking analysis identified icaritin as an orthosteric ligand for M2R. Furthermore, all three compounds, icariin and its two metabolites, successfully mitigated MK-801-induced schizophrenia-like symptoms, including deficits in prepulse inhibition and social interaction, in mice. In summary, these findings highlight the potential of icariin and its metabolites as promising lead structures for the discovery of new drugs targeting cognitive and neurodegenerative disorders.

Icariside II mitigates myocardial infarction by balancing mitochondrial dynamics and reducing oxidative stress through the activation of Nrf2/SIRT3 signaling pathway.

Nuclear factor erythroid 2-related factor 2 (Nrf2)/silent mating type information regulation 2 homolog 3 (SIRT3) signaling pathway plays a pivotal role in regulating mitochondrial dynamics and oxidative stress, which are considered to be the principal pathogenesis of myocardial infarction (MI). Our previous study proved that pretreatment with icariside II (ICS II), a major active ingredient of Herbal Epimedii, exerts cardioprotective effect on MI, however, whether post-treatment with ICS II can alleviate MI and its underlying mechanism are still uncertain. Therefore, the present study was designed to investigate the therapeutic effect and the possible mechanism of ICS II on MI both in vivo and in vitro. The results revealed that post-treatment with ICS II markedly ameliorated myocardial injury in MI-induced mice and mitigated oxygen and glucose deprivation (OGD)-elicited cardiomyocyte injury. Further researches showed that ICS II promoted mitochondrial fusion, and suppressed mitochondrial fission and oxidative stress, which were achieved by facilitating the nuclear translocation of Nrf2 and activation of SIRT3. In summary, our findings indicate that ICS II mitigates MI-induced mitochondrial dynamics disorder and oxidative stress via activating the Nrf2/SIRT3 signaling pathway.

UPLC-MS/MS method for Icariin and metabolites in whole blood of C57 mice: development, validation, and pharmacokinetics study.

Icariin, a Chinese medicinal herb with significant effects on Alzheimer's disease, lacks pharmacokinetic data in mice. To address this, a UPLC-MS/MS method was developed and validated for quantifying Icariin and its metabolites, Icariside I and Icariside II, in the whole blood of mice. The method processed micro-whole blood from serial collections of the same C57 mouse, with well-fitted linearity (0.25-800 ng mL-1) and intra- and inter-day precision and accuracy within 15%. Short-time and autosampler stability were verified, with acceptable extraction recoveries and matrix effects over 74.55%. After intravenous administration (15 mg kg-1) of Icariin in C57 mice, Icariside I and Icariside II were detected within 2 min. However, after the intragastric administration (30, 90, and 150 mg kg-1) of Icariin in C57 mice, Icariin and Icariside I were not detected, and Icariin was rapidly converted into Icariside II. Furthermore, the Cmax and AUC0-t of three doses (30, 90, and 150 mg kg-1) of Icariside II increased as the dose increased. In conclusion, this method improves the traditional method of collecting only one blood sample from each mouse, detecting Icariin and its metabolites in the whole blood of mice, especially for serial collection of micro-whole blood.

Network pharmacology analysis of Icariside II against bladder cancer.

As a global health threat, bladder cancer (BC) is a common urological disease characterized by a high risk of progression and recurrence. Icariside II (ICA-II), a flavonol glycoside, exhibits antitumor ability in various tumors. However, there is no systematic study exploring the pharmacological mechanism of ICA-II in BC. We used public databases to obtain potential targets of ICA-II and related genes in BC. Bioinformatics analysis and molecular docking were used to identify potential targets and signaling pathways. Then, MTT, cell cycle assays and western blot (WB) were used to validate the predicted pathways in bladder cell lines, and in situ bladder cancer models were also established to verify the effect of ICA-II. Our research demonstrated that these ICA-II hub genes were related to the cell cycle. Then, our molecular docking analysis confirmed the interaction between ICA-II and CCNB1. In addition, our in vitro experiment demonstrated that ICA-II restrained the proliferation of BC cells mainly by blocking the cell cycle. WB also verified that ICA-II decreased the expression levels of CCNB1. In situ BC models showed that ICA-II had no hepatotoxicity or nephrotoxicity and could suppress the growth of in situ BC. In summary, during this study, we found that ICA-II had low toxicity in the kidney and liver. Network pharmacology was used, and both cell and animal experiments verified that ICA-II has a good therapeutic effect on bladder cancer, which may inhibit the proliferation and progression of bladder cancer by blocking the cell cycle of BC cells.

