Human-induced pluripotent stem cell-derived neural stem cell exosomes improve blood-brain barrier function after intracerebral hemorrhage by activating astrocytes via PI3K/AKT/MCP-1 axis.

JOURNAL/nrgr/04.03/01300535-202502000-00029/figure1/v/2024-05-28T214302Z/r/image-tiff Cerebral edema caused by blood-brain barrier injury after intracerebral hemorrhage is an important factor leading to poor prognosis. Human-induced pluripotent stem cell-derived neural stem cell exosomes (hiPSC-NSC-Exos) have shown potential for brain injury repair in central nervous system diseases. In this study, we explored the impact of hiPSC-NSC-Exos on blood-brain barrier preservation and the underlying mechanism. Our results indicated that intranasal delivery of hiPSC-NSC-Exos mitigated neurological deficits, enhanced blood-brain barrier integrity, and reduced leukocyte infiltration in a mouse model of intracerebral hemorrhage. Additionally, hiPSC-NSC-Exos decreased immune cell infiltration, activated astrocytes, and decreased the secretion of inflammatory cytokines like monocyte chemoattractant protein-1, macrophage inflammatory protein-1α, and tumor necrosis factor-α post-intracerebral hemorrhage, thereby improving the inflammatory microenvironment. RNA sequencing indicated that hiPSC-NSC-Exo activated the PI3K/AKT signaling pathway in astrocytes and decreased monocyte chemoattractant protein-1 secretion, thereby improving blood-brain barrier integrity. Treatment with the PI3K/AKT inhibitor LY294002 or the monocyte chemoattractant protein-1 neutralizing agent C1142 abolished these effects. In summary, our findings suggest that hiPSC-NSC-Exos maintains blood-brain barrier integrity, in part by downregulating monocyte chemoattractant protein-1 secretion through activation of the PI3K/AKT signaling pathway in astrocytes.

Identification of toll-like receptor 2 as a key regulator of neuronal apoptosis in vascular dementia by bioinformatics analysis and experimental validation.

BACKGROUND: Vascular dementia (VaD), the second most prevalent type of dementia, lacks a well-defined cause and effective treatment. Our objective was to utilize bioinformatics analysis to discover the fundamental disease-causing genes and pathological mechanisms in individuals diagnosed with VaD. METHODS: To identify potential pathogenic genes associated with VaD, we conducted weighted gene co-expression network analysis (WGCNA), differential expression analysis, and protein-protein interaction (PPI) analysis. The exploration of potential biological mechanisms involved the utilization of Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis. Moreover, a bilateral common carotid artery stenosis (BCAS) mouse model of VaD was established, and the expression of the hub gene, its relationship with cognitive function and its potential pathogenic mechanism were verified by cognitive behavior tests, cerebral blood flow measurement, Western blotting, and immunofluorescence experiments. RESULTS: This study identified 293 DEGs from the brain cortex of VaD patients and healthy controls, among these genes, the Toll-like receptor 2 (TLR2) gene was identified as hub gene, and it was associated with the apoptosis-related pathway PI3K/AKT.The BCAS model demonstrated that the use of TLR2 inhibitors greatly enhanced the cognitive function of the mice (p < 0.05). Additionally, there was a notable decrease in the number of apoptotic cells in the brain cortex of the mice (p < 0.01). Moreover, significant alterations in the levels of proteins related to the PI3K/AKT pathway and cleaved-caspase3 proteins were detected (p < 0.05). CONCLUSIONS: TLR2 plays a role in the pathophysiology of VaD by enhancing the neuronal apoptotic pathway, suggesting it could be a promising therapeutic target.

Dietary emulsifier polysorbate 80 exposure accelerates age-related cognitive decline.

