CI/R occurs when sudden recovery of blood supply after ischemic stroke, leading to neuronal apoptosis, microglia-mediated neuroinflammation and disruption of the NVU. NVU serves as the complex structural and functional unit in the brain composed by neurons, microglia, astrocytes and endothelial cells. Recent reports have been indicated that impaired NVU components after CI/R contributed primarily to brain damage. In addition, new therapeutic strategies to restore brain function after CI/R with Traditional Chinese medicine (TCM) treatment have been verified to exert protective role in the NVU cell components. The treatment of CI/R through protection and regeneration of NVU has been considered as a novel focus. Our previous studies have demonstrated that Alisol A confers neuroprotective effects against CI/R injury via inhibiting the microglia-mediated inflammatory factors production, indicating that Alisol A could potentially be a novel alternative option for improving NVU after CI/R.
Inflammation and apoptosis are the important contributors to the destruction of NVU, which aggravate the development of CI/R injury. Tight junctions form a virtually impermeable barrier between endothelial cells. ZO-1 and Occludin are critical components of TJs, which is relevant in maintaining BBB integrity and homeostasis of the central nervous system. Following CI/R, sustained release of inflammatory cytokines results in local brain tissue inflammatory infiltration, which further aggravates BBB damage [21]. Angiogenesis is an important protective and restorative mechanism in response to CI/R injury, which plays a critical role in prognostic outcome after CI/R. CD31 (an angiogenesis marker), is mainly expressed in brain microvascular endothelial cells (BMECs), which is associated with the integrity of the BBB. It was observed that increased CD31 in endothelial cells could restore BBB strength and endothelial cell morphology after CI/R [22]. Consistent with the previous studies, CD31 level markedly decreased after GCI/R injury [23]. Alisol A treatment increased TJs and CD31 expression and decreased IL-6, IL-1β levels, indicating that Alisol A may protect BBB integrity by reducing inflammatory factors production and promoting TJs and CD31 expression.
Astrocytes are the most abundant glial cells in the central nervous system, providing structural, trophic and metabolic support to neurons. Inflammatory factors released by overactivated astrocytes are involved in various neurological dysfunction diseases, which is known as “reactive astrogliosis”. Reactive astrogliosis is considered as a marker of structural injury, damages the NVU homeostasis by altering their morphology and metabolism [24]. Microglia-mediated neuroinflammation after CI/R affects basal lamina integrity and leads to an increase in BBB permeability, ultimately results in a significant amelioration on the NVU permeability. Neuronal injury leads to the release of the intracellular content, which in turn stimulates the secretion of inflammatory cytokines from overactivated microglia [25]. Our study showed that astrocytes and microglia significantly activated after GCI/R. Treatment with Alisol A inhibited the activation of glial cells and reduced the production of inflammatory factors after GCI/R, suggesting that the positive effects of Alisol A on NVU dysfunction may be partly through its anti-inflammatory property.
MRS is a non-invasive and highly sensitive technique which provide metabolic information in various neurological disorders, including ischemic stroke. Several representative neurometabolic markers such as Cho, NAA, Cr, MI can be assessed using MRS to reflect neuronal/glial metabolism. Cho concentration in glial cells have 2-3-fold higher than that in the neurons, which may be used as a marker of gliosis [26]. NAA, found predominantly in neurons, has been used as a specific biochemical marker to assess neuronal metabolism. Decreased NAA has been identified as a strong signal for neuronal loss [27]. MI has been considered as a putative glial marker, because it’s primarily stored in glial cells and functions as an osmolyte that maintains glial cell volumes [28]. Meanwhile, MI may also be a marker of gliosis for it's present at a much higher concentration in glial cells than in neurons [20]. We found that GCI/R induced the up-regulation of Cho MI, and down-regulation of NAA. Alisol A decreased Cho, MI concentrations in the hippocampal CA1 region, while increased the levels of NAA, implicating that protective effect of Alisol A against NVU injury may be partly contributed to the regulation of neuronal/glial metabolism.
Apoptosis is critical for the regulation of NVU homeostasis. Neuroinflammation mediated by excessive production of pro-inflammatory cytokines aggravates neuronal apoptosis in hippocampal CA1 region [29]. Bcl-2 and BAX are essential proteins participating in apoptotic regulation, and neuronal survival in CI/R depends deeply on the balance between Bcl-2 and BAX. Bcl-2 exerts an anti-apoptosis effect by inhibiting the expression of BAX. Therefore, neuronal apoptosis is inhibited by down-regulating BAX/Bcl-2 ratio, which primarily explained the mechanism of neuroprotection [30]. Nissl staining and NeuN immunostaining exhibited that GCI/R decreased the number of neurons in hippocampal CA1 region. Moreover, our results indicated that GCI/R increased the ratio of BAX to Bcl-2, which partially indicated that apoptosis was aggravated after GCI/R attributed to the up-regulation of BAX/Bcl-2 ratio. Alisol A treatment reduced neuron apoptosis in hippocampal CA1, up-regulated the anti-apoptotic factor Bcl-2 and down-regulated the pro-apoptotic factor BAX, indicating the neuroprotective and anti-apoptotic effects of Alisol A on neuron.
Neurons, astrocytes, microglia, and vascular endothelial cells are the cellular components of NVU. Among these components, neuron acts as a pivotal determinant of NVU and have been studied in the past decades. Thus, we focus on the components other than neurons after GCI/R. Ultrastructural alterations of NVU after GCI/R were observed by TEM in our study, including neuronal morphological destroy, thickened capillary walls edema between endothelial cells and the basement membrane, swelling of astrocytic end-foot processes, which were consistent with previous study [31]. It has been reported that swollen astrocytic end-foot processes and perivascular edema result in BBB breakdown [32]. Moreover, discontinuous endothelium basement membrane was observed in our study, which is related to the down-regulated ZO-1, Occludin [33]. However, these damages were markedly alleviated by Alisol A treatment, demonstrating that Alisol A treatment may attenuate NVU injury.
Alisol A, a triterpene isolated from Alismatis Rhizoma, has been reported to attenuate the expression of pro-inflammatory cytokines [34]. Inflammasome IL-1β and TNF-α levels have been down-regulated by inactivating GSK3β through phosphorylation [35]. GSK3β is a pivotal downstream element of PI3K/AKT. Phosphorylated GSK3β is mediated by AKT, and activation of AKT/GSK3β exerts a protective effect on neurons survival in hippocampal CA1 region after GCI/R [36]. However, oxygen-glucose deprivation decreases the phosphorylation of AKT, subsequently induces the decreased GSK3β phosphorylation [37]. It is demonstrated that activated GSK3β contribute to neuronal apoptosis. Recent studies have shown that attenuation of inflammation and apoptosis induced by cerebral ischemia is critical for NVU protection [38]. AKT/GSK3β signal pathway has been confirmed to has neuroprotective effects and maintain BBB integrity [39]. Considering the protective effects of AKT/GSK3β on NVU dysfunction, we speculated that Alisol A-mediated NVU protection is achieved by anti-inflammation and anti-apoptosis through activated AKT/GSK3β. In this study, the phosphorylation of AKT and GSK3β levels decreased after GCI/R, but increased after Alisol A treatment. AKT inhibitor GSK690693 was used to verify our hypothesis that AKT/GSK3β is involved in Alisol A-mediated NVU protection. As shown in our study, inhibition of AKT abolished the protective effects of Alisol A on cellular components of NVU, providing evidence that AKT/GSK3β signal pathway is involved in the mechanisms underlying the therapeutic effect of Alisol A on NVU injury during CI/R.