TMT-based quantitative proteomics revealed protective efficacy of Icariside II against airway inflammation and remodeling via inhibiting LAMP2, CTSD and CTSS expression in OVA-induced chronic asthma mice.

BACKGROUND: Asthma is a chronic inflammatory disorder in airways with typical pathologic features of airflow limitation, airway inflammation and remodeling. Icariside II (IS), derived from herbal medicine Herba Epimedii, exerts an anti-inflammatory property. However, underlying mechanisms with specifically targeted molecular expression by IS in asthma have not been fully understood, and whether IS could inhibit remodeling and EMT still remains unclear. PURPOSE: The study aimed to clarify therapeutic efficacy of IS for attenuating airway inflammation and remodeling in asthma, and illustrate IS-regulated specific pathway and target proteins through TMT-based quantitative proteomics. STUDY DESIGN AND METHODS: Murine model of chronic asthma was constructed with ovalbumin (OVA) sensitization and then challenge for 8 weeks. Pulmonary function, leukocyte count in bronchoalveolar lavage fluid (BALF), lung histopathology, inflammatory and fibrotic cytokines, and markers of epithelial-mesenchymal transition (EMT) were evaluated. TMT-based quantitative proteomics were performed on lung tissues to explore IS-regulated proteins. RESULTS: IS contributed to alleviative airway hyperresponsiveness (AHR) evidenced by declined RL and increased Cdyn. After IS treatment, we observed a remarked down-regulation of leukocyte count, inflammatory cytokines in BALF, and peribronchial inflammation infiltration. Goblet cell hyperplasia, mucus secretion and peribronchial collagen deposition were attenuated, with the level of TGF-ß and MMP-9 in BALF declined. Furthermore, IS induced a rise of Occludin and E-cadherin and a decline of N-cadherin and α-SMA in lung tissues. These results proved the protective property of IS against airway inflammation, remodeling and EMT. To further investigate underlying mechanisms of IS in asthma treatment, TMT-based quantitative proteomics were performed and 102 overlapped DEPs regulated by IS were identified. KEGG enrichment exhibited these DEPs were enriched in lysosome, phagosome and autophagy, in which LAMP2, CTSD and CTSS were common DEPs. WB, q-PCR and IHC results proofed expressional alteration of these proteins. Besides, IS could decrease Beclin-1 and LC3B expression with increasing p62 expression thus inhibiting autophagy. CONCLUSIONS: The study demonstrated IS could ameliorate AHR, airway inflammation, remodeling and EMT in OVA-induced chronic asthma mice. Our research was the first to reveal that inhibition of LAMP2, CTSD and CTSS expression in autophagy contributed to the therapeutic efficacy of IS to asthma.

Icariside II, a Prenyl-Flavonol, Alleviates Inflammatory and Neuropathic Pain by Inhibiting T-Type Calcium Channels and USP5-Cav3.2 Interactions.

