Zhonghua wei zhong bing ji jiu yi xue · 2026
[Effect and regulatory mechanism of nuclear fragility X mental retardation interaction protein 1 on apoptosis of dendritic cells in sepsis].
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Zheng Liyu, Yao Renqi, Dong Ning, Wu Yao, Yao Yongming
Аннотация
OBJECTIVE: To investigate the regulatory effect and mechanism of nuclear fragile X mental retardation-interacting protein 1 (NUFIP1) on the apoptosis of dendritic cells (DCs) in septic mice.
METHODS: Nufip1+/- C57BL/6J mice were generated, and Western blotting was performed to verify the protein expression level of NUFIP1 in splenic DCs. A total of 48 SPF male wild-type (WT) mice and Nufip1+/- mice were divided into the sham group (18 mice) and the sepsis group (30 mice). Three independent replicate experiments were conducted, and mice were grouped using a random number table in each trial. The cecal ligation and puncture (CLP) procedure was performed to establish the sepsis model in the sepsis group; mice in the Sham group only underwent laparotomy and cecal separation without ligation. Twenty-four hours after operation, orbital blood was collected. The mice were then sacrificed, and heart, lung, liver, kidney tissues as well as spleens were harvested for isolation and extraction of splenic DCs. Flow cytometry was applied to detect the apoptosis rate of splenic DCs. Western blotting was used to determine the protein expression levels of apoptosis-related proteins including caspase-3, Bcl-2, heat shock protein 5 (HSPA5), eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3), phosphorylated EIF2AK3 (p-EIF2AK3), eukaryotic translation initiation factor 2A (EIF2A), phosphorylated EIF2A (p-EIF2A), activating transcription factor 4 (ATF4) and DNA damage-inducible transcript 3 protein (DDIT3) in splenic DCs. Enzyme-linked immunosorbent assay (ELISA) was adopted to detect the serum levels of interleukins (IL-2, IL-4, IL-10), transforming growth factor-β (TGF-β) and interferon-γ (IFN-γ). Hematoxylin-eosin (HE) staining was performed to observe the injury of heart, lung, liver and kidney under light microscope. The endoplasmic reticulum (ER) morphology of splenic DCs was observed under a laser confocal microscope. An additional 20 WT mice and 20 Nufip1+/- mice were respectively assigned to the sham group and sepsis group, with 10 animals in each subgroup. The 7-day survival rate after CLP was observed in all groups.
RESULTS: Western blotting results showed that the protein expression level of NUFIP1 in splenic DCs of Nufip1+/- mice was lower than that of WT mice (NUFIP1/β-actin: 1.080±0.233 vs. 2.178±0.440, P<0.05), indicating the successful construction of NUFIP1 gene-deficient mice. Flow cytometry results revealed that the apoptosis rate of splenic DCs at 24 h after CLP in the Nufip1+/- sepsis group was higher than that in the WT sepsis group [(43.41±1.70)% vs. (30.43±4.99)%, P<0.05]. Meanwhile, compared with the WT sepsis group, the protein expression level of caspase-3 in splenic DCs was increased, while the protein expression level of Bcl-2 was decreased in the Nufip1+/- sepsis group (caspase-3/β-actin: 1.23±0.17 vs. 0.59±0.14, Bcl-2/β-actin: 0.46±0.13 vs. 0.72±0.08, all P<0.05). Compared with the WT sepsis group, the serum levels of IL-2 and IFN-γ in the Nufip1+/- sepsis group were decreased [IL-2 (ng/L): 8.54±0.27 vs. 209.70±1.56; IFN-γ (ng/L): 391.60±9.24 vs. 512.70±31.50, all P<0.05], whereas the serum levels of IL-4, IL-10 and TGF-β were increased [IL-4 (ng/L): 73.19±2.38 vs. 28.52±0.98; IL-10 (ng/L): 359.20±2.02 vs. 267.60±5.05; TGF-β (ng/L): 826.20±26.73 vs. 497.30±3.33, all P<0.05], and the IFN-γ/IL-4 ratio was decreased (7.98±0.46 vs. 16.59±0.40, P<0.05). These findings suggested that peripheral immunosuppression was further aggravated in Nufip1+/- mice during sepsis relative to WT mice. HE staining demonstrated that the injury of heart, lung, liver, kidney and other tissues in the Nufip1+/- sepsis group was markedly severer than that in the WT sepsis group, and the 7-day survival rate was lower in Nufip1+/- mice [20% (2/10) vs. 50% (5/10), P<0.05). Compared with the WT sepsis group, the protein expression levels of HSPA5, the ratio of p-EIF2AK3 to EIF2AK3, the ratio of p-EIF2A to EIF2A, ATF4 and DDIT3 in splenic DCs after CLP were all up-regulated in the Nufip1+/- sepsis group (HSPA5/β-actin: 0.93±0.38 vs. 0.34±0.02, p-EIF2AK3/EIF2AK3 ratio: 1.67±0.28 vs. 0.59±0.05, p-EIF2A/EIF2A ratio: 0.90±0.09 vs. 0.61±0.08, ATF4/β-actin: 1.71±0.77 vs. 1.60±0.48, DDIT3/β-actin: 1.18±0.12 vs. 0.56±0.34, all P<0.05). Laser confocal microscopy showed that the morphological changes of ER in splenic DCs of the Nufip1+/- sepsis group were more obvious than those in the WT sepsis group, presenting irregular distortion, swelling and fragmentation.
