| Objective:Anti-tumor drugs are difficult to“enter the brain and enter the tumor”because of the blood-brain barrier/cerebral blood-tumor barrier?BBB/BBTB?,which makes the treatment of glioma become a difficult problem in the medical field.The combination of antineoplastic drugs and meridians drugs to increase the concentration of therapeutic drug target areas is an effective method to improve the therapeutic effect of glioma.In this paper,Ligusticum chuanxiong,which is frequently used in traditional Chinese medicine,was selected as the research object,and the volatile oil with strong activity in Ligusticum chuanxiong was selected to treat the antitumor effect of temozolamine in vivo and to the cytotoxicity of glioma in vitro.From the point of view of pharmacodynamics to study whether the volatile oil of Chuanxiong has the effect of improving the compatibility of drugs against glioma;To compare the concentration difference of temozolamine in the intracellular and external fluid of HUVEC after single administration and combined administration,and to elucidate the mechanism of action by Western blot assay.Method:1?Establish the animal model of C6 glioma,and verify whether the model is established successfully by observing the survival state of rats and histopathological examination.2?After the animal model of C6 glioma was successfully established,drug therapy was given to investigate the body weight and survival status of the model rats,to detect the changes of tumor volume in each group,and to calculate the tumor inhibition rate.3?In vitro cell experiments,to investigate the toxicity of temozolamide single administration,chuanxiong volatile oil and temozolamide combined administration to the epidermal cells HUVEC and U87-MG glioma cells;The contents of temozolomide in intracellular and external fluid of HUVEC were detected by HPLC after single administration of temozolomide and ligusticum chuanxiong volatile oil combined with temozolomide,and statistical analysis.4?Western blot assay was used to detect the expression of GLUT-1,occludin,P-gPand Caveolin-1 protein after single administration of TMZ and combined administration of volatile oil from Ligusticum chuanxiong with TMZ.Results:1?This paper established the rat glioma model,the method is feasible,reproducibility,,the rate of tumor formation has certain security,the period of tumorigenesis is short,the model has good reliability,high stability.It is close to human brain glioma in biological characteristics.It is an ideal animal model of glioma.2?The tumor volume of the temozolomide grouPand the ligusticum chuanxiong grouPwas significantly smaller than that of the model grouP?P<0.05?.the volatile oil of Ligusticum chuanxiong combined with temozolomide?1:5 group;1:10 group?was significantly different from that of the model grouP?P<0.01,P<0.05?.Comparison between the 1:5 grouPand the montemozolomide grouPsignificant difference,P<0.05.3?The survival rates of HUVEC cells in the range of 20-200?g·mL-1 TMZ,1.250×10-3?g·mL-1 tween-80 and 6.250×10-3?g·mL-1 ligusticum chuanxiong volatile oil were all>90%,indicating that there was no effect on normal cells within the concentration range.Compared with the control group,the concentration of TMZ at 20?g·mL-1 was non-toxic?P>0.05?.When the concentration increased to 40?g·mL-1 or more,it could kill glioma cells significantly?P<0.01?.The higher the concentration,the stronger the killing effect.Compared with the temozolomide group,the volatile oil of Ligusticum Chuanxiong could significantly enhance the killing effect of temozolomide on glioma cells?P<0.01?,and the synergistic effect was stronger with the increase of the concentration of volatile oil.6.250×10-3?g·mL-1 volatile oil of Ligusticum Chuanxiong can significantly increased the concentration of temozolomide in the intracellular fluid of HUVEC?P<0.05?.4?Western blot results showed that the protein expression of GLUT-1,claudin-5,occludin and P-g Pwas significantly decreased when the volatile oil of Ligusticum Chuanxiong was combined with temozolomide.Compared with temozolomide group,claudin-5,occludin and P-gPprotein expression were significantly decreased in Temozolomide and Ligusticum chuanxiong volatile oil(3.125×10-3?g·mL-1,6.250×10-3?g·mL-1)groups?P<0.01?.Compared with blank control grouPand temozolomide group,the expression of GLUT-1 protein in Temozolomide Combined with volatile oil of Ligusticum Chuanxiong(1.563,3.125,6.250×10-3?g·mL-1)grouPdecreased significantly?P<0.01?.And with the increase of volatile oil concentration,the more protein expression decreases.Conclusion:The volatile oil of Ligusticum Chuanxiong can enhance the inhibitory effect of temozolomide on glioma in vivo,and the synergistic effect is stronger with the increase of the concentration of volatile oil.On the one hand,the volatile oil of Ligusticum Chuanxiong can promote temozolomide into the interior of the cell through the cell membrane,while capable of inhibiting the expression of GLUT-1,claudin-5,occludin and P-gPprotein.It suggests that the volatile oil of Ligusticum Chuanxiong can down-regulate the expression of GLUT-1 protein,which resulted in the decrease of glucose intake,insufficient energy supply,and affect the function of claudin-5,occludin and P-gp.impact barrier function BBB/BBTB improve the permeability of endothelial cells,while reducing the amount of the drug is discharged from the P-gp,promoting temozolomide into glioma cells,enhance the therapeutic effect of glioma.Decreased expression of claudin-5,occludin and P-gPprotein,destroying the structure and function of tight junction,affecting the barrier function of BBB/BBTB and improving the permeability of endothelial cells,while reducing the amount of the drug is discharged from the P-gp,promoting temozolomide into glioma cells,enhance the therapeutic effect of glioma.With the increase of the concentration of volatile oil in Ligusticum chuanxiong,the inhibitory effect on protein expression was stronger. |
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