Osthole Research Articles

Osthole attenuates asthma-induced airway epithelial cell apoptosis and inflammation by suppressing TSLP/NF-κB-mediated inhibition of Th2 differentiation

ObjectiveThe aim of this study was to investigate the influence of osthole (OS) on asthma-induced airway epithelial cell apoptosis and inflammation by restraining Th2 differentiation through suppressing TSLP/NF-κB.MethodsAn asthma mouse model and an inflammation cell model were constructed with ovalbumin (OVA) and lipopolysaccharide (LPS), respectively. CD4 + T cells were treated with IL-4 to induce Th2 differentiation. Model mice were treated with OS (15,40 mg/kg) for 7 days, and 10 µg/mL OS was added to cell treatment groups. The levels of relevant indices were detected by RT‒qPCR, HE and Masson staining, Western blotting, ELISA and flow cytometry.ResultsIn a mouse asthma model, TSLP expression was elevated, and the NF-κB pathway was activated. Therefore, OS could restrain the apoptosis and inflammation of airway epithelial cells. Downstream mechanistic studies revealed that OS can suppress Th2 differentiation by restraining the level of TSLP and NF-κB nuclear translocation, thus facilitating the proliferation of airway epithelial cells, restraining their apoptosis and inflammation, and alleviating airway inflammation in asthmatic mice.ConclusionOS can inhibit Th2 differentiation by inhibiting the TSLP and NF-κB pathways, which can reduce the apoptosis and inflammation of airway epithelial cells caused by asthma.

Tumor-Specific Nano-Herb Delivery System with High L-Arginine Loading for Synergistic Chemo and Gas Therapy against Cervical Cancer.

Cancer metastasis poses significant challenges in current clinical therapy. Osthole (OST) has demonstrated efficacy in treating cervical cancer and inhibiting metastasis. Despite these positive results, its limited solubility, poor oral absorption, low bioavailability, and photosensitivity hinder its clinical application. To address this limitation, a glutathione (GSH)-responded nano-herb delivery system (HA/MOS@OST&L-Arg nanoparticles,HMOA NPs) is devised for the targeted delivery of OST with cascade-activatable nitric oxide (NO) release. The HMOA NPs system is engineered utilizing enhanced permeability and retention (EPR) effects and active targeting mediated by hyaluronic acid (HA) binding to glycoprotein CD44. The cargoes, including OST and L-Arginine (L-Arg), are released rapidly due to the degradation of GSH-responsive mesoporous organic silica (MOS). Then abundant reactive oxygen species (ROS) are produced from OST in the presence of high concentrations of NAD(P)H quinone oxidoreductase 1 (NQO1), resulting in the generation of NO and subsequently highly toxic peroxynitrite (ONOO-) by catalyzing guanidine groups of L-Arg. These ROS, NO, and ONOO- molecules have a direct impact on mitochondrial function by reducing mitochondrial membrane potential and inhibiting adenosine triphosphate (ATP) production, thereby promoting increased apoptosis and inhibiting metastasis. Overall, the results indicated that HMOA NPs has great potential as a promising alternative for the clinical treatment of cervical cancer.

Synergistic effect of osthole and notopterol combination against Alzheimer's disease and osteoporosis by applying zebrafish AD/OP comorbidity model

Alzheimer's disease (AD) and osteoporosis (OP) are both serious degenerative diseases, with the potential for concurrent occurrence in clinical settings, and they share certain pathological correlations. Osthole (OST) and notopterol (NOT) are the main active ingredients in traditional Chinese medicine, Angelica pubescens and Notopterygium incisum, respectively, and they exhibit neuroprotective and osteoprotective effects. However, whether the combination of OST and NOT produces a synergistic effect against AD and/or OP remains unclear. The aim of this study was to investigate whether the combination of OST and NOT could produce synergistic anti-AD and/or OP effects using the previously constructed zebrafish AD/OP comorbidity model. Active compounds with anti-AD and OP effects were screened from Angelica pubescens and Notopterygium incisum through network pharmacology, identifying OST and NOT, respectively. Then, the AlCl3-induced (Aluminum chloride, AlCl3) AD combined with OP zebrafish model, in conjunction with the Chou-Talalay synergy evaluation model, was employed to assess whether the OST and NOT combination produced synergistic effects against AD and/or OP. Furthermore, a CuSO4-induced (Copper sulfate, CuSO4) inflammation zebrafish model was used to investigate whether the combination of OST and NOT produced synergistic anti-inflammatory effects, thereby resulting in synergistic anti-AD and/or OP effects. The results demonstrated that the OST-NOT combined treatment produced a synergistic anti-AD and OP effect. Moreover, the combined treatment of OST and NOT significantly inhibited nitric oxide (NO) and reactive oxygen species (ROS) release more effectively than OST or NOT alone, indicating a synergistic anti-inflammatory effect of the OST and NOT combined treatment.

