Effects of osthole on renal oxidative stress injury in aged spontaneously hypertensive rats by regulating PI3K/Akt/mTOR signaling pathway / 中国临床药理学与治疗学

AIM: To explore the mechanism of osthole on elderly spontaneously hypertensive rats. METHODS: 20-month-old spontaneously hypertensive rats (SHRs) and healthy Wistar-Kyoto (WKY) rats were purchased. SHRs were treated with osthole (i.g.) for 8 weeks. The systolic blood pressure and diastolic blood pressure of rats were monitored. Hematoxylin-eosin staining (H&E), periodic acid-schiff staining (PAS) and Masson staining were used to observe the pathological changes of rat kidney tissues. The activity of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH) in rat kidney was detected by ELISA kit. PI3K/Akt/mTOR signaling pathway related proteins were detected by western blot. RESULTS: Osthole reduced the systolic and diastolic blood pressure of SHRs, improved the histopathological changes of SHRs kidney, reduced the activity of MDA in SHRs kidney, and increased the activity of SOD and GSH. Osthole reduced the levels of p-PI3K, p-Akt and p-mTOR. CONCLUSION: Osthole reduces the activity of PI3K/Akt/mTOR signaling pathway and exerts a protective effect on renal oxidative stress injury in aged spontaneously hypertensive rats.

Effect and Mechanism of Osthole on Proliferation and Apoptosis in Human Intrahepatic Cholangiocarcinoma HuCCT1 Cells / 中国实验方剂学杂志

ObjectiveTo investigate the effect and mechanism of osthole on the proliferation and apoptosis in human intrahepatic cholangiocarcinoma HuCCT1 cells. MethodThe effect of 10， 20， 40， 80， and 120 μmol·L-1 osthole on the proliferation of HuCCT1 cells was detected by the cell counting kit-8 （CCK-8）. A blank group， and low-， medium-， and high-dose osthole groups （16， 32， and 64 μmol·L-1） were set up. The effect of osthole on cell clone formation rate was detected by colony formation assay. The effect of osthole on cell cycle and apoptosis was detected by flow cytometry. The effect of osthole on cell apoptotic morphology was detected by Hoechst 33342 fluorescent staining. The effect of osthole on cell cycle protein cyclin B1， proliferating cell nuclear antigen （PCNA）， cysteine-aspartic acid protease （Caspase）-9， Caspase-3， cleaved Caspase-9， cleaved Caspase-3， cleaved poly（ADP-ribose） polymerase （cleaved PARP）， B-cell lymphoma-2 （Bcl-2）， phosphorylated protein kinase B （p-Akt）， phosphorylated mammalian target of rapamycin （p-mTOR）， and phosphorylated ribosomal protein S6 （p-RPS6） was detected by Western blot. ResultThe cell viability in the osthole group（40，80，120 μmol·L-1） decreased （P<0.05，P<0.01）， with the half maximal inhibitory concentration （IC50） of 63.8 μmol·L-1 as compared with that in the blank group. Compared with the blank group， the osthole groups（32，64 μmol·L-1）showed reduced clone formation rate （P<0.01）， increased number of cells in the G2 phase （P<0.05，P<0.01）， decreased number of cells， increased pyknosis and fragmentation， increased apoptosis rate （P<0.05，P<0.01）， down-regulated expression of cyclin B1， PCNA， Bcl-2， Caspase-3， Caspase-9， p-Akt， p-mTOR， and p-RPS6 （P<0.05，P<0.01）， and up-regulated expression of cleaved Caspase-3， cleaved Caspase-9， and cleaved PARP （P<0.05，P<0.01）. ConclusionOsthole can inhibit the proliferation and promote the apoptosis of HuCCT1 cells， and its mechanism may be related to the Akt/mTOR signaling pathway.

