Role Of Lysosomal Damage In Senescence Of SH-SY5Y Cells Induced By Methylmercury And Intervention Of Melatonin

Major:Health Toxicology Candidate:Song Shuang Supervisor:Chen XiongObjective:Methylmercury（MeHg）is a heavy metal with strong toxicity to the central nervous system,which can easily lead to the occurrence of neurodevelopment and neurodegenerative diseases.The latest research shows that lysosome-targeted therapy may be effective in neurodegenerative diseases.disease is a useful adjunct therapy.Whether methylmercury can cause cell senescence and the specific mechanism of lysosome function damage in methylmercury-induced aging SH-SY5Y cells is still unclear.Melatonin（MT）is a major secretion product of the pineal gland,which has a protective effect on the nervous system,and the intervention effect of melatonin in it is still unclear.Methods:1.Concentration and grouping（1）MeHg exposure experiment:SH-SY5Y cells were treated with different doses of MeHg(0,0.125,0.25,0.5,1,2,4μmol L^-1)for 48 h,and the cell viability detection kit（CCK-8 method）was used to detect cell viability and calculate the survival rate,andβ-galactosidase staining method（SA-β-gal）was used to detect the condition of senescent cells and observe the cell shape,so as to screen out cells that could obviously cause SH-SY5Y cell senescence.MeHg dose(0.5μmol·L^-1),and its 1/2 and 1/4 dose(0.25 and 0.125μmol·L^-1)were used as the middle and low dose groups,respectively.（2）TFEB activator 1 intervention experiment:SH-SY5Y cells were divided into control group（0.1%DMSO）,MeHg exposure group(0.5μmol L^-1 MeHg),TFEB activator 1 group(1μmol L^-1 TFEB activator 1 pretreated cells for 12 h),TFEB activator 1+MeHg group(1μmol L^-1 TFEB activator 1 pretreated cells for 12 h,then0.5μmol L^-1 MeHg exposure for 48 h).（3）Chloroquine（CQ）intervention experiment:SH-SY5Y cells were divided into control group（0.1%DMSO）,MeHg exposure group(0.5μmol L^-1 MeHg),CQ group(10μmol L^-1CQ pretreatment cells for 1 h),CQ+MeHg group(10μmol L^-1 CQ pretreated cells for 1 h,and then 0.5μmol L^-1 MeHg was exposed for 48 h).（4）Melatonin intervention experiment:SH-SY5Y cells were divided into 4groups:control group（0.1%DMSO）,MeHg staining group(0.5μmol L^-1 MeHg),MT group(1 mmol L^-1 MT pretreated cells for 24 h),MT+MeHg group(1 mmol·L^-1 MT pretreated cells for 24 h and then 0.5μmol·L^-1 MeHg exposure for 48 h).2.Research methods（1）β-galactosidase staining to observe the aging changes of SH-SY5Y cells（2）Western blot to detect the expression levels of aging-related protein p16;cycle-related protein E2F1;autophagy-related proteins p62,LC3,and lysosome-related proteins LAMP1,LAMP2,and TFEB（3）Cell cycle detection by flow cytometry（4）Lysosomal red fluorescent probe staining to detect the number of lysosomes;Lyso Sensor Green DND-189 staining to detect lysosomal p H changes（5）Observation of morphological changes of lysosomes by electron microscope Results:1.MeHg can induce SH-SY5Y cell senescenceThe results of CCK-8 showed that the survival rate of cells decreased with the increase of exposure concentration;compared with the control group,the rate ofβ-galactosidase blue-stained cells in the 0.5μmol·L^-1 MeHg exposure group increased significantly by 48%（P<0.01）,the SH-SY5Y cells in the control group were spindle-shaped under the optical microscope,while the cells in the 0.5μmol·L^-1 MeHg-treated group were flat and enlarged,showing a senescent phenotype.Compared with the control group,the expression level of p16 protein in the 0.25 and 0.5μmol·L^-1 MeHg exposure groups gradually increased（P<0.05）,the cell cycle was arrested in G0/G1phase（P<0.05）,and the expression level of E2F1 protein Gradually decreased（P<0.05）.2.MeHg-induced SH-SY5Y cell senescence is related to lysosome function damageAfter MeHg(0.125,0.25,0.5μmol L^-1)treated SH-SY5Y cells for 48 h,compared with the control group,the expression of autophagy-related protein LC3Ⅱin the 0.5μmol L^-1 MeHg treatment group increased significantly,and the difference was Statistically