Ulinastatin alleviates early brain injury after intracerebral hemorrhage by inhibiting necroptosis and neuroinflammation via MAPK/NF-κB signaling pathway

Abstract

Purpose: Spontaneous intracerebral hemorrhage (ICH) is a major public health problem with a huge economic burden worldwide. Ulinastatin (UTI), a serine protease inhibitor, has been reported to be anti-inflammatory, immune regulation, and organ protection by reducing reactive oxygen species production, and inflammation. Necroptosis is a programmed cell death mechanism that plays a vital role in neuronal cell death after ICH. However, the neuroprotection of UTI in ICH has not been confirmed, and the potential mechanism is unclear. The present study aimed to investigate the neuroprotection and potential molecular mechanisms of UTI in ICH-induced EBI in a C57BL/6 mouse model.

Methods: The neurological score, brain water content, neuroinflammatory cytokine levels, and neuronal damage were evaluated. The anti-inflammation effectiveness of UTI in ICH patients also was evaluated.

Results: UTI treatment markedly increased the neurological score, alleviate the brain edema, decreased the inflammatory cytokine TNF-α, interleukin‑1β (IL‑1β), IL‑6, NF‑κB levels, and RIP1/RIP3, which indicated that UTI-mediated inhibition of neuroinflammation, and necroptosis alleviated neuronal damage after ICH. UTI also can decrease the inflammatory cytokine of ICH patients. The neuroprotective capacity of UTI is partly dependent on the MAPK/NF-κB signaling pathway.

Conclusions: UTI improves neurological outcomes in mice and reduces neuronal death by protecting against neural neuroinflammation, and necroptosis.

Figure 1. UTI alleviates neurological deficits and mortality after ICH. (a) Comparison of the mortality between the three groups, the mortality increased significantly after ICH, Mortality rates in the Sham group (5%), ICH group rate (35%)1, the ICH + UTI group rate (15%); (b) Neurological scores of mice in the sham group, ICH group, and ICH group treated with UTI at 72 h (n = 10, ^*p < 0.05 vs. Sham; ^#p < 0.05 vs. ICH; ANOVA; means ± SD).

Figure 2. UTI alleviate brain edema and BBB permeability after ICH. (a) UTI alleviates brain water content after ICH; (b) UTI alleviates BBB permeability after ICH (n = 5, ^*p < 0.05 vs. Sham; ^#p < 0.05 vs. ICH; ANOVA; means ± SD).

Figure 3. UTI alleviates neuronal necroptosis after ICH. (a) TUNEL staining showed that UTI alleviated neuronal apoptosis in the hippocampus at 72 h after ICH, and representative images of apoptotic neurons are shown. Scale bar = 50 μm; (b) Levels of RIP1 and RIP3 in the brain cortex of mice after TBI were determined using Western blotting; (c) Quantification of RIP1 levels in the brain cortex relative to β-actin, the loading control; (d) Quantification of RIP3 levels in the brain cortex relative to β-actin. DAPI, 4’,6-diamidino-2-phenylindole; SAH, subarachnoid hemorrhage; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 4. UTI alleviates neuroinflammation after ICH. UTI significantly reduced hippocampal (a) TNF-α; (b) interleukin-1β (IL-1β); (c) IL-6; and (d) NF-κB levels at 72 h after ICH (n = 5, ^*p < 0.05 vs. Sham; ^# p < 0.05 vs. ICH, ANOVA; means ± SD).

Figure 5. UTI regulates necroptosis and neuroinflammation by modulating the MAPK/ NF-κB signaling pathway after ICH. (a) Levels of MAPK, and NF-κB in the brain cortex of mice after ICH were determined using Western blotting; (b) Quantification of NF-κB levels in the brain cortex relative to β-actin, the loading control; (c) Quantification of MAPK levels in the brain cortex relative to β-actin; (d) Levels of NF-κB mRNA in the brain of ICH mice weremeasured by real-time PCR; (e) Levels of MAPK mRNA in the brain of ICH mice were measured by real-timePCR (n = 5, Data are presented as the mean ± SD, ^*p < 0.05 vs. Sham; ^#p < 0.05 vs. ICH).

Figure 6. UTI alleviates neuroinflammation in ICH patients. UTI significantly reduced ICH patients’ serum (a) TNF-α; (b) interleukin-1β (IL-1β) levels at 72 h after ICH (n = 10, p < 0.01, ANOVA; means ± SD).

Figure 7. A diagram of the proposed model explaining the observations of MAPK/NF-κB -mediated regulation of necroptosis, and neuroinflammation after ICH and potential mechanisms underlying the effect of the UTI intervention.