Middle cerebral artery occlusion is a common model for ischemic stroke. In this study, the modified Longa method,22 which allows stable and reproducible modeling, is simple and can simulate clinical stroke caused by ischemia well, was used to prepare a permanent middle cerebral artery occlusion (pMCAO) model.
The modified neurological severity score test is a commonly used and preferred method for assessing the extent of neurological impairment in permanent and transient middle cerebral artery occlusion models. In this experiment, pMCAO rats showed obvious symptoms of neurological damage, while NBP improved the degree of neurological damage in pMCAO rats to some extent. Taken together with the pathological staining results in brain tissues, these data show that NBP can significantly reduce the area of brain damage area in this model, which is consistent with the existing research results.23
LA-ICP MSI is a new method of mass spectrometry imaging. Several metal-labeled antibodies bind specific proteins on a tissue slice, and then the tissue is ablated by a laser to produce aerosol, which is transported by a carrier gas into plasma to complete ionization. Then, the ions are detected by mass spectrometry, and a distribution map of various proteins in a single test is obtained. LA-ICP MSI is simple, fast, high-throughput, and highly accurate and has a low operating cost, and it is gradually becoming a widely used imaging method.24
The mechanisms of brain damage in ischemia mainly involve the consumption of oxygen and energy (within a few minutes), the release of excitatory amino acids (within a few hours), the inflammatory response and apoptosis. These biochemical reactions mediate the death of nerve cells. Accordingly, one of the important early treatment methods for cerebral infarction is preventing the biochemical reactions of these processes, thereby blocking the death of nerve cells in brain tissue after cerebral vascular occlusion.
Phospholipids are important components of cell membranes and can indicate apoptosis, necrosis and the occurrence of excessive inflammation in cerebral ischemia. We used MALDI-TOF MSI technology to detect the distribution of phospholipid molecules in the brains of pMCAO rat models, which was very important for our study of the effect of NBP on the brain.25 We found that PA (16:0/18:1), PA (18:0/22:6), PE (16:0/22:6), PE (P-18:0/22:6), PE (18:0/22:6), PS (18:0/22:6), PI (18:0/20:4) and other phospholipids were decreased in the ischemic area (right cortex and striatum), indicating that cell death occurred in ischemic cells; however, NBP alleviated cell death in ischemic cells. It is noteworthy that phospholipid PE (18:0) is abnormally increased in the ischemic area of the pMCAO model and that it is susceptible to oxidative stress for the formation of oxidized phospholipids. Oxidized phospholipids have a strong proinflammatory effect, indicating that NBP may inhibit the inflammatory reaction by indirectly affecting the excessive production of oxidized phospholipids.
The neuroinflammatory response promotes damage in cerebral ischemia. After cerebral ischemia, there is a severe inflammatory reaction in the brain tissue. In addition to inducing cell pyroptosis, excessive inflammatory factors promote apoptosis and necrosis.26 The NLRP3 inflammasome in neurons and glial cells may play an important role in detecting cell damage and mediating inflammatory responses.27, 28 During the innate immune response, the NLRP3 inflammasome regulates the activation of Caspase-1, which promotes the maturation of the cytokine pro-IL-1β. Additionally, the NLRP3 inflammasome regulates Caspase-1-dependent pyroptosis and induces cell death under pathological conditions of inflammation and stress.29 Immunohistochemistry showed that NBP reduced the levels of NLRP3, IL-1β and Caspase-1 in the ischemic brain area, indicating that NBP can inhibit the inflammatory response mediated by the NLRP3 pathway. Similarly, Yang et al. suggested that NBP reduces neurovascular inflammation and ischemic brain injury in mice. This effect is associated with reduced infiltration of myeloid cells into the brain and improved cerebral blood flow after reperfusion. 30
Regulatory T (Treg) cells are a special subset of T cells that are essential for inducing and maintaining the stability and tolerance of the immune environment.13, 31 Treg cells prevent the overactivation of the immune system in inflammatory diseases.32 Foxp3-positive T cells are typical anti-inflammatory cells that control and limit antigen-specific immune responses and immune tolerance.33, 34 A previous study reported staining for Foxp3 (a Treg marker) in the striatal and cortical regions of the brain after ischemia and reperfusion in an MCAO-induced stroke model.35 In our study, this protein was observed using LA-ICP MSI technology, which can evaluate the expression of several different proteins in a single test. We found for the first time that NBP significantly increases the content of Foxp3 in the ischemic area in an animal stroke model. Additionally, NBP can play an anti-inflammatory and neuroprotective role by maintaining the function of Foxp3-positive T cells.
The most well-recognized mechanisms of action of NBP are improvements in the microcirculation, the promotion of angiogenesis, and increased cerebral blood flow in the ischemic area. Using LA-ICP MSI, we also found that NBP can increase the distribution of Ki-67 and pCREB in the ischemic cortex and striatum. Ki-67 is a nuclear protein involved in cell proliferation and can indicate vessel density.36 Our study first explored the level of Ki-67 after NBP administration in an animal stroke model. The increase in the Ki-67 level in the ischemic area induced by NBP illustrated that NBP can improve angiogenesis in the ischemic region of the pMCAO model. NBP treatment also promotes the expression of vascular endothelial growth factor and angiopoietin-1 and induces angiogenesis.37 Li et al. found that the Ang-1/Ang-2/Tie-2 signaling axis is altered in the cortex of chronic cerebral hypoperfusion rats and that NBP treatment can regulate this angiopoietin/Tie signaling axis in a timely manner to promote neovascularization in early stages.38
Cyclic-AMP response element-binding protein (CREB) is a protein that regulates gene transcription and is involved in synaptic plasticity, memory, and cognition.39 The activation of the CREB pathway promotes nerve regeneration after cerebral ischemia-reperfusion in rats.40 Liu B et al found Gadd45b-RNAi significantly decreased the expression levels of both BDNF and cAMP/PKA/phosphorylated cAMP response element-binding protein (pCREB) pathway and promoted ROCK expression. They conclude that Gadd45b stimulates recovery after stroke by enhancing axonal plasticity required for brain repair.41 LA-ICP MSI showed that NBP can increase the level of phosphorylated CREB (pCREB) in the ischemic cortex and striatum, which is consistent with the results of Yang et al., indicating that NBP can improve nerve regeneration in the pMCAO model.42 Nevertheless, how NBP exerts nerve regeneration and protects cognitive function deserves further research.
We demonstrated that NBP can reduce inflammatory damage, maintain immune tolerance, improve vascular density, and promote nerve cell regeneration through mechanisms that are shown in Fig. 5.