EGCG恢复Sirt-1拮抗高糖诱导的PC12细胞凋亡
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摘要
目的:探讨表没食子儿茶素没食子酸酯(EGCG)对高糖诱导的PC12细胞损伤的保护作用及Sirt-1的介导机制。方法:CCK-8法检测PC12细胞活力; ELISA法检测Bcl-2的活性;免疫荧光法检测Caspase-3在胞质的活性及胞核内DAPI的表达; WB检测Sirt-1的表达。结果:高浓度的葡萄糖(15、30、45 mmol/L)诱导了PC12细胞的损伤,细胞活性较对照组分别下降了14%、32%及57%,凋亡保护蛋白Bcl-2的表达较对照组下降了8%、30%及35%; EGCG(10、20、40μmol/L)对高糖诱导的PC12细胞凋亡提供了保护作用,细胞活性较高糖损伤组分别提升了2%、20%及24%,Bcl-2的表达提升了10%、22%和25%;阻断Sirt-1通路逆转了EGCG提供的保护作用,同时加入阻断剂组的细胞活性较EGCG保护组下降了8%,Bcl-2的表达下降了16%。结论:EGCG通过恢复Sirt-1活性拮抗高糖诱导的PC12细胞的凋亡。
Objective: To observe the protective effect of epigallocatechin gallate( EGCG) on PC12 cells from high glucose-induced apoptosis by regulation of Sirt-1.Methods: CCK-8 assay was performed to detect PC12 cell viability.ELISA was used to determine Bcl-2 expression,and immol/Lunofluorescence to detect the expression of Caspase-3 and DAPI.Western blotting was performed to determine Sirt-1 expression.Results: High-glucose dose( 15,30,and 45 mmol/L,respectively) induced apoptosis of PC12 cells.The cell viability and Bcl-2 expression were reduced by 14%,32% and 57%; and by 8%,30%and 35%,respectively,compared to the control group.Contrarily,EGCG in dose of 10,20 and 40 μmol/L was able to produce protective effects on PC12 cells,leading to increased cell activity by 2%,20% and 24%; and up-regulated Bcl-2 expression by 10%,22% and 25%,respectively,as compared with the injury group.Blocking of Sirt-1 reversed the protective effect of EGCG,and the cell viability and Bcl-2 expression were decreased by 8% and by 16%following treatment of blocking agent compared to EGCG protection group.Conclusion: EGCG can antagonize high glucose-induce apoptosis by up-regulating Sirt-1 in PC12 cells.
Objective: To observe the protective effect of epigallocatechin gallate( EGCG) on PC12 cells from high glucose-induced apoptosis by regulation of Sirt-1.Methods: CCK-8 assay was performed to detect PC12 cell viability.ELISA was used to determine Bcl-2 expression,and immol/Lunofluorescence to detect the expression of Caspase-3 and DAPI.Western blotting was performed to determine Sirt-1 expression.Results: High-glucose dose( 15,30,and 45 mmol/L,respectively) induced apoptosis of PC12 cells.The cell viability and Bcl-2 expression were reduced by 14%,32% and 57%; and by 8%,30%and 35%,respectively,compared to the control group.Contrarily,EGCG in dose of 10,20 and 40 μmol/L was able to produce protective effects on PC12 cells,leading to increased cell activity by 2%,20% and 24%; and up-regulated Bcl-2 expression by 10%,22% and 25%,respectively,as compared with the injury group.Blocking of Sirt-1 reversed the protective effect of EGCG,and the cell viability and Bcl-2 expression were decreased by 8% and by 16%following treatment of blocking agent compared to EGCG protection group.Conclusion: EGCG can antagonize high glucose-induce apoptosis by up-regulating Sirt-1 in PC12 cells.
引文
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[10]RAHMAN M,HALADE GV,BHATTACHARYA A,et al.The fat-1transgene in mice increases antioxidant potential,reduces pro-inflammol/Latory cytokine levels,and enhances PPAR-gammol/La and SIRT-1 expression on a calorie restricted diet[J]. Oxidative Medicine and Cellular Longevity,2009,2(5):307-316.
[11] SCHEIBYE-KNUDSEN M. Neurodegeneration in accelerated aging[J].Danish Medical Journal,2016(11):63.
[2] OTHMAN AI,EL-SAWI MR,EL-MISSIRY MA,et al.Epigallocatechin-3-gallate protects against diabetic cardiomyopathy through modulating the cardiometabolic risk factors,oxidative stress,inflammol/Lation,cell death and fibrosis in streptozotocin-nicotinamideinduced diabetic rats[J]. Biomedicine&Pharmacotherapy,2017(94):362-373.
[3] TAKAHASHI H,SUZUKI Y,MOHAMED JS,et al. Epigallocatechin-3-gallate increases autophagy signaling in resting and unloaded plantaris muscles but selectively suppresses autophagy protein abundance in reloaded muscles of aged rats[J].Experimental Berontology,2017(92):56-66.
[4] NAGAR H,JUNG SB,RYU MJ,et al.CR6-interacting factor 1 deficiency impairs vascular function by inhibiting the sirt1-endothelial nitric oxide synthase pathway[J].Antioxidants&Redox Signaling,2017,27(4):234-249.
[5] LU H,WANG B.SIRT1 exerts neuroprotective effects by attenuating cerebral ischemia/reperfusion-induced injury via targeting p53/microRNA-22[J]. International Journal of Molecular Medicine,2017,39(1):208-216.
[6] VAZQUEZ-CALVO A,JIMENEZ DE OYA N,MARTIN-ACEBES MA,et al.Antiviral properties of the natural polyphenols delphinidin and epigallocatechin gallate against the flaviviruses west nile virus[J].Zika Virus,and Dengue Virus.Frontiers in Microbiology,2017(8):1314.
[7] HALLMAN K,ALECK K,QUIGLEY M,et al. The regulation of steroid receptors by epigallocatechin-3-gallate in breast cancer cells[J]. Breast Cancer(Dove Medical Press),2017(9):365-373.
[8] DE LOS RIOS C,CANO-ABAD MF,VILLARROYA M,et al.Chromaffin cells as a model to evaluate mechanisms of cell death and neuroprotective compounds[J].Pflugers Archiv:European Journal of Physiology,2018,470(1):187-198.
[9] SAVITSKAYA MA,ONISHCHENK GE.Mechanisms of Apoptosis[J].Biochemistry Biokhimiia,2015,80(11):1393-1405.
[10]RAHMAN M,HALADE GV,BHATTACHARYA A,et al.The fat-1transgene in mice increases antioxidant potential,reduces pro-inflammol/Latory cytokine levels,and enhances PPAR-gammol/La and SIRT-1 expression on a calorie restricted diet[J]. Oxidative Medicine and Cellular Longevity,2009,2(5):307-316.
[11] SCHEIBYE-KNUDSEN M. Neurodegeneration in accelerated aging[J].Danish Medical Journal,2016(11):63.