| In recent years,as the world’s population continues to age,the incidence of age-related diseases has also gradually increased.These diseases have become a serious threat to the life and health of the elderly.In order to improve the life quality of the elderly people and to reduce the social burden,we must accelerate the research on the mechanism of aging and the development of drugs for the treatment of aging-related diseases.Studies have found that there are a large number of senescent cells in the elderly.These accumulated senescent cells will affect the normal function of adjacent cells through autocrine and paracrine pathways,and then lead to the loss of tissue homeostasis,which will eventually cause the occurrence of aging-related diseases.Is it possible to reduce the incidence or slow the progress of the related diseases by removing accumulated senescent cells in the body.Studies have found that targeted elimination of senescent cells in aging mice can improve aging-related characteristics and prolong lifespan in mice.This study suggests that targeted elimination of senescent cells to regain health is feasible.Studies have found that FOXO4 protein in senescent cells interacts with p53 protein,which is not found in normal cells,and that blocking the interaction in senescent cells will induce cell apoptosis.Therefore,based on this molecular mechanism,this research designed and constructed a screening model for FOXO4-p53 interaction inhibitors.This model was used to screen the small molecular compounds that could inhibit FOXO4-p53 interaction,and verified the apoptosis-inducing effect of the compounds on senescent cells,with a view to providing drug lead compounds for the development of drugs to clear senescent cells.We first used the HOSEpiC human ovarian epithelial cell cDNA as a template to amplify the FOXO4 gene coding fragment,and inseted into the pACT vector to successfully construct the pACT-FOXO4 expression vector,which was co-transfected with pBind-p53 and pG51uc vectors into HEK293T cells,the detection of luciferase activity showed that luciferase was highly expressed in the cells,indicating that the FOXO4-p53 interaction screening model was successfully constructed.This model was used to screen the small-molecule compound library in laboratory,and 14 small-molecule compounds that could inhibit FOXO4-p53 interaction were successfully screened out.Secondly,we used H2O2 to induce 2BS in human embryonic lung fibroblasts,and identified that H2O2 induced senescence in 2BS cells by aging-relatedβ-galactosidase staining.We also examined the cell cycle and the expression of the transcription factor p21,and found that H2O2 treated cells had undergone cycle arrest and the expression of the senescence marker p21 was up-regulated.These results indicated that we have successfully constructed a senescent cell model.Finally,we used the constructed senescent cell model to verify the effect of the 14 compounds screened out.First,the IC10 value of the compounds on the viability of 2BS cells was measured by MTT method to obtain the non-toxic concentration of the compounds on the cells.It was found that there are 10 kinds of compound which IC10 values were greater than 5μg/mL,so we treated senescent cells and normal cells at 5μg/mL concentration,and detected the effect of the compounds on the apoptosis of normal and senescent 2BS cells by DAPI staining,TUNEL staining and flow cytometry.The results showed that the senescent 2BS cells treated with the compounds A3-37,A3-40,A4-47,A5-71,A6-56,A6-69 partly undergo apoptosis,indicating that the compounds have promotive effect on the apoptosis of senescent cells.In this study,we successfully constructed a screening model for senescent cell scavengers,and screened out six small molecule compounds that can induce senescent cell apoptosis by blocking FOXO4-p53 interaction.Therefore,this study provides an effective drug screening model for the development of aging cell scavengers,and also provided lead compounds for the development of anti-aging drugs. |
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