Roles Of Ceramide In H₂O₂-induced Senescence In HFL-1 Cells

With the aging of the population,the prevention and treatment of aging-related diseases have become an important research area.Cell senescence is considered as the basis of individual aging.General aging of cells will lead to individual aging,and it is also the culprit for a variety of aging-related diseases.Therefore,understanding the biological basis of cell senescence and its related molecular mechanisms are very important for the diagnosis and treatment of aging-related diseases.Ceramide is a bioactive sphingolipid with unique structure,and it is also one of the central molecules in sphingolipid metabolism.It plays a key role in regulating cell functions such as cell proliferation,death,migration and aging.Ceramide can affect the process of cell senescence through various pathways.It has been found that the level of endogenous ceramide was increased in senescent cells,and the addition of exogenous ceramide induced cell senescence.However,in our study,mass spectrometry(MS)results showed that in the human fetal lung fibroblast-1(HFL-1)premature aging model induced by sub-lethal H2O2 stimulation,the level of ceramide was declining during the aging period.In addition,the regulation of intracellular ceramide levels can affect cell senescence.Therefore,the relationship between ceramide and H2O2-induced cell senescence was explored in this study.The main research findings of this study:(1)Intracellular ceramide level decreased during cell senescence.Mass spectrometry results showed that during the senescence of HFL-1 cells induced by H2O2,the intracellular ceramide levels continued decreasing.Morever,mass-spectrometry and reverse transcription-polymerase chain reaction(RT-PCR)results showed that the synthesis of ceramide increased during early cell senescence,including the de novo synthesis pathway,the sphingomyelin pathway and the Hexosylceramide(HexCer)pathway.However,due to the continuous synthesis of ceramide downstream derivatives such as sphingomyelin,the consumption of ceramides increased,which leads to the decrease of intracellular ceramide levels during cellular senescence.(2)Ceramide could cooperate with H2O2 to regulate aging,but H2O2 did not regulate aging through ceramide metabolism.The reduction of ceramide levels by H2O2 may be its negative regulation mechanism of aging.Myriocin blocked intracellular ceramide synthesis by inhibiting the activity of serine palmitoyl-CoA transferase(SPT)in the de novo synthesis pathway of ceramide.Palmitic acid(PA)as a substrate for ceramide synthesis could greatly promote the synthesis of ceramide in cells.MYR and PA were used to reduce and increase the level of ceramide in the cells and then induce HFL-1 cell senescence with H2O2.MTT assay showed that reducing intracellular ceramide levels reduced H2O2-induced oxidative damage,while increasing intracellular ceramide levels increased H2O2-induced oxidative damage.Senescence-associated ?-galactosidase(SA-?-gal)staining and flow cytometry and Western blot(WB)assays revealed that the number of SA-?-gal positive cells,the number of cells in the G2 phase,the levels of p21 and Cyclin-D1 protein in cells with low ceramide levels were lower than those in control cells,while the cells with high ceramide levels were completely the opposite.This suggested that reducing the level of ceramide in cells can delay the aging process of cells,and increasing the level of ceramide in cells will promote the process of cell senescence.Moreover,H2O2 did not regulate cell senescence through ceramide,and the reduced ceramide levels by H2O2 may be its negative regulation mechanism of aging.(3)Ceramide-induced cell senescence is not associated with protein phosphatase 2A(PP2A).After discovering the role of ceramide in cell senescence,the mechanism by which ceramide regulated the process of cellular senescence was explored.WB detection revealed no significant changes in intracellular p21 and Cyclin-D1 protein levels after activation or inhibition of PP2A.This suggested that PP2A was not a key factor in ceramide regulation of cell senescence.Ceramide may play a role through other targets such as PKC and PP1,further research is needed to determine the molecular mechanism for ceramide regulation of cell senescence.Conclusion:During cell senescence caused by sub-lethal H2O2,cells reduced ceramide levels through various sphingolipid metabolism pathways.Ceramide could cooperate with H2O2 to regulate aging,but H2O2 did not regulate aging through ceramide metabolism,the reduced ceramide levels by H2O2 may be its negative regulation mechanism of aging.In addition,ceramide-induced cell senescence was not associated with PP2A.This study revealed that reducing ceramide levels in cells may be a new target for delaying cell senescence.