Study on method of osthole loaded by exosomes / 中国药理学通报

Aim To construct a drug delivery system of osthole loaded by exosomes. Methods Osthole could inhibit the proliferation of ovarian cancer cells by literature. SKOV3 cells were treated with 80 (µnol • L

Mechanism of autophagy induced by osthole through PINK1/Parkin pathway in HeLa cells / 中国药理学通报

Aim To investigate the potential mechanism of osthole promoting autophagy in cervical cancer HeLa cells. Methods HeLa cells were treated with various concentrations of Osthole(0,10,20,40,80,160,240,320 mg·L-1). MTT was used to detect cell vitality. Transmission electron microscopy(TEM)was used to observe the morphology of HeLa cells after osthole intervention. Mondane sulfonyl cadaverine(MDC)staining was used to dectect the level of autophagy. Western blot was employed to analyze the expression levels of mitochondrial protein MFN1 and DPR1. JC-1 flourescence probe was applied to detect mitochondrial membrane potential. Flow cytometry was used to deteminet the release of reactive oxygen species(ROS). A transplanted tumor model of cervical cancer was established in vivo in nude mice. Western blot was used to detect the protein expression levels of PINK1,Parkin and LC3Ⅱ/. Results Osthole could inhibit the proliferation of HeLa cells significantly. Transmission electron microscopy showed that typical autophagosomes were formed in HeLa cells after osthole intervention. The fluorescence intensity of MDC was enhanced. The expression of mitochondrial fusion protein MFN1 was down-regulated after HeLa cells pretreated with osthole,and mitochondrial fission protein DRP1 was up-regulated. Mitochondrial membrane potential decreased. ROS production of HeLa cells was increased by flow cytometry,which could be reversed by autophagy inhibitor 3-MA. Tumor weight in nude mice was inhibited by osthole obviously,which might restrain cervical cancer. Western blot result indicated that the key factors of mitochondrial autophagy PINK1,Parkin and LC3Ⅱ/ratio were up-regulated in HeLa cells. Conclusions Osthole could induce autophagy in HeLa cells and its mechanism may be related to ROS production and PINK1/Parkin pathway.

Extraction Technology of Volatile Oil from Wenjing Huoxue Cataplasm / 药学实践杂志

Objective To optimize the supercritical CO2 extraction conditions of volatile oil from Wenjing Huoxue cataplasm. Methods On the basis of single factor investigation on the comprehensive score of extraction yield , osthole content and isoimperatorin, the effects of extraction temperature, pressure and time on the comprehensive score of extracted volatile oil were optimized by orthogonal design. Results In the single factor experiment, the factors that had a great influence on the comprehensive score of the extracted volatile oil were extraction temperature, extraction pressure and extraction time. The orthogonal experiment results showed that the extraction temperature and extraction pressure had a significant influence on the comprehensive score of volatile oil. The optimized extraction process was as follows: extraction temperature at 55 ℃, extraction pressure as 30 MPa, and extraction time as 2 h. Conclusion The extraction process optimized in this experiment is stable and feasible, which could be used for the extraction and preparation of the volatile oil.

Osthole attenuates diabetes-induced renal injury by regulating NF-κB and p38/MAPK pathway mediated inflammatory responses / 中国临床药理学与治疗学

To investigate the therapeutic effects of oral osthole on streptozotocin (STZ)-induced type 1 diabetes mellitus(T1DM) mice and explore its internal mechanism. METHODS: The diabetes model induced by STZ was established. Mice were randomly divided into control group, STZ model group, STZ+osthole group (20 mg/kg). Body weight, blood glucose, urine protein, blood urea nitrogen and creatinine were observed to detect renal function. The degree of renal tissue damage was detected by H&E staining and PAS staining, and the degree of renal fibrosis was detected by Sirius Red staining. CD68 and F4/80 immunofluorescence staining was used to observe the infiltration of macrophages in kidney tissue. The mRNA expressive levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) in renal tissue were detected by RT-qPCR. The protein expressive levels of phospho-NF-κB p65, NF-κB p65, IκBα, phospho-IκBα, phospho-p38 and p38 were detected by Western blot in renal tissue. RESULTS: Compared with the STZ model group, the levels of urinary protein, blood urea nitrogen, creatinine were significantly decreased after osthole treatment (P<0.05 or P<0.01). The renal structure disorder, mesangial matrix area, collagen fiber accumulation, and macrophage infiltration were significantly improved (P<0.05 or P<0.01). The expression of mRNA of pro-inflammatory cytokines TNF-α and IL-6 were significantly decreased (P<0.05 or P<0.001). The expression of phospho-NF-κB p65, phospho-IκBα and phospho-p38 were significantly down-regulated (P<0.05 or P<0.01), while the protein expression level of NF-κB p65, IκBα was up-regulated (P<0.05). CONCLUSION: Osthole has a protective effect on kidney injury caused by diabetes and inhibits NF-κB and p38/MAPK signaling pathway.

