| Objective:In this study,we will observe the effect of 2,5-hexanedione(HD)on autophay of VSC4.1 cells and explore its mechanism.Materials and Methods:VSC4.1 cells were pre-treated with DMEM containing 15% FBS for 24 h,then the cells were divided into four groups:(1)Control group: no HD was added in the medium;(2)HD exposure group: VSC4.1 cells were exposed to 0 m M,5 m M,15 m M and 25 m M HD for 24 h;(3)Autophagic inhibitor group: VSC4.1 cells were treated with 0 m M,25 m M HD for 24 h,and then 5 μM PIK-III was added the cells for 24 h;(4)Agonist group: VSC4.1 cells were treated with 25 m M HD for 24 h,and then the cells were treated with 30 μM 740Y-P,14 μM SC79 and 120 μM 3BDO for 12 h.The autophagy of VSC4.1 cells was detected by LC3-DAPI immunofluorescence double staining;The number of autophagic vacuoles was detected by electron microscopy;The protein expressions of LC3,p62,Akt,p-Akt,m TOR,p-m TOR,ULK1 and p-ULK1 was detected by Western-Blotting;LDH was used to detect cell death;SPSS13.0 statistical software was used to analyze all the results.Results: HD-induced excessive autophagy of VSC4.1 cells: LC3-DAPI double staining showed that LC3 punctated foci was significantly increased that with increasing concentration of HD(p<0.05).The number of autophagic vacuoles were significantly increased with increasing concentration of HD by electron microscopy(p<0.05).The expression of LC3-II/I was significantly increased(p<0.05),and the expression of p62 protein was significantly decreased(p<0.05)with increasing concentration of HD by Western-Blotting;HD induced autophagic death in VSC4.1 cells: LDH showed that rate of cell death was significantly increased with increasing concentration of HD(p<0.05),PIK-III intervention group was significantly lower than that in HD group(p<0.05).Pearson correlation analysis showed that positive correlation between rate of cell death and cells with LC3 punctated foci.LC3-DAPI double staining showed that LC3 punctated foci was significantly increased in HD group,while PIK-III intervention group was significantly decreased(p<0.05).The expression ratio of LC3-II/I in cells was detected by Western-Blotting,the expression ratio of LC3-II/I in HD group was significantly higher than that in control group(p<0.05),PIK-III intervention group was significantly lower than that in HD group(p<0.05);Effects of HD on the level of Akt phosphorylation in VSC4.1 cells: The protein expression was detected by Western-Blotting,the protein expression of p-Akt was significantly decreased with increasing concentration of HD(p<0.05),the expression of p-Akt in 740Y-P intervention group was significantly higher than that in HD group(p<0.05),while the total protein of Akt had no significant change after HD administration(p>0.05);Effects of HD on the level of m TOR phosphorylation in VSC4.1 cells: The protein expression was detected by Western-Blotting,the protein expression of p-m TOR was significantly decreased with increasing concentration of HD(p<0.05),the expression of p-m TOR in 740Y-P or SC79 intervention group was significantly higher than that in HD group(p<0.05),while the total protein of m TOR had no significant change after HD administration(p>0.05);Effects of HD on the level of ULK1 phosphorylation in VSC4.1 cells: The protein expression was detected by Western-Blotting,the protein expression of p-ULK1 was significantly decreased with increasing concentration of HD(p<0.05),the expression of p-ULK in SC79 or 3BDO intervention group was significantly higher than that in HD group(p<0.05),while the total protein of m TOR had no significant change after HD administration(p>0.05);Effects of HD on autophagy of VSC4.1 cells via PI3K/Akt/m TOR signaling pathway: LC3-DAPI double staining showed that LC3 punctated foci in HD group was significantly higher than that in control group(p<0.05),while that in 740Y-P or SC79 or 3BDO intervention group was significantly lower than that in HD group(p<0.05).The numbers of autophagic vacuoles in HD group were significantly higher than that in control group by electron microscopy(p<0.05),but that in 740Y-P or SC79 or 3BDO intervention group was significantly lower than that in HD group(p<0.05).The protein expression was detected by Western-Blotting,the protein expression of LC3-II/I in HD group was significantly higher than that in control group(p<0.05),and that in 740Y-P,SC79 or 3BDO intervention group was significantly lower than that in HD group(p<0.05).The expression of p62 protein in HD group was significantly lower than that in control group(p<0.05),and that in 740Y-P,SC79 or 3BDO intervention group was significantly higher than that in HD group(p<0.05).Conclusion: HD induced excessive autophagy of VSC4.1 cells in a dose-dependent manner;HD induced autophagic death of VSC4.1 cells;HD induced excessive autophagy of VSC4.1 cells is associated with PI3K/Akt/m TOR signaling pathway. |
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