The Effect of Osthole on Transient Receptor Potential Channels: A Possible Alternative Therapy for Atopic Dermatitis.

Chronic recurrent skin inflammation and severe itching in patients with atopic dermatitis (AD) significantly impair their quality of life. The H4 histamine receptor plays a key role in histamine-induced itching. During the skin inflammation associated with AD, pro-inflammatory mediators (interleukins, cytokines) are released from neurons. Ultimately, a cascade of reactions leads to the activation and sensitization of transient receptor potential channels (TRP), which exacerbate the inflammation and itching associated with AD. Osthole (OST) is a natural coumarin with a proven versatile pharmacological effect: anti-cancer, anti-inflammatory and immunomodulatory. However, the molecular mechanism of OST in relieving inflammation in histamine-mediated itching is not yet clear. In the studies presented, the possible effect of the OST action on the inhibition of the gene expression of the histamine H4 receptor and the key genes of the TRP channels as well as on the concentration of proinflammatory interleukins was analyzed. Inflammation was induced in a 3D skin model and a keratinocyte cell line Normal Human Epidermal Keratinocytes (NHEK) identical to that of AD, and then OST was administered at various doses. The concentrations of IL-4/-13 were determined by ELISA. RNA was isolated from the 3D skin cells and the NHEK cell line, and the qPCR method was used to determine the expression of: IL-4α, H4R, TRPV1, TRPV4, TRPM8 analyzed. The study showed that OST significantly reduced the secretion of IL-4/-13 in a keratinocyte cell line and in a 3D skin model. In addition, OST was found to significantly decrease the gene expression of IL-4α, H4R, TRPV1, TRPV4 and increase TRPM8 in both the NHEK cell line and the organotypic 3D skin model. The data obtained provide the first in vitro evidence of itch relief following the application of OST to atopic skin. Research on the use of OST as an active component of emollients in the treatment of AD should be continued in the future.

Designing nanostructured lipid carriers modified with folate-conjugated chitosan for targeted delivery of osthole to HT-29 colon cancer cells: investigation of anticancer, antioxidant, and antibacterial activities

AbstractThe present study proposed to design nanostructured lipid carriers (NLC) coated with chitosan (CS) conjugated folate (FA) for the targeted delivery of Osthole (OST) to the HT-29 colon cancer cell line and improve its anticancer capability. To assess the physicochemical characteristics of OST-loaded NLC decorated with CS-conjugated FA (OST-NCF-NPS), several techniques, including DLS, SEM, and FTIR, were applied. After determining the encapsulation efficiency of OST in CSFA-modified NLC-NPs, an MTT test was conducted to evaluate the cytotoxic effects of this nano platform on the HT-29 cancer cell line in comparison to normal HFF cells. Possible mechanisms of apoptosis in cancer cells treated with OST-NCF-NPs were examined using qPCR, flow cytometry, and AO/PI fluorescent staining methods. Moreover, the antioxidant capacity of these biosynthesized nanocarriers was determined using ABTS and DPPH methods, and their antibacterial potential was measured through disk diffusion, MIC, and MBC assays. According to the findings, OST-NCF-NPS had the ideal average size of 179.19 nm, low polydispersity (PI = 0.23), acceptable physical stability (ζ-potential = + 18.99 mV), and high entrapment efficiency (83.5%). The MTT data demonstrated the selective cytotoxicity of NPs toward cancerous cells compared to normal cells. Cell cycle and Annexin V/Propidium Iodide (AnV/PI) analysis indicated that OST-NCF-NPs increased the sub-G1 population and AnV/PI-positive cells. The occurrence of programmed cell death in the treated cells was also verified by altered expression of proapoptotic (BAX and caspase-3) and antiapoptotic (Bcl-2) genes. Furthermore, the NPs exhibited strong antibacterial activity, particularly against gram-negative bacteria, and high antioxidant effects in reducing ABTS and DPPH-free radicals. Graphical Abstract