Cav3.2 channels play an important role in the afferent nociceptive pathway, which is responsible for both physiological and pathological pain transmission. Cav3.2 channels are upregulated during neuropathic pain or peripheral inflammation in part due to an increased association with the deubiquitinase USP5. In this study, we investigated nine naturally occurring flavonoid derivatives which we tested for their abilities to inhibit transiently expressed Cav3.2 channels and their interactions with USP5. Icariside II (ICA-II), one of the flavonols studied, inhibited the biochemical interactions between USP5 and Cav3.2 and concomitantly and effectively blocked Cav3.2 channels. Molecular docking analysis predicts that ICA-II binds to the cUBP domain and the Cav3.2 interaction region. In addition, ICA-II was predicted to interact with residues in close proximity to the Cav3.2 channel's fenestrations, thus accounting for the observed blocking activity. In mice with inflammatory and neuropathic pain, ICA-II inhibited both phases of the formalin-induced nocifensive responses and abolished thermal hyperalgesia induced by injection of complete Freund's adjuvant (CFA) into the hind paw. Furthermore, ICA-II produced significant and long-lasting thermal anti-hyperalgesia in female mice, whereas Cav3.2 null mice were resistant to the action of ICA-II. Altogether, our data show that ICA-II has analgesic activity via an action on Cav3.2 channels.

Therapeutic effects of icariin and icariside II on diabetes mellitus and its complications.

Diabetes mellitus (DM) is a global health issue in the twenty-first century, and there are numerous challenges in preventing and alleviating its chronic complications. The herb Epimedium has beneficial therapeutic effects on various human diseases, including DM. Its major flavonoid component, icariin, has significant anti-DM activity and may help improve pancreatic ß-cell dysfunction and insulin resistance. Furthermore, preclinical evidence has shown that icariin and its in vivo bioactive form, icariside II, have preventive and therapeutic effects on several diabetic complications, including diabetic cardiomyopathy, diabetic vascular endothelial disorder, diabetic nephropathy, and diabetic erectile dysfunction. In this review, we present the general and toxicological information concerning icariin and icariside II and review the anti-DM effects of icariin from a molecular perspective. Additionally, we discuss the potential benefits of icariin and icariside II on the important pathological mechanisms of various diabetic complications. Despite positive preclinical evidence, additional investigations are needed before relevant clinical studies can be conducted. Therefore, we conclude with suggestions for future research. Hopefully, this review will provide a comprehensive molecular perspective for future research and product development related to icariin and icariside II in treating DM and diabetic complications.

Icariside II potentiates the anti-PD-1 antitumor effect by reducing chemotactic infiltration of myeloid-derived suppressor cells into the tumor microenvironment via ROS-mediated inactivation of the SRC/ERK/STAT3 signaling pathways.

BACKGROUND: Immune checkpoint blockade agents, such as anti-PD-1 antibodies, show promising antitumor efficacy but only a limited response in patients with non-small cell lung cancer (NSCLC). Icariside II (IS), a metabolite of Herba Epimedii, is a COX-2 and EGFR inhibitor that can enhance the anti-PD-1 effect. This study aimed to evaluate the antitumor effect of IS in combination with anti-PD-1 and explore the underlying mechanism. METHODS: Tumor growth was assessed in Lewis Lung Cancer (LLC) tumor-bearing mice in seven groups (control, IS 20 mg/kg, IS 40 mg/kg, anti-PD-1, IS 20 mg/kg+anti-PD-1, IS 40 mg/kg+anti-PD-1, ERK inhibitor+anti-PD-1). Tumor-infiltrating immune cells were measured by flow cytometry. The mechanisms were explored by tumor RNA-seq and validated in LLC cells through molecular biological experiments using qRT‒PCR, ELISA, and western blotting. RESULTS: Animal experiments showed that IS in combination with anti-PD-1 further inhibited tumor growth and remarkably reduced the infiltration of myeloid-derived suppressor cells (MDSCs) into the tumor compared with anti-PD-1 monotherapy. RNA-seq and in vitro experiments showed that IS suppressed the chemotactic migration of MDSCs by downregulating the expression of CXC chemokine ligands 2 (CXCL2) and CXCL3. Moreover, IS promoted reactive oxygen species (ROS) generation and inhibited the activation of SRC/ERK/STAT3 in LLC cells, which are upstream signaling pathways of these chemokines. CONCLUSION: IS potentiates the anti-PD-1 anti-tumor effect by reducing chemotactic infiltration of the myeloid-derived suppressor cell into the tumor microenvironment, via ROS-mediated inactivation of SRC/ERK/STAT3 signaling pathways.

