AMPK对过氧化氢诱导细胞衰老的阻遏研究

doi:10.16431/j.cnki.1671-7236.2023.06.009

Abstract

Abstract: 【Objective】 This study was aimed to construct a stable expression of adenosine 5'-monophosphate-activated protein kinase (AMPK)/AMPK-T172D NIH3T3 cell lines, and investigate the effect of AMPK on hydrogen peroxide (H₂O₂)-induced cell senescence.【Method】 AMPK gene and its mutant form AMPK-T172D were amplified by PCR and cloned into lentiviral vector pLVX-IRES-Puro.The pLVX-IRES-AMPK/AMPK-T172D recombinant plasmids were constructed and double digestion was verified.The constructed recombinant plasmid was packaged into Lentivirus, infected with NIH3T3 cells, and puromycin screening was used to obtain stable cell lines.Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression of AMPK in stable cell lines, respectively.The AMPK/AMPK-T172D overexpression of NIH3T3 cell lines was treated with 8.8 mmol/L H₂O₂, and after 2 days of culture, cellular senescence was detected by senescence-associated-β-galactosidase (SA-β-Gal) and the expression of p53, p21 and IL-6 genes were detected by Real-time quantitative PCR.【Result】 The results of double digestion verification showed that compared with control group, the pLVX-IRES-AMPK/AMPK-T172D expression plasmid was successfully constructed.Real-time quantitative PCR and Western blotting results showed that the expressions of mRNA and protein of AMPK and AMPK-T172D were extremely significantly or significantly increased (P<0.01 or P<0.05).The mRNA expression of carnitine palmitoyltransferase-1 (CPT-1) and fatty acid synthase (FAS) in AMPK/AMPK-T172D overexpressing NIH3T3 cells were significantly or extremely significantly increased (P<0.05 or P<0.01).The cell positive rate of SA-β-Gal was extremely significantly decreased (P<0.01). The mRNA expression of p53, p21 and IL-6 genes were significantly or extremely significantly decreased in AMPK/AMPK-T172D overexpressed NIH3T3 cells(P<0.05 or P<0.01).【Conclusion】 The stable expression of AMPK/AMPK-T172D overexpression NIH3T3 stable cell lines was successfully obtained, and AMPK activation could reduce the cell positive rate of SA-β-Gal and the expression of aging-related factors (p53, p21 and IL-6) in H₂O₂-induced senescent cells.The results would provide a basis for the research on the role of AMPK in aging, possible molecular mechanisms and anti-aging treatment strategies.

Key words: AMPK gene; sustained activation type; stable cell line; NIH3T3 cell; aging

CLC Number:

Q255

CHEN Cui, GONG Lei, XU Zhe, WANG Xiaobo. Study on the Inhibition of Hydrogen Peroxide-induced Cellular Aging by AMPK[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2255-2264.

