Intracellular Reactive Oxygen Species And Calcium Ion Concentration In Endothelial Progenitor Cells And Protection Of Geniposide In Diabetes

Objective:To approach the changes of intracellular reactive oxygen species and calcium ion concentration in endothelial progenitor cells in diabetes and controls, and protection of Geniposide.Methods:we choosed the20hospitalized patients with type2diabetes in Nanjing Military Region, Nanjing General Hospital from July2011to September2011,and20healthy volunteers in the control group. In40cases, there were16female patients, and24males. Their average age was (55±3) years old. Two groups of patients with general information was no significant difference (P>0.05).Experimental and control groups were set to four groups:experimental groups:group Al was blank group (without drug intervention), Bl was drug intervention group with geniposide concentration of12.5mg/L, Cl group’s drug concentration of geniposide was25mg/L, Dl group’s drug concentration of geniposide was50mg/L. The control groups were divided into A2, B2, C2, D2in the same way.We collected15ml peripheral blood of human, and isolated mononuclear cells using lymphocyte separation medium. Cells were seeded in culture plates with HFN. We used the Ml99medium to culture the cells37o C incubator. The growth status of the cells were observed under the microscope every day. When cells growth to7days,we were identified with Dil-ac-LDL and FITC-UEA-1,to determine the cells were or not differentiated endothelial progenitor cells. The red cells were ac-LDL positive cells, and the green cells were UEA-1positive cells. The double staining of cells are endothelial progenitor cells which being differentiated. When the cell growth to the sixth day, we conducted the drug intervention. The first step,we must prove that the geniposide drugs within a certain concentration range was toxic effects to the EPCs with a method of MTT. In accordance with the the MTT instructions, we used a microplate reader the cells’activity.When cells cultured for7days (after drug intervention), we determined the levels of ROS by flow cytometry, and observed the fluorescence intensity of intracellular calcium in cells by fluorescence microscope. Each group of experiments were performed three independent experiments. The experiments data was computed with SPSS17.0software. The experimental results expressed as the form of mean±standard deviation (x±s). The statistical methods were single-factor analysis of variance and T test. P<0.05was considered statistically significant.Results:1. The MTT method for the determination of cell activity:Geniposide could significantly improve the activity of EPCs in patients with DM (P<0.01), the difference was statistically significant. And100mg/L or less concentration of geniposide on EPCs was non-toxic. So we choosed below drug concentration of100mg/L in the following experimental groups to exclude the geniposide drug itself toxic effects on EPCs interfere with the experimental results.2. The result of intracellular ROS levels by flow cytometry:the ROS levels of endothelial progenitor cells of the diabetic group was significantly higher than the normal control group. The geniposide intervention could significantly reduce the level of intracellular reactive oxygen species, and the average level of intracellular reactive oxygen species decreased from1247.8to680.6in the group of12.5mg/L drug concentration.The fluorescence intensity of calcium within the cells by the fluorescence microscope and the image analysis using Imagepro plus6.0software to calculate the average fluorescence intensity, the results showed:calcium ions of the diabetic group the mean fluorescence intensity higher than that in the control group. After geniposide drug intervention,the calcium fluorescence intensity was decreased. In the group of12.5mg/L, calcium fluorescence intensity of the DM group decreased from0.103to Conclusion:The growth of EPCs of peripheral blood in diabetic patients was slow. The level of intracellular ROS was increasing, and calcium overload. However geniposide could significantly increase the viability of peripheral blood EPCs in diabetic patients, reducing the level of intracellular ROS, significantly alleviate EPCs calcium overload. The geniposide could maintain the ionic homeostasis of intracellular calcium, reduce cell toxicity induced by the oxidative stress. It provided an evidence of geniposide for the prevention and treatment of vascular lesions in diabetic patients.