Icariside II preconditioning evokes robust neuroprotection against ischaemic stroke, by targeting Nrf2 and the OXPHOS/NF-κB/ferroptosis pathway.

BACKGROUND AND PURPOSE: Astrocytic nuclear factor erythroid-derived 2-related factor 2 (Nrf2) is a potential therapeutic target of ischaemic preconditioning (IPC). Icariside II (ICS II) is a naturally occurring flavonoid derived from Herba Epimedii with Nrf2 induction potency. This study was designed to clarify if exposure to ICS II mimicks IPC neuroprotection and if Nrf2 from astrocytes contributes to ICS II preconditioning against ischaemic stroke. EXPERIMENTAL APPROACH: Mice with transient middle cerebral artery occlusion (MCAO)-induced focal cerebral ischaemia and primary astrocytes challenged with oxygen-glucose deprivation (OGD) were used to explore the neuroprotective effect of ICS II preconditioning. Additionally, Nrf2-deficient mice were pretreated with ICS II to determine whether ICS II exerts its neuroprotection by activating Nrf2. KEY RESULTS: ICS II pretreatment mitigated cerebral injury in the mouse model of ischaemic stroke along with improving long-term recovery. Furthermore, proteomics screening identified Nrf2 as a crucial gene evoked by ICS II treatment and required for the anti-oxidative effect and anti-inflammatory effect of ICS II. Also, ICS II directly bound to Nrf2 and reinforced the transcriptional activity of Nrf2 after MCAO. Moreover, ICS II pretreatment exerted cytoprotective effects on astrocyte cultures following lethal OGD exposure, by promoting Nrf2 nuclear translocation and activating the OXPHOS/NF-κB/ferroptosis axis, while neuroprotection was decreased in Nrf2-deficient mice and Nrf2 siRNA blocked effects of ICS II. CONCLUSION AND IMPLICATIONS: ICS II preconditioning provides robust neuroprotection against ischaemic stroke via the astrocytic Nrf2-mediated OXPHOS/NF-κB/ferroptosis axis. Thus, ICS II could be a promising Nrf2 activator to treat ischaemic stroke.

Icariside II induces rapid phosphorylation of endothelial nitric oxide synthase via multiple signaling pathways.

Icariside II, as a favonoid compound derived from epimedium, has been proved to involed in a variety of biological and pharmacological effects such as anti-inflammatory, anti-osteoporosis, anti-oxidation, anti-aging, and anti-cancer but its mechanism is unclear, especially in terms of its effect on post-transcriptional modification of endothelial nitric oxide synthase (eNOS). Phosphorylation of eNOS plays an important role in the synthesis of nitric oxide in endothelial cells, which is closely related to erectile dysfunction, atherosclerosis, Alzheimer's disease, and other diseases. Our study aims to investigate the effect and mechanism of Icariside II on the rapid phosphorylation of eNOS. In this study, human umbilical vein endothelial cells (HUVECs) were stimulated with Icariside II in the presence or absence of multiple inhibitors (1 µM), including LY294002 (PI3K-inhibitor), MK-2206 (AKT-inhibitor), Bisindolylmaleimide X (AMPK-inhibitor), H-89 (CaMKII-inhibitor), KN-62 (PKA-inhibitor), Dorsomorphin (PKC-inhibitor). The proliferation of HUVECs was assessed using cell counting kit-8 (CCK-8). The release of nitric oxide (NO) within HUVECs was detected via fluorescence probe (DAF-FM). Western blot was used to examine the effect of Icariside II on the expression of eNOS, phosphorylation of eNOS, and common signaling pathways proteins. In this study, Icariside II was found to promote the cell proliferation and rapid NO release in HUVECs. The phosphorylation of eNOS-Ser1177 was significantly increased after Icariside II stimulation and reached a peak at 10 min (p < 0.05). Meanwhile, the phosphorylation of eNOS-Thr495 was significantly decreased after 45 min of stimulation (p < 0.05). Following the intervention with multiple inhibitors, it was found that MK-2206 (AKT inhibitor), LY294002 (PI3K inhibitor), KN-62 (AMPK inhibitor), and Bisindolylmaleimide X (PKC inhibitor) could significantly inhibit the phosphorylation of eNOS-Ser1177 caused by Icariside II (p < 0.05), while MK-2206, LY294002, and Bisindolylmaleimide X reversed the alleviated phosphorylation of eNOS-Thr495. We concluded that Icariside can regulate rapid phosphorylation of eNOS- Ser1177 and eNOS-Thr495 via multiple signaling pathways, resulting in the up-regulation of eNOS and the increased release of NO.

