Osthole Treatment Research Articles

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.

Oral administration of osthole mitigates maladaptive behaviors through PPARα activation in mice subjected to repeated social defeat stress

Psychological stress induces neuroinflammatory responses, which are associated with the pathogenesis of various psychiatric disorders, such as posttraumatic stress disorder and anxiety. Osthole—a natural coumarin isolated from the seeds of the Chinese herb Cnidium monnieri—exerts anti-inflammatory and antioxidative effects on the central nervous system. However, the therapeutic benefits of osthole against psychiatric disorders remain largely unknown. We previously demonstrated that mice subjected to repeated social defeat stress (RSDS) in the presence of aggressor mice exhibited symptoms of posttraumatic stress disorder, such as social avoidance and anxiety-like behaviors. In this study, we investigated the therapeutic effects of osthole and the underlying molecular mechanisms. Osthole exerted therapeutic effects on cognitive behaviors, mitigating anxiety-like behaviors and social avoidance in a mouse model of RSDS. The anti-inflammatory response induced by the oral administration of osthole was strengthened through the upregulation of heme oxygenase-1 expression. The expression of PPARα was inhibited in mice subjected to RSDS. Nonetheless, osthole treatment reversed the inhibition of PPARα expression. We identified a positive correlation between heme oxygenase-1 expression and PPARα expression in osthole-treated mice. In conclusion, osthole has potential as a Chinese herbal medicine for anxiety disorders. When designing novel drugs for psychiatric disorders, researchers should consider targeting the activation of PPARα.

Osthole Activates the Cholinergic Anti-Inflammatory Pathway via α7nAChR Upregulation to Alleviate Inflammatory Responses.

Osthole (also known as Osthol) is the main anti-inflammatory coumarin found in Cnidium monnieri and severs as the exclusive quality-controlled component according the Chinese Pharmacopoeia. However, its underlying anti-inflammatory mechanism remains unknown. In this study, we demonstrated that Osthole treatment significantly inhibited the generation of TNF-α, but not IL-6 in the classical LPS-stimulated RAW264.7 macrophage model. In addition, LPS induced the activation of both MAPK and NF-κB signalling pathways, of which the former was dose-dependently restrained by Osthole via suppressing the phosphorylation of JNK and P38 proteins, while the phosphorylation of IκB and P65 proteins remained unaffected. Interestingly, Osthole dose-dependently up-regulated the expression of the key cholinergic anti-inflammatory pathway regulator α7nAChR, and the TNF-α inhibition effect of Osthole was also significantly alleviated by the treatment of α7nAChR antagonist methylbetaine. These results demonstrate that Osthole may regulate TNF-α by promoting the expression of α7nAChR, thereby activate the vagus nerve-dependent cholinergic anti-inflammatory pathway.

Comparative physiological and transcriptome analysis provide insights into the inhibitory effect of osthole on Penicillium choerospondiatis

Blue mold induced by Penicillium choerospondiatis is a primary cause of growth and postharvest losses in the fruit of Phyllanthus emblica. There is an urgent need to explore novel and safe fungicides to control this disease. Here, we demonstrated osthole, a natural coumarin compound isolated from Cnidium monnieri, exhibited a strong inhibitory effect on mycelia growth, conidial germination rate and germ tube length of P. choerospondiatis, and effectively suppressed the blue mold development in postharvest fruit of P. emblica. The median effective concentration of osthole was 9.86 mg/L. Osthole treatment resulted in cellular structural disruption, reactive oxygen species (ROS) accumulation, and induced autophagic vacuoles containing cytoplasmic components in fungal cells. Transcriptome analysis revealed that osthole treatment led to the differentially expressed genes mainly enriched in the cell wall synthesis, TCA cycle, glycolysis/ gluconeogenesis, oxidative phosphorylation. Moreover, osthole treatment led to increase genes expression involved in peroxisome, autophagy and endocytosis. Particularly, the autophagy pathway related genes (PcATG1, PcATG3, PcATG15, PcATG27, PcYPT7 and PcSEC18) were prominently up-regulated by osthole. Summarily, these results revealed the potential antifungal mechanism of osthole against P. choerospondiatis. Osthole has potentials to develop as a natural antifungal agent for controlling blue mold disease in postharvest fruits.