Osthole attenuates pulmonary arterial hypertension by modulation of phospholipid metabolism / 中国药理学与毒理学杂志

OBJECTIVE Pulmonary arterial hypertension (PAH) is a malignant pulmonary vascular disease lacking efficacy therapeutics. Therefore, it urgently needs to develop safe and effective drugs for PAH treatment. Osthole derived from Cnidium monnieri (L.) Cusson (Shechuangzi) or Angelica pubescens Maxim (Duhuo) has the capacity to alleviate PAH by decreasing pulmonary arterial pressure and alleviating pulmonary vascular remodeling in rats, which is a candi?date drug for the prevention of PAH, but the underlying modulatory mechanism is still unclear. Our study aims at investi?gating the metabolic modulatory mechanism of osthole against PAH employing functional metabolomics strategy. METH?ODS PAH model rats were successfully established with MCT, following osthole administration, then functional metabo?lomics based on untargeted metabolomics assay, targeted lipidomics analysis, qRT-PCR, Western blotting and ELISA were performed to investigate the modulatory mechanism of osthole against pulmonary arterial pressure and pulmonary vascular remodeling in PAH. RESULTS Untargeted metabolomics results found that sphingosine 1-phosphate (S1P) was the differential metabolites characterized PAH and reversed by osthole treatment. S1P is a crucial sphingolipid metabolite catalyzed by sphingosine kinases1 (Sphk1) and functions as promoting PASMCs proliferation contributing to pulmonary vascular remodeling and pulmonary arterial pressure increase. We revealed that osthole reversed high level of S1P by modulating metabolic enzyme Sphk1 via inactivating microRNA-21-PI3K/Akt/mTOR signal pathway to decrease pulmonary arterial pressure in rats with PAH. Then, targeted phospholipid metabolomics results uncovered that decadienyl-L-carnitine (C10:2) was the differential metabolite characterized PAH and corrected by osthole treatment in rat with PAH. C10:2 is the intermediate metabolite of fatty acid oxidation (FAO), and C10:2 accumulation indicated mitochondrial dysfunction and FAO increase. CONCLUSION Osthole could block lipid metabolic reprogramming through functional modulating the expression of fatty acid translocase, fatty acid synthase, phospholipase A2, carnitine palmitoyltransferase 1A to inhibit C10:2, thus to improve mitochondrial dysfunction and inhibit utilizing lipid to biosyn?thesize necessary essence for pulmonary artery smooth muscle cells (PASMCs) proliferation. Moreover, we delineated that C10:2 and metabolic reprogramming enzymes were modulated by miRNA-22-3p which was involved in PASMCs proliferation and pulmonary vascular remodeling. Therefore, osthole inhibited miRNA-22-3p mediated lipid metabolic reprogramming to ameliorate pulmonary vascular remodeling.

