Inhbition Effect Of Total Flavonions From Inula Brtannica Flower On Senecence L929 Induced By Advanced Glycation End Products

Part one Effects of advanced glycation end products on aging of L929Objective To establish the aging model of NCTC clone 929(L929)induced by advanced glycation end products(AGEs)and to explore its biological mechanism.Methods In this study,L929 was randomly divided into six groups:blank control group(cultivated L929 with common incubation media)?0.025g/L AGEs group,0.05g/L AGEs group,0.1g/L AGEs group,0.2g/L AGEs group,positive control group(D-galactose,10g/L).Cell proliferation was detected by CCK-8 at 24,48 h and 72 hours.The establishment of aging model in L929 were evaluated by senescence aging ?-galactosidase(SA-?-gal)staining and cell cycle assay,and the expression of p16 and Cyclin D1 were detected by western blot;The levels of reactive oxygen species associated with oxidative stress in the cells,and aging indicators such as superoxide dismutase(SOD),malondialdehyde(MDA)and catalase(CAT)were examined.Results CCK8 showed that AGEs could inhibit L929 proliferation and cell viability decreased significantly after cultivated for 48 hours.Compared with blank control group,AGEs with dose-dependent could increase the activity of ?-galactosidase(P<0.01,Resp)and the blocking rate of phase G1(P<0.01,Resp).AGEs decreased the activity of SOD and CAT(P<0.01,Resp)and increased the level of MDA and reactive oxygen species(P<0.01,Resp).The number of cells in phase G1 and ?-galactosidase positive staining cells in experimental group A was significantly lower than positive control group(P<0.01,Resp).There was no significant differences between 0.025g/L AGEs group and blank control group.The activity of SOD and CAT were significantly higher than positive control group(P<0.05,Resp),while the level of MDA and reactive oxygen species were significantly lower than positive control group(P<0.05,Resp).There were no significant differences between AGEs(0.1 and 0.2 g/L)and positive control group in these respects(P>0.05).Conclusion 0.1g/L and 0.2g/L AGEs can establish aging L929 in vitro model,which may be related to the changes of oxidative stress.Part two Inula britannica flower total flavonoids inhibiting the expression of RAGE in aging L929 induced by Advanced glycation end productsObjective To investigate the protective effect of Inula britannica flower total flavonoids(IBFTF)on aging L929,which induced by Advanced glycation end products(AGEs),and the mechanism of expression of Receptor advanced glycation end products(RAGE).Methods The senescence L929 model was established using 0.1 g/L AGEs to stimulate L929 for 48 hours,and different concentration of IBFTF(0.1 g/L?0.2 g/L and 0.4 g/L)was given to treatment group continue to culture 6 hours,and 0.1 g/L aminoguanidine(AG)was given to positive group continue to culture 6 hours,and then blank group was cultured with common cultured medium.The levels of reactive oxygen species associated with oxidative stress in the cells,and aging indicators such as superoxide dismutase(SOD),malondialdehyde(MDA)were also examined.The fluorescent intensity of RAGE was detected by immunofluorescence.The expression of RAGE protein and m RNA was detected by Western blot and q PCR respectively.Results IBFTF significantly inhibited AGEs-induced L929 senescence and decreased RAGE protein and m RNA expression in a dose-dependent manner(P<0.01,Resp).Different concentrations of IBFTF could significantly increase the SOD activity and reduce the MDA content andeliminate the reactive oxygen species in aging L929(P<0.01,Resp).Conclusion Protective effect of IBFTF on aging L929 induced by AGEs may be related to its effect on the surpressing the expression of RAGE.