RESUMO
Objective:To investigate the role and significance of NUFIP-1-mediated ribophagy in apoptosis of dendritic cells (DCs) stimulated by lipopolysaccharide (LPS).Methods:Cultured mouse dendritic cell line DC2.4 were divided into the blank control group and LPS stimulation groups for 6, 12, 24, 48 and 72 h ( n=5). LPS subgroups were consistently cultured with 1 μg/mL LPS for the corresponding incubation time. Western blot was adopted to detect the expression levels of NUFIP-1 and autophagy-related proteins p62 and LC3B across groups. Laser scanning confocal microscopy (LSCM) was applied to detect the expression and cellular localization of NUFIP-1, with its co-localization with Lyso-tracker and LC3B, respectively. The silencing blank vector NS and silencing virus vector NUFIP-1 siRNA were transferred into DC2.4 ( n=3) and stimulated with 1 μg/mL LPS for 24 h. The apoptosis of DC2.4 was measured by flow cytometry analysis. The expression levels of apoptosis-related proteins were determined using Western blot, including cleaved caspase-3 and Bcl-2. One-way analysis of variance (ANOVA) was applied for comparison among multiple groups, and LSD-t method was used for subsequent pairwise comparison. A P<0.05 was considered statistically significant. Results:The results of Western blot showed that expression level of NUFIP-1 in DC2.4 revealed a trend of first increasing and subsequent decreasing upon LPS stimulation for different times (6, 12, 24, 48 and 72 h), and the expression level of NUFIP-1 in the LPS 24 h group was significantly higher than that in the blank control group [blank control group: (0.6786 ± 0.0820); LPS 24 h group: (1.4830 ± 0.1170); P<0.01]. Meanwhile, p62 expression in the LPS 24 h group was significantly lower than that in the blank control group [blank control group: (0.9087 ± 0.1235); LPS 24 h group: (0.3113 ± 0.5571); P<0.01]. Moreover, the conversion from LC3B-I to LC3B-II in the LPS 24 h group was significantly higher than that in the blank control group [blank control group: (0.5542 ± 0.1248); LPS 24 h group: (2.5310 ± 0.3119); P<0.01]. LSCM indicated that NUFIP-1 was predominantly located in the nucleus and perinuclear area in DC2.4. The fluorescence intensity of NUFIP-1 increased in a time-dependent manner from 6 h to 24 h after LPS stimulation, whereas a significant reduction could be observed at 48 h and 72 h after LPS stimulation. Meanwhile, the co-localization of NUFIP-1 with Lyso-tracker and LC3B was substantially reinforced in comparison with the blank control group. Transfection of NUFIP-1 siRNA through lentivirus transfection technology significantly down-regulated the expression level of NUFIP-1 in DC2.4, with statistical differences compared with the blank control group and empty vector group [blank control group: (0.6627 ± 0.1707); empty vector group: (0.6966 ± 0.1107); siRNA group: (0.1428 ± 0.0296); P<0.05]. Flow cytometry analysis revealed that the apoptotic rate of LPS-stimulated DC2.4 was significantly higher in the NUFIP-1 siRNA transfection group than that in the blank control group and empty vector group [blank control LPS 24 h group: (47.91% ± 1.006%); empty vector LPS 24 h group: (70.26% ± 1.011%); siRNA LPS 24 h group: (80.23% ± 2.094); P<0.01]. Western blot analysis of apoptosis-related protein further confirmed that the expression level of cleaved caspase-3 was significantly elevated in the NUFIP-1 siRNA transfection group compared to those of the blank control group and empty vector group under LPS challenge [blank control LPS 24 h group: (0.4748 ± 0.0876); empty vector LPS 24 h group: (0.2849 ± 0.0418); siRNA LPS 24 h group: (0.9733 ± 0.0525); P<0.01]. Likewise, expression of Bcl-2, an anti-apoptotic protein was significantly down-regulated in the siRNA LPS 24 h group [blank control LPS 24 h group: (0.7810 ± 0.0490); empty vector LPS 24 h group: (0.8292 ± 0.0729); siRNA LPS 24 h group: (0.3957 ± 0.0838); P<0.05]. Conclusions:NUFIP-1-mediated ribophagy is significantly activated in DC2.4 upon LPS stimulation, exerting an underlying protective effect on apoptosis.