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.

GelMA Hydrogel as a Promising Delivery System for Osthole in the Treatment of Rheumatoid Arthritis: Targeting the miR-1224-3p/AGO1 Axis.

Rheumatoid arthritis (RA) is a multifaceted, chronic, progressive autoimmune disease. This study aims to explore the potential benefits of an enhanced drug delivery system utilizing optimized Gelatin Methacryloyl (GelMA) vectors in RA management. We evaluated the levels of miR-1124-3p and AGO1 in RA tissues and cell lines using qPCR, WB, and immunofluorescence. The effects of osthole on inflammatory response and joint morphology were determined by qPCR, H&E staining, and micro-CT. The data showed that miR-1224-3p was downregulated in RA tissues and HUM-iCell-s010RA cells, while the overexpression of miR-1224-3p in HUM-iCell-s010RA cells reduced the expression of IL-6 and IL-1β. Luciferase assay demonstrated that AGO1 was a direct target gene of miR-1224-3p. Additionally, osthole treatment increased miR-1224-3p levels and decreased AGO1 expression. The release data showed that osthole loaded on GelMA was released at a slower rate than free osthole. Further studies in a mouse model of CIA confirmed that osthole-loaded GelMA was more effective in attenuating osteopenia in RA as well as alleviating autoimmune arthritis. These findings suggest that osthole can regulate the miR-1224-3p/AGO1 axis in RASFs cells and has the potential to be developed as a clinical anti-RA drug. GelMA could provide a new approach to long-term RA treatment.

Osthole inhibits malignant phenotypes and induces ferroptosis in KRAS-mutant colorectal cancer cells via suppressing AMPK/Akt signaling.

Ferroptosis is a form of cell death driven by iron-dependent lipid peroxidation. Intriguingly, KRAS-mutant cancers are particularly vulnerable to ferroptosis. Osthole is a natural coumarin extracted from Cnidium spp. and other Apiaceous plants. In the present study, we explored the antitumor potential of osthole in KRAS-mutant colorectal cancer (CRC) cells. Cell viability assay, EdU incorporation assay, flow cytometry, tumor xenograft model, western blot, immunochemistry staining, immunofluorescence, transcriptome RNA sequencing and quantitative reverse transcription-PCR were performed to evaluate the influence of osthole treatment on KRAS-mutant CRC cells. We found that osthole treatment suppressed proliferation and tumor growth of KRAS-mutant CRC cell lines HCT116 and SW480. Moreover, osthole treatment increased ROS production and induced ferroptosis. Osthole treatment also promoted autophagy, but inhibition of autophagy by ATG7 knockdown or 3-MA showed no influence on osthole-induced ferroptosis. In comparison, osthole increased lysosomal activation, and co-treatment with lysosome inhibitor Baf-A1 attenuated osthole-induced ferroptosis. Besides, osthole treatment reduced the phosphorylation of AMPK, Akt and mTOR in HCT116 and SW480 cells, while restored AMPK signaling by AMPK agonist AICAR partially abrogated ferroptosis induced by osthole treatment. Finally, co-treatment with osthole increased the cytotoxicity of cetuximab in KRAS-mutant CRC cells in vitro and in vivo. Our results suggested that the natural product osthole exerted its anticancer effects in KRAS-mutant CRC cells via inducing ferroptosis, and this was partially through inhibiting AMPK/Akt/mTOR signaling. Our results may expand our current knowledge for the use of osthole as an anticancer agent.