Osthole-infused polyurethane flexible coatings for enhanced underwater drag reduction and robust anti-biofouling

Excessive underwater drag, coupled with a heavy marine fouling, not only adversely affects the operation and speed of ships but also leads to increased energy and fuel consumption. Inspired by the remarkable drag reduction and anti-biofouling capabilities of dolphin skin, we have developed a flexible polyurethane (PU) coating infused with osthole (OST). This coating not only exhibits exceptional drag reduction by postponing the boundary layer transition from laminar to turbulent flow but also demonstrates robust anti-biofouling properties under both dynamic and static scenarios. The resultant PUx/OSTy composite coating achieved the drag reduction efficiency of 8.20 % to 12.65 % with mean flow velocities ranging from 1 to 6 m/s. The PUx/OSTy surfaces also displayed highly enhanced anti-biofouling performances, with ∼98.4 % and 97.5 % reduction for green algae Chlorella and the diatom N. closterium, respectively, as well as ∼99.4 % reduction against P. pantotrophus. Furthermore, the PUx/OSTy coating had amazing durability in extreme environments, such as light aging, cycle sand abrasion, thermal aging, ultrasonic treatment, and tape-peeling. Therefore, this study underscores the potential of the bioinspired flexible coating for the application of underwater devices with prominent drag reduction and antifouling performance.

Osthole ameliorates myonecrosis caused by Clostridium perfringens type A infection in mice

This study aimed to investigate the protective effect of the nature product osthole (OST) against Clostridium perfringens type A infection-caused myonecrosis in a mouse model. Male mice were divided into (1) control, (2) infected, (3) OST50 and (4) OST100 treatment groups. In the infected groups, mice were intramuscularly injected with 1 × 108 CFU of C. perfringens per day for 6 days. Mice in the OST50 and OST100 groups were administrated intraperitoneally with OST at the doses of 50 or 100 mg/kg per day post C. perfringens infection. Our results showed that C. perfringens infection caused marked necrosis and inflammatory cell infiltration in the muscle tissues of mice. Mice in the OST50 and OST100 treatment groups displayed significantly attenuated C. perfringens infection-induced lipid peroxidation, oxidative stress, and apoptosis in their muscle tissue. Furthermore, OST treatment significantly downregulated the expressions of NF-κB, IL-1β, and TNF-α mRNA and protein levels, while concomitantly upregulating the expressions of Nrf2 and HO-1 mRNA and protein. OST treatments also inhibited the expression of phosphorylation (p)-p38, p-mTOR, and p-Erk1/2 proteins, and upregulated LC3II and Beclin1 proteins. In summary, our results reveal that OST therapy confers a protective effect against C. perfringens infection-induced oxidative stress and inflammation in muscle tissue, via activation of Nrf2/HO-1 and autophagy pathways and inhibition of p38, Erk1/2 and NF-κB pathways.

Osthole protects H9c2 cardiomyocytes against trastuzumab-induced damage by enhancing autophagy through the p38MAPK/mTOR signaling pathway

Trastuzumab (TRZ) is a novel targeted anti-tumor agent that significantly improve the survival of patients with human epidermal growth factor receptor (HER2) positive breast cancer. However, its clinical application is limited due to the side effects of cardiotoxicity. Osthole (OST), a coumarin derivative isolated from Cnidium monnieri (L.) Cusson, has previously demonstrated cardioprotective effects. The aim of this study was to observe the protective effect of OST on TRZ-induced cardiomyocytes damage and to explore its potential mechanism. The results showed that OST pretreatment could significantly inhibit TRZ-induced cardiomyocytes damage, markedly increase the ratio of LC3II/I and Beclin-1 protein expression, and reduce the protein expression of p62. OST pretreatment significantly attenuated oxidative stress and apoptosis induced by TRZ, as evidenced by reducing intracellular ROS level, the Bax/Bcl-2 ratio, and Caspase-3 protein expression. Additionally, OST markedly increased the phosphorylation level of p38MAPK and decreased the mTOR phosphorylation level. However, the effects of OST on enhancing autophagy, reducing oxidative stress, apoptosis, and the phosphorylation level of mTOR were reversed after the addition of 3-MA or SB203580. Molecular docking results indicated that OST exerted a good binding ability with the p38MAPK protein. Our findings suggested that OST could protect TRZ-induced cardiomyocytes damage by enhancing autophagy via the p38MAPK/mTOR signaling pathway.

