The miR-9-5p/CXCL11 pathway is a key target of hydrogen sulfide-mediated inhibition of neuroinflammation in hypoxic ischemic brain injury.

We previously showed that hydrogen sulfide (H2S) has a neuroprotective effect in the context of hypoxic ischemic brain injury in neonatal mice. However, the precise mechanism underlying the role of H2S in this situation remains unclear. In this study, we used a neonatal mouse model of hypoxic ischemic brain injury and a lipopolysaccharide-stimulated BV2 cell model and found that treatment with L-cysteine, a H2S precursor, attenuated the cerebral infarction and cerebral atrophy induced by hypoxia and ischemia and increased the expression of miR-9-5p and cystathionine ß synthase (a major H2S synthetase in the brain) in the prefrontal cortex. We also found that an miR-9-5p inhibitor blocked the expression of cystathionine ß synthase in the prefrontal cortex in mice with brain injury caused by hypoxia and ischemia. Furthermore, miR-9-5p overexpression increased cystathionine-ß-synthase and H2S expression in the injured prefrontal cortex of mice with hypoxic ischemic brain injury. L-cysteine decreased the expression of CXCL11, an miR-9-5p target gene, in the prefrontal cortex of the mouse model and in lipopolysaccharide-stimulated BV-2 cells and increased the levels of proinflammatory cytokines BNIP3, FSTL1, SOCS2 and SOCS5, while treatment with an miR-9-5p inhibitor reversed these changes. These findings suggest that H2S can reduce neuroinflammation in a neonatal mouse model of hypoxic ischemic brain injury through regulating the miR-9-5p/CXCL11 axis and restoring ß-synthase expression, thereby playing a role in reducing neuroinflammation in hypoxic ischemic brain injury.

Mitochondria-targeted cerium vanadate nanozyme suppressed hypoxia-ischemia injury in neonatal mice via intranasal administration.

Oxidative stress is a major obstacle for neurological functional recovery after hypoxia-ischemia (HI) brain damage. Nanozymes with robust anti-oxidative stress properties offer a therapeutic option for HI injury. However, insufficiency of nanozyme accumulation in the HI brain by noninvasive administration hinders their application. Herein, we reported a cerium vanadate (CeVO4) nanozyme to realize a noninvasive therapy for HI brain in neonatal mice by targeting brain neuron mitochondria. CeVO4 nanozyme with superoxide dismutase activity mainly co-located with neuronal mitochondria 1 h after administration. Pre- and post-HI administrations of CeVO4 nanozyme were able to attenuate acute brain injury, by inhibiting caspase-3 activation, microglia activation, and proinflammation cytokine production in the lesioned cortex 2 d after HI injury. Moreover, CeVO4 nanozyme administration led to short- and long-term functional recovery following HI insult without any potential toxicities in peripheral organs of mice even after prolonged delivery for 4 weeks. These beneficial effects of CeVO4 nanozyme were associated with suppressed oxidative stress and up-regulated nuclear factor erythroid-2-related factor 2 (Nrf2) expression. Finally, we found that Nrf2 inhibition with ML385 abolished the protective effects of CeVO4 nanozyme on HI injury. Collectively, this strategy may provide an applicative perspective for CeVO4 nanozyme therapy in HI brain damage via noninvasive delivery.

Procyanidin improves experimental colitis by regulating macrophage polarization.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic disease with an unclear pathogenesis for which successful treatments are still lacking. It has been reported that procyanidin, a natural antioxidant, relieves colitis, but the specific mechanism is elusive. PURPOSE: Our present study was designed to investigate the effects of procyanidin on colitis and the regulation of the M1 macrophage phenotype and related signaling pathways. METHODS: In vivo, we used two classic colitis models to observe the effect of procyanidin on macrophage polarization. In vitro, we further validated the therapeutic effect of procyanidin in the RAW264.7 cell line and peritoneal macrophages. RESULTS: The current findings provide new evidence that procyanidin ameliorated dextran sulfate sodium (DSS)-induced colitis by preventing the polarization of macrophages to the M1 type and downregulating the levels of proinflammatory factors in cells. We also showed that procyanidin prevented lipopolysaccharide (LPS)-induced elevation of inflammatory cytokines and the activation of proinflammatory macrophages, which was achieved by activating the STAT3 and NF-κB pathways. CONCLUSIONS: This is the first study to demonstrate that procyanidin alleviates experimental colitis by inhibiting the polarization of proinflammatory macrophages. These data reveal new ideas for the pathogenesis and treatment of inflammatory diseases.