Effects of Osthole on Apoptosis and Autophagy of Human Tongue Cancer Tca8113 Cells / 中国医学科学院学报

Objective To investigate the effects of osthole on the proliferation,apoptosis,and autophagy of human tongue cancer Tca8113 cells and explore its possible mechanism of action. Methods Tca8113 cells were cultured

Evaluation of in vitro antifungal activity of osthole against Talaromyces marneffei in yeast phase / 中华皮肤科杂志

Objective To evaluate the in vitro antifungal activity of osthole against Talaromyces marneffei (TM) in yeast phase,in order to provide an experimental reference for the clinical treatment of TM infection with Chinese medicine.Methods There were 20 TM strains,including 1 standard strain,2 fluconazole-spontaneously resistant strains,11 clinical isolates,and 6 isolates from wild bamboo rats.A microdilution method was used to prepare 96-well antifungal sensitivity test plates containing osthole,fluconazole,amphotericin B,itraconazole and voriconazole at different concentrations,which were incubated with (1-5) × 103 CFU/ml of tested TM strain suspensions at 37 ℃ for 48 hours.Meanwhile,TM strains cultured in the media without antifungal drugs served as positive (growth) control group,and culture media served as negative group.The minimum inhibitory concentrations (MICs) of antifungal drugs against yeasts were determined using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution susceptibility method (M27-A2 Document).Fluconazole MIC was defined as the lowest drug concentration that resulted in ≥ 80％ growth inhibition,and MICs of other antifungal drugs were the lowest drug concentrations that resulted in 100％ growth inhibition,compared with growth control wells.Results The MICs among the quality control strains were all within the reference range,and TM grew well in the positive control wells.The MIC ranges of fluconazole,amphotericin B,itraconazole and voriconazole against TM strains were 2.0-8.0 mg/L,1.0-4.0 mg/L,0.03-0.25 mg/L and 0.06-0.25 mg/L respectively,and the MIC of fluconazole against fluconazole-spontaneously resistant strains was 128 mg/L.The MICs of osthole against the TM standard strain (FRR2161),fluconazole-spontaneously resistant strains and 1 isolate from wild bamboo rats were 16,32 and 128 mg/L respectively,and the MIC range of osthole against other 16 TM strains was 16-64 mg/L.The MICs of osthole at which 90％ and 50％ of the TM strains were inhibited were 64 and 32 mg/L respectively.Conclusion Osthole exhibits the antifungal activity against the yeast form of TM.

Analysis of Transport Mechanism of Cyperotundone in Caco-2 Cell Model / 中国实验方剂学杂志

Objective: To investigate transport mechanism of cyperotundone in Caco-2 cell model and provide experimental basis for clinical application of Cyperi Rhizoma. Method: The toxicity of cyperotundone with different concentrations to Caco-2 cells was investigated by methyl thiazolyl tetrazolium (MTT) colorimetry, in order to determine the concentration of administration in transport test. The content of cyperotundone was determined by liquid chromatography-mass spectrometry (LC-MS) with apparent permeability coefficient (Papp) and cumulative transport capacity as indexes. The chromatographic conditions were as following:mobile phase of acetonitrile (A)-water (B) for gradient elution (0-1.5 min, 35%A; 1.5-2 min, 35%-90%A; 2-4 min, 90%A; 4-4.1, 90%-35%A; 4.1-8 min, 35%A), the flow rate at 0.3 mL · min-1, injection volume of 1 μL, and temperature of column at 30℃. The mass spectrometric conditions was electrospray ionization (ESI) and positive ion mode, the detection ions of cyperotundone and osthole (internal standard substance) were m/z 219.2-110.9 and m/z 245.0-189.0, respectively. Effect of concentration of cyperotundone, administration time, ethylenediamine tetraacetic acid (EDTA) and P-glycoprotein (P-gp) inhibitor on the transmembrane transport of cyperotundone on in vitro cell model were investigated. Result: Cyperotundone didn't have significant toxicity to Caco-2 cells at 3-90 mg · L-1 after incubation for 4 h. The transportion of cyperotundone in Caco-2 cell model was related to the concentration and time to a certain extent, its Papp was higher than 1×10-6 cm · s-1, which indicated that absorption of cyperotundone was good, the efflux rate (ER) of cyperotundone was 0.5-1.5.There was no significant difference in bidirectional Papp of cyperotundone after the addition of cell bypass transport inhibitor (EDTA) and P-gp transport inhibitor (verapamil). Conclusion: The transport mechanism of cyperotundone in Caco-2 cell model is mainly passive diffusion, and cell bypass transport and P-gp are not involved in its transport.