Development of Osthole-Loaded Microemulsions as a Prospective Ocular Delivery System for the Treatment of Corneal Neovascularization: In Vitro and In Vivo Assessments.

Osthole (OST), a natural coumarin compound, has shown a significant inhibitory effect on corneal neovascularization (CNV). But, its effect on treating CNV is restricted by its water insolubility. To overcome this limitation, an OST-loaded microemulsion (OST-ME) was created to improve the drug's therapeutic effect on CNV after topical administration. The OST-ME formulation comprised Capryol-90 (CP-90), Cremophor® EL (EL-35), Transcutol-P (TSP) and water, and sodium hyaluronate (SH) was also included to increase viscosity. The OST-ME had a droplet size of 16.18 ± 0.02 nm and a low polydispersity index (0.09 ± 0.00). In vitro drug release from OST-ME fitted well to the Higuchi release kinetics model. Cytotoxicity assays demonstrated that OST-ME was not notably toxic to human corneal epithelial cells (HCECs), and the formulation had no irritation to rabbit eyes. Ocular pharmacokinetics studies showed that the areas under the concentration-time curves (AUC0-t) in the cornea and conjunctiva were 19.74 and 63.96 μg/g*min after the administration of OST-ME, both of which were 28.2- and 102.34-fold higher than those after the administration of OST suspension (OST-Susp). Moreover, OST-ME (0.1%) presented a similar therapeutic effect to commercially available dexamethasone eye drops (0.025%) on CNV in mouse models. In conclusion, the optimized OST-ME exhibited good tolerance and enhanced 28.2- and 102.34-fold bioavailability in the cornea and conjunctiva tissues compared with suspensions in rabbit eyes. The OST-ME is a potential ocular drug delivery for anti-CNV.

Multifunctional Gomisin B enhances cognitive function in APP/PS1 transgenic mice by regulating Aβ clearance and neuronal apoptosis

This study aimed to investigate the potential effects of Gomisin B, a natural compound known for its inhibition of CYP3A4, on cognitive dysfunction in APP/PS1 transgenic mice with Alzheimer's disease (AD). Additionally, the study explored the combined effects of Gomisin B and Osthole (OST). The research involved male wild-type (WT) mice and 7-month-old APP/PS1 transgenic AD mice. The assessment of behavioral changes included the use of the open field test (OFT) and the Morris water maze (MWM). OST levels in brain tissue were quantified using LC-MS/MS, while levels of oxidative stress were measured through an assay kit. Neuronal apoptosis was studied using Nissl staining, RT-qPCR, and immunofluorescence. Amyloid plaque clearance was assessed using thioflavine-S (Th-S) staining, RT-qPCR, and ELISA. The results of the study revealed that Gomisin B led to a significant improvement in cognitive dysfunction in APP/PS1 mice. Moreover, the simultaneous administration of OST and Gomisin B demonstrated enhanced therapeutic effects. These effects were attributed to the inhibition of β-site APP-Cleaving Enzyme 1 (BACE1) and oxidative stress by Gomisin B, along with its anti-apoptotic properties. The combined use of OST and Gomisin B exhibited a synergistic impact, resulting in more pronounced anti-oxidant and anti-apoptotic effects. In summary, this study pioneers the exploration of Gomisin B's multifunctional anti-AD properties in APP/PS1 mice. The findings provide a solid groundwork for the development of anti-Alzheimer's drugs based on natural active ingredients.