Mechanistic Insights into the Role of OPN in Mediating Brain Damage via Triggering Lysosomal Damage in Microglia/Macrophage.

We previously found that osteopontin (OPN) played a role in hypoxia-ischemia (HI) brain damage. However, its underlying mechanism is still unknown. Bioinformatics analysis revealed that the OPN protein was linked to the lysosomal cathepsin B (CTSB) and galectin-3 (GAL-3) proteins after HI exposure. In the present study, we tested the hypothesis that OPN was able to play a critical role in the lysosomal damage of microglia/macrophages following HI insult in neonatal mice. The results showed that OPN expression was enhanced, especially in microglia/macrophages, and colocalized with lysosomal-associated membrane protein 1 (LAMP1) and GAL-3; this was accompanied by increased LAMP1 and GAL-3 expression, CTSB leakage, as well as impairment of autophagic flux in the early stage of the HI process. In addition, the knockdown of OPN expression markedly restored lysosomal function with significant improvements in the autophagic flux after HI insult. Interestingly, cleavage of OPN was observed in the ipsilateral cortex following HI. The wild-type OPN and C-terminal OPN (Leu152-Asn294), rather than N-terminal OPN (Met1-Gly151), interacted with GAL-3 to induce lysosomal damage. Furthermore, the secreted OPN stimulated lysosomal damage by binding to CD44 in microglia in vitro. Collectively, this study demonstrated that upregulated OPN in microglia/macrophages and its cleavage product was able to interact with GAL-3, and secreted OPN combined with CD44, leading to lysosomal damage and exacerbating autophagosome accumulation after HI exposure.

Blocking osteopontin expression attenuates neuroinflammation and mitigates LPS-induced depressive-like behavior in mice.

INTRODUCTION: Neuroinflammation plays an important role in the development of major depressive disorder (MDD). Osteopontin (OPN) is one of the key molecules involved in neuroinflammation. We demonstrate here for the first time a key role of OPN in lipopolysaccharide (LPS)-induced depressive-like behavioral syndrome. METHODS: Systemic administration of LPS (5 mg/kg) mimics distinct depressive-like behavior, which could significantly upregulate OPN expression in microglia/macrophage in the hippocampus. The neurobehavioral assessments, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), Western blot, immunofluorescent staining, flow cytometry cell staining and Golgi staining were performed. RESULTS: Similar to fluoxetine treatment (the positive control), OPN knockdown with shRNA lentivirus markedly reversed LPS-induced depressive-like behavior. Moreover, knockdown of OPN suppressed LPS-induced proinflammatory cytokine expression, microglial activation, dendritic spines loss, as well as unregulated PSD-95 and BDNF in the hippocampus. CONCLUSION: We demonstrated that targeting OPN expression in microglia/macrophage might help to rescue LPS-induced depressive-like behavior. The underlying mechanism may relate to the modulation of neuroinflammation, BDNF signaling and synaptic structural complexity.

3D Gelatin Microsphere Scaffolds Promote Functional Recovery after Spinal Cord Hemisection in Rats.

