| Along with China entering an aging society,the rapid development of aging population drives medical treatment,social insurance and other society prominent problems to be highlighted.Aging is closely related with a variety of chronic diseases,such as cancer,diabetes,Parkinson's disease,dementia,acute coronary syndromes,cataracts,etc.Although aging cannot be avoided,appropriate methods can retard the aging.Therefore,deeply studying the aging mechanism and exploring new anti-aging targets and measures are of great significance.Autophagy,a conservative catabolism process in the evolution,mediates longevity protein and function disorder in eukaryotic cells or degrades extra and damaged organelles.In addition,the regulation of autophagy plays an important role in cell quality control.More and more studies have shown that autophagy is closely related with aging.WI-38 cells,derived from human embryonic lung tissue with limited passage numbers,are diploid fibroblasts,and they are generally accepted as models for studying aging.This paper creates a model of D-galactose inducing cells aging through D-galactose inducing WI-38(PDL30-40)cell aging,and then explores the molecular mechanism of inducing aging and finds the corresponding anti-aging targets on the basis of previous experiments.The effects of different concentrations of D-galactose(0,1.25,2.5,5,10,20 g/L)on the vitality of WI-38 cells were evaluated by MTT method.The aging status of different concentrations of D-galactose(0,1.25,2.5,5,10 g/L)constantly processing WI-38 cells after 14 days was detected by SA-?-Gal staining.Western Blot method was used to detect the expression of cell cycle restraining protein P21 and P16 and aging marker ?-gal.DCFH-DA method was used to determine the effect that D-galactose had an on the total ROS level in the WI-38 cells.Western Blot was used to detect the expression of antioxidant enzyme,autophagy marker protein and autophagy related pathway protein.On the model of D-galactose inducing cells aging,different concentrations of Mito Q was be used to intervene,and using DCFH-DA method further accesses the total cell ROS,and Western Blot further tested autoxidation protein and expression of autophagy protein,and MDC method wasused to detect autophagy,and SA-?-Gal dyeing tested aging condition,and Western Blot further monitored expression of aging related protein.The main results were as follows:1.The establishment for model of D-galactose inducing WI-38 cells aging.(1)D-galactose had a certain inhibitory effect on the viability of WI-38 cells,and the cell vitality decreased significantly with the increasing.(2)D-galactose could induce WI-38 cells aging;After 14 days,under influence of different concentrations of D-galactose,the positive rate of SA-?-Gal staining cells rose as the concentration increases,reaching the highest level in 5g/L concentration.(3)After 14 days,under the influence of different concentrations of D-galactose,the expression of P21?P16and ?-gal gradually increased as D-galactose concentration increased,the expression of P16 and ?-gal with the highest concentration in 5g/L.2.Research on the aging mechanism of the D-galactose inducing WI-38 cells.(1)Under the influence of different concentrations of D-galactose,after 24 hours,the total ROS of the cells increased with the increase of the concentration,and at the concentration of 5g/L and the subsequent concentrations the significant differences occurred.(2)After 14 days under the influence of different concentrations of D-galactose,the expression of antioxidant enzyme SOD2,SOD1 and CAT was decreased with the increase of D-galactose concentration.In addition,the expression of antioxidant enzyme SOD2,SOD1,GPX1 and CAT in the control group decreased significantly at the concentration of 5g/L.(3)Under the influence of different concentrations of D-galactose,after 14 days,different concentrations of D-galactose sequential processing WI-38 after 14 d cells,in 5 g/L concentration the levels of autophagy marker protein LC3 II lowered,the levels of P62 increased;at the same time the levels of autophagy pathway protein AMPK?(Thr172)? P-ULK1(Ser555)? P-ULK1(Ser555)decreased,and the levels of P-m TOR(Ser2448)and P-m TOR(Ser2481)rose.Cell aging was associated with these changes.(4)Mito Q could inhibit the ROS produced by D-galactose inducing WI-38 cells.(5)The expression of antioxidant enzyme SOD2,SOD1,GPX1 and CAT increased compared with the aging group.(6)After the intervention of Mito Q,compared with the aging group the autophagy's level increased,and the autophagy marker proteinLC3 II was significantly higher,while the level of P62 decreased significantly.The MDC results shown that the fluorescence intensity decreased significantly compared with control group,while the fluorescence intensity was significantly enhanced after the intervention.(7)After Mito Q intervenes,the degree of aging could be lowered.Compared with the group of inducing aging,the result of constantly processed 14 d SA-?-Gal shown that cell positive rate lowered significantly after Mito Q intervenes.Western Blot further shown that the expression of P21,P16 and ?-gal decreased obviously compared with aging group after Mito Q intervenes.The result shown that D-galactose had certain inhibitory effect on WI-38 cells,and D-galactose could induce WI-38 cell aging.Under this experimental condition,5g/L D-galactose continuously processed after 14 days was the best condition to induce WI-38 aging.WI-38 cells processed by D-galactose made ROS level rise,expression of antioxidant enzymes lower,and autophagy level reduce.The AMPK-ULK1 pathway and m TOR-ULK1 pathway might play a role in the induction of aging by D-galactose.Mitochondrial targeting antioxidant Mito Q could effectively reduced ROS,improved antioxidant ability,improved autophagy level and play an anti-aging role. |
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