Matrine combined with Osthole inhibited the PERK apoptosis of splenic lymphocytes in PCV2-infected mice model

BackgroundPorcine circovirus type 2 (PCV2) is one of the major pathogens commonly found in pigs, which causes immunosuppression and apoptosis. Vaccination and a single drug cannot totally prevent and treat PCV2 infection. Our previous in vitro study reported that the synergistic anti-PCV2 effect of Matrine and Osthole was better than that of Matrine or Osthole alone, This study was aimed to evaluate the synergistic anti-PCV2 effect as well as the underline molecular mechanism of Matrine and Osthole in Kunming (KM) mice model infected with PCV2. KM mice were randomly divided into 8 groups namely control group, PCV2 infected, Matrine combined with Osthole high dose treatment (40 mg/kg + 12 mg/kg), medium dose treatment (20 mg/kg + 6 mg/kg), low dose treatment (10 mg/kg + 3 mg/kg), Matrine treatment (40 mg/kg), Osthole treatment (12 mg/kg) and Ribavirin positive control (40 mg/kg) groups. PCV2 was intraperitoneally (i.p.) injected in all mice except the control group. 5 days of post-infection (dpi), mice in different treatment groups were injected i.p. with various doses of Matrine, Osthole and Ribavirin once daily for the next 5 consecutive days.ResultsThe synergistic inhibitory effect of Matrine and Osthole on PCV2 replication in mouse liver was significantly heigher than that of Matrine and Osthole alone. The expression of GRP78, p-PERK, p-eIF2α, ATF4, CHOP, cleaved caspase-3 and Bax proteins were significantly reduced, while that of Bcl-2 was significantly increased in Matrine combined with Osthole groups, which alleviated the pathological changes caused by PCV2, such as interstitial pneumonia, loss of spleen lymphocytes, infiltration of macrophages and eosinophils.ConclusionsThe synergistic anti-apoptotic effect of Matrine and Osthole was better than their alone effect, Both Matrine and Osthole had directly inhibited the expression of PCV2 Cap and the apoptosis of spleen cells induced by PCV2 Cap through the PERK pathway activated by endoplasmic reticulum (ER) GRP78. These results provided a new insight to control PCV2 infection and provide good component prescription candidate for the development of novel anti-PCV2 drugs.

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.

Osthole Alleviates D-Galactose-Induced Liver Injury In Vivo via the TLR4/MAPK/NF-κB Pathways

Osthole, a coumarin derivative, is found in several medicinal herbs. However, the protective effects of osthole against D-galactose (D-Gal)-induced liver injury still remain unclear. In this study, osthole treatment effectively reversed D-Gal-induced liver injury, according to the results of liver HE staining, and improved ALT and AST activities. Feeding with D-Gal significantly increased MDA content, and reduced the level or activity of SOD, CAT and GSH-Px, which were all alleviated by osthole intervention. Meanwhile, osthole treatment significantly inhibited the D-Gal-induced secretion of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6, in both serum and liver tissue. Investigations revealed that osthole ameliorated the D-Gal-induced activation of TLR4, MYD88 and its downstream signaling pathways of MAPK (p38 and JNK) and NF-κB (nucleus p65). Therefore, osthole mediates a protective effect against D-Gal-induced liver injury via the TLR4/MAPK/NF-κB pathways, and this coumarin derivative could be developed as a candidate bioactive component for functional food.

Untargeted UPLC-MS-based metabolomics analysis reveals the metabolic profile of intrahepatic cholangiocarcinoma process and the intervention effect of Osthole in mice