Spinal cord injury (SCI) damages signal connections and conductions, with the result that neuronal circuits are disrupted leading to neural dysfunctions. Such injuries represent a serious and relatively common central nervous system condition and current treatments have limited success in the reconstruction of nerve connections in injured areas, especially where sizeable gaps are present. Biomaterial scaffolds have become an effective alternative to nerve transplantation in filling these gaps and provide the foundation for simulating the 3D structure of solid organs. However, there remain some limitations with the application of 3D bioprinting for preparation of biomaterial scaffolds. Here, the approach in constructing and testing mini-tissue building blocks and self-assembly, solid 3D gelatin microsphere (GM) scaffolds with multiple voids as based on the convenient preparation of gelatin microspheres by microfluidic devices is described. These 3D GM scaffolds demonstrate suitable biocompatibility, biodegradation, porosity, low preparation costs, and relative ease of production. Moreover, 3D GM scaffolds can effectively bridge injury gaps, establish nerve connections and signal transductions, mitigate inflammatory microenvironments, and reduce glial scar formation. Accordingly, these 3D GM scaffolds can serve as a novel and effective bridging method to promote nerve regeneration and reconstruction and thus recovery of nerve function after SCI.

The delivery of miR-21a-5p by extracellular vesicles induces microglial polarization via the STAT3 pathway following hypoxia-ischemia in neonatal mice.

Extracellular vesicles (EVs) from mesenchymal stromal cells (MSCs) have previously been shown to protect against brain injury caused by hypoxia-ischemia (HI). The neuroprotective effects have been found to relate to the anti-inflammatory effects of EVs. However, the underlying mechanisms have not previously been determined. In this study, we induced oxygen-glucose deprivation in BV-2 cells (a microglia cell line), which mimics HI in vitro, and found that treatment with MSCs-EVs increased the cell viability. The treatment was also found to reduce the expression of pro-inflammatory cytokines, induce the polarization of microglia towards the M2 phenotype, and suppress the phosphorylation of selective signal transducer and activator of transcription 3 (STAT3) in the microglia. These results were also obtained in vivo using neonatal mice with induced HI. We investigated the potential role of miR-21a-5p in mediating these effects, as it is the most highly expressed miRNA in MSCs-EVs and interacts with the STAT3 pathway. We found that treatment with MSCs-EVs increased the levels of miR-21a-5p in BV-2 cells, which had been lowered following oxygen-glucose deprivation. When the level of miR-21a-5p in the MSCs-EVs was reduced, the effects on microglial polarization and STAT3 phosphorylation were reduced, for both the in vitro and in vivo HI models. These results indicate that MSCs-EVs attenuate HI brain injury in neonatal mice by shuttling miR-21a-5p, which induces microglial M2 polarization by targeting STAT3.

L-Cysteine attenuates osteopontin-mediated neuroinflammation following hypoxia-ischemia insult in neonatal mice by inducing S-sulfhydration of Stat3.

We previously show that L-Cysteine administration significantly suppresses hypoxia-ischemia (HI)-induced neuroinflammation in neonatal mice through releasing H2S. In this study we conducted proteomics analysis to explore the potential biomarkers or molecular therapeutic targets associated with anti-inflammatory effect of L-Cysteine in neonatal mice following HI insult. HI brain injury was induced in postnatal day 7 (P7) neonatal mice. The pups were administered L-Cysteine (5 mg/kg) at 24, 48, and 72 h post-HI. By conducting TMT-based proteomics analysis, we confirmed that osteopontin (OPN) was the most upregulated protein in ipsilateral cortex 72 h following HI insult. Moreover, OPN was expressed in CD11b+/CD45low cells and infiltrating CD11b+/CD45high cells after HI exposure. Intracerebroventricular injection of OPN antibody blocked OPN expression, significantly attenuated brain damage, reduced pro-inflammatory cytokine levels and suppressed cerebral recruitment of CD11b+/CD45high immune cells following HI insult. L-Cysteine administration reduced OPN expression in CD11b+/CD45high immune cells, concomitant with improving the behavior in Y-maze test and suppressing cerebral recruitment of CD11b+/CD45high immune cells post-HI insult. Moreover, L-Cysteine administration suppressed the Stat3 activation by inducing S-sulfhydration of Stat3. Intracerebroventricular injection of Stat3 siRNA not only decreased OPN expression, but also reversed HI brain damage. Our data demonstrate that L-Cysteine administration effectively attenuates the OPN-mediated neuroinflammation by inducing S-sulfhydration of Stat3, which contributes to its anti-inflammatory effect following HI insult in neonatal mice. Blocking OPN expression may serve as a new target for therapeutic intervention for perinatal HI brain injury.