Icariside II Exerts Anti-Type 2 Diabetic Effect by Targeting PPARα/γ: Involvement of ROS/NF-κB/IRS1 Signaling Pathway.

Type 2 diabetes mellitus (T2DM) is a multisystem and complex metabolic disorder which is associated with insulin resistance and impairments of pancreatic ß-cells. Previous studies have shown that icariside II (ICS II), one of the main active ingredients of Herba Epimedii, exerts potent anti-inflammatory and anti-oxidative properties. In this study, we investigated whether ICS II exerted anti-T2DM profile and further explored its possible underlying mechanism both in vivo and in vitro. db/db mice were administered ICS II (10, 20, 40 mg·kg-1) for 7 weeks. We found that ICS II dose-dependently attenuated hyperglycemia and dyslipidemia, as well as inhibited hepatic steatosis and islet architecture damage in db/db mice. Moreover, ICS II not only dramatically reduced inflammatory cytokines and oxidative stress, but also up-regulated PPARα/γ protein expressions, phosphorylation of Akt, GSK3ß and IR, meanwhile, down-regulated phosphorylation of NF-κB(p65) and IRS1 in db/db mice. In palmitic acid (PA)-treated HepG2 or MIN6 cells, ICS II (5-20 µM) concentration-dependently promoted the cell viability via mediating PPARα/γ/NF-κB signaling pathway. PPARα/γ knockout by CRISPR-Cas9 system partly abolished the protective effects of ICS II on HepG2 or MIN6 cells following PA insults. These findings reveal that ICS II effectively confer anti-T2DM property by targeting PPARα/γ through mediation of ROS/NF-κB/IRS1 signaling pathway.

Pharmacological mechanism and therapeutic efficacy of Icariside II in the treatment of acute ischemic stroke: a systematic review and network pharmacological analysis.

BACKGROUND AND OBJECTIVE: Epimedii has long been used as a traditional medicine in Asia for the treatment of various common diseases, including Alzheimer's disease, cancer, erectile dysfunction, and stroke. Studies have reported the ameliorative effects of Icariside II (ICS II), a major metabolite of Epimedii, on acute ischemic stroke (AIS) in animal models. Based on network pharmacology, molecular docking, and molecular dynamics (MD) simulations, we conducted a systematic review to evaluate the effects and neuroprotective mechanisms of ICS II on AIS. METHODS: First, we have searched 6 databases using studies with ICS II treatment on AIS animal models to explore the efficacy of ICS II on AIS in preclinical studies. The literature retrieval time ended on March 8, 2022 (Systematic Review Registration ID: CRD42022306291). There were no restrictions on the language of the search strategy. Systematic review follows the Patient, Intervention, Comparison and Outcome (PICO) methodology and framework. SYCLE's RoB tool was used to evaluate the the risk of bias. In network pharmacology, AIS-related genes were identified and the target-pathway network was constructed. Then, these targets were used in the enrichments of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and gene ontology (GO). Molecular docking and MD simulation were finally employed between ICS II and the potential target genes. RESULTS: Twelve publications were included describing outcomes of 1993 animals. The literature details, animal strains, induction models, doses administered, duration of administration, and outcome measures were extracted from the 12 included studies. ICS II has a good protective effect against AIS. Most of the studies in this systematic review had the appropriate methodological quality, but some did not clearly state the controlling for bias of potential study. Network pharmacology identified 246 targets with SRC, CTNNB1, HSP90AA1, MAPK1, and RELA as the core target proteins. Besides, 215 potential pathways of ICS II were identified, such as PI3K-Akt, MAPK, and cGMP-PKG signaling pathway. GO enrichment analysis showed that ICS II was significantly enriched in subsequent regulation such as MAPK cascade. Molecular docking and MD simulations showed that ICS II can closely bind with important targets. CONCLUSIONS: ICS II is a promising drug in the treatment of AIS. However, this systematic review reveals key knowledge gaps (i.e., the protective role of ICS II in women) that ICS II must address before it can be used for the treatment of human AIS. Our study shows that ICS II plays a protective role in AIS through multi-target and multi-pathway characteristics, providing ideas for the development of drugs for the treatment of AIS.

