| Objective: To investigate whether the combination of formononetin and temozolomide(TMZ)has a synergistic effect on glioma C6 cells,and to explore the molecular mechanism of the synergistic effect of two drugs combined with synergistic anti-glioma.Then a rat glioma model was established to explore the in vivo efficacy of the two drugs combined.Methods:(1)The rat glioma C6 cells were used to explore the anti-proliferative effects of the drugs in vitro.In this study,the solvent control group,the formononetin group,the TMZ group and the combination group were respectively set up.The effect of each group on the viability of C6 cells after administration was detected by MTT assay.The combined index(CI)of formononetin and TMZ was calculated according to the Chou-Talalay theory,and the optimal combination of two drugs was selected according to the ci value.(2)HE staining was used to observe the effect of formononetin and TMZ in combination on the morphological changes of rat glioma C6 cells compared with single drug after 48 hours.(3)Flow cytometry was used to compare the effects of the two drugs combined with single drug treatment on the apoptosis of rat glioma C6 cells.(4)Wound-healing assay and Transwell experiments were used to investigate the effects of two drugs combined with single drug on the migration of rat glioma C6 cells.(5)The rat glioma C6 cell line was injected through the skull through a microinjection into the rat brain to establish a rat orthotopic brain glioma model.The scalp of the rat which fixed on the stereotaxic apparatus after anesthesia was incised and then positioned in the caudate nucleus region of the left cerebellum.Ten microliters of C6 cells were slowly injected into the caudate nucleus region of the left brain within 10 minutes with a microsyringe.The needle were pulled slowly and suture the wound.After the rats were awake,they were observed for one week.The rats with successful rat model of orthotopic glioma were selected and administered,and then investigating the in vitro efficacy of the combination of formononetin and TMZ.(6)A glioma U251 cell suspension was injected under the right forelimb of the mouse to establish a mouse ectopic subcutaneous glioma model.The tumor growth of mice was observed after the establishment.Then the tumor-bearing mice were selected and randomly grouped for in vitro pharmacodynamic exploration of drug combination for later use.Results:(1)Formononetin mediates the anti-proliferative effect of glioma C6 cells in a concentration-dependent manner.After combined treatment of glioma C6 cells for 48 hours with a specific concentration range of formononetin and TMZ,the proliferation inhibition of cells has a synergistic effect,and when the concentration of formononetin is 80 ?M and the concentration of temozolomide is 500 ?M,the synergistic effect produced by the two drugs is the strongest.(2)For 48 hours after combined treatment of formononetin and TMZ for C6 cells,the morphology of the combination of formononetin and TMZ cells significantly changed compared with the morphology of the control group and single drug group.The number of cells was significantly reduced,and the cells became smaller and the chromatin condensed.(3)By flow cytometry,C6 cells were treated with formononetin and TMZ for 48 hours,the apoptosis rate of the cells was significantly higher than that of solvent control group and single drug group,p<0.05.The results of Cleaved Caspase showed that the protein level of Bcl-2 in the combination group was lower than that in the control group and the single drug group.The expression level of Bax protein was higher in the combination group than in the control group and the single drug group.The expression of Caspase-3 and Caspase-9 in the combination group was also higher than the control group and the single drug group.(4)Wound-healing assay and Transwell experiments showed that combination of formononetin and TMZ increased the inhibition of migration of glioma C6 cells compared to the single drug group.Inhibition can be achieved by down-regulating the protein expression levels of MMP-2 and MMP-9.(5)The rat brain glioma in situ model and mouse glioma subcutaneous heterotopic model were successfully established.Conclusion: Formononetin has antiproliferative effects on rat glioma C6 cells in a concentration-dependent manner.Formononetin and TMZ combined in a certain concentration range have a synergistic effect on glioma C6 cells.For combined use of formononetin and temozolomide can promote the apoptosis of glioma C6 cells through down-regulation of Bcl-2 protein expression and up-regulation of Bax,Caspase-3,and Caspase-9.The migration of cells was inhibited through down-regulating the protein expression levels of MMP-2,MMP-9.The rat model of orthotopic glioma was successfully established to facilitate the in vivo pharmacodynamic study of the synergistic effect of formononetin and TMZ for later use. |
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