Corrigendum to "Neuroprotective Effects of the Sonic Hedgehog Signaling Pathway in Ischemic Injury through Promotion of Synaptic and Neuronal Health".

[This corrects the article DOI: 10.1155/2020/8815195.].

H2S prevents peripheral immune cell invasion, increasing [Ca2+]i and excessive phagocytosis following hypoxia-ischemia injury in neonatal mice.

We previously reported that L-Cysteine, H2S donor, remarkably attenuated neuroinflammation following hypoxia-ischemia (HI) brain injury in neonatal mice. However, its anti-inflammatory mechanism for HI insult is still unknown. The study focus on the effects of L-Cysteine on immune cell populations, Ca2+ mobilization and phagocytosis after neonatal HI. We found that L-Cysteine treatment skewed CD11b+/CD45low microglia and CD11b+/CD45high brain monocytes/macrophages towards a more anti-inflammatory property 72 h after HI-injured brain. Moreover, L-Cysteine treatment reduced cerebral infiltration of CD4 T cells 7 days following HI insult. Furthermore, CD4 T cell subset analysis revealed that L-Cysteine treatment decreased Th1 and Th2 counts, while increased Th17/Th2 ratio. Moreover, L-Cysteine treatment suppressed LPS-induced cytosolic Ca2+ and LPS-stimulated phagocytosis in primary microglia. The anti-inflammatory effect of L-Cysteine was associated with improving neurobehavioral impairment following HI insult. Our results demonstrate L-Cysteine treatment suppressed the invasion of peripheral immune cells, increasing [Ca2+]i and excessive phagocytosis to improve neurobehavioral deficits following hypoxia-ischemia injury in neonatal mice by H2S release.

Mesenchymal stem cell-derived extracellular vesicles promote microglial M2 polarization after subarachnoid hemorrhage in rats and involve the AMPK/NF-κB signaling pathway.

Subarachnoid hemorrhage (SAH) is an acute and severe disease with high disability and mortality. Inflammatory reactions have been proven to occur throughout SAH. Extracellular vesicles derived from mesenchymal stem cells (MSCs-EVs) have shown broad potential for the treatment of brain dysfunction and neuroprotective effects through neurogenesis and angiogenesis after stroke. However, the mechanisms of EVs in neuroinflammation during the acute phase of SAH are not well known. Our present study was designed to investigate the effects of MSCs-EVs on neuroinflammation and the polarization regulation of microglia to the M2 phenotype and related signaling pathways after SAH in rats. The SAH model was induced by an improved method of intravascular perforation, and MSCs-EVs were injected via the tail vein. Post-SAH assessments included neurobehavioral tests as well as brain water content, immunohistochemistry, PCR and Western blot analyses. Our results showed that MSCs-EVs alleviated the expression of inflammatory cytokines in the parietal cortex and hippocampus 24 h and 48 h after SAH and that MSCs-EVs inhibited NF-κB and activated AMPK to reduce inflammation after SAH. Furthermore, MSC-EVs regulated the polarization of microglia toward the M2 phenotype by downregulating interleukin-1ß, cluster of differentiation 16, cluster of differentiation 11b, and inducible nitric oxide synthase and upregulating the expression of cluster of differentiation 206 and arginase-1. Additionally, MSCs-EVs inhibited the neuroinflammatory response and had neuroprotective effects in the brain tissues of rats after SAH. This study may support their use as a potential treatment strategy for early SAH in the future.