Osthole inhibits proliferation and induces apoptosis of hepatocellular carcinoma cell line HepG2 via upregulating PTEN / 中国免疫学杂志

Objective: To investigate the effects and mechanism of osthole on proliferation and apoptosis in a hepatocellular carcinoma cell ( HCC) line Hep G2. Methods: Treated cells with osthole at different concentrations. Cell viability was measured by CCK8 assay and apoptosis was detected by flow cytometry. Western blot was performed for calculating the expression levels of proliferation-related, apoptosis-related proteins and PTEN. After pretreatment with bp V ( HOpic), cell proliferation and apoptosis were measured again. Results: Treatment with osthole ( 100 μmol/L) for 4 and 5 days inhibited cell viability of HCC markedly ( P＜0. 05, P＜0. 01). Osthole ( 150 μmol/L) decreased cell viability of HCC with a time-dependently manner and also decreased the expressions of Ki67 and PCNA ( P＜0. 05, P＜0. 01). Meanwhile, treatment with osthole ( 100 μmol/L, 150 μmol/L) induced apoptosis of HCC significantly coupled with increasing Bax and decreasing Bcl-2 ( P＜0. 05, P＜0. 01). In addition, osthole ( 100 μmol/L, 150 μmol/L) up-regulated the protein level of PTEN ( P＜0. 05, P＜ 0. 01). Furthermore, pretreatment with bp V ( HOpic) ( 1 μmol/L) notably reversed the inhibitory effect on proliferation and promotive effect on apoptosis of osthole ( P＜0. 05). Conclusion: Osthole inhibits cell proliferation and induces apoptosis of HCC by up-regulating the protein level of PTEN.

Preparation of molecularly imprinted osthole on surface-modified quartz sand / 中草药

Objective: The molecularly imprinted osthole on surface-modified quartz sand was prepared and characterized by SEM and FT-IR. The adsorption properties of molecularly imprinted materials were investigated. Methods: The morphology of MIP was prepared using N-(β-aminoethyl)-γ-aminopropylmethylbimethoxy silane (KH-602) modified quartz sand as supporter, osthole as template, methacrylic acid (MAA) as functional monomer, ethylene glycol dimethacrylate (EGDMA) as crosslinker, axodiisobutyronitrile (AIBN) as initiator, and methanol as porogen, observed by SEM, and the chemical structure of MIP was characterized by FT-IR. Results: The dynamic adsorption experiment showed that the adsorption capacity of molecularly imprinted materials to osthole gradually reached saturation with the increase of time. The maximum adsorption capacity of molecularly imprinted materials for osthole was studied by static adsorption experiment. The selective adsorption experiment was adopted to study the binding properties and molecule recognition characters of molecularly imprinted materials for osthole. Conclusion: The experimental results showed that MIP had specific recognition selectivity, excellent binding affinity and elution property for ractopamine. The MIP on the adsorption ability of osthole was significantly better than xanthotoxin and imperatorin.

Comparative study of preparation, characterization and pharmacokinetics of three preparations of osthole / 中草药

