敲低硒蛋白N1基因对L929细胞衰老的影响及其机制

doi:10.13481/j.1671-587X.20260304

摘要/Abstract

摘要：

目的探讨硒蛋白N1（SEPN1）基因在D-半乳糖（D-gal）诱导的L929细胞衰老中的作用，并阐明其对细胞增殖、氧化应激、内质网应激、线粒体功能和细胞外基质（ECM）合成影响的信号机制。方法采用D-gal处理L929细胞建立细胞衰老模型。实验分为对照组和D-gal诱导组（D-gal组），利用小干扰RNA（siRNA）敲低SEPN1基因，分为阴性对照组（si-NC组）和si-SEPN1组。采用β-半乳糖苷酶（SA-β-gal）染色法观察各组L929细胞染色情况并计算SA-β-gal阳性细胞百分率，Western blotting法检测各组L929细胞中SEPN1、P21、P16、Ⅰ型胶原蛋白（Col Ⅰ）、Ⅲ型胶原蛋白（Col Ⅲ）、葡萄糖调节蛋白78（GRP78）和C/EBP同源蛋白（CHOP）蛋白表达水平，2'-7'-二氯荧光素二乙酸（DCFH-DA）荧光探针法检测各组细胞中活性氧（ROS）水平，采用试剂盒检测各组细胞线粒体膜电位水平，采用Fluo-4 AM钙离子（Ca²⁺）荧光探针检测各组L929细胞中Ca2?水平，5-乙炔基-2'-脱氧尿苷（EdU）染色检测各组L929细胞增殖活性，免疫荧光法检测各组L929细胞中Col Ⅰ和Col Ⅲ蛋白表达水平。结果 SA-β-gal染色法，与对照组比较，D-gal组L929细胞中SA-β-gal阳性细胞百分率明显升高（P<0.01）；与si-NC组比较，si-SEPN1组SA-β-gal阳性细胞百分率明显升高（P<0.01）。Western blotting法，与对照组比较，D-gal组L929细胞中P21和P16蛋白表达水平明显升高（P<0.01），SEPN1蛋白表达水平明显降低（P<0.01）。EdU染色，与对照组比较，D-gal组L929细胞增殖活性明显降低（P<0.01）；与si-NC组比较，si-SEPN1组L929细胞增殖活性明显降低（P<0.01）。DCFH-DA荧光探针法，与对照组比较，D-gal组L929细胞中ROS水平明显升高（P<0.01）；与si-NC组比较，si-SEPN1组细胞中ROS水平明显升高（P<0.05）。Western blotting法，与si-NC组比较，si-SEPN1组细胞中SEPN1、Col Ⅰ和Col Ⅲ蛋白表达水平明显降低（P<0.05或P<0.01），P21、P16、GRP78和CHOP蛋白表达水平均明显升高（P<0.05或P<0.01）。与si-NC组比较，si-SEPN1组细胞线粒体膜电位水平明显降低（P<0.01）；与si-NC组比较，si-SEPN1组细胞中Ca2?水平明显升高（P<0.05）。与si-NC组比较，si-SEPN1组L929细胞中Col Ⅰ和Col Ⅲ蛋白表达水平均明显降低（P<0.05或P<0.01）。结论敲低SEPN1可促进L929细胞衰老，抑制细胞增殖以及Col Ⅰ和Col Ⅲ合成，其作用机制可能与诱导内质网应激、损伤线粒体功能、Ca²⁺超载和细胞内ROS增加有关。

关键词: 盆底功能障碍性疾病, 硒蛋白N1, 细胞衰老, 内质网应激, 线粒体功能障碍, 成纤维细胞

Abstract:

Objective To discuss the role of selenoprotein N1 （SEPN1） gene in D-galactose （D-gal）- induced senescence of L929 cells， and to clarify its signaling mechanism on cell proliferation， oxidative stress， endoplasmic reticulum stress， mitochondrial function， and extracellular matrix synthesis（ECM）. Methods D-gal was used to treat the L929 cells to establish a cell senescence model. The experiment was divided into control group and D-gal-induced group （D-gal group）. Small interfering RNA （siRNA） was used to knock down SEPN1 gene， and the cells were divided into negative control group （si-NC group） and si-SEPN1 group. SA-β-gal staining was used to observe the staining condition of L929 cells in various groups and calculate the percentage of SA-β-gal positive cells； Western blotting method was used to detect the expression levels of SEPN1， P21， P16， collagen type Ⅰ（Col Ⅰ）， collagen type Ⅲ（Col Ⅲ）， glucose-regulated protein 78 （GRP78）， and C/EBP homologous protein （CHOP） proteins in the L929 cells in various groups； 2'-7'- dichlorofluorescein diacetate （DCFH-DA） fluorescent probe method was used to detect the levels of reactive oxygen species （ROS） in the cells in various groups； kits were used to detect the mitochondrial membrane potential levels in the cells in various groups； Fluo-4 AM calcium ion （Ca²⁺） fluorescent probe was used to detect the Ca²⁺ levels in the L929 cells in various groups； 5-ethynyl-2'- deoxyuridine （EdU） staining was used to detect the proliferation activity of L929 cells in various groups； immunofluorescence method was used to detect the expression levels of Col Ⅰ and Col Ⅲ proteins in the L929 cells in various groups. Results The SA-β-gal staining results showed that compared with control group， the percentage of SA-β-gal positive cells in L929 cells in D-gal group was significantly increased （P<0.01）； compared with si-NC group， the percentage of SA-β-gal positive cells in si-SEPN1 group was significantly increased （P<0.01）. The Western blotting method results showed that compared with control group， the expression levels of P21 and P16 proteins in L929 cells in D-gal group were significantly increased （P<0.01）， and the expression level of SEPN1 protein was significantly decreased （P<0.01）. The EdU staining results showed that compared with control group， the proliferation activity of L929 cells in D-gal group was significantly decreased （P<0.01）； compared with si-NC group， the proliferation activity of L929 cells in si-SEPN1 group was significantly decreased （P<0.01）. The DCFH-DA fluorescent probe method results showed that compared with control group， the ROS level in L929 cells in D-gal group was significantly increased （P<0.01）； compared with si-NC group， the ROS level in the cells in si-SEPN1 group was significantly increased （P<0.05）. The Western blotting method results showed that compared with si-NC group， the expression levels of SEPN1， Col Ⅰ and Col Ⅲ proteins in the cells in si-SEPN1 group were significantly decreased （P<0.05 or P<0.01）， and the expression levels of P21， P16， GRP78 and CHOP proteins were significantly increased （P<0.05 or P<0.01）. The mitochondrial membrane potential assay results showed that compared with si-NC group， the level of mitochondrial membrane potential in the cells in si-SEPN1 group was significantly decreased （P<0.01）. The Fluo-4 AM calcium ion fluorescent probe results showed that compared with si-NC group， the Ca²⁺ level in the cells in si-SEPN1 group was significantly increased （P<0.05）. The immunofluorescence method results showed that compared with si-NC group， the expression levels of Col Ⅰ and Col Ⅲ proteins in the L929 cells in si-SEPN1 group were significantly decreased （P<0.05 or P<0.01）. Conclusion Knockdown of SEPN1 can promote senescence of L929 cells， and inhibit cell proliferation and synthesis of Col Ⅰ and Col Ⅲ； its mechanism may be related to induction of endoplasmic reticulum stress， impairment of mitochondrial function， Ca²⁺ overload， and increase of intracellular ROS.

Key words: Pelvic floor dysfunction diseases, Selenoprotein N1, Cell senescence, Endoplasmic reticulum stress, Mitochondrial dysfunction, Fibroblasts

中图分类号:

R711.4

蒋诺,张舒飞,洪莉. 敲低硒蛋白N1基因对L929细胞衰老的影响及其机制[J]. 吉林大学学报(医学版), 2026, 52(3): 612-620.

Nuo JIANG,Shufei ZHANG,Li HONG. Effect of selenoprotein N1 gene on senescence of L929 cells and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2026, 52(3): 612-620.