MSCs-extracellular vesicles attenuated neuroinflammation, synapse damage and microglial phagocytosis after hypoxia-ischemia injury by preventing osteopontin expression.

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) significantly suppressed hypoxia-ischemia (HI)-induced neuroinflammation in neonatal mice. However, its underlying mechanism is still unknown. Osteopontin (OPN) is one of the key molecules involved in neuroinflammation. We demonstrate here for the first time a key role of OPN in EVs-mediated neuroinflammation following HI. Firstly, HI exposure upregulated OPN expression in Iba-1+/ TMEM119+ microglia and Iba-1+/TMEM119- monocytes/macrophages. Blocking OPN mRNA expression with LV-shOPN attenuated edema, infarct volumes, and the levels of inflammatory cytokines following HI exposure. MSCs-EVs treatment remarkably restored synaptic reorganization and up-regulated synaptic protein expression post-HI, concomitant with reducing OPN levels. Moreover, MSCs-EVs treatment rescued microglial phagocytosis of viable neurons following HI, concomitant with decreasing OPN expression. In addition, blocking NF-κB activation with pyrrolidine dithiocarbamate (PDTC, NF-κB inhibitor) or MSCs-EVs attenuated HI-induced OPN expression in the ipsilateral cortex. This study demonstrates that upregulation of OPN expression in cerebral immune cells aggravated brain damage and inflammation following HI insult. MSCs-EVs suppressed neuroinflammation, synaptic damage and microglial phagocytosis after HI injury by preventing NF-κB-mediated OPN expression in neonate mice.

Serum KIAA1199 is an advanced-stage prognostic biomarker and metastatic oncogene in cholangiocarcinoma.

BACKGROUND: Cell proliferation and migration are the determinants of malignant tumor progression, and a better understanding of related genes will lead to the identification of new targets aimed at preventing the spread of cancer. Some studies have shown that KIAA1199 (CEMIP) is a transmembrane protein expressed in many types of noncancerous cells and cancer cells. However, the potential role of KIAA1199 in the progression of cholangiocarcinoma (CCA) remains unclear. RESULTS: Analysis of cancer-related databases showed that KIAA1199 is overexpressed in CCA. ELISA, immunohistochemistry, Western blotting and qPCR indicated high expression levels of KIAA1199 in serum, CCA tissues and CCA cell lines. In the serum (n = 41) and large sample validation (n = 177) cohorts, higher KIAA1199 expression was associated with shorter overall survival and disease-free survival times. At the cellular level, KIAA1199 overexpression (OE) promoted CCA growth and metastasis. Subcutaneous tumor xenograft experiments showed that KIAA1199 enhances CCA cell proliferation. Additionally, the expression levels of components in the EMT-related TGF-ß pathway changed significantly after KIAA1199 upregulation and silencing. CONCLUSION: KIAA1199 is a promising new diagnostic molecule and therapeutic target in CCA. The serum KIAA1199 level can be used as a promising clinical tool for predicting the overall postoperative outcomes of patients with CCA. METHODS: CCA-related KIAA1199 data were downloaded from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. To assess the prognostic impact of KIAA1199, an enzyme-linked immunosorbent assay (ELISA) was used to measure the serum level of KIAA1199 in 41 patients who underwent surgical resection. Immunohistochemical staining, Western blotting and qPCR were used to verify and retrospectively review the expression levels of KIAA1199 in cancer tissue specimens from 177 CCA patients. The effect of KIAA1199 on CCA was evaluated by cell-based functional assays and subcutaneous tumor xenograft experiments. The expression levels of proteins associated with epithelial-mesenchymal transition (EMT) and activation of relevant signaling pathways were measured via Western blotting.

Hydrogen-Rich Saline Regulates Microglial Phagocytosis and Restores Behavioral Deficits Following Hypoxia-Ischemia Injury in Neonatal Mice via the Akt Pathway.