Modulating endoplasmic reticulum stress in APP/PS1 mice by Gomisin B and Osthole in Bushen-Yizhi formula: Synergistic effects and therapeutic implications for Alzheimer's disease

BackgroundAlzheimer's disease (AD) is a prevalent neurodegenerative disorder with no effective cure. Targeting endoplasmic reticulum (ER) stress pathway may offer a novel approach to ameliorate cognitive deficits in AD. Bushen-Yizhi formula (BSYZ), a traditional Chinese medicine (TCM) prescription, has shown potential benefits for AD. To facilitate the development of new therapeutic agents for AD, it is important to identify the active components and the underlying mechanisms of BSYZ against AD. PurposeThe aim of this study was to systematically screen the active components of BSYZ that could improve learning and memory impairment in AD by modulating ER stress pathway. MethodsA drug-target (D-T) network was constructed to analyze the herbal components of BSYZ. Network proximity method was used to identify the potential anti-AD components that targeted ER stress and evaluate their synergistic effects. The absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties and the literature evidence were considered to select promising candidates for further validation. The selected components were tested in vitro using an AD cell model (APPswe-SH-SY5Y). In vivo anti-AD effects of the components were assessed in APP/PS1 double-transgenic mice. Results58 potential anti-AD components targeting ER stress were detected by network proximity analysis, and 13 out of them were selected based on ADMET properties and literature evidence. In vitro experiments confirmed that 5 components, namely gomisin B, β-Carotene, imperatorin, chrysophanol, and osthole (OST), exhibited anti-AD effects on the APPswe-SH-SY5Y model. Moreover, network proximity analysis suggested that OST and Gomisin B might have synergistic effects on modulating ER stress. In vivo experiments demonstrated that OST, Gomisin B, OST+Gomisin B, and BSYZ all improved learning and memory function in APP/PS1 mice. Gomisin B and OST also restored cellular morphology and tissue structure in APP/PS1 mice. Thioflavine-S (Th-S) staining revealed that they reduced amyloid plaque deposition in the brain tissue of AD model mice. The qPCR results indicated that BSYZ, OST, and Gomisin B differentially regulated IRE1α, PERK, EIF2α, DDIT3, and Caspase 12 expression levels, while the OST and Gomisin B co-administration group showed better efficacy. This trend was further confirmed by immunofluorescence experiments. ConclusionThis study identified the active components of BSYZ that could ameliorate learning and memory impairment in AD by targeting ER stress pathway. OST and Gomisin B exhibited synergistic effects on modulating ER stress and reducing amyloid plaque deposition in vivo. Overall, our study elucidated the molecular mechanisms of BSYZ and its active components in attenuating AD symptoms which suggested the therapeutic potential of TCM for AD.

Covalent modification of proteins by osthole reactive metabolites using proteomic approaches.

Osthole (OST) is a bioactive natural coumarin derived from the plant Cnidium monnieri (L.) Cusson fruit (She Chuang Zi), which has various pharmacological and biological activities. OST contains an α,β-unsaturated lactone, which is an electrophilic group that tends to be metabolized into reactive metabolites (RMs). Then, RMs are able to covalently modify nucleophilic amino acid (AA) residues of target proteins. However, few researchers considered the contribution of the covalent modification induced by OST or its metabolites. This study aims to investigate the metabolic profile and the metabolites-protein modification of OST. The metabolites of OST were qualitatively identified using UHPLC-Q-TOF-MS. The RMs modification patterns and potentially modified AA residues were confirmed by UHPLC-Q-TOF-MS using rat liver microsomes (RLMs) and model AAs. Finally, the modified peptides derived from high-abundance microsomal peptides were separated via nano-LC-Orbitrap-MS, and then RM-modified proteins were identified using a proteome discoverer. In the presence of RLMs, OST could rapidly be metabolized within 1 h and hardly identified at 4 h. We detected 10 OST metabolites, 13 OST metabolites-NAC (N-acetyl cysteine) adducts, 3 NAL (N-acetyl lysine) adducts, and 11 GSH (glutathione) adducts. Furthermore, 16 RM-modified protein targets were identified, many of which are included in the essential biological processes of OST's anti-Alzheimer's disease (AD) and anti-tumor. This study provides a novel perspective on the molecular mechanism of OST's pharmacological activities, as well as identifies potential targets for further development and application of OST and other Natural products (NPs).

