| 【Objective】D-galactose induced PC12 cell senescence model was established and treated with metformin.At the same time,AMPK and ERK1/2 inhibitors were given to study the possible role and mechanism of metformin in AMPK and ERK1/2 signaling pathways.【Methods】(1)PC12 cell line was treated with D-galactose of different concentration gradients for48 hours.CCK-8 was used to detect the cell activity,and the concentration of 15 mg/ml was selected to establish a senescence model.(2)The following groupings are performed according to different processing methods: 1)Group N: control group,cultured in normal medium for 48 hours without special treatment; 2)Group D: D-galactose group,cultured for 48 h with D-galactose medium at a concentration of 15 mg/ml; 3)Group D+M: metformin intervention group,the cells were pretreated with metformin for 1 h,and then continued to culture with 15 mg/ml D-galactose medium for 48 h; 4)Group D+M+U: U0126 inhibition group,U0126 was used to inhibit ERK1/2 activation before establishment of senescence model,followed by treatment with metformin and D-galactose.5)Group D+M+CC: Compound C inhibition group,AMPK activation was inhibited with Compound C prior to establishment of the senescence model,followed by treatment with metformin and D-galactose.(3)The morphology and ultrastructure of cells were observed by optical microscopy andtransmission electron microscopy respectively;the positive rate of senescent cells was detected by β-galactosidase senescence staining;the mitochondrial DNA deletion rate(CD)was detected by RT-PCR;The apoptosis rate was detected by Annexin V-PI method and JC-1 probe method;the cell mitochondrial membrane potential was detected by confocal microscopy;The contents of GSH,MDA and ATP,and the activity of SOD and CAT were detected by colorimetric assay;Western blot was used to detect the expression levels of ERK1/2,P-ERK1/2,AMPK,P-AMPK,HSP90 and GRP78.【Results】Electron microscopy showed,in the control group the nucleus of PC12 cells was round,the nuclear membrane was intact,and the cytoplasm was rich in mitochondria and normal morphology,whereas,in group D,the nucleus were irregular,with some pyknosis,chromatin concentration,mitochondrial swelling and vacuolation.After the intervention of metformin,the ultrastructural damage of cells in group D was slightly improved,which showed that metformin could alleviate the cell damage caused by D-gal.The protective effect of metformin can be reversed by using ERK1/2 and AMPK inhibitors in advance.The positive rate of beta-galactosidase staining was significantly increased after D-galactose induction.Pretreatment with metformin could slightly reduce the positive rate of beta-galactosidase staining.The rate of apoptotic cells in D-gal group was significantly higher than that in control group.Pretreatment with metformin could reduce the apoptotic level caused by aging.Inhibition of ERK1/2 and AMPK phosphorylation could reverse the protective effect of metformin on apoptotic cells.D-gal could increase the expression level of abnormal mitochondrial membrane potential.Pretreatment with metformin could reduce the abnormal mitochondrial membrane potential level.When ERK1/2 and AMPK phosphorylation were inhibited,the protective effect of metformin could be reversed.Western blot analysis showed that the phosphorylation of ERK1/2 and AMPK protein increased,and the expression of HSP90,GRP78 and cleaved-caspase 3 increased too.Metformin pretreatment furtherincreased the expression of P-ERK1/2,P-AMPK and H90,and decreased the expression of GRP78.However,when AMPK and ERK1/2 inhibitors were applied,the protective effect of metformin was counteracted,especially when AMPK was inhibited.【Conclusion】(1)High concentration of D-galactose was used in PC12 cells could increase the content of reactive oxygen species,increase the rate of CD deletion and degenerate the ultrastructure of PC12 cells within a certain period of time,which indicates that we have successfully established a senescence model of PC12 cells.(2)D-galactose can induce aging of PC12 cells with neuronal characteristics.Metformin can delay aging and prolong life by reducing oxidative stress and apoptotic levels related to aging.The protective effect of metformin is partly mediated by AMPK and ERK1/2 signaling pathways. |
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