References

[1] 陈建, 夏豪.米诺地尔对血管平滑肌细胞衰老的影响及相关信号机制[J].中华老年心脑血管病杂志, 2022, 24(4):418-421. CHEN J, XIA H.Effect of minoxidil on senescence of vascular smooth muscle cells and its signal mechanism[J].Chinese Journal of Geriatric Heart Brain and Vessel Diseases, 2022, 24(4):418-421.(in Chinese)
[2] SEN P, SHAH P P, NATIVIO R, et al.Epigenetic mechanisms of longevity and aging[J].Cell, 2016, 166(4):822-839.
[3] HERNANDEZ-SEGURA A, NEHME J, DEMARIA M.Hallmarks of cellular senescence[J].Trends in Cell Biology, 2018, 28(6):436-453.
[4] LUO J, MILLS K, LE CESSIE S, et al.Ageing, age-related diseases and oxidative stress:What to do next?[J].Ageing Research Reviews, 2020, 57:100982.
[5] ZUO S Q, LI C, LIU Y L, et al.Cordycepin inhibits cell senescence by ameliorating lysosomal dysfunction and inducing autophagy through the AMPK and mTOR-p70S6K pathway[J].FEBS Open Bio, 2021, 11(10):2705-2714.
[6] JANG H J, YANG K E, OK W K, et al.Nectandrin B-mediated activation of the AMPK pathway prevents cellular senescence in human diploid fibroblasts by reducing intracellular ROS levels[J].Aging (Albany NY), 2019, 11(11):3731-3749.
[7] CHENG X Y, LI Y Y, HUANG C, et al.AMP-activated protein kinase reduces inflammatory responses and cellular senescence in pulmonary emphysema[J]. Oncotarget, 2017, 8(14):22513-22523.
[8] TSUBOI T, DA SILVA XAVIER G, LECLERC I, et al.5'-AMP-activated protein kinase controls insulin-containing secretory vesicle dynamics[J].Journal of Biological Chemistry, 2003, 278(52):52042-52051.
[9] TSUCHIHASHI N A, HAYASHI K, DAN K, et al.Autophagy through 4EBP1 and AMPK regulates oxidative stress-induced premature senescence in auditory cells[J].Oncotarget, 2015, 6(6):3644-3655.
[10] IDO Y, DURANTON A, LAN F, et al.Acute activation of AMP-activated protein kinase prevents H₂O₂-induced premature senescence in primary human keratinocytes[J].PLoS One, 2012, 7(4):e35092.
[11] HAN X, TAI H, WANG X, et al.AMPK activation protects cells from oxidative stress-induced senescence via autophagic flux restoration and intracellular NAD⁺ elevation[J]. Aging Cell, 2016, 15(3):416-427.
[12] GUO L, XIE B, MAO Z.Autophagy in premature senescent cells is activated via AMPK pathway[J]. International Journal of Molecular Sciences, 2012, 13(3):3563-3582.
[13] HERZIG S, SHAW R J.AMPK:Guardian of metabolism and mitochondrial homeostasis[J].Nature Neviews Nolecular Nell Niology, 2018, 19(2):121-135.
[14] SALMINEN A, KAARNIRANT K.AMP-activated protein kinase (AMPK) controls the aging process via an integrated signaling network[J].Ageing Research Reviews, 2012, 11(2):230-241.
[15] DING Y, CHEN J, OKON I S, et al.Absence of AMPKα2 accelerates cellular senescence via p16 induction in mouse embryonic fibroblasts[J].The International Journal of Biochemistry & Cell Biology, 2016, 71:72-80.
[16] SUNG J Y, KIM S G, KIM J R, et al.Prednisolone suppresses adriamycin-induced vascular smooth muscle cell senescence and inflammatory response via the SIRT1-AMPK signaling pathway[J].PLoS One, 2020, 15(9):e0239976.
[17] 汤晗, 聂建云, 李文斌, 等.慢病毒载体系统的发展及在肿瘤基因治疗中的应用[J].现代肿瘤医学, 2017, 25(10):1659-1662. TANG H, NIE J Y, LI W B, et al.Research progress of Lentiviral vectors system based on HIV-1[J].Modern Oncology, 2017, 25(10):1659-1662.(in Chinese)
[18] 王江, 段君君, 张小玉, 等.SOX9慢病毒表达载体的构建及其在LNcap细胞中的稳定表达[J].昆明医科大学学报, 2018, 39(2):5-9. WANG J, DUAN J J, ZHANG X Y, et al.Construction of Lentiviral expression vector carrying SOX9 gene and its stable expression in LNcap cell[J].Journal of Kunming Medical University, 2018, 39(2):5-9.(in Chinese)
[19] LOPEZ-OTIN C, BLASCO M A, PARTRIDE L, et al.The hallmarks of aging[J].Cell, 2013, 153(6):1194-1217.
[20] HAN X J, CHEN H H, ZHOU J, et al.The inhibitory effect in Fraxinellone on oxidative stress-induced senescence correlates with AMP-activated protein kinase-dependent autophagy restoration[J].Journal of Cellular Physiology, 2018, 233(5):3945-3954.
[21] 张配颖, 宋鹏琰, 周营, 等.基于RNA-Seq数据分析葡萄糖诱导绵羊卵泡颗粒细胞衰老的差异表达基因[J].中国畜牧兽医, 2022, 49(2):631-640. ZHANG P Y, SONG P Y, ZHOU Y, et al.Analysis of differentially expressed genes in glucose-Induced senescence of sheep follicular granulosa cells based on RNA-Seq data[J].China Animal Husbandry & Veterinary Medicine, 2022, 49(2):631-640.(in Chinese)
[22] WANG Z, CHEN Z, JIANG Z, et al.Cordycepin prevents radiation ulcer by inhibiting cell senescence via NRF2 and AMPK in rodents[J].Nature Communications, 2019, 10(1):2538.
[23] BERGER F, RAMIREZ-HERNANDEZ M H, ZIEGLER M.The new life of a centenarian:Signalling functions of NAD(P)[J].Trends in Biochemical Sciences, 2004, 29(3):111-118.
[24] IDO Y, DURANTON A, LAN F, et al.Resveratrol prevents oxidative stress-induced senescence and proliferative dysfunction by activating the AMPK-FOXO3 cascade in cultured primary human keratinocytes[J]. PLoS One, 2015, 10(2):e0115341.
[25] MAHARAJAN N, GANESAN CD, MOON C, et al.Licochalcone D ameliorates oxidative stress-Induced senescence via AMPK activation[J].International Journal of Molecular Sciences, 2021, 22(14):7324.

Study on the Inhibition of Hydrogen Peroxide-induced Cellular Aging by AMPK

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