Study On Intervention Effects And Mechanisms Of Lycium Barbarum Polysaccharide On Cellular Senescence Induced By PM_2.5 In HUVECs

BackgroundFine particulate matter(PM2.5)is a risk factor that poses a significant risk to public health,and PM2.5 pollution causes many health problems.PM2.5 exposure is strongly associated with vascular endothelial senescence.However,the underlying mechanism by which PM2.5 triggers vascular endothelial senescence is unclear.In this study,human umbilical vein endothelial cells(HUVECs)exposed to PM2.5 for a short period of time were used as the research model to investigate the toxic effect of PM2.5 on HUVECs and the potential mechanism of its influence on endothelial cell senescence.On this basis,the potential protective effect and mechanism of Lycium barbarum polysaccharide(LBP)on PM2.5-induced HUVECs damage and senescence were investigated.To provide basic information for in-depth exploration of the possible molecular mechanism of vascular endothelium induced by air pollution,and to provide new ideas and insights for the toxic effects and mechanisms of cardiovascular disease induced by ambient air pollution.Methods1.Toxic effects of exposure to different concentrations of PM2.5 on HUVECsWeigh a certain amount of PM2.5 particles and prepare 20mg/mL PM2.5 stock solution for later use.CCK-8 experiment was conducted by gradient dilution of PM2.5 stock solution.Compared with the control group,the group with statistical difference for the first time was set as the medium dose group with low,medium and high experimental doses,and subsequent experiments were carried out.LDH activity and cell proliferation capacity were detected in control group,low,medium and high dose group PM2.5.Cell morphology was observed by microscope,and reactive oxygen species(ROS),H2O2,MDA,SOD and other indicators of cell oxidative damage were detected by relevant kits.2.Effect of LBP on cellular senescence of HUVECs induced by PM2.5 exposure20 mg/mL of PM2.5 stock solution was diluted in serum-free medium to prepare three doses of low-dose,medium-dose and high-dose venoms.5 mg/mL of LBP stock solution was diluted with serum-free medium to prepare the desired working solution concentration.HUVECs were cultured in corresponding groups for 24 h.The kit measured cell β-galactosidase activity after PM2.5 exposure.Cell cycle was measured by flow cytometry.Senescence associated secretory phenotype(SASP)and markers of cellular senescence,p16(CDKN2A)、p21(CDKN2A)and p53(TP53),were measured by RT-qPCR and Western blot.HUVECs were cultured with LBP for 24h,and CCK-8 experiment was carried out.No observable effect level was selected as the intervention dose of LBP follow-up experiment,and these experiments were repeated with the increase of LBP group and LBP+PM2.5 group.3.LBP alleviates autophagy disorders against PM2.5-induced cellular senescence on HUVECs(1)The effect of PM2.5 on autophagy and the intervention effect of LBPFirst,HUVECs were grouped into control groups and low,medium and high PM2.5 exposure doses.Western blot was used to detect the expression content of autophagy-related proteins including LC3,Beclin 1 and p62 after PM2.5 exposure.The mRNA levels of BECN1 and SQSTM1 were detected by RT-qPCR.Secondly,after stable transfection of LC3 by Ad-mCherry-EGFP-LC3 adenovirus,the autophagy flow of HUVECs was observed by confocal fluorescence microscopy.Finally,the experiments were repeated with LBP group and LBP+PM2.5 group.(2)LBP alleviates PM2.5-induced cellular senescence in HUVECs by alleviating autophagy disordersHUVECs were treated with the autophagy inhibitors 3-methyladenine(3-MA)and Bafilomycin A(Baf Al)in combination with PM2.5,respectively,and the results were compared with those of the PM2.5+LBP group.The changes of key autophagy proteins were detected by Western blot to evaluate the alleviation of autophagy disorder,and the expression levels of cellular senescence marker proteins were detected to evaluate the situation of cellular senescence,so as to judge the cellular senescence after alleviation of autophagy disorder.Results1.Toxic effects of exposure to different concentrations of PM2.5 on HUVECs(1)The results of CCK-8 showed a dose-dependent decrease in cell viability in HUVECs after PM2.5 exposure.The Lowest observed adverse effect level(LOAEL),25 μg/mL dose,was selected as the medium dose for PM2.5 exposure.12.5 μg/mL and 50 μg/mL were used as low-dose and high-dose groups for follow-up experiments,respectively.In subsequent experiments,LDH release was significantly increased in PM2.5 medium and high dose groups compared with control groups(P<0.01).The morphological changes of HUVECs after PM2.5 exposure were observed microscopically,and it was seen that with the increase of PM2.5 dose,the cell length gradually increased significantly,and vacuoleization manifested obviously.The EdU cell proliferation assay showed that PM2.5 exposure significantly reduced the proliferation capacity of cells,and the PM2.5 exposure dose was significantly different than that of the control group(P<0.01).