An integrated supramolecular fungicide nanoplatform based on pH-sensitive metal–organic frameworks

The construction of an integrated nanoplatform with controlled fungicide delivery features in the specific microenvironment produced by fungal pathogens is a highly desirable strategy to improve the utilization of fungicides. Herein, we report a supramolecular fungicide delivery system based on benzimidazole-modified NH2-MIL-101(Fe) metal–organic frameworks (B-MIL-101(Fe) MOFs) as carriers loaded with osthole (OS), and β-cyclodextrin (β-CD) as nanovalves to form β-CD@B-MIL-101(Fe)-OS. The nanoplatform can release the loaded OS for fungus control through self-degradation of the MOFs skeleton in an oxalic acid microenvironment produced by Botrytis cinerea. The experimental results exhibit that the constructed supramolecular fungicide delivery system could effectively inhibit mycelial growth and protect the tomatoes from infection by B. cinerea during the ripening stage. This strategy constructs a facile and integrated supramolecular drug delivery system for B. cinerea control and opens up a new avenue for the sustainable development of modern agriculture.

Osthole regulates N6-methyladenosine-modified TGM2 to inhibit the progression of rheumatoid arthritis and associated interstitial lung disease.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease, and RA interstitial lung disease (ILD) is a severe complication of RA. This investigation aims to determine the effect and underlying mechanism of osthole (OS), which could be extracted from Cnidium, Angelica, and Citrus plants and evaluate the role of transglutaminase 2 (TGM2) in RA and RA-ILD. In this work, OS downregulated TGM2 to exert its additive effect with methotrexate and suppress the proliferation, migration, and invasion of RA-fibroblast-like synoviocytes (FLS) by attenuating NF-κB signaling, resulting in the suppression of RA progression. Interestingly, WTAP-mediated N6-methyladenosine modification of TGM2 and Myc-mediated WTAP transcription cooperatively contributed to the formation of a TGM2/Myc/WTAP-positive feedback loop through upregulating NF-κB signaling. Moreover, OS could downregulate the activation of the TGM2/Myc/WTAP-positive feedback circuit. Furthermore, OS restrained the proliferation and polarization of M2 macrophages to inhibit the aggregation of lung interstitial CD11b+ macrophages, and the effectiveness and non-toxicity of OS in suppressing RA and RA-ILD progression were verified in vivo. Finally, bioinformatics analyses validated the importance and the clinical significance of the OS-regulated molecular network. Taken together, our work emphasized OS as an effective drug candidate and TGM2 as a promising target for RA and RA-ILD treatment.

Osthole Inhibits M1 Macrophage Polarization and Attenuates Osteolysis in a Mouse Skull Model.

Excessive bone resorption due to increased inflammatory factors is a common feature of inflammatory lytic bone diseases. This group of diseases is effectively treated with drugs. In recent years, many studies have reported that traditional Chinese medicine herbs have substantial effects on inflammation, osteoclast differentiation and maturation, and bone destruction. Herein, we investigated the effects of osthole (OST) on lipopolysaccharide- (LPS-) induced macrophage polarization, inflammatory responses, and osteolysis. In vitro, we used immunofluorescence and quantitative real-time polymerase chain reaction assays to confirm whether bone marrow-derived macrophages showed an increased expression of inflammatory factors, such as interleukin-6, iNOS, CCR7, and CD86, in the presence of LPS. However, we found that such expression was suppressed and that the M2 macrophage expression increased in the presence of OST. OST reduced LPS- and RANKL-induced intracellular reactive oxygen species production in the bone marrow-derived macrophages. Further, it potently suppressed osteoclast differentiation and osteoclast-specific gene expression by suppressing the P38/MAPK and NF-κB pathways. Consistent with the in vitro observations, OST greatly ameliorated LPS-induced bone resorption and modulated the ratio of macrophages at the site of osteolysis. Taken together, OST has great potential for use in the management of osteolytic diseases.

UPLC-Q-TOF/MS-Based Metabolomics Approach Reveals Osthole Intervention in Breast Cancer 4T1 Cells.