INTRODUCTION: We have reported previously that hydrogen-rich saline (HS) plays a neuroprotective role in hypoxia-ischemia (HI) brain damage in newborn mice. However, the mechanisms for this neuroprotection resulting from HS remain unknown. In this study, we examined the potential for HS to exert effects upon microglial phagocytosis via involvement of the Akt signaling pathway as one of the neuroprotective mechanisms in response to neonatal HI. METHODS: The HI brain injury model was performed on postnatal day (PND) 7 (modified Vannucci model). The acute brain damage was detected at 3 days after HI exposure. The behavioral and functional screening of the pups at PND11 and PND13 and their long-term outcomes (PND35, 28-days post-HI) were evaluated sensorimotor performance and cognitive functions, respectively. RESULTS: The result showed that HS administration alleviated HI-induced edema, infract volume and cellular apoptosis within the cortex of neonatal mice. Accompanying these indices of neuroprotection from HS were reductions in HI-induced phagocytosis in microglia as demonstrated in vivo and in vitro, effects that were associated with increasing levels of Akt phosphorylation and improvements in neurobehavioral responses. These beneficial effects of HS were abolished in mice treated with an Akt inhibitor. DISCUSSION: These results demonstrate that HS treatment attenuates neurobehavioral deficits and apoptosis resulting from HI, effects which were associated with reductions in phagocytosis and appear to involve the Akt signaling pathway.

Neuroprotective Effect of Mesenchymal Stromal Cell-Derived Extracellular Vesicles Against Cerebral Ischemia-Reperfusion-Induced Neural Functional Injury: A Pivotal Role for AMPK and JAK2/STAT3/NF-κB Signaling Pathway Modulation.

INTRODUCTION: Cerebral ischemia-reperfusion injury (CIRI) is the main factor that leads to poor prognosis of cerebral ischemia. Apoptosis has been shown to occur during the process of CIRI. Extracellular vesicles derived from mesenchymal stromal cells (MSCs-EVs) have shown broad potential for treating brain dysfunction and eliciting neuroprotective effects after stroke through neurogenesis and angiogenesis. However, the mechanism of action of extracellular vesicles during CIRI is not well known. METHODS: A middle cerebral artery occlusion (MCAO) model was induced by the modified Longa method, and MSCs-EVs were injected via the tail vein. RESULTS: Our results showed that MSCs-EVs significantly alleviated neurological deficits, reduced the volume of cerebral infarction and brain water content, improved pathological lesions in cortical brain tissue, and attenuated neuronal apoptosis in the cortex at 24 h and 48 h after MCAO in rats. Western blotting analysis showed that MSCs-EVs significantly upregulated p-AMPK and downregulated p-JAK2, p-STAT3 and p-NF-κB. In addition, an AMPK pathway blocker reversed the effect of MSCs-EVs on brain damage. CONCLUSION: These results indicate that MSCs-EVs protected MCAO-injured rats, possibly by regulating the AMPK and JAK2/STAT3/NF-κB signaling pathways. This study supports the use of MSCs-EVs as a potential treatment strategy for MCAO in the future.

Hydrogen sulfide-modified extracellular vesicles from mesenchymal stem cells for treatment of hypoxic-ischemic brain injury.

