Mechanism Of Lactate Attenuating Endothelial Cell Senescence

Cellular senescence is a state characterized by permanent cell cycle arrest.Telomere attrition,metabolic perturbation and epigenetic modification alterations are the main incentives.In addition to participating in the metabolic processes,some metabolic enzymes and metabolites also play non-metabolic functions in cells,such as serving as epigenetic modifying enzymes and modification substrates to affect the epigenetic modifications of DNA,histone and nonhistone proteins.Therefore,the connection between cell metabolism and epigenetic modifications plays a crucial role in regulating gene expression and cellular senescence.The relationship between various metabolites and senescence have been reported widely.For instance,acetate can promote cell senescence.But the relationship between lactate as well as protein lactylation in cellular senescence is remain unknow.In our study,we found that in senescent HUVECs,intracellular lactate concentration and total protein lactylation level were significantly decreased.The cellular senescence phenotype was attenuated after exogenous Nala treatment,suggesting that lactate is closely related to endothelial cell senescence.We also identified the downstream target for lactate and uncovered the underlying mechanism.The following are the specific results:1.During the aging process of the human umbilical vein endothelial cell(HUVEC,P6 VS P27),the level of lactate dehydrogenase-A was significantly reduced,the concentration of intracellular lactate was significantly reduced and the level of total protein lactylation was significantly reduced.Cell cycle suppressor protein P16 was significantly increased,indicating that lactate and lactylation are closely related to cell senescence.2.0 mM,2 mM,5 mM,10 mM,and 20 mM Nala(sodium lactate)was added to HUVEC,and Na Cl(sodium chloride)was added as a control.It was found that exogenous Nala treatment could reduce senescence associated β-galactosidase(SA-β-Gal)staining positive rate,and the effect was the most significant at the concentration of 10 mM,while the level of total protein lactylation was increased in a dose-dependent manner.After 10 mM Nala treatment,significantly reduction of cell cycle suppressor proteins p16,p21 and p53,as well as IL6 and IL8 in SASP(senescence associated secretion phenotype)were detected by q RT-PCR;SAHF(senescence associated heterochromatin foci)showed significant reduction of the percentage of senescent cells;the growth experiment also found that lactate can promote cell proliferation.These results showed that exogenous lactate could attenuate endothelial cell senescence and promote cell proliferation.3.We knocked down the expression of LDHA to reduce the production of intracellular lactate.SA-β-Gal staining showed that the percentage of senescent cells was significantly increased after LDHA knockdown;the transcription levels of p16,p21,p53,IL6 and IL8 were significantly increased when LDHA was knockdown.Furthermore,exogenous Nala treatment could reduce the percentage of senescent cells after LDHA knockdown.These results suggested that LDHA knockdown can promote cell senescence by reducing endogenous lactate level.4.To identify the downstream targets of lactylation,we used LC-MS(liquid chromatography-mass spectrometry)to detect the proteins with the highest level of lactylation and identified PKM2 as a potential target.To investigate whether PKM2 is the downstream target of lactylation,we immunoprecipitated PKM2 from cells and found that PKM2 can indeed undergo lactylation.Using Co-IP,we found there is an interaction between PKM2 and LDHA.The PKM2 lactylation level was decreased after LDHA knockdown.Furthermore,we found that p300 was the modifying enzyme that catalyzes the lactylation of PKM2.These results indicated that LDHA could interact with PKM2,produce lactate and modify PKM2 with lactylation through p300.5.To verify whether lactate attenuats cell senescence through PKM2,we constructed the cell line with stable PKM2 knockdown and conducted senescence phenotype experiments.The results showed that SA-β-Gal staining and SAHF senescent cell positive rate were significantly increased after PKM2 knockdown;the senescent cell positive rate was significantly reduced after Nala treatment in the p LKO.1-control cells,but not in the p LKO.1-PKM2 cells.These results indicated that PKM2 is the key downstream target of lactate in attenuating cell senescence.In summary,our study found that lactate can serve as an epigenetic modification precursor for PKM2 lactylation by p300,which then attenuates cell senescence.Our research thus provides an important theoretical basis for the development of antisenescence small-molecule metabolic drugs.