Osthole (OST) is a simple coumarin derivative with pharmacological effects in many types of cancer cells. However, its role and its mechanism of action in breast cancer 4T1 cells remain unclear. In this study, we explored the effects and potential mechanisms of action of OST in 4T1 cells. The MTT, PI, and Annexin V-FITC/PI methods were used to evaluate the effects of OST-treated and untreated 4T1 cells on viability, cell cycle, and apoptosis, respectively. UPLC-Q-TOF/MS combined with multivariate data analysis was used to screen potential biomarkers relevant to the therapeutic mechanisms of OST. Additionally, mTOR, SREBP1, and FASN protein levels were detected using western blotting in OST-treated and untreated 4T1 cells. OST inhibited 4T1 cell proliferation, blocked the cells from remaining in S-phase, and induced apoptosis. In 4T1 cells, OST mainly affected the phospholipid biosynthesis, methyl histidine metabolism, pyrimidine metabolism, and β-oxidation of very long chain fatty acid pathways, suggesting that metabolic changes related to lipid metabolism-mediated signaling systems were the most influential pathways, possibly via inhibition of mTOR/SREBP1/FASN signaling. Our findings reveal biomarkers with potential therapeutic effects in breast cancer and provide insight into the therapeutic and metabolic mechanisms of OST in 4T1 cells.

Osthole Antagonizes Microglial Activation in an NRF2-Dependent Manner

Microglia are neuroglia in the brain with an innate immune function and participate in the progress of neurodegenerative diseases. Osthole (OST) is a coumarin derivative extracted from Cnidium monnieri and bears a microglia-antagonizing ability. However, the underlying mechanism of the antagonism is not clear. The lipopolysaccharides-induced microglial BV2 cell line and amyloid-overexpressing fruit fly were used as models to study OST treatment. We found that OST treatment is sufficient to evoke NRF2 cascade under an LPS-induced inflammatory environment, and silencing NRF2 is sufficient to abolish the process. Moreover, we found that OST is sufficient to antagonize microglial activation in both LPS-induced BV2 cells and Aβ-overexpressing fruit flies, and silencing NRF2 abolishes OST's antagonism. Furthermore, OST treatment rescued survival, climbing, and the learning ability of Aβ-overexpressing fruit flies and relieved oxidative stress. In conclusion, we proved that OST antagonizes microglial activation induced by either LPS or Aβ and that NRF2 is necessary for OST's antagonism.

Computational Screening of the Natural Product Osthole and Its Derivates for Anti-Inflammatory Activity.

Osthole (OS) is a natural coumarin with a long history of medicinal use in a variety of diseases, such as itch and menstrual disorders. In recent years, OS has been shown to treat inflammation and reduce the expression and activity of NF-κB, although its mechanism of action is still unclear. Overexpression of inflammatory cytokines can have many negative effects in the body, including inducing preterm labor; thus, the modulation of inflammation by OS and its derivatives may be able to delay preterm birth, increasing neonatal survival rates. The objectives of this study were to screen and identify the derivatives of OS with the highest potential for binding capacity to inflammatory mediators NF-κB, TNF-α, and ERK1, and to measure the drug-like properties of these compounds. GLIDE docking in Schrodinger Maestro software was used to calculate docking scores for a variety of semi-synthetic OS derivatives against three proteins involved in inflammation: NF-κB, TNF-α, and ERK1. Schrodinger Qikprop was also used to measure the pharmaceutically relevant properties of the compounds. The protonated demethoxy osthole 1 showed the highest docking of all the proteins tested, while the deprotonated demethoxy osthole 2 consistently had the lowest scores, denoting the importance of pH in the binding activity of this derivative. The lowest docking was at NF-κB, suggesting that this is less likely to be the primary target of OS. All of the screened derivatives showed high drug potential, based on their Qikprop properties. OS and its derivatives showed potential to bind to multiple proteins that regulate the inflammatory response and are prospective candidates for delaying preterm birth.

Osthole Regulates Secretion of Pro-Inflammatory Cytokines and Expression of TLR2 and NF-κB in Normal Human Keratinocytes and Fibroblasts.