We previously reported that preconditioning of mesenchymal stem cells (MSCs) with hydrogen sulfide (H2S) improved their therapeutic potential in cerebral ischemia. However, the mechanisms involved with this effect have not been determined. As one approach to address this issue, we focused on a neuroprotective role of modification of MSCs-derived extracellular vesicles (EVs) with H2S treatment, and further examined the underlying mechanisms during hypoxia-ischemia (HI) injury in neonatal mice. At 24 h following HI insult, neonatal mice received either systemically administered EVs (derived from MSCs) or H2S-EVs (derived from NaHS-preconditioned MSCs). Both treatments reached the injured region of the ipsilateral hemisphere within 2 h after administration and were incorporated into microglia and neurons. Mice receiving H2S-EVs exhibited substantially lower amounts of brain tissue loss, decreased levels of pro-inflammatory mediators, and a skewed distribution of CD45low microglia and CD45high brain mononuclear phagocytes toward a more anti-inflammatory condition as compared with that in mice receiving only EVs. Moreover, these neuroprotective and anti-inflammatory effects of H2S-EVs were accompanied with long-term preservation of cognitive and memory functions, in contrast to the functional deficits observed in mice receiving only EVs. This H2S preconditioning upregulated miR-7b-5p levels in EVs as determined with next-generation sequencing, while knockdown analyses revealed that inducing miR-7b-5p expression and targeting FOS in the ipsilateral cortex were essential for the neuroprotective and anti-inflammatory effects of H2S-EVs following HI exposure. Taken together, these results demonstrate that miR-7b-5p transferred by H2S-EVs into the ipsilateral hemisphere further induced miR-7b-5p expression, which promoted CD45low microglia and CD45high brain mononuclear phagocytes toward a beneficial phenotype and improved HI-induced cognitive impairments in neonatal mice.

Neuroprotective Effects of the Sonic Hedgehog Signaling Pathway in Ischemic Injury through Promotion of Synaptic and Neuronal Health.

Cerebral ischemia is a common cerebrovascular condition which often induces neuronal apoptosis, leading to brain damage. The sonic hedgehog (Shh) signaling pathway has been reported to be involved in ischemic stroke, but the underlying mechanisms have not been fully elucidated. In the present study, we demonstrated that expressions of Shh, Ptch, and Gli-1 were significantly downregulated at 24 h following oxygen-glucose deprivation (OGD) injury in neurons in vitro, effects which were associated with increasing numbers of apoptotic cells and reactive oxygen species generation. In addition, expressions of synaptic proteins (neuroligin and neurexin) were significantly downregulated at 8 h following OGD, also associated with concomitant neuronal apoptosis. Treatment with purmorphamine, a Shh agonist, increased Gli-1 in the nucleus of neurons and protected against OGD injury, whereas the Shh inhibitor, cyclopamine, produced the opposite effects. Activation of Shh signals promoted CREB and Akt phosphorylation; upregulated the expressions of BDNF, neuroligin, and neurexin; and decreased NF-κB phosphorylation following OGD. Notably, this activation of Shh signals was accompanied by improved neurobehavioral responses along with attenuations in edema and apoptosis at 48 h postischemic insult in rats. Taken together, these results demonstrate that activation of the Shh signaling pathway played a neuroprotective role in response to ischemic exposure via promotion of synaptic and neuronal health.

Mesenchymal stromal cell-derived extracellular vesicles modulate microglia/macrophage polarization and protect the brain against hypoxia-ischemic injury in neonatal mice by targeting delivery of miR-21a-5p.

Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) (MSC-EVs) exhibit protective effects in damaged or diseased tissues. However, the role of EVs secreted by MSC in hypoxia-ischemic (HI) injury in neonatal mice remains unknown. Systemic administration of MSC-EVs attenuated acute brain damage and neuroinflammation, and skewed CD11b+/CD45low microglia and CD11b+/CD45high brain monocyte/macrophage towards a more anti-inflammatory property as determined at 72 h post-HI. In addition, MSC-EVs remarkably improve the injury outcomes pups prior to weaning (P21), while no effect on long-term memory impairment (P42). Importantly, these effects were preceded by incorporation of MSC-EVs into a large number of neurons and microglia within HI group. Abundant levels of miR-21a-5p were present in EVs as determined with next-generation sequencing. Notably, MSC-EVs treatment further increased miR-21a-5p levels at 72 h post HI. Knockdown analyses revealed that miR-21a-5p, and its target-Timp3, were essential for this neuroprotective property of MSC-EVs following HI exposure as demonstrated in both in vitro and in vivo models. These findings suggest that a systemic administration of EVs derived from MSC, have the capacity to incorporated into neurons and microglia where they can then exert neuroprotection against HI-induced injury in neonates through the delivery of miR-21a-5p.

