SENP1 attenuates hypoxia‑reoxygenation injury in liver sinusoid endothelial cells by relying on the HIF‑1α signaling pathway

Abstract

Liver sinusoidal endothelial cells (LSECs) have an important role in hepatic ischemia‑reperfusion injury (I/R), but the specific molecular mechanism of action is unknown. LSEC proliferation is regulated and fenestration is maintained via the Sentrin/SUMO‑specific protease 1 (SENP1)/hypoxia‑inducible factor‑1α (HIF‑1α) signaling axis under hypoxic conditions. In the present study, a hypoxia‑reoxygenation (H‑R) injury model was established using mouse LSECs to explore the relationship between SENP1 and H‑R injury in vitro, and the specific underlying mechanism was identified, revealing new targets for the clinical attenuation of hepatic I/R injury. Following the culture of LSECs under H‑R conditions, it was demonstrated that the expression of SENP1 was upregulated by reverse transcription‑quantitative polymerase chain reaction and western blotting (WB). In addition, scanning electron microscopy indicated that fenestrae damage was increased, a Cell Counting Kit‑8 assay demonstrated that the proliferation of cells was impaired and flow cytometry showed that apoptosis was increased. After silencing SENP1 expression with short interfering RNA, the proliferation activity of LSECs decreased, the fenestrae damage increased, the apoptosis rate increased and the expression levels of SENP1, HIF‑1α, heme oxygenase and Bcl‑2 were downregulated (as demonstrated by WB), while the expression levels of apoptosis‑related proteins, cleaved‑caspase‑3 and Bax, were upregulated. Enzyme‑linked immunosorbent assay detection showed that the level of vascular endothelial growth factor in the supernatant decreased and the level of IL‑6 and TNF‑α increased. Following the administration of an HIF‑1α signaling pathway agonist, the situation was reversed. These results therefore suggested that SENP1 attenuated the reduction in proliferation, apoptosis and fenestration of LSECs observed following H‑R injury through the HIF‑1α signaling pathway. In conclusion, SENP1 may attenuate H‑R injury in LSECs in a HIF‑1α signaling pathway‑dependent manner.

Keywords: Sentrin/SUMO- specific protease 1; hypoxia-reoxygenation injury; hypoxia‑inducible factor‑1α; liver sinusoidal endothelial cells.

Figure 1.

Expression of SENP1 in liver sinusoidal endothelial cells following H-R. (A and B) Western blotting analysis. (C) Reverse transcription-quantitative polymerase chain reaction analysis. Data were presented as the mean ± SD (n=3); ***P<0.001, ****P<0.0001 vs. the control (normoxic) group. H-R, hypoxia-reoxygenation; SENP1, Sentrin/SUMO-specific protease 1.

Figure 2.

Effects of SENP1 expression downregulation and reactivation on the extent of fenestration damage and the reduction in the viability of H-R injured LSECs. (A) Representative scanning electron microscopy images showing fenestrae in each treatment group. (B) Scanning electron microscopy images showing the number of fenestrae in LSECs. (C) Cell Counting Kit-8 assay results. Data were presented as the mean ± SD (n=3); *P<0.05 vs. the control (normoxic) group; ****P<0.0001 vs. the H-R + si-NC group; ***P<0.001, ****P<0.0001 vs. the H-R + si-SENP1 group. H-R, hypoxia-reoxygenation; LSECs, liver sinusoidal endothelial cells; NC, negative control; SENP1, Sentrin/SUMO-specific protease 1; si(RNA), short interfering RNA.

Figure 3.

Effects of SENP1 expression downregulation and reactivation on the apoptosis rate of H-R injured liver sinusoidal endothelial cells. (A and B) Flow cytometry plots. (C) Apoptosis rates of the normoxic and H-R groups. Data were presented as the mean ± SD (n=3); ^#P<0.05 vs. the normoxic group; ****P<0.0001 vs. the si-NC group; ***P<0.001 and *P<0.05 vs. the rescue group. H-R, hypoxia-reoxygenation; NC, negative control; SENP1, Sentrin/SUMO-specific protease 1; si(RNA), short interfering RNA.

Figure 4.

SENP1, HIF-1α, HO-1, cleaved-caspase-3, Bax and Bcl-2 protein expression levels in SENP1-knockdown H-R-injured liver sinusoidal endothelial cells. (A) Representative western blots and ratio of SENP1, HIF-1α vs. GAPDH expression determined from the western blot images in the normoxia group. (B) Representative western blots and ratio of SENP1, HIF-1α in the H-R group. (C) Representative western blots and ratio of Bax and cleaved-caspase-3 in the normoxia group. (D) Representative western blots and ratio of Bax and cleaved-caspase-3 in the H-R group. (E) Representative western blots and ratio of Bcl-2 and HO-1 in the normoxia group. (F) Representative western blots and ratio of Bcl-2 and HO-1 in the H-R group. Date were shown as the mean ± SD (n=3); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. the si-NC or si-SENP1 group. HIF-1α, hypoxia-inducible transcription factor-1α; HO-1, heme oxygenase; NC, negative control; SENP1, Sentrin/SUMO-specific protease 1; si(RNA), short interfering RNA; H-R, hypoxia-reoxygenation.

Figure 5.

VEGF, IL-6 and TNF-α protein levels in SENP1-knockdown hypoxia-reoxygenation-injured LSECs. (A) ELISA of VEGF expression in all LSEC groups. (B and C) ELISAs of IL-6 and TNF-α expression in all LSEC groups. Data were presented as the mean ± SD (n=5); **P<0.01, ***P<0.001, ****P<0.0001 vs. the si-NC or si-SENP1 groups; ^##P<0.01, ^###P<0.001 vs. the normoxic group. ELISA, enzyme-linked immunosorbent assay; LSECs, liver sinusoidal endothelial cells; NC, negative control; VEGF, vascular endothelial growth factor; SENP1, Sentrin/SUMO-specific protease 1; si(RNA), short interfering RNA.