IntroductionOsthole (OST), an active compound isolated from Cnidium monnieri, is used in traditional Chinese medicine to treat a variety of human diseases. Although OST has a good therapeutic effect, the underlying mechanism of its action in inflammatory skin diseases in humans is still unknown.PurposeThe present study aimed to test the hypothesis that OST can be used as an herbal substance that minimizes skin inflammation and barrier dysfunction. In this study, histamine and LPS were used to induce inflammation in skin keratinocytes and fibroblasts to test whether OST can inhibit their responses.MethodsCell migration was analyzed using a wound healing assay. Changes in cell monolayer integrity were assessed by the measurement of transepithelial electrical resistance. Secretion of IL-1β, IL-6, IL-8, TNF-α, CCL2/MCP-1, CCL5/RANTES, and COX-2 was measured by ELISA, while expression of TLR2, NF-κB, and COX-2 was analyzed by qPCR.ResultsOST decreased the level of IL-1β, TNF-α, CCL2/MCP-1 and CCL5/RANTES, and expression of TLR2, NF-κB and COX-2 during histamine/LPS-induced inflammation in human keratinocytes and fibroblasts. OST also improved cell migration and cell barrier function.ConclusionOur results suggest that OST suppresses inflammatory responses via regulation of IL-1β, TNF-α, CCL2/MCP-1 and CCL5/RANTES secretion, and TLR2, and COX-2 expression.

Screening and verification of CYP3A4 inhibitors from Bushen-Yizhi formula to enhance the bioavailability of osthole in rat plasma

Ethnopharmacological relevanceWith the features of multiple-components and targets as well as multifunction, traditional Chinese medicine (TCM) has been widely used in the prevention and treatment of various diseases for a long time. During the application of TCM, the researches about bioavailability enhancement of the bioactive constituents in formula are flourishing. Bushen-Yizhi formula (BSYZ), a TCM prescription with osthole (OST) as one of the main bioactive ingredients, have been widely used to treat kidney deficiency, mental retardation and Alzheimer's disease. However, the underlying biological mechanism and compound-enzyme interaction mediated bioavailability enhancement of OST are still not clearly illuminated. Aim of the studyThe aim of this study is to explore the material basis and molecular mechanism from BSYZ in the bioavailability enhancement of OST. Screening the potential CYP3A4 inhibitors using theoretical prediction and then verifying them in vitro, and pharmacokinetics study of OST in rat plasma under co-administrated of screened CYP3A4 inhibitors and BSYZ were also scarcely reported. Materials and methodsScreening of CYP3A4 inhibitors from BSYZ was performed with molecular docking simulation from systems pharmacology database. The screened compounds were verified by using P450-Glo Screening Systems. A multiple reaction monitoring (MRM) mass spectrometry method was established for OST quantification. Male Sprague-Dawley rats divided into four groups and six rats in each group were employed in the pharmacokinetics study of OST. The administrated conditions were group I, OST (20 mg/kg); group II, BSYZ (containing OST 1 mg/mL, at the dose of 20 mg/kg OST in BSYZ); group III, co-administration of ketoconazole (Ket, 75 mg/kg) and OST (20 mg/kg); group IV, co-administration of CYP3A4 inhibitor (10 mg/kg) and OST (20 mg/kg). They were determined by using HPLC-MS/MS (MRM) and statistical analysis was performed using student’s t-test with p < 0.05 as the level of significance. Results21 potential CYP3A4 inhibitors were screened from BSYZ compounds library. From the results of verification in vitro, we found 4 compounds with better CYP3A4 inhibition efficiency including Oleic acid, 1,2,3,4,6-O-Pentagalloylglucose, Rutin, and Schisantherin B. Under further verification, Schisantherin B exhibited the best inhibitory effect on CYP3A4 (IC50 = 0.339 μM), and even better than the clinically used drug (Ket) at the concentration of 5 μM. In the study of pharmacokinetics, the area under the curve (AUC, ng/L*h) of OST after oral administration of BSYZ, Ket and Schisantherin B (2196.23 ± 581.33, 462.90 ± 92.30 and 1053.03 ± 263.62, respectively) were significantly higher than that of pure OST treatment (227.89 ± 107.90, p < 0.01). ConclusionsSchisantherin B, a profoundly effective CYP3A4 inhibitor screened from BSYZ antagonized the metabolism of CYP3A4 on OST via activity inhibition, therefore significantly enhanced the bioavailability of OST in rat plasma. The results of this study will be helpful to explain the rationality of the compatibility in TCM formula, and also to develop new TCM formula with more reasonable drug compatibility.