Objective: To prepare osthole solid dispersions (Ost-SD), osthole phospholipids complex (Ost-PC), and osthole nanosuspensions (Ost-NS), and compare their effects on the pharmacokinetics in SD rats in vivo. Methods: Solvent evaporation method was used to prepare Ost-SD and Ost-PC. Their existential state of Ost in Ost-SD and Ost-PC were analyzed by X-ray power diffraction (XRPD). High pressure homogenization method was employed to prepare Ost-NS, its particle size and Zeta potential were studied. The dissolution in vitro of Ost-SD, Ost-PC, and Ost-NS were also studied compared to Ost suspension. SD rats in each group were ig administered with Ost, Ost-SD, Ost-PC, and Ost-NS, respectively. The concentration of Ost in blood was analyzed by HPLC, and the main pharmacokinetic parameters were obtained. The pharmacokinetic behavior and bioavailability were also been compared. Results: The results of XRPD indicated that Ost showed an amorphous state in Ost-SD and Ost-PC. The average particle size and Zeta potential of Ost-NS were (161.37 ± 3.77) nm and (-29.16 ± 1.83) mV, respectively. The results of dissolution in vitro indicated that the dissolution of Ost was improved greatly by Ost-SD, Ost-PC, and Ost-NS. The results of pharmacokinetics in vivo showed that Cmax, AUC0~t and AUC0~∞ of Ost-SD, Ost-PC, and Ost-NS were enhanced greatly compared to Ost. The bioavailability of Ost-SD, Ost-PC,and Ost-NS were enhanced to 165.92%, 138.46%, and 259.35%, respectively. Conclusion: Ost-SD, Ost-PC, and Ost-NS can enhance the bioavailability of Ost in SD rats notably. In addition, Ost-NS can give a better effect.

Antitumor effect of osthole on human B-cell acute lymphoblastic leukemia 697 cells and its mechanism / 肿瘤

Objective: To investigate the antitumor effect of osthole on human B-cell acute lymphoblastic leukemia (B-ALL) 697 cells and its possible mechanism. Methods: After B-ALL 697 cells were treated with different concentrations (8, 16, 32, 64 and 128 μmol/L) of osthole, the inhibition rate of cell proliferation was detected by CCK-8 assay. After B-ALL 697 cells were treated with 8 and 32 μmol/L osthole, the apoptosis was detected by FCM, the expressions of apoptosis-associated molecule Bcl-2 and Bax were detected by real-time fluorescent quantitative PCR and Western blotting. After B-ALL 697 cells were treated with 8 and 32 μmol/L osthole alone or in combination with autophagy inhibitor 3-methyladenine (3-MA), the intracellular mean fluorescent intensity (MFI) was detected by FCM [stained by monodansylcadaverine (MDC)] to reflect the autophagy. The expressions of autophagy-associated molecule Beclin 1 mRNA and protein was detected by real-time fluorescent quantitative PCR and Western blotting, respectively. Results: The proliferation of B-ALL 697 cells in 8, 16, 32, 64 or 128 μmol/L osthole treatment group was significantly inhibited in a dose-and time-dependent manner (all P 0.05). Conclusion: Osthole inhibits the proliferation, and induces the apoptosis and autophagy of B-ALL 697 cells. The mechanism of promoting apoptosis may be related to the up-regulation of Bax expression and the down-regulation of Bcl-2 expression. Beclin 1 participates in the autophagy.

Optimization of Extraction Technology of Osthole from Pine Needles of Cedrus deodara by Box-Behnken Design-response Surface Methodology / 中国药房

OBJECTIVE： To optimize the extraction technology of osthole from the pine needles of Cedrus deodara. METHODS： HPLC method was adopted to determine the content of osthole. Based on single factor test， ethanol volume fraction， extraction time and material-solvent ratio were selected as influential factors， and the content of osthole was selected as response value. Box-Behnken design-response surface methodology was used to optimize the extraction technology of osthole in pine needles of C. deodara. Validation test was conducted. RESULTS： The optimal extraction technology was as follows as ethanol volume fraction of 88％， material-solvent ratio of 1 ∶ 20 （g/mL）， extracting for 2 times， lasting for 57 min each time. Under this technology， average content of osthole was 0.675 7 mg/g （RSD＝1.78％， n＝3）， and the relative error of which to predicted value 0.680 9 mg/g was 0.59％. CONCLUSIONS： The optimal extraction technology is simple and feasible，and it can be used for the extraction of osthole from the pine needles of C. deodara.  