Icariside II, a Naturally Occurring SIRT3 Agonist, Protects against Myocardial Infarction through the AMPK/PGC-1α/Apoptosis Signaling Pathway.

Myocardial infarction (MI) refers to the death of cardiomyocytes triggered by a lack of energy due to myocardial ischemia and hypoxia, and silent mating type information regulation 2 homolog 3 (SIRT3) plays an essential role in protecting against myocardial oxidative stress and apoptosis, which are deemed to be the principal causes of MI. Icariside II (ICS II), one of the main active ingredients of Herbal Epimedii, possesses extensive pharmacological activities. However, whether ICS II can protect against MI is still unknown. Therefore, this study was designed to investigate the effect and possible underlying mechanism of ICS II on MI both in vivo and in vitro. The results showed that pretreatment with ICS II not only dramatically mitigated MI-induced myocardial damage in mice but also alleviated H9c2 cardiomyocyte injury elicited by oxygen and glucose deprivation (OGD), which were achieved by suppressing mitochondrial oxidative stress and apoptosis. Furthermore, ICS II elevated the phosphorylation level of adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) expression, thereby activating SIRT3. However, these protective effects of ICS II on MI injury were largely abolished in SIRT3-deficient mice, manifesting that ICS II-mediated cardioprotective effects are, at least partly, due to the presence of SIRT3. Most interestingly, ICS II directly bound with SIRT3, as reflected by molecular docking, which indicated that SIRT3 might be a promising therapeutic target for ICS II-elicited cardioprotection in MI. In conclusion, our findings illustrate that ICS II protects against MI-induced oxidative injury and apoptosis by targeting SIRT3 through regulating the AMPK/PGC-1α pathway.

Bioavailability Improvement Strategies for Icariin and Its Derivates: A Review.

In recent years, there has been considerable interest in icariin (ICA) and its derivates, icariside II (ICS) and icaritin (ICT), due to their wide range of potential applications in preventing cancer, cardiovascular disease, osteoporosis, delaying the effects of Alzheimer's disease, treating erectile dysfunction, etc. However, their poor water solubility and membrane permeability, resulting in low bioavailability, dampens their potential beneficial effects. In this regard, several strategies have been developed, such as pharmaceutical technologies, structural transformations, and absorption enhancers. All these strategies manage to improve the bioavailability of the above-mentioned flavonoids, thus increasing their concentration in the desired places. This paper focuses on gathering the latest knowledge on strategies to improve bioavailability for enhancing the efficacy of icariin, icariside II, and icaritin. We conclude that there is an opportunity for many further improvements in this field. To the best of our knowledge, no such review articles scoping the bioavailability improvement of icariin and its derivates have been published to date. Therefore, this paper can be a good starting point for all those who want to deepen their understanding of the field.