Ulinastatin alleviates early brain injury after traumatic brain injury by inhibiting oxidative stress and apoptosis

Abstract

Purpose: Traumatic brain injury (TBI) remains a major public health problem and cause of death. Ulinastatin (UTI), a serine protease inhibitor, has been reported to have an anti-inflammatory effect and play a role in immunoregulation and organ protection by reducing reactive oxygen species (ROS) production, oxidative stress and inflammation. However, the neuroprotective of UTI in TBI has not been confirmed. Therefore, this study aimed to investigate the neuroprotection and potential molecular mechanisms of UTI in TBI-induced EBI in a C57BL/6 mouse model.

Methods: The neurological score and brain water content were evaluated. Enzyme-linked immunosorbent assay was used to detect neuroinflammatory cytokine levels, ROS and malondialdehyde detection to evaluate oxidative stress levels, and TUNEL staining and western blotting to examine neuronal damages and their related mechanisms.

Results: Treatment with UTI markedly increased the neurological score; alleviated brain oedema; decreased the inflammatory cytokine tumour necrosis factor a, interleukin-1β (IL-1β), IL-6 and nuclear factor kappa B (NF-kB) levels; inhibited oxidative stress; decreased caspase-3 and Bax protein expressions; and increased the Bcl-2 levels, indicating that UTI-mediated inhibition of neuroinflammation, oxidative stress and apoptosis ameliorated neuronal death after TBI. The neuroprotective capacity of UTI is partly dependent on the TLR4/NF-kB/p65 signalling pathway.

Conclusions: Therefore, this study reveals that UTI improves neurological outcomes in mice and reduces neuronal death by protecting against neural neuroinflammation, oxidative stress and apoptosis.

Figure 1. Ulinastatin alleviates neurological deficits and brain oedema after TBI. (a) Comparison of the brain water content among the three groups (n = 5, p < 0.05). (b) Neurological scores of mice in the sham, TBI and TBI groups treated with UTI at 72 h after TBI (n = 10, p < 0.05). ANOVA; means ± SEM.

Figure 2. Ulinastatin alleviates neuronal apoptosis after TBI. (a) TUNEL staining showed that UTI alleviated neuronal apoptosis in the hippocampus at 72 h after TBI, and representative images of apoptotic neurons are shown. Scale bar = 50 μm. DAPI, 4´,6-diamidino-2-phenylindole; SAH, subarachnoid haemorrhage; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling. (b) Levels of caspase-3, Bcl-2 and Bax in the brain cortex of mice after TBI were determined using Western blotting. (c-e): Quantification of caspase-3, Bcl-2 and Bax in the brain cortex relative to b-actin, the loading control. UTI reduced Bax (c) and caspase-3 (e) levels in mice with TBI and increased the Bcl-2 level (d). (n = 5, *p < 0.05 vs. the sham group; #p < 0.05 vs. the TBI group; ANOVA; means ± SEM).

Figure 3. Ulinastatin alleviates neuroinflammation post TBI. UTI significantly reduced hippocampal (a) TNF-a, (b) interleukin-1β (IL-1β), (c) IL-6 and (d) NF-kB levels 72 h after TBI (n = 5, ^*p < 0.05 vs. the sham group; ^#p < 0.05 vs. the TBI group, ANOVA; means ± SEM).

Figure 4. Ulinastatin inhibits TBI-induced oxidative stress in the hippocampus. (a) The ROS level by the DCF-DA method. (b-d) The MDA, GSH and SOD levels were quantified using commercial kits (n = 5, data are presented as the mean ± SEM, ^*p <0.05 vs. the sham group; ^#p < 0.05 vs. the TBI group).

Figure 5. Ulinastatin regulates oxidative stress and apoptosis by modulating the TLR4/NF-kB/p65 signalling pathway after TBI. (a) TLR4 and NF-kB levels in the brain cortex of mice after TBI were determined using western blotting; (b) Quantification of NF-kB levels in the brain cortex relative to β-actin, the loading control; (c) Quantification of TLR4 levels in the brain cortex relative to β-actin; (d) NF-kB mRNA levels in the brain of TBI mice were measured by real-time PCR; (e) TLR4 mRNA levels in the brain of TBI mice were measured by real-time PCR (n = 5, data are presented as the mean ± SEM, ^*p < 0.05 vs. the sham group; ^#p < 0.05 vs. the TBI group).

Figure 6. A diagram of the proposed model explaining the observations of TLR4/NF-kB/p65-mediated regulation of oxidative stress, neuroinflammation and apoptosis after TBI and potential mechanisms underlying the effects of the UTI intervention.