Study On The Mechanism Of Diosmetin Treatment Of Glioma Based On PI3K/AKT/mTOR Signaling Pathway

Background:Gliomas are one of the most common malignancies,and its morbidity and mortality continue to increase every year.The current treatment methods for gliomas include surgical resection,chemotherapy,and radiation therapy.However,due to the highly invasive characteristic of gliomas,the boundary with surrounding normal tissues is blurred;the brain is rich in nerves and there are many restricted areas for surgical;and the blood-brain barrier,blood-tumor barrier and other interfering factors that hinder the delivery of therapeutic drugs,these make the treatment of gliomas difficult,ineffective,poor prognosis,tumor recurrence and short median survival,Which indicates that the treatment of gliomas is still a huge challenge.Therefore,it is urgent to explore and develop new methods for treating gliomas with high efficiency and low toxicity.Natural flavonoids are widely studied as anti-tumor drugs due to their excellent anti-tumor activity and low biotoxicity.Diosmetin is a natural flavonoid compound.Studies have shown that diosmetin can promote the apoptosis of various tumor cells such as glioma cells and thus play an anti-tumor role.Therefore,diosmetin is considered to have great potential application prospects in the treatment of gliomas.However,there are few reports on the treatment of glioma and its mechanism by diosmetin.The PI3K/AKT/m TOR signaling pathway is one of the classic signal transduction pathways in cells,it is widely present in various cells and participates in many physiological and pathological activities in cell.It plays a vital role in inhibiting apoptosis,promoting cell proliferation,promoting differentiation,promoting migration and so on,and it is also closely related to the occurrence and development of various tumors such as glioma.This paper will use in vitro cell experiments to prove whether diosmetin exerts an anti-glioma effect through the PI3K/AKT/m TOR signaling pathway to explore its potential mechanism of action.Objective:We screened glioma cell lines most sensitive to diosmetin from gliomas C6 cells,U118 cells,and TG98 cells,and from the perspective of PI3K/AKT/m TOR signaling pathway,to preliminary explore whether the anti-glioma effect of diosmetin is related to its interference with the PI3K/AKT/m TOR signaling pathway in glioma cells,thus providing a certain theoretical basis for the application of diosmetin in the treatment of gliomas.Method:(1)The CCK-8 method was used to detect the effect of diosmetin on glioma cell C6 cells,U118 cells,TG98 cells and normal mouse brain astrocytes cells C8-D1 A activity,and to select the most sensitive glioma cell to preliminarily explore the mechanism of diosmetin inhibiting gliomas.(2)Calcein AM staining was used to further verified that diosmetin inhibited C6 cells proliferation.(3)Fluorescent probe DCFH-DA was used to detect the effect of diosmetin on ROS in C6 cells.(4)Hoechst 33342/PI double staining method and inverted microscope was used to detect the effect of diosmetin on apoptosis and morphology of C6 cells.(5)Flow cytometry(PI/RNase staining)was used to detect the effect of diosmetin on C6 cells cycle.(6)Cell scratch test and Transwell migration test was used to detect the effect of diosmetin on the migration ability of C6 cells.(7)Cellular immunohistochemistry and Western blot method was used to detect the effect of diosmetin on the expression of molecular p-PI3 K,p-AKT,and p-m TOR and the phosphorylation of PI3 K,AKT,m TOR in PI3K/AKT/m TOR signaling pathway in C6 cells.Result:(1)The results of CCK-8 method and Calcein AM staining showed that:compared with the blank control group,the concentrations of 0 μM,12.5 μM,50μM,and 200 μM diosmetin have certain inhibitory effects on C6 cells,U118 cells,and TG98 cells,and shows a certain concentration and time dependence,Among them,C6 cells are the most sensitive to diosmetin;Diosmetin has no significant inhibitory effect on mouse brain astrocytes C8-D1 A.(2)The results of the fluorescent probe DCFH-DA showed that: the concentration of 12.5 μM,50 μM,and 200 μM diosmetin acted on C6 cells for 24 h,48 h,and 72 h,respectively.Compared with the blank control group,diosmetin can significantly reduce the intracellular ROS level,and with the extension of the action time of diosmetin or the increase of the concentration,the less the content of ROS in the cells.(3)The results of Hoechst 33342/PI double staining method and inverted microscope showed that: C6 cells were treated with 12.5 μM,50 μM,and 200 μM diosmetin,compared with the blank control group,we can see that the number of cells decreased,the gap became larger,and the volume became smaller,the number of debris increased,and the apoptosis rate increased.(4)The results of cell cycle detection showed that: the concentrations of 12.5μM,50 μM,and 200 μM diosmetin acted on C6 cells for 12 h,compared with the blank control group,diosmetin can induce the stagnation of C6 cell cycle at the G0/G1 phase,and it was positively correlated with the concentration.(5)The results of cell scratch test and Transwell migration test showed that:C6 cells were treated with 12.5 μM,50 μM,and 200 μM diosmetin,compared with the blank control group,the greater the concentration of diosmetin,the stronger its ability to inhibit C6 cell migration.(6)The results of cell immunohistochemistry and Western blot showed that:the concentrations of 12.5 μM,50 μM,and 200 μM diosmetin acted on C6 cells for 24 h,compared with the blank control group,the expression levels of p-PI3 K,p-AKT,and p-m TOR protein in the cells decreased,and the phosphorylation level of PI3 K,AKT,m TOR decreased.Conclusion:(1)The detection of the activity of diosmetin on glioma cells and normal mouse brain astrocytes shows that diosmetin has the characteristics of high efficiency and low toxicity.(2)Diosmetin may inhibit the activity of the PI3K/AKT/m TOR signaling pathway in C6 cells,thereby exerting the effect of inhibiting the growth and proliferation of C6 cells,inducing apoptosis,blocking the cell cycle to stay in G0/G1 phase,and inhibiting cell migration,which provides a certain idea,method and theoretical basis for the research of diosmetin in the treatment of gliomas.