The Function And The Underlying Mechanism Of EVs In SASP

BackgroundSenescence is an unavoidable biological process,which is often accompanied with the persistent and low-level secretion of pro-inflammatory factors(Senescenceassociated secretory phenotype,SASP),the process called inflammaging.The SASP attribute to a persistent inflammatory state,which could induce age-related diseases(ARDs)and geriatric syndromes(GS),and is the key risk factors for geriatric morbidity and mortality.Although inflammatory factor of SASP can transmit pro-senescence messages to adjacent,nearby or surrounding cells and tissues in an autocrine,paracrine or endocrine manner;the mechanisms underlying the persistence of low levels of inflammatory factor of SASP remain unclear and therefore,it is of great social significance and economic value to explore the occurrence and development mechanism of inflammaging.Cells release Extracellular Vesicles(EVs)encapsulating bioactive substances through cytosolic vomiting,plasma membrane blowing or plasma membrane shedding,and EVs can shuttle between cells for signaling through their good histocompatibility,participating in pathophysiological processes such as wound healing,regulation of stem cell differentiation and development,tumor angiogenesis and metastasis,immune response,and atherosclerosis.However,the role of EVs as information carriers in inflammatory senescence(SASP)was rarely reported.Therefore,the purpose of this study is to explore the role and mechanism of EVs in inflammaging and transmission of pro-senescence information.Methods1.Cell senescence model was established by continuous passage.Early passage THP-1 cells(≤35 generations)were used as young cells,and late passage THP-1 cells(≥75 generations)were used as senescent cells,and large number of young and senescent THP-1 cells were harvested by continuous passage,simultaneously.Flow cytometry,Flow microsphere array(CBA),Western Blot and other methods were used to analyze senescence markers such as:SA-β-gal activity,P16INK4a expression,cell cycle arrest,inflammatory factor secretion,etc.2.We implemented differential centrifugation and ultracentrifugation to obtain EVs from culture supernatants of young and senescent TPH-1 cells.Quantitative proteomics was used to analyze the differentially expressed proteins between EVs derived from senescent cells and young cells.Bioinformatics analysis was implemented to explore whether EVs derived from senescent cells carry biological information related to senescence.3.Young THP-1 cells or senescent THP-1 cells were co-incubated with EVs derived from senescent cells.Immunoassay,Flow cytometry,CBA,Q-RT-PCR and other methods were employed to analyze SA-β-Gal activity,P16INK4a expression,cell cycle arrest,and inflammatory factor secretion to investigate whether EVs derived from senescencent cells were able to propagate senescence signals.4.Young THP-1 cells or senescent THP-1 cells were co-incubated with EVs derived from senescent cells and the expression levels of TREX1,DNase2,and STING were detected by Western Blot to investigate whether EVs could transmit the senescent information through activation of the STING signal pathway.Results1.In vitro,by continuous culture,the young THP-1 cells(≤35 generations)and the senescent THP-1 cells(≥75 generations)were successfully obtained.Both the expression levels of P16INK4a and the activity of SA-β-gal were increased in the senescent THP-1 cells.2.TMT-labelling Quantitative Proteomics result shows that there are a total of 276 differentially expressed proteins between EVs from the young and senescent groups.Among them,132 proteins were significantly up-regulated and 144 proteins were significantly down-regulated in the senescent group compared to the young group(with fold change>1.3 and P<0.05).Gene ontology(GO)and KEGG analysis show that the up-regulated proteins such as ZFP91(Q96JP5),NFKB2(Q00653),AIMP1(Q12904),and SASH1(094885)are involved in DNA damage repair,cell structure stabilization,metabolism,and inflammation response,and the down-regulated proteins such as GNL3(Q9BVP2),NAT10(Q9HOAO),COL6A1(P12109),VWF(P04275)and C3(P01024)are involved in coagulation,complement system and cell proliferation.3.After co-incubation of young THP-1 cells with EVs derived from senescent cells,CBA assay results showed that the secretion of inflammatory factors IL-1β,IL-6 and IL-8 were increased significantly in the co-incubated young THP-1 cells,compared with un-incubated young THP-1 cells;however,Q-PCR results showed that the mRNA expression levels of the above-mentioned factors was not different between the two groups.In addition,after co-incubation of senescent THP-1 cells with senescent EVs,CBA results showed that the secretion of inflammatory factors IL-1β,IL-6,IL-8,TNF and IL-10 factors were increased significantly in the co-incubated senescent THP-1 cells;similarly,Q-PCR results showed that there was no difference in the mRNA expression of the above inflammatory factors.The dynamic detection results of mRNA expression of inflammatory factors showed that the change trend of mRNA expression of each inflammatory factor over time was not the same.4.Western Blot results showed that the expression of cGAS,STING and NF-κB proteins were elevated in young THP-1 cells after co-incubation with senescent EVs(40 μg/ml);the expression of cGAS,STING and NF-κB were elevated in senescent THP-1 cells after co-incubation with senescent EVs(20 μg/ml or 40 μg/ml).ConclusionsIn vitro,by continuous culture,the senescent cell model was established successfully,when the cell passage number≥75.EVs from senescent cells carry active substances associated with inflammaging.The EVs from senescent THP-1 cells deliver pro-inflammatory senescence substances and induce inflammatory senescence(SASP)in target cells.EVs can promote senescence by activating CGAS-STING and NF-κB signaling pathway.