Effect of Different Isorhamnetin of Different Density Cultured Rat C6 Glioma Cells in Vitro

Jinhua Yang, Zhuanni Sun


Objective To explore the influence of different concentrations of isorhamnetin on C6 glioma cell morphology. Methods Set the blank control group, blank solvent control group and reagent group of four concentration, the growth of cells were observed under microscope; MTT assay was used to test the effect of isorhamnetin on cultured C6 glioma cells, as well as calculate the cell inhibition rate and survival rate; flow cytometry was used to check the detection peak and detection rate of Isorhamnetin group and negative control apoptosis group, and analyzed the relationship between different concentrations of isorhamnetin and C6 glioma cell apoptosis rate; total protein was extracted from cells, and used Western blotting to detected total AKT protein and Ser473 AKT protein loci in cells; used SD rats to construct brain glioma model, feed isorhamnetin plain to them for five days, and then used HPLC to detect plasma, liver, brain tissue content. Results Under the observation of inverted microscope and image analysis, after using Isorhamnetin, tumor cells appear apoptosis and necrosis change. Display with different Isorhamnetin MTT colorimetric method shows that the higher the concentration of added Isorhamnetin, the worse the growth rate of C6 glioma cells in vitro, and the higher the Inhibitory rate, the lower survival rate. The flow cytometric detection shows the C6 glioma cells which is added 40 ug/ul Isorhamnetin have the highest rate of apoptosis. After adding 80 μ g/ μ l concentration of the isorhamnetin, C6 glioma cells have the lowest survival rate. Western blot test shows the AKT protein and Ser473 total site AKT protein density is in reverse proportion to the increase of the concentration of the isorhamnetin. High performance liquid chromatographic method has determined that there are isorhamnetin in both the rat plasma and brain tissue, which shows that the plasma and tissue all have different isorhamnetin  distribution,  and  isorhamnetin mainly exist in the brain tissue. Conclusion Low concentration  of  isorhamnetin  can  induce apoptosis  of  C6  glioma  cells,  and  high concentration of isorhamnetin can lead to apoptosis and necrosis of C6 glioma cells in vitro, which has obvious inhibitory effect on the growth of glioma cells, and the mechanism is closely related to PI3K/AKT pathway, and in SD rat brain glioma model , the high performance liquid chromatography was used to detect the content of plasma and brain tissue, which indicated the isorhamnetin has target in brain tissue, which provided  experimental  evidence  for  the development and utilization of isorhamnetin in mice.

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