Intrahepatic cholangiocarcinoma (ICC) is the second most common malignant liver tumor after hepatocellular carcinoma (HCC). It poses a significant risk to human health as it is highly malignant, insidious, and prone to recurrence after surgery. However, current diagnostic techniques and treatments for ICC are limited. Therefore, it is critical to investigate biomarkers of ICC for the discovery of novel drugs that can effectively treat ICC. In a previous study on the molecular mechanisms of ICC, proto-oncogenes AKT and Yap were found to often be overexpressed or activated in ICC, and the co-expression of AKT/YapS127A could promote the formation of ICC in mice. In this study, co-expression of AKT/YapS127A in an ICC mouse model was established via hydrodynamic transfection. To identify ICC biomarkers, we investigated the inhibitory effect of Osthole on the formation of ICC in mice induced with the co-expression of AKT/YapS127A. Furthermore, we preliminarily established the pathogenesis of ICC and the mechanism of Osthole. The metabolomic profiles for different stages in ICC formation process and Osthole treatment were examined via UHPLC-Q-TOF-MS/MS. The morphology (hematoxylin and eosin and Sirius red staining) and immunohistochemistry (CK19, Ki67, Vimentin, and α-SMA) results demonstrated that the ICC mouse model was useful. The ICC formation process was divided into three stages: early (1–2 weeks), formative (3–4 weeks), and late (5–7 weeks) stages. For the metabolomic profiles of ICC formation process, 11 differential metabolite species were identified at the early stage, 14 at the formative stage, and 20 at the late stage. The model group mice mainly showed dysregulation of amino acids and lipid metabolism compared with the control group mice. For the metabolomic profiles of Osthole treatment, 14 potential biomarkers with reduced levels were preliminarily identified, which were also linked to the dysregulation of amino acids and lipid metabolism. Combined with the results of Western Blotting experiments, it was found that Osthole can stop tumors from progressing by altering the AKT/mTOR signaling pathway with changes in amino acid and lipid metabolism. This study can provide a potentially valuable metabolic basis for the early detection, treatment, and prognosis of ICC, as well as the clinical application of Osthole in ICC.

The Role of Osthole on TGF-β-Induced Lung Epithelium Apoptosis Injury and Epithelial-Mesenchymal Transition-Mediated Airway Remodeling in Pediatric Asthma.

Osthole, a coumarin compound derived from Fructus Cnidii, exerts anti-inflammatory effects in an asthma model. But the effect of osthole on epithelial injury and epithelial-mesenchymal transition (EMT) in asthma remains unclear. 16HBE cells were incubated with TGF-β1 with or without osthole in vitro. Ovalbumin (OVA)-induced asthmatic mouse model was established in vivo. Cell counting kit-8 was carried out to evaluate the viability of 16HBE cells. The impact of osthole on TGF-β1-evoked cell apoptosis and EMT process was measured by flow cytometry based on Annexin V-FITC/PI staining, transwell assay, immunofluorescence, and Western blot. The regulatory role of osthole in TGF-β1/Smad and p38, ERK1/2, and JNK MAPK signaling was detected via Western blot. Osthole treatment significantly suppressed TGF-β1-induced 16HBE cell apoptosis, verified by a reduced percentage of apoptotic cells, decreased expression of proapoptotic proteins (cleaved-caspase3 and Bax), and enhanced antiapoptotic factor (Bcl-2) expression. In addition, the promotive impact of TGF-β1 on the migration of 16HBE cells was reversed by osthole, accompanied by elevated E-cadherin expression and reduced Snail and N-cadherin expression. The activation of the Smad2/3 and MAPKs pathway evoked by TGF-β1 was inhibited by osthole in 16HBE cells. We also found that osthole mitigated airway epithelium injury and subepithelial fibrosis in OVA-challenged asthmatic mice in vivo. Osthole could mitigate TGF-β1-induced epithelial cell injury and EMT process by suppressing the activation of MAPK and Smad2/3 pathways separately. Our present study showed a new insight into understanding the underlying mechanism of osthole injury on epithelium injury and subepithelial fibrosis in airway remodeling. Asthma, epithelial injury, epithelial-mesenchymal transition, and airway remodeling are the effects of osthole on airway remodeling.

Integrated 16S rRNA Gene Sequencing and Metabolomics Analysis to Investigate the Important Role of Osthole on Gut Microbiota and Serum Metabolites in Neuropathic Pain Mice.