CONCLUSIONS: NUFIP1 gene deficiency induces extensive apoptosis of splenic DCs in the early stage of sepsis, further aggravate peripheral immunosuppression, exacerbate multiple organ damage and worsen survival outcomes in septic animals.
METHODS: Nufip1+/- C57BL/6J mice were generated, and Western blotting was performed to verify the protein expression level of NUFIP1 in splenic DCs. A total of 48 SPF male wild-type (WT) mice and Nufip1+/- mice were divided into the sham group (18 mice) and the sepsis group (30 mice). Three independent replicate experiments were conducted, and mice were grouped using a random number table in each trial. The cecal ligation and puncture (CLP) procedure was performed to establish the sepsis model in the sepsis group; mice in the Sham group only underwent laparotomy and cecal separation without ligation. Twenty-four hours after operation, orbital blood was collected. The mice were then sacrificed, and heart, lung, liver, kidney tissues as well as spleens were harvested for isolation and extraction of splenic DCs. Flow cytometry was applied to detect the apoptosis rate of splenic DCs. Western blotting was used to determine the protein expression levels of apoptosis-related proteins including caspase-3, Bcl-2, heat shock protein 5 (HSPA5), eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3), phosphorylated EIF2AK3 (p-EIF2AK3), eukaryotic translation initiation factor 2A (EIF2A), phosphorylated EIF2A (p-EIF2A), activating transcription factor 4 (ATF4) and DNA damage-inducible transcript 3 protein (DDIT3) in splenic DCs. Enzyme-linked immunosorbent assay (ELISA) was adopted to detect the serum levels of interleukins (IL-2, IL-4, IL-10), transforming growth factor-β (TGF-β) and interferon-γ (IFN-γ). Hematoxylin-eosin (HE) staining was performed to observe the injury of heart, lung, liver and kidney under light microscope. The endoplasmic reticulum (ER) morphology of splenic DCs was observed under a laser confocal microscope. An additional 20 WT mice and 20 Nufip1+/- mice were respectively assigned to the sham group and sepsis group, with 10 animals in each subgroup. The 7-day survival rate after CLP was observed in all groups.
RESULTS: Western blotting results showed that the protein expression level of NUFIP1 in splenic DCs of Nufip1+/- mice was lower than that of WT mice (NUFIP1/β-actin: 1.080±0.233 vs. 2.178±0.440, P<0.05), indicating the successful construction of NUFIP1 gene-deficient mice. Flow cytometry results revealed that the apoptosis rate of splenic DCs at 24 h after CLP in the Nufip1+/- sepsis group was higher than that in the WT sepsis group [(43.41±1.70)% vs. (30.43±4.99)%, P<0.05]. Meanwhile, compared with the WT sepsis group, the protein expression level of caspase-3 in splenic DCs was increased, while the protein expression level of Bcl-2 was decreased in the Nufip1+/- sepsis group (caspase-3/β-actin: 1.23±0.17 vs. 0.59±0.14, Bcl-2/β-actin: 0.46±0.13 vs. 0.72±0.08, all P<0.05). Compared with the WT sepsis group, the serum levels of IL-2 and IFN-γ in the Nufip1+/- sepsis group were decreased [IL-2 (ng/L): 8.54±0.27 vs. 209.70±1.56; IFN-γ (ng/L): 391.60±9.24 vs. 512.70±31.50, all P<0.05], whereas the serum levels of IL-4, IL-10 and TGF-β were increased [IL-4 (ng/L): 73.19±2.38 vs. 28.52±0.98; IL-10 (ng/L): 359.20±2.02 vs. 267.60±5.05; TGF-β (ng/L): 826.20±26.73 vs. 497.30±3.33, all P<0.05], and the IFN-γ/IL-4 ratio was decreased (7.98±0.46 vs. 16.59±0.40, P<0.05). These findings suggested that peripheral immunosuppression was further aggravated in Nufip1+/- mice during sepsis relative to WT mice. HE staining demonstrated that the injury of heart, lung, liver, kidney and other tissues in the Nufip1+/- sepsis group was markedly severer than that in the WT sepsis group, and the 7-day survival rate was lower in Nufip1+/- mice [20% (2/10) vs. 50% (5/10), P<0.05). Compared with the WT sepsis group, the protein expression levels of HSPA5, the ratio of p-EIF2AK3 to EIF2AK3, the ratio of p-EIF2A to EIF2A, ATF4 and DDIT3 in splenic DCs after CLP were all up-regulated in the Nufip1+/- sepsis group (HSPA5/β-actin: 0.93±0.38 vs. 0.34±0.02, p-EIF2AK3/EIF2AK3 ratio: 1.67±0.28 vs. 0.59±0.05, p-EIF2A/EIF2A ratio: 0.90±0.09 vs. 0.61±0.08, ATF4/β-actin: 1.71±0.77 vs. 1.60±0.48, DDIT3/β-actin: 1.18±0.12 vs. 0.56±0.34, all P<0.05). Laser confocal microscopy showed that the morphological changes of ER in splenic DCs of the Nufip1+/- sepsis group were more obvious than those in the WT sepsis group, presenting irregular distortion, swelling and fragmentation.
CONCLUSIONS: NUFIP1 gene deficiency induces extensive apoptosis of splenic DCs in the early stage of sepsis, further aggravate peripheral immunosuppression, exacerbate multiple organ damage and worsen survival outcomes in septic animals.