Effect Of Inula Britannica Flower Total Flavonoids On The Autophagy Of Senescent L929 Cells

PART ? ESTABLISHMENT OF D-GALACTOSE-INDUCED CELL SENESCENCE MODELObject:To investigate the optimal concentration of D-galactose,which is used to establish the senescent L929 cell model.Methods : L929 cells were treated with D-galactose at different concentrations(0,5,10,20,40g/L),cell viability was detected by MTT assays,the ?-galactosidase activity was detected by galactosidase staining assays,and cell apoptosis was detected by mitochondrial membrane potential assays.Results:Compared with the blank control group,cell viability was significantly decreased in the D-galactose group(P<0.01),and decreased with the increase of D-galactose concentration(P<0.01).Compared with the blank control group,the activity of ?-galactosidase in cells of the D-galactose treatment group increased,and the number of blue staining was significantly higher than that of the blank control group(P<0.01).When the D-galactose concentrations exceeded 20g/L,the number of blue staining was not significant(P=0.22).Compared with the blankcontrol group,the mitochondrial membrane potential was significantly reduced in the D-galactose treatment group(P<0.05),which was concentration-dependent.When the D-galactose concentration exceeded20g/L,the mitochondrial membrane potential was significantly reduced(P<0.01).Conclusion : D-galactose could induce the cell senescence,and increase cell apoptosis.PART ? THE IBFTF DELAYED CELL SENESCENCEObjective:To investigate the effect of Inula britannica flower total flavonoids(IBFTF)on delay cell senescence.Methods: Different concentrations of IBFTF were applied to 20g/L D-galactose-induced senescent L929 cells,and the experiments were divided into blank control group(1640 complete medium),model group(20g/L D-galactose),and IBFTF low concentration group.(20g/L D-galactose + 25mg/L IBFTF),IBFTF medium concentration group(20g/L D-galactose + 50mg/L IBFTF),IBFTF high concentration group(20g/L D-galactose + 100mg/L)IBFTF).After L929 cell culture was completed,cell viability was detected by MTT assay,SOD and ROS content in cells were detected,and the expression of P53 protein in cells was detected by immunofluorescence,and the level of cell senescence was identified.Results:Compared with the blank control group,the cell viability of the model group was significantly decreased(P<0.05).Compared with the model group,the cell viability of IBFTF-treated cells increased.When the concentration of IBFTF exceeded 100 mg/L,the cell viability began to decrease(P<0.05)..Compared with the blank control group,the intracellular ROS content in the model group was significantly increased(P<0.01),the P53 protein expression was significantly increased(P<0.01),and the intracellular SOD content was significantly decreased(P<0.01).Compared with the model group,IBFTF The intracellular ROS content was significantly decreased(P<0.01),P53 protein expression was significantly decreased(P<0.05),intracellular SOD content was significantly increased(P<0.01),and concentration-dependent,when the IBFTF concentration was 100 mg/At L,intracellular ROS content(P<0.01),P53 expression level was the lowest(P<0.05),and SOD content was the highest(P<0.01).Conclusions : The IBFTF could delay cell senescence in a concentration-dependent manner.PART ? THE IBFTF INDUCED CELL AUTOPHAGYObject : To investigate the effect of IBFTF on intracellular autophagyMethods : After applying the gradient concentration IBFTF to senescent L929 cells for 24 h,the experiments were divided into blank control group(1640 complete medium),model group(20 g/L D-galactose),and IBFTF low concentration group(20 g/L D-half).Lactose +25 mg / L IBFTF),IBFTF medium concentration group(20g /L D-galactose + 50 mg / L IBFTF),IBFTF high concentration group(20g / L D-galactose + 100 mg / L IBFTF).The expression of Beclin 1,P62 and LC3 protein was detected by Western Blot,and the concentration of lysosome in cells was detected by OA staining.Results:Compared with the blank control group,the expression of LC3 and Beclin1 protein in L929 cells treated with D-galactose was significantly decreased(P<0.01),and the expression of P62 protein in cells was significantly increased(P<0.01).Intracellular lysosomes The number decreased(P<0.01).Compared with the model group,the expression of LC3 and Beclin1 protein in L929 cells was significantly increased after IBFTF treatment(P<0.01),P62 protein expression was significantly decreased(P<0.01),and the number of lysosomes was decreased.The increase was(P<0.05),and the process was concentration-dependent.When the concentration of IBFTF was 100mg/L,the expression levels of LC3,Beclin1 and P62 and the number of intracellular lysosomes were the highest(P<0.01).Conclusions:IBFTF could increase the protein levels of Beclin1 and P62,and enhance the lysosome concentration,then resulted in the intracellular autophagy of senescent L929 cells.PART ? ASSOCIATION OF CELL SENESCENCE AND IBFTF-MEDIATED AUTOPHAGYObjective:To investigate the relationship between cell senescence and IBFTF-mediated autophagy.Methods : Dealing with 1640 medium,20 g/L D-galactose,100?g/Lchloroquine,100 mg/L IBFTF,40 ng/L rapamycin for senescence L929 cells for 24 h,the cells were grouped into : blank control group(control group),model group(20 g/L D-galactose),negative control group(20 g/L D-galactose + 100 ?g/L chloroquine),IBFTF group(20g/L D-galactose + 100 mg/L IBFTF),positive control group(20 g/L D-galactose + 40 ng/L rapamycin).The expression of LC3 protein was detected by immunohistochemistry.The expression of P16,P19,P21,P53,LC3,Beclin1 and P62 proteins was detected by Western-Blot.Results:The expressions of P19,P16,P21,P53 and P62 proteins in L929 cells treated with D-galactose were significantly higher than those in the control group(P<0.01),and LC3 in the model group and negative control group.The expression of Beclin1 was significantly decreased(P<0.01).The intracellular expression of LC3 and Beclin1 in the IBFTF-treated group and the positive control group was significantly higher than that in the model group and the negative control group,while the intracellular P19,P16,P21,P53 and P62 protein relative models were observed.The content of the control group and the negative control group were significantly lower,and the differences were statistically significant(P<0.01).Conclusions:The IBFTF could delay cell senescence,the cellular autophagy of senescent cell was induced by IBFTF.Therefore,the effect of delay cell senescence by IBFTF treatment may be related to the increase of intracellular autophagy.