Accumulating evidence suggests that neuropathic pain (NP) is closely connected to the metabolic disorder of gut microbiota, and natural products could relieve NP by regulating gut microbiota. The purpose of this study is to investigate the important regulatory effects of osthole on gut microbiota and serum metabolites in mice with chronic constriction injury (CCI). Mice’s intestinal contents and serum metabolites were collected from the sham group, CCI group, and osthole treatment CCI group. The 16S rRNA gene sequencing was analyzed, based on Illumina NovaSeq platform, and ANOVA analysis were used to analyze the composition variety and screen differential expression of intestinal bacteria in the three groups. Ultra-high-performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry (UHPLC-Q-TOF-MS) was used for analyzing the data obtained from serum specimens, and KEGG enrichment analysis was used to identify pathways of differential metabolites in the treatment of neuralgia mice. Furthermore, the Pearson method and Cytoscape soft were used to analyze the correlation network of differential metabolites, gut microbiota, and disease genes. The analysis results of 16S rRNA gene sequencing displayed that Bacteroidetes, Firmicutes, and Verrucomicrobia were highly correlated with NP after osthole treatment at the phylum level. Akkermansia, Lachnospiraceae_unclassified, Lachnospiraceae_NK4A136_group, Bacteroides, Lactobacillus, and Clostridiales_unclassified exhibited higher relative abundance and were considered important microbial members at genus level in neuralgia mice. Serum metabolomics results showed that 131 metabolites were considered to be significantly different in the CCI group compared to the sham group, and 44 metabolites were significantly expressed between the osthole treatment group and the CCI group. At the same time, we found that 29 differential metabolites in the two comparison groups were overlapping. Integrated analysis results showed that many intestinal microorganisms and metabolites have a strong positive correlation. The correlation network diagram displays that 10 genes were involved in the process of osthole alleviating NP through a metabolic pathway and gut microbiota, including IGF2, GDAP1, MYLK, IL18, CD55, MIR331, FHIT, F3, ERBB4, and ITGB3. Our findings have preliminarily confirmed that NP is closely related to metabolism and intestinal microbial imbalance, and osthole can improve the metabolic disorder of NP by acting on gut microbiota.

Effects of Osthole on Inflammatory Gene Expression and Cytokine Secretion in Histamine-Induced Inflammation in the Caco-2 Cell Line.

Hyperactivity of the immune system in the gastrointestinal tract leads to the development of chronic, inflammation-associated disorders. Such diseases, including inflammatory bowel disease, are not completely curable, but the specific line of treatment may reduce its symptoms. However, the response to treatment varies among patients, creating a necessity to uncover the pathophysiological basis of immune-mediated diseases and apply novel therapeutic strategies. The present study describes the anti-inflammatory properties of osthole during histamine-induced inflammation in the intestinal Caco-2 cell line. Osthole reduced the secretion of cytokines (CKs) and the expression level of inflammation-associated genes, which were increased after a histamine treatment. We have shown that the secretion of pro-inflammatory CKs (IL-1β, IL-6, IL-8, and TNF-α) during inflammation may be mediated by NFκB, and, after osthole treatment, this signaling pathway was disrupted. Our results suggest a possible role for osthole in the protection against inflammation in the gastrointestinal tract; thus, osthole may be considered as an anti-inflammatory modulator.

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.

Osthole suppresses the proliferation and induces apoptosis via inhibiting the PI3K/AKT signaling pathway of endometrial cancer JEC cells.

Osthole, a natural product extracted mainly from fruits of Fructus Cnidii, possesses multiple pharmacological functions, including anti-inflammatory, anti-convulsant and anticancer effects. However, the effects of osthole in endometrial cancer (EC) is not fully understood. In the present study, EC cell lines, including JEC, KLE and Ishikawa cells and normal human cervical epithelial cells (HcerEpic) were applied to detect the anticancer effect of osthole. The present study demonstrated that osthole inhibited the proliferation of JEC, KLE and Ishikawa cells, but had no cytotoxic effect on HcerEpic. Furthermore, treatment of osthole induced JEC cell apoptosis, while osthole promoted the release of pro-apoptotic proteins, Bax and activated the cleaved caspase-3, caspase-9 and PARP. Additionally, osthole significantly increased the expression of PETN and decreased the phosphorylated form of PI3K and AKT in a concentration-dependent manner. Furthermore, osthole treatment suppressed the JEC tumor cell growth in a nude mouse xenograft model in vivo, and neither renal toxicity nor hepatotoxicity was induced by the indicated concentration. Taken together, the results of the present study suggested that osthole may be a novel and potential therapeutic agent of EC.