(2)The oxidative damage index showed that PM2.5 exposure increased the accumulation of HUVECs reactive oxygen species(ROS),and the difference was significant in the medium and high dose groups(P<0.01).Compared with the control group,the levels of MDA and H2O2 increased in a dose-dependent manner,and compared with the control group,the MDA content in the high-dose group of PM2.5 was statistically different(P<0.01),and the level of H2O2 in the medium-and high-dose groups was statistically different(P<0.01).Superoxide levels were dose-dependently reduced,and there was a statistically significant difference between the high-dose group and the control group(P<0.01).2.PM2.5 induces cellular senescence and remission of LBP in HUVECs(1)HUVECs were cultured with the medium containing 1000,2000,3000 and 4000 μg/mL LBP for 24 h.The results of CCK-8 showed that the cell viability of LBP cells in 1000 and 2000μg/mL groups was not statistically different from that in the control group.The cell viability of LBP in the 3000 μg/mL group was significantly different from that in the control group(P<0.05),and the difference was significant in the 4000 μg/mL group(P<0.01).Therefore,No Observed Adverse Effect Level(NOAEL),2000μg/mL,was used as the intervention dose of LBP.The cell viability was measured by 2000 μg/mL LBP co-cultured with each dose of PM2.5 for 24 h,and it was found that LBP could significantly improve the cell viability of HUVECs exposed to 50 and 100 μg/mL PM2.5.Follow-up experiments were conducted in control group,PM2.5 high-dose group,LBP group and PM2.5+LBP group.With the intervention of LBP,LDH release and proliferation capacity of HUVECs were significantly alleviated(P<0.01).LBP significantly reduced ROS production after PM2.5 exposure(P<0.01),significantly reduced the increase of MDA and H2O2 levels caused by PM2.5(P<0.01),and increased the decrease of superoxide levels caused by PM2.5(P<0.01),indicating that LBP could effectively reduce the oxidative damage induced by PM2.5.(2)The proportion of β-galactosidase-positive cells after PM2.5 exposure increased dose-dependently,and the difference between the medium and high dose groups was significant compared with the control group(P<0.01).The results of cell cycle detection showed that the proportion of cells in the G0/G1 phase increased from 54.01%of the control group to 68.35%,71.38%and 72.14%after the exposure of low,medium and high doses of PM2.5,and the proportion of cells in the G0/G1 phase increased significantly(P<0.01),while the proportion of cells in the S phase decreased from 35.83%in the control group to 19.34%,15.84%and 14.61%.The reduction was statistically significant(P<0.01),indicating that PM2.5 exposure led to cell cycle arrest in the G0/G1 phase.After PM2.5 exposure,the expression level of PAI-1 protein increased dose-dependently,and the intermediate and high dose groups were significantly different compared with the control group(P<0.01),IL-1β,IL-6,TNF-α mRNA expression levels were significantly increased(P<0.01),and the expression levels of senescence markers,p16(CDKN2A)、p21(CDKN2A)and p53(TP53),were also significantly increased(P<0.01).(3)After LBP intervention,PM2.5-induced increased β-galactosidase activity(P<0.01)and G0/G1 phase cell arrest(P<0.05)were significantly reduced.At the same time,compared with the PM2.5 exposure group,PM2.5+LBP reduced the expression level of PAI-1 protein(P<0.01),the secretory phenotype associated with senescence(IL-1β,IL-6,TNF-α)and the expression levels of senescence markers,p16(CDKN2A)、p21(CDKN2A)and p53(TP53),were also significantly increased(P<0.01).3.Role of autophagy in LBP inhibition of PM2.5-induced cellular senescence(1)The effect of PM2.5 on autophagy and the intervention effect of LBPThe results of Western blot showed that the ratio of LC3Ⅱ to LC3Ⅰ increased compared with the control group due to PM2.5 exposure,and the ratio of LC3Ⅱ/LC3Ⅰin the high-dose group increased significantly compared with the control group(P<0.05),and the protein expression of Beclin 1 and p62 showed an upward trend after PM2.5 infection,and was dose-dependent(P<0.05).The results of BECN1 and SQSTMl at the transcription level were consistent with the protein expression levels.The detection of mCherry-EGFP-LC3 by double-standard adenovirus showed excessive accumulation of autophagies,which once again indicated that PM2.5 led to autophagy disorders in HUVECs.After LBP intervention,the expression levels of LC3Ⅱ/LC3Ⅰ,Beclin 1(BECN1)and p62(SQSTM1)decreased significantly,and the excessive accumulation of autophagies was alleviated,indicating that LBP could alleviate PM2.s-induced autophagy disorders.(2)LBP alleviates PM2.5-induced cellular senescence in HUVECs by alleviating autophagy disordersAutophagy inhibitors 3-MA and Baf A1 were co-treated with PM2.5,respectively,to detect the expression of cellular senescence markers p16 and 21.Western blot showed that,compared with the control group,autophagy was inhibited in HUVECs cultured with 3-MA and Baf A1 alone,and the protein levels of p16 and 21 were increased,suggesting that the inhibition of autophagy induced the occurrence of cellular senescence.Compared with PM2.5 exposure group,p16 and p21 showed a downward trend after 3-MA intervention,and the protein level of p21 was significantly decreased(P<0.05).After Baf A1 intervention in PM2.5 exposure group,p16 and p21 did not show a decreasing trend,suggesting that inhibition of the binding of autophagosome and lysosome could not alleviate the occurrence of cellular senescence.In conclusion,LBP may alleviate autophagy disorders by reducing excessive accumulation of autophagosomes,thus alleviating the occurrence of cellular senescence.ConclusionIn this study,we explored the toxicity and potential mechanism of PM2.5 on vascular endothelial cells,and found that PM2.5 could induce oxidative stress and oxidative damage in HUVECs,resulting in autophagy disorder and cellular senescence.While LBP could reduce the toxic effects and alleviate cellular senescence,and the effect of LBP on alleviating cellular senescence may be achieved by improving the autophagy disorder.Our results provide new ideas and directions for air pollution control from the perspectives of environmental toxicology and nutritional toxicology.