significant（P<0.05）;the expression of p62 protein in the 0.25 and 0.5μmol·L^-1 dose exposure groups was higher than that in the control group,and there was a dose-effect relationship（P<0.05）.Compared with the control group,the intensities of lysosomal red fluorescent probe and lysosomal green fluorescent probe in each MeHg treatment group were weakened,and the most obvious after 0.5μmol·L^-1 MeHg treatment;compared with the control group,LAMP1,LAMP2 protein expression level gradually decreased,the most obvious in the 0.5μmol L^-1 MeHg exposure group（P<0.05）;after 0.5μmol L^-1 MeHg treatment,TFEB,an important regulator of lysosomes,was comparable to that in the control group Compared with the control group,the TFEB protein content in the cytoplasm was significantly increased（P<0.05）.Compared with the control group,the volume of lysosomes after 0.5μmol·L^-1 MeHg treatment increase.Compared with the blank control group,the protein expression levels of p16and E2F1 in the CQ group were significantly increased（P<0.05）;compared with the mercury-stained group,the protein expression levels of p16 and E2F1 in the CQ+MeHg group were significantly higher,and the protein expression levels were statistically significant（P<0.05）.Scientific significance（P<0.05）.Compared with the blank control group,the rate ofβ-galactosidase blue-stained cells in the CQ group was significantly increased by 51%,which was statistically significant（P<0.01）;The rate of enzyme blue stained cells increased significantly by 18%（P<0.05）.3.Promoting TFEB into nucleus can interfere with MeHg can induce SH-SY5Y cell senescenceThe content of TFEB protein in the cytoplasm and nucleus was detected by Western blot experiment,indicating that pretreatment of SH-SY5Y cells with 1μmol L^-1 TFEB activator 1 for 12 hours can effectively promote the nuclear translocation of Flag-TFEB,and the test was statistically significant（P<0.05）;then we proved that the same dose and time of TFEB activator 1 pretreatment can increase the number of lysosomes by lysosomal red fluorescent probe staining test;Compared with the TFEB activator 1+MeHg treatment group,the aging-related protein p16 was significantly decreased,and the test was statistically significant（P<0.05）.Compared with the blank control group,the rate ofβ-galactosidase blue-stained cells in the TFEB activator1+MeHg treatment group was significantly reduced by 21%,which was statistically significant（P<0.05）.4.Melatonin can restore lysosomal function damage and promote TFEB into the nucleus,thereby interfering with MeHg can induce SH-SY5Y cell senescenceAfter being treated with 1 mmol L^-1 MT for 24 h,compared with the 0.5μmol L^-1MeHg treatment group,the rate ofβ-galactosidase blue-stained cells in the MT+MeHg group was significantly reduced by 19%（P<0.01）,the expression of senescence-related protein p16 was significantly reduced（P<0.05）,which promoted the transition of cells from G0/G1 phase to S phase（P<0.05）,the expression level of E2F1 protein was significantly increased（P<0.05）,and the expression level of autophagy-related protein LC3ⅡThe expression levels of protein and p62/SQSTM were significantly decreased（P<0.05）.Lysosome red fluorescent probe staining and Lyso Sensor Green DND-189 staining experiments proved that the number of lysosomes in the MT+MeHg group increased and the p H value decreased.The expression levels of body-associated proteins LAMP1 and LAMP2 were significantly increased（P<0.05）,and the amount of TFEB into the nucleus was significantly increased（P<0.05）.Conclusions:MeHg may inhibit lysosomal biogenesis and damage lysosomal activity,thereby causing autophagosome degradation and inducing SH-SY5Y cell senescence;while melatonin may promote lysosomal biogenesis and induce SH-SY5Y cell senescence by increasing TFEB into the nucleus.Promote the degradation of autophagosomes and alleviate the senescence effect of MeHg on SH-SY5Y cells.This study can provide an experimental basis for intervening in MeHg-induced nerve cell damage.