Ameliorative Effects of Osthole on Experimental Renal Fibrosis in vivo and in vitro by Inhibiting IL-11/ERK1/2 Signaling.

Objectives: Natural product, osthole, has been proven to have a protective effect on organ fibrosis, including renal fibrosis. All of these studies are mainly focused on the regulation of TGF-β/Smad signaling pathway. However, due to the pleiotropic roles of TGF-β/Smad signaling, direct TGF-β-targeted treatments are unlikely to be therapeutically feasible in clinic. Recently, the downstream IL-11/ERK1/2 signaling of TGF-β has become an attractive therapeutic target without upstream disadvantages. Based on that, this study was designed to identify the potential effects of osthole on IL-11/ERK1/2 signaling pathway in renal fibrosis. Methods: The renal fibrosis model was established in vivo and in vitro, we investigated the effects of osthole on unilateral ureteral obstruction (UUO)-induced renal fibrosis and TGF-β-induced HK-2 cells. After preliminarily confirming the antifibrogenic effects of osthole and the link between its antifibrogenic effects and the inhibition of IL-11/ERK1/2 signaling, we applied a direct IL-11-induced HK-2 cells fibrosis model to further explore the inhibitory effects of osthole on IL-11/ERK1/2 signaling pathway. Results: Our results confirmed that osthole can decrease the secretion of fibrosis proteins, such as α-smooth muscle actin (α-SMA), collagen I, and fibronectin, ameliorate experimental renal fibrosis in vivo and in vitro, and the effect was associated with suppressing TGF-β1/Smad signaling. More importantly, we found that IL-11/ERK1/2 signaling in UUO-induced renal fibrosis and TGF-β-induced HK-2 cell model was obviously upregulated, and osthole treatment also significantly inhibited the abnormal IL-11/ERK1/2 signaling activation. Given the direct link between TGF-β/Smad signaling and IL-11/ERK1/2 signaling pathway, we have verified that osthole has a direct inhibitory effect on IL-11/ERK1/2 signaling independent of TGF-β signaling by using an IL-11-induced HK-2 cells fibrosis model. Osthole treatment decreased the protein expression of α-SMA, collagen I and fibronectin without changing their mRNA levels in IL-11-induced HK-2 cells. Moreover, it was observed that the IL-11/ERK1/2 inhibitor, U0126, partly blocked the antifibrogenic effects of osthole. Conclusion: In this study, we found that osthole has a previously unrecognized role in inhibiting IL-11/ERK1/2 signaling pathway. Our work demonstrated that the antifibrogenic effect of osthole is not only mediated by TGF-β/Smad2/3 signaling, but also directly mediated by IL-11/ERK1/2 signaling pathway independent of TGF-β1 signaling.

Osthole Ameliorates Estrogen Deficiency-Induced Cognitive Impairment in Female Mice.

Loss of endogenous estrogen and dysregulation of the estrogen receptor signaling pathways are associated with an increase in risk for cognitive deficit and depression in women after menopause. Estrogen therapy for menopause increases the risk of breast and ovarian cancers, and stroke. Therefore, it is critical to find an alternate treatment for menopausal women. Osthole (OST), a coumarin, has been reported to have neuroprotective effects. This study examined whether OST improves ovariectomy (OVX)-induced cognitive impairment, and alleviates anxiety- and depression-like behaviors induced by OVX in mice. Adult female C57BL/6J mice were ovariectomized and then treated with OST at a dose of 30 mg/kg for 14 days. At the end of the treatment period, behavioral tests were used to evaluate spatial learning and memory, recognition memory, anxiety- and depression-like behaviors. A cohort of the mice were sacrificed after 14 days of OST treatment and their hippocampi were collected for measurement of the proteins of interest using western blot. OVX-induced alteration in the levels of proteins was accompanied by cognitive deficit, anxiety- and depression-like behaviors. OST treatment improved cognitive deficit, alleviated anxiety- and depression-like behaviors induced by OVX, and reversed OVX-induced alterations in the levels of synaptic proteins and ERα, BDNF, TrKB, p-CREB, p-Akt and Rac1 in the hippocampus. Therefore, reversal of OVX-induced decrease in the levels of hippocampal proteins by OST might contribute to the effects of OST on improving cognitive deficit and alleviating anxiety- and depression-like behaviors induced by OVX.

