The Study Of Proliferation Of Daphnoretin To Neuroblastoma Cell And Its Metabolism In Cell

Backgrounds: Neuroblastoma is a rare malignancy that occurs in children under 5 years of age.There are few drugs currently available to treat the disease,so it is necessary to find effective drugs.Daphnoretin,chemical name 7-Hydroxy-6-methoxy-3,7'-dicouvenyl ester,also known as double-white daphnoretin,has a bis-coumarin derived from ether bond.The structure of the material,in the Compositae,Ruixiang Branch and other plants have been found.The study found that Daphnoretin has anti-tumor,anti-inflammatory,anti-bacterial,anti-virus,anti-anxiety and other pharmacological effects,so in this study using the drug to explore its ability to neuroblastoma cells effective.Objective: To investigate whether the drug plays a role in the proliferation of neuroblastoma cells by inhibiting the proliferation,apoptosis and cycle of SH-SY5 Y.The method of HPLC for the determination of daphnoretin in neuroblastoma cells was established.The uptake and elimination of daphnoretin in neuroblastoma cells provide a preliminary theoretical basis for the study of cell pharmacokinetic study of daphnoretin.Through the study exploring whether daphnoretin can be a new drug for the treatment of human neuroblastoma and help the treatment of the disease.Methods: The MTT(tetrazolium salt)method was used to calculate the survival rate of daphnoretin in different tumor cells at different concentrations,and the inhibitory effect of daphnoretin on the proliferation of tumor cells was evaluated.Using Annexin V labeled FITC as a fluorescent probe to bind to phosphatidylserine on early apoptotic neuroblastoma cells and PI(propidium iodide)nucleic acid dye to the apoptotic cells and necrotic cells in the nucleus.The simultaneous use of both dyes can distinguish early apoptosis from late and necrotic cells and then use fluorescence microscopy to observe the effect of different concentrations of daphnoretinon the apoptosis of neuroblastoma cells.After 24 hours of cell administration,PI staining was performed and the effect of daphnoretin on cell cycle of neuroblastoma cell was detected by flow cytometry.The changes of mitochondrial membrane potential in the cells were detected by JC-1 staining,and the effect of daphnoretin on mitochondrial membrane potential was measured.The effect of daphnoretin on the migration ability of neuroblastoma cellwas detected by cell scratches.To establish a HPLC method for the determination of daphnoretinin neuroblastoma cell and verify the methodology.The uptake and elimination of daphnoretin in neuroblastoma cell in a given period of time were determined by the established method.To investigate whether serum and verapamil have an effect on the content of daphnoretin in SH-SY5 Y.Results: MTT assay showed that daphnoretin had inhibitory effect on proliferation of SH-SY5 Y.The experiment of apoptosis of neuroblastoma cell showed that daphnoretin could induce cell apoptosis.Cell cycle test showed that the proportion of neuroblastoma cells in the S phase increased,indicating that daphnoretin can arrest the cell cycle,mainly blocking cells from S to G2 phase.The results of JC-1 staining showed that daphnoretin could decrease the mitochondrial membrane potential and further confirm that daphnoretin could induce apoptosis of neuroblastoma cell.Cell scratches have demonstrated that daphnoretin can inhibit the migration of neuroblastoma cells.Cell Pharmacokinetic Methods Test Results : Chromatographic conditions: Phenomenex C18 column(250 mm x 4.6 mm,5 ?m),the mobile phase is methanol : phosphoric acid aqueous solution(55: 45,v/v,pH ? 3.0),flow rate is 1.0 mL·min-1.Detection wavelength 345 nm,column temperature 30 ?separation effect is good.The linear range was 0.02 ?g·mL-1,the lowest linearity was 0.05 ?g·mL-1,the standard linear equation was Y = 80620 X + 5962,R2 was greater than 0.99.The RSD of the intra-day precision of the first day of daphnoretin 1.0,5.0,10.0 ?g · mL-1 was 9.34%,2.97%,2.24%,respectively.The second day was 9.55%,2.27%,4.60%,and the third day was 6.35%,1.54% and 2.74%.The day precision was 8.91%,2.41%,3.61%.The RSD of the accuracy was 5.69%,6.11%,3.35% respectively.The stability of room temperature immediately treated group,room temperature for 24 h,4 ?24h test results were 14.85%,6.03%,8.29%,11.97%,3.16%,5.54%,11.11%,4.67% and 5.02%,respectively.The RSD values of extraction recovery rate of different concentrations were 2.18%,1.65%,3.49%,respectively.All RSD% were less than 15%,indicating that the established HPLC method was available.The results of ultrasonic lysis showed that the drug content was the highest at 30 min.The uptake of daphnoretin is time-dependent and the uptake of daphnoretin in serum-containing conditions is higher than that in serum-free conditions.The content of the drug in the cell gradually decreases with time,and verapamil does not affect the uptake of the drug in the cell.The results showed that there were no significant differences in the content of daphnoretin in SH-SY5 Y between the groups treated with 10?M and 50?M of verapamil and without the verapamil,indicating that there was no efflux of P-gp effect in the process of the transport of daphnoretin.Discussion: The experiment found that the drug on the neuroblastoma cell has a certain proliferation inhibition and can induce the analysis of apoptosis,also have certain impacts on cell cycle and migration capacity.The HPLC method for the detection of daphnoretin in neuroblastoma cell in this experiment has high efficiency,high specificity and good sensitivity,and can provide the basis for the study of cell pharmacokinetics.The uptake and elimination of daphnoretin in cell can provide a basis for exploring the mechanisms of action of drug on cell.