敲低硒蛋白N1基因对L929细胞衰老的影响及其机制OA

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.