Osthole suppresses BACE-1 expression by up-regulating miR-9 in Alzheimer's disease / 中国药理学通报

Aim: To explore the mechanism of osthole regulating the expression of miR-9 to delay the occurance of Alzheimer' s disease (AD). Methods: The miRNA which expressed differently with osthole treatment was selected by microarray; the target gene binding to miRNA-9 was verified by databases and Cytoscape; the SH-SY5Y cells with over expression of APP were established using Lipofectamine2000. The cell viability was determined by MTT assay. The miRNA-9 inhibitor was transfected into cells, and the expression of miRNA-9 and BACE-1 mRNA was determined by RT-PCR; the changes of BACE-1 protein were determined by Western blot. Results: The results of database showed that osthole-mediated miRNA-9 was combined with BACE-1 genes. Our lab had established SH-SY5Y cells with over expression of APP. The results of MTT assay showed that 50 p,mol · L-1 osthole had a protective effect on cells. Osthole could increase the expression of miRNA-9, and the expression of miR- 9 was lowest in inhibitor group determined by RT- PCR. Osthole decreased the expression of BACE-1 mRNA and protein compared with APP group, and the expression of BACE-1 was highest in inhibitor group. Conclusion: Osthole plays a protective role in AD partly through suppressing the expression of BACE-1 by up-regulating miRNA-9.

Osthole inhibits HIV gpl20 induced neuropathic pain by down-regulating P2X3 receptor in DRG / 中国药理学通报

Aim To investigate whether osthole can alleviate neuropathic pain induced by HIV gpl20 and its mechanism. Methods The paw withdrawal threshold and the paw withdrawal latency were observed to assess pain behaviors in four groups of the rats, including sham group, sham combined with osthole treatment group, gpl20 treatment group, and gpl20 combined with osthole treatment group. The protein expression levels of the P2X3 receptor, tumor necrosis factor-a receptor (TNF-aR), ERK, p-ERK in the L4-L6 dorsal root ganglia (DRG) were measured by Western blot. The mRNA expression level of P2X3 receptor was assessed by real-time quantitative polymerase chain reaction ( qPCR). The whole path clamp recording was used to measure HEK293 cell current activated by ATP. Results Osthole attenuated the mechanical and thermal hyperalgesia in gpl20 treated rats and down-regulated P2X3 receptor mRNA and protein expression in L4-L6 DRGs of gpl20 treated rats. Additionally, osthole simultaneously decreased the expression of TNF-ctR protein in L4-L6 DRGs and inhibited the phosphorylated ERK1/2 protein expression. Moreover, osthole reduced ATP activated current of HEK293 cells transfected with hP2X3R. Conclusion Osthole decreases the mechanical and thermal hyperalgesia induced by gpl20 through inhibiting P2X3R in DRG.

Osthole induces apoptosis of vulvar squamous cell carcinoma SW962 through PI3K/Akt signaling pathway / 中国药理学通报

Aim: To investigate the effect of osthole on inducing apoptosis and inhibiting the proliferation of vulvar squamous cell carcinoma SW962 cells through PDK/Akt signaling pathway and changes of PDK/Akt pathway protein in the coexistence of osthole and IGF-1. Methods: Vulvar squamous cell carcinoma SW962 cells were cultured in vitro and different concentrations of osthole intervention were given. The effect of osthole on the proliferation of SW962 cells was determined by MTT. Cell apoptosis was detected by flow cytometry. DAPI staining revealed the changes in cellular morphology. The expressions of cleaved-caspase-3, PI3K, p-Akt, Bcl-2, Bax protein in SW962 cells were assessed by Western blot. Results: MTT results showed that osthole could inhibit the proliferation of SW962 cells in a concentration-dependent manner. The results of DAPI staining and FCM by Annexin V-FITC and PI staining indicated osthole induced apoptosis of SW962 cells in a concentration-dependent manner. Western blot showed that osthole increased the expression of Bax and cleaved caspase-3 and decreased the expression of Bcl-2, PI3K, p-Akt. IGF-1 induced increased expression of PI3K, p-Akt and Bcl-2, while osthole reversed IGF-1 and down-regulated the expression of three proteins. Conclusions: Osthole induces the apoptosis and inhibits the proliferation of SW962 in vulvar squamous cell carcinoma by inhibiting PI3K/Akt signaling pathway.