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参考文献 29

[1]	COLLINS S， LEWICKY-GAUPP C. Pelvic organ prolapse［J］. Gastroenterol Clin North Am， 2022， 51（1）： 177-193.
[2]	SIVALINGARAJAH R， BALASINGAM B， URUTHIRAKUMAR P， et al. Gynecological morbidity in geriatric women： findings from a cross-sectional study in a tertiary care center in northern Sri Lanka［J］. Cureus， 2025， 17（6）： e85416.
[3]	LI P P， MA H Y， ZHANG J， et al. Global trends in prevalence and future projections of pelvic organ prolapse： a 30-year epidemiological study［J］. Int Urogynecol J， 2025， 36（5）： 991-1002.
[4]	GULER Z， ROOVERS J P. Role of fibroblasts and myofibroblasts on the pathogenesis and treatment of pelvic organ prolapse［J］. Biomolecules， 2022， 12（1）： 94.
[5]	CHILDS B G， GLUSCEVIC M， BAKER D J， et al. Senescent cells： an emerging target for diseases of ageing［J］. Nat Rev Drug Discov， 2017， 16（10）： 718-735.
[6]	HUANG G X， SUN S N， LIAO M D， et al. METTL3-mediated m6A modification regulates D-galactose-induced skin fibroblast senescence through miR-208a-5p［J］. Front Immunol， 2025， 16： 1577783.
[7]	LÓPEZ-OTÍN C， BLASCO M A， PARTRIDGE L， et al. Hallmarks of aging： an expanding universe［J］. Cell， 2023， 186（2）： 243-278.
[8]	BARAZZUOL L， GIAMOGANTE F， CALÌ T. Mitochondria associated membranes （MAMs）： architecture and physiopathological role［J］. Cell Calcium， 2021， 94： 102343.
[9]	MAKIO T， CHEN J S， SIMMEN T. ER stress as a sentinel mechanism for ER Ca²⁺ homeostasis［J］. Cell Calcium， 2024， 124： 102961.
[10]	GERMANI S， VAN HO A T， CHERUBINI A， et al. SEPN1-related myopathy depends on the oxidoreductase ERO1A and is druggable with the chemical chaperone TUDCA［J］. Cell Rep Med， 2024， 5（3）： 101439.
[11]	RUI B J， RUI G H， YANG Y F. Unveiling pathogenesis of pelvic organ prolapse through transcriptomic and bioinformatic analyses in uterosacral ligament tissues of postmenopausal women［J］. Front Genet， 2025， 16： 1588278.
[12]	TAPPY E， SHI H L， PRUSZYNSKI J， et al. Use of the senolytics dasatinib and quercetin for prevention of pelvic organ prolapse in a mouse animal model［J］. Aging， 2024， 16（19）： 12685-12696.
[13]	HUANG L W， ZHAO Z W， WEN J R， et al. Cellular senescence： a pathogenic mechanism of pelvic organ prolapse （Review）［J］. Mol Med Rep， 2020， 22（3）： 2155-2162.
[14]	NAN L， GUO P C， HUI W， et al. Recent advances in dermal fibroblast senescence and skin aging： unraveling mechanisms and pioneering therapeutic strategies［J］. Front Pharmacol， 2025， 16： 1592596.
[15]	MA Z J， DING Y J， DING X F， et al. PDK4 rescues high-glucose-induced senescent fibroblasts and promotes diabetic wound healing through enhancing glycolysis and regulating YAP and JNK pathway［J］. Cell Death Discov， 2023， 9（1）： 424.
[16]	MIAO Z R， WANG W， MIAO Z Y， et al. Role of Selenoprotein W in participating in the progression of non-alcoholic fatty liver disease［J］. Redox Biol， 2024， 71： 103114.
[17]	POTHION H， JEHAN C， TOSTIVINT H， et al. Selenoprotein T： an essential oxidoreductase serving as a guardian of endoplasmic reticulum homeostasis［J］. Antioxid Redox Signal， 2020， 33（17）： 1257-1275.
[18]	OUYANG P， CAI Z Y， PENG J Y， et al. SELENOK-dependent CD36 palmitoylation regulates microglial functions and Aβ phagocytosis［J］. Redox Biol， 2024， 70： 103064.
[19]	KIM B S， JUNG J S， JANG J H， et al. Nuclear Argonaute 2 regulates adipose tissue-derived stem cell survival through direct control of miR10b and selenoprotein N1 expression［J］. Aging Cell， 2011， 10（2）： 277-291.
[20]	VARONE E， POZZER D， DI MODICA S， et al. SELENON （SEPN1） protects skeletal muscle from saturated fatty acid-induced ER stress and insulin resistance［J］. Redox Biol， 2019， 24： 101176.
[21]	CHEN X， CUBILLOS-RUIZ J R. Endoplasmic reticulum stress signals in the tumour and its microenvironment［J］. Nat Rev Cancer， 2021， 21（2）： 71-88.
[22]	REN J， BI Y G， SOWERS J R， et al. Endoplasmic reticulum stress and unfolded protein response in cardiovascular diseases［J］. Nat Rev Cardiol， 2021， 18（7）： 499-521.
[23]	GAO S Y， CHEN T， DENG C L， et al. An endoplasmic reticulum stress-responsive nanocomposite hydrogel for diabetic wound healing through a fibroblast-immune cell dual regulation hub［J］. J Nanobiotechnology， 2025， 23（1）： 689.
[24]	FARHAN H， ZAHOOR M， TAUER J T， et al. The endoplasmic reticulum proteostasis network and bone disease［J］. Trends Mol Med， 2026， 32（2）： 119-132.
[25]	SANG M D， LI X D， CHEN M， et al. Role of mitochondria-associated ER in apoptosis［J］. Cell Biochem Funct， 2025， 43（7）： e70105.
[26]	MADREITER-SOKOLOWSKI C T， THOMAS C， RISTOW M. Interrelation between ROS and Ca²⁺ in aging and age-related diseases［J］. Redox Biol， 2020， 36： 101678.
[27]	KONG M， WANG Z， HAO Y， et al. Role of the integrin-β1/TGF-β1 signaling pathway in the pathogenesis of pelvic organ prolapse： a study on vaginal wall tissue alterations and molecular dysfunction［J］. Mol Med Rep， 2025， 31（4）： 104.
[28]	ZHENG Y J， LEI J D， ZHANG A， et al. Regulatory mechanisms of transforming growth factor-β in senescence of fibroblast associated with refractory skin diseases［J］. Exp Gerontol， 2025， 211： 112900.
[29]	YAN Y F， PENG J， HE W， et al. SGK1 inhibits oxidative injury and extracellular matrix degradation by activating the GSK-3β （Ser9）/Fyn/NRF2 pathway in pelvic organ prolapse［J］. Life Sci， 2025， 380： 123939.