A Preliminary Study On The Mechanism Of Selective Elimination Of Senescent Cells And Inhibition Of SASP Expression By Cycloastragenol

With the development of living standards and medical technology and the extension of the average life expectancy of the population,China has begun to enter an aging society.Aging is the primary risk factor for many chronic diseases.The heavy morbidity has brought great pain to the lives of the elderly,and it has also imposed a heavy burden on families and society.At the overall level,aging is manifested by the gradual decline in the function of various organs of the body,the level of hormone regulation decreases,and degenerative diseases appear in each organ,such as cataracts,osteoporosis,diabetes,cardiovascular disease,neurodegenerative diseases and tumor and so on.The accumulation of senescent cells is considered to be one of the important culprits leading to aging of the body.Senescent cells not only accumulate in the body,but also secrete senescence-associated secretory phenotype（SASP）through autocrine and paracrine methods,which further leads to the aging of surrounding cells and tissues,and ultimately leads to individual aging and senile diseases happened.Studies on senescent cells have found that injecting senescent cells into young mice will induce the aging of young mice and lead to aging-related diseases.Another study found that the elimination of senescent cells in aging mice by transgenic methods can extend the lifespan of mice and reduce the occurrence of aging-related diseases.Therefore,senescent cells may become a new target for anti-aging.In pharmacology,representative"senolytic"are:Dasatinib+Quercetin,ABT263 can eliminate senescent cells;The JAK inhibitor—ruxolitinib can inhibite the expression of SASP.However,Dasatinib can cause pulmonary edema;ABT263 can cause transient platelets and neutropenia;As an anticancer drug,rusotinib can cause a decrease in platelets,red blood cells or white blood cells for a long time.Therefore,it is particularly important to develop and research compounds that are safe and effective in eliminating senescent cells.In order to further verify the clearance effect of CAG on senescent cells,we used theβ-galactosidase-labeled probe C12FDG to label senescent cells and then used flow cytometry to detect the number of senescent cells.The results showed that the number of senescent cells in the CAG treatment group was significantly reduced.Further validated the elimination of CAG on senescent cells.Therefore,CAG may become a safer"senolytics".Eliminating senescent cells and inhibiting the secretion of SASP require a multi-target and multi-path intervention for the different characteristics of senescent cells as a whole.Traditional Chinese medicine,which is guided by a holistic concept and dialectical treatment,has received much attention because of its long-lasting efficacy,multi-target activity,and few toxic side effects.Therefore,we first used DNA damage agent—etoposide to treat human lung fibroblast W2 to construct a DNA damage-induced senescence cell model.On this basis,the MTT method was used to screen a variety of small molecule compounds with anti-aging activity to eliminate senescent cells.The results showed that the treatment of senescent W2 with the monomeric component of Cycloastragenol（CAG）from the traditional Chinese medicine Astragalus mongholicus,it reduced the number of senescent cells by more than 80%and had no significant effect on non-senescent cells,suggesting that CAG can selectively eliminate senescent W2 cells.In order to further verify the elimination effect of CAG on senescent cells,we used the C₁₂FDG probe labeled withβ-galactosidase to label senescent cells and then used flow cytometry to detect the number of senescent cells.The results showed that the number of senescent cells in the CAG treatment group was significantly reduced,further validated the elimination of CAG on senescent cells.Therefore,CAG may become a safer"senolytics".We saw that a large number of cells died after the treatment of senescent W2 by CAG through the microscope,in order to investigate whether this phenomenon is related to apoptosis.We used the Annexin V/PI double staining method to detect the effect of CAG on apoptosis of senescent W2.The results showed that compared with the control group,the proportion of senescent cells in the CAG-treated group increased by more than 30%.At the same time,our detection of anti-apoptotic protein Bcl2,pro-apoptotic protein Bax and PARP showed that the Bax/Bcl2 ratio was significantly increased,the PARP cleavage band was more obvious.It further proves that CAG promotes apoptosis of senescent W2cells.In addition,the pro-survival pathway PI3K/Akt/mTOR is often up-regulated during cell senescence.After treatment with CAG,we found that the expression of p-PI3K,p-Akt,and p-mTOR decreased,indicating that the PI3K/Akt/mTOR signaling pathway is also involved in the eliminate of senescent W2 cells by CAG.A large amount of SASP is secreted during cell enescence,and SASP is the main component of the aging microenvironment,which affects the tissue repair ability of stem cells,and then promotes the body’s aging process,forming a vicious circle.Therefore,blocking SASP is also an important means of targeting senescent cells.We first studied the effect of CAG on the expression of SASP in senescent W2 cells through Real-time PCR.The results showed that CAG reduced the m RNA expression of IL6,IL10,IL1β,CXCL5,CXCL10,and IGBP7.In addition,we detected several key proteins related to the expression of SASP,CAG inhibited the expression of JAK2/STAT3,p38MAPK/NF-κB,indicating that CAG inhibition of SASP expression may be related to JAK2/STAT3,p38MAPK/NF-κB.In summary,CAG may inhibit the expression of anti-apoptotic protein Bcl2 and down-regulate PI3K/AKT/mTOR promotes apoptosis of senescent cells and eliminates senescent cells,while down-regulating JAK/STAT3 and p38MAPK/NF-κB pathways to inhibit SASP expression.