Simultaneous Determination of Isoalantolactone,Alantolactone,Xanthotoxol,Oxypeucedanin, Imperator-in and Osthole in Kangfu Ointment by HPLC / 中国药师

Objective:To establish an HPLC gradient elution method for the determination of isoalantolactone,alantolactone,xan-thotoxol,oxypeucedanin,imperatorin and osthole in Kangfu ointment simultaneously. Methods:Isoalantolactone,alantolactone,xan-thotoxol,oxypeucedanin,imperatorin and osthole were determined by HPLC with a chromatographic column of Agilent TC-C18(250 mm ×4.6 mm,5 μm),the mobile phase was methanol-0.1% formic acid solution with gradient elution at a flow rate of 0.9 ml·min-1. The detection wavelengths were 220 nm and 300 nm. The column temperature was 35℃. Results: The linear range of isoalantolac-tone,alantolactone,xanthotoxol,oxypeucedanin,imperatorin and osthole was 6.16-123.20 μg·ml-1,3.78-75.60 μg·ml-1,1.87-37.40 μg·ml-1,4.06-81.20 μg·ml-1,9.27-185.40 μg·ml-1and 13.89-277.80 μg·ml-1,respectively. The correlation coef-ficient was 0.999 4,0.999 8,0.999 6,0.999 5,0.999 9 and 0.999 7, respectively. The average recovery was 98.04% (RSD=1.06%),97.10%(RSD = 1.53%), 96.73% (RSD = 0.90%), 98.92% (RSD = 1.36%), 99.12% (RSD = 0.83%) and 100.27%(RSD=0.58%),respectively. Conclusion:The method is simple and accurate,which can be used to improve the quality standard for Kangfu ointment.

Effect of osthole on p38 MAPK signaling related pathway of rat model with inflammatory radicular pain induce by nucleus pulposus in the spinal horn / 中国药理学通报

Aim To observe the effect of epidurally application of osthole on the model of nucleus pulposusinduced inflammatory radicular pain and the expression of p38 MAPK signaling related pathway in the spinal dorsal horn of rats.Methods The model of radicular pain was generated by putting nucleus pulposus to the L5 dorsal root ganglion (DRG).50％ MWT was measured using Von Frey filaments to calculate mechanical pain threshold before and after operation.50 μL of 20 g · L-1 osthole was administered epidurally in group Ost and 50 μL of 100 mL · L-1 DMSO in group DMSO at postoperative day (POD).The expression of phosphorylated p38 (p-p38),IL-18 and IL-18R in the lumbar spinal dorsal horn was detected by Western blot.IL-18 mRNA was assessed by real-time PCR.Results The mechanical pain threshold significantly decreased after operation (P ＜ 0.05),while the expression of protein p-p38 MAPK,IL-18,IL-18R and IL-18 mRNA was significantly different.Compared with DMSO group,50％ MWT was significantly increased and accompanied with the decrease of protein p-p38,IL-18,IL-lgR and IL-18 mRNA in Ost group after drug administration (P ＜ 0.05).The correlation analysis between protein concentration of p38 MAPK and IL-18 mRNA showed that the Spearman correlation coefficient was 0.9 (P ＜ 0.05).Conclusion p-p38 and IL-18 of spinal dorsal horn participate in the rat model with inflammatory radicular pain induced by nucleus pulposus,and IL-18R plays a role in maintenance of the pain.Osthole administered epidurally in the early stage of pain could alleviate the pain for a long time,which may be related with inhibiting p38 MAPK signaling related pathways.