Baicalein Promotes Autophagy And Apoptosis And Related Mechanisms On Glima Cells

Objective: 1.To investigate the autophagy of baicalein(BAI)on glioma U251 cells and the apoptosis during autophagy.2.To explore the pathways of autophagy of U251 cells activated by baicalein.Methods : 1.Ad-m Cherry-GFP-LC3 B adenovirus was transfected into U251 cells and the expression of LC3 protein was observed in BAI at different time.2.Western blot was used to detect the expression of LC3,p-AMPK and cleaved caspase-3 protein on glioma cells treated with BAI at different concentrations(0,10,20,40,80 μmol/L)for 24 h.3.Western blot was used to detect the expression of LC3,p-AMPK and cleaved caspase-3 protein at different time(0,6,12,24,36 and 48 h)after treatment with BAI(80 μmol/L).4.The expression of LC3 protein was detected by Western blot in BAI(80 μmol / L)alone and in combination witn CQ.5.The immunofluorescence was used to detect the changes of LC3 protein in BAI(80 μmol/L)at diferent time(0,6,24 and 36 h).6.The immunofluorescence was used to detect the changes of LC3 protein in BAI(80 μmol/L)combination with CQ.7.The mitochondrial membrane potential was observed by DAPI staining and JC-1 staining were used to observed the change of mitochondrial membrane potential under the conditions of different time(0,24,36,48 h)of BAI(80 μmol/L).8.The growth of glioma cells was observed under light microscope,the fragmentation of nuclei was observed by DAPI staining and JC-1 staining was used to observe the change of mitochondrial membrane potential after BAI(80 μmol/L)alone and in combination with inhibitor compound C.9.The expression of LC3,p-AMPK and cleaved caspase-3 protein was detected by Western blot in BAI(80 μmol/L)alone and in combination with compound C.10.The CCK-8 method was used to detect cell viability in different concentrations of BAI(20,40,80,100,160 μmol/L),different concentrations of TMZ(100,200,300,400,500 μmol/L)under different time.11.The CCK-8 method was used to detect cell viability in BAI(100 μmol/L)combinationwith different concentrations of TMZ(100,200,300,400,500 μmol/L)under different time.Results : 1.Ad-m Cherry-GFP-LC3 B adenovirus was transfected into U251 cells,Ad-m Cherry-GFP-LC3 B showed diffuse yellow fluorescence-dotted yellow fluorescence-spots with the increase of BAI drug time,indicating the change of LC3 protein after adding BAI.2.Western blot showed that the expression of LC3 protein increased gradually with the increase of BAI concentration and treatment.After adding CQ,the expression of LC3 protein was increased.Immunofluorescence assay of LC3 protein confirmed this result.3.DAPI staining observed the nucleus was broken at 24 h,with the increase of time fragmentation increased;JC-1 staining showed that the mitochondrial membrane potential changes in cells and has time-dependent;western blot showed that the expression of cleaved caspase-3 protein was positively correlated with the time and BAI concentration.4.The expression of p-AMPK protein was increased during autophagy by western blot.After adding compound C,we found that cell growth was inhibited,the levels of LC3 and cleaved caspase-3 were also decreased.In addition,DAPI and JC-1 showed that the apoptosis was also alleviated.5.The results of CCK-8 showed that cell viability of U251 cells was decreased with the increase of drug concentration and time of action.The cell viability was significantly decreased with the increase of TMZ concentration and the prolongation of the time of TMZ and BAI.Conclusion : 1.Baicalein could induce autophagy and apoptosis in glioma U251 cells.2.BAI participates in BAI-induced autophagic death of gliomas by AMPK.3.BAI promotes the anti-glioma effect of TMZ.