Extracellular vesicle-mediated transfer of miR-21-5p from mesenchymal stromal cells to neurons alleviates early brain injury to improve cognitive function via the PTEN/Akt pathway after subarachnoid hemorrhage.

Patients with subarachnoid hemorrhage (SAH) often suffer from cognitive function impairments even when they have received proper treatment, such as the clipping or coiling of aneurysms, and this causes problems with returning to work and burdens the family. Increasing attention has been paid to mesenchymal stem cell (MSC)-derived extracellular vesicle (MSC-EV) as promising therapeutic vesicles for stroke management. In this study, we explored the potential role of MSC-EV in a rat model of SAH. We observed that MSC-EV ameliorated early brain injury (EBI) after SAH by reducing the apoptosis of neurons and that SAH induced an increase in the expression level of miR-21 in the prefrontal cortex and hippocampus. In addition, using miRNA profiling and CSF sequencing data from the exRNA Atlas, we demonstrated that EV-derived miR-21 protected neurons from apoptosis and alleviated SAH-induced cognitive dysfunction. The neuroprotective role of MSC-EV was abrogated by miR-21 knockdown or the administration of MK2206, a PTEN/Akt inhibitor. Overall, our results suggest that MSC-EV promotes neuronal survival and alleviates EBI after SAH through transferring miR-21 to recipient neurons.

Neuroprotective mechanism of L-cysteine after subarachnoid hemorrhage.

Hydrogen sulfide, which can be generated in the central nervous system from the sulfhydryl-containing amino acid, L-cysteine, by cystathionine-ß-synthase, may exert protective effects in experimental subarachnoid hemorrhage; however, the mechanism underlying this effect is unknown. This study explored the mechanism using a subarachnoid hemorrhage rat model induced by an endovascular perforation technique. Rats were treated with an intraperitoneal injection of 100 mM L-cysteine (30 µL) 30 minutes after subarachnoid hemorrhage. At 48 hours after subarachnoid hemorrhage, hematoxylin-eosin staining was used to detect changes in prefrontal cortex cells. L-cysteine significantly reduced cell edema. Neurological function was assessed using a modified Garcia score. Brain water content was measured by the wet-dry method. L-cysteine significantly reduced neurological deficits and cerebral edema after subarachnoid hemorrhage. Immunofluorescence was used to detect the number of activated microglia. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the levels of interleukin 1ß and CD86 mRNA in the prefrontal cortex. L-cysteine inhibited microglial activation in the prefrontal cortex and reduced the mRNA levels of interleukin 1ß and CD86. RT-PCR and western blot analysis of the complement system showed that L-cysteine reduced expression of the complement factors, C1q, C3α and its receptor C3aR1, and the deposition of C1q in the prefrontal cortex. Dihydroethidium staining was applied to detect changes in reactive oxygen species, and immunohistochemistry was used to detect the number of NRF2- and HO-1-positive cells. L-cysteine reduced the level of reactive oxygen species in the prefrontal cortex and the number of NRF2- and HO-1-positive cells. Western blot assays and immunohistochemistry were used to detect the protein levels of CHOP and GRP78 in the prefrontal cortex and the number of CHOP- and GRP78-positive cells. L-cysteine reduced CHOP and GRP78 levels and the number of CHOP- and GRP78-positive cells. The cystathionine-ß-synthase inhibitor, aminooxyacetic acid, significantly reversed the above neuroprotective effects of L-cysteine. Taken together, L-cysteine can play a neuroprotective role by regulating neuroinflammation, complement deposition, oxidative stress and endoplasmic reticulum stress. The study was approved by the Animals Ethics Committee of Shandong University, China on February 22, 2016 (approval No. LL-201602022).