Osthole ameliorates simulated microgravity-induced bone loss through down-regulation of miR-34c-5p

Bone loss severely threatens the heath of astronauts in long-term spaceflight and the effects of the developed countermeasures are limited. Emerging studies have revealed the positive role of osthole (OST) in induction of osteogenic differentiation and bone formation. In this study, the effects of OST on bone metabolism were investigated in osteoblasts and rats treated with random positioning machine and hindlimb unloading, respectively. The results showed that OST treatment promoted the cellular proliferation and elevated the expression levels of RUNX2 and BMP2 in osteoblasts under simulated microgravity. In addition, hindlimb unloading rats administered with OST (5 mg kg/d, i.g.) exhibited improved bone mass, bone strength, expression of bone formation markers (BALP and OCN), and decreased expression of bone resorption markers (CTX-1 and TRACP-5b) in serum compared with rats treated with placebo (0.9% saline). Notably, we found that miR-34c-5p expression level was significantly up-regulated when exposed to simulated microgravity both in vitro and in vivo, and suppression of miR-34c-5p by antagomiR in rats obviously rescued the damages of BMD and trabecular bone microstructures caused by simulated microgravity. Importantly, OST treatment reversed miR-34c-5p expression level in hindlimb unloading rats, and ectopic expression of miR-34c-5p by transfection of agomiR in osteoblasts treated with OST decreased the expression levels of bone formation genes COL1α1, RUNX2, and BMP2. Overall, these data suggest an undisclosed role for OST in regulating microgravity-induced bone loss via control of miR-34c-5p expression, which may help to countermeasure development to overcome bone loss in spaceflight.

Coumarins modulate the anti-glioma properties of temozolomide

In the recent years, coumarin bioactive compounds have been identified to posess anticancer properties. Therefore, the aim of the present study was to investigate for the first time the efficacy of osthole, umbelliferone, esculin, and 4-hydroxycoumarin, alone and in combination with Temozolomide, in the elimination of deadly brain tumors, anaplastic astrocytoma (AA) and glioblastoma multiforme (GBM) cells via programmed death. Our results indicated that osthole, umbelliferone, esculin, and 4-hydroxycoumarin initiated mainly apoptosis in the T98G and MOGGCCM cells. Osthole was the most effective. It also initiated autophagy in a small percentage of the cell population. The co-incubation with Temozolomide did not increase the pro-apoptotic potential of natural compounds but decreased the level of autophagy in the T98G cells. Apoptosis was associated with reduced mitochondrial membrane potential, activation of caspase 3, inhibition of Bcl-2 expression and the presence of a Bcl-2/Beclin 1. Blocking of Bcl-2 expression resulted in promotion of apoptosis, but not autophagy, in the MOGGCCM and T98G lines. It also sensitized astrocytoma cells, but not GBM, to the combined osthole and TMZ treatment, which was correlated with a reduced level of Beclin 1 and increased expression of caspase 3. Osthole and TMZ, alone and in combination, inhibited the migratory phenotype of the GBM and AA cells. In summary, our results indicated that osthole effectively eliminated glioma cells via apoptosis, what was correlated with Bcl-2/Beclin 1 complex formation. Considering the anti-migratory effect, osthole and Temozolomide display antiglioma potential but it needs further extensive studies.