Gut microbial homeostasis is crucial for the health of cognition in elderly. Previous study revealed that polysorbate 80 (P80) as a widely used emulsifier in food industries and pharmaceutical formulations could directly alter the human gut microbiota compositions. However, whether long-term exposure to P80 could accelerate age-related cognitive decline via gut-brain axis is still unknown. Accordingly, in this study, we used the senescence accelerated mouse prone 8 (SAMP8) mouse model to investigate the effects of the emulsifier P80 intake (1 % P80 in drinking water for 12 weeks) on gut microbiota and cognitive function. Our results indicated that P80 intake significantly exacerbated cognitive decline in SAMP8 mice, along with increased brain pathological proteins deposition, disruption of the blood-brain barrier and activation of microglia and neurotoxic astrocytes. Besides, P80 intake could also induce gut microbiota dysbiosis, especially the increased abundance of secondary bile acids producing bacteria, such as Ruminococcaceae, Lachnospiraceae, and Clostridium scindens. Moreover, fecal microbiota transplantation from P80 mice into 16-week-old SAMP8 mice could also exacerbated cognitive decline, microglia activation and intestinal barrier impairment. Intriguingly, the alterations of gut microbial composition significantly affected bile acid metabolism profiles after P80 exposure, with markedly elevated levels of deoxycholic acid (DCA) in serum and brain tissue. Mechanically, DCA could activate microglial and promote senescence-associated secretory phenotype production through adenosine triphosphate-binding cassette transporter A1 (ABCA1) importing lysosomal cholesterol. Altogether, the emulsifier P80 accelerated cognitive decline of aging mice by inducing gut dysbiosis, bile acid metabolism alteration, intestinal barrier and blood brain barrier disruption as well as neuroinflammation. This study provides strong evidence that dietary-induced gut microbiota dysbiosis may be a risk factor for age-related cognitive decline.

Mesenchymal stromal cell treatment attenuates repetitive mild traumatic brain injury-induced persistent cognitive deficits via suppressing ferroptosis.

The incidence of repetitive mild traumatic brain injury (rmTBI), one of the main risk factors for predicting neurodegenerative disorders, is increasing; however, its underlying mechanism remains unclear. As suggested by several studies, ferroptosis is possibly related to TBI pathophysiology, but its effect on rmTBI is rarely studied. Mesenchymal stromal cells (MSCs), the most studied experimental cells in stem cell therapy, exert many beneficial effects on diseases of the central nervous system, yet evidence regarding the role of MSCs in ferroptosis and post-rmTBI neurodegeneration is unavailable. Our study showed that rmTBI resulted in time-dependent alterations in ferroptosis-related biomarker levels, such as abnormal iron metabolism, glutathione peroxidase (GPx) inactivation, decrease in GPx4 levels, and increase in lipid peroxidation. Furthermore, MSC treatment markedly decreased the aforementioned rmTBI-mediated alterations, neuronal damage, pathological protein deposition, and improved cognitive function compared with vehicle control. Similarly, liproxstatin-1, a ferroptosis inhibitor, showed similar effects. Collectively, based on the above observations, MSCs ameliorate cognitive impairment following rmTBI, partially via suppressing ferroptosis, which could be a therapeutic target for rmTBI.

Hydrogen improves cell viability partly through inhibition of autophagy and activation of PI3K/Akt/GSK3ß signal pathway in a microvascular endothelial cell model of traumatic brain injury.

Objective：Traumatic brain injury (TBI) is one of the most serious public health problems in the world. Hydrogen (H2), a flammable, colorless, and odorless gas, has been observed to have preventive and therapeutic effects on brain trauma and other neurological disorders, but its exact mechanism has not been fully clarified.Methods: To further study the mechanism underlying the role of hydrogen gas in alleviating BBB damage after TBI, we performed the scratch injury model on cultured brain microvascular endothelial cells (bEnd.3), which formed the microvascular endothelial barrier - an integral part of the highly specialized BBB.Results: In the case of TBI, hydrogen was able to improve the decline of cell viability induced by TBI. More importantly, inhibition of PI3 K/Akt/GSK3ß signal pathway or activation of autophagy reduced the protective effect of hydrogen on cell viability, indicating that such protective effect was regulated by PI3 K/Akt/GSK3ß signal pathway and was related to the inhibition of autophagy.Conclusion: So we concluded that hydrogen improved the cell viability in a microvascular endothelial cell model of TBI partly through inhibition of autophagy, and inhibitory effect of hydrogen on autophagy was exerted by activating PI3 K/Akt/GSK3ß signal pathway. These findings enriched our knowledge about the mechanism of hydrogen therapy against TBI.

Hydrogen exerts neuroprotection by activation of the miR-21/PI3K/AKT/GSK-3ß pathway in an in vitro model of traumatic brain injury.

Few studies have explored the effect of hydrogen on neuronal apoptosis or impaired nerve regeneration after traumatic brain injury, and the mechanisms involved in these processes are unclear. In this study, we explored neuroprotection of hydrogen-rich medium through activation of the miR-21/PI3K/AKT/GSK-3ß pathway in an in vitro model of traumatic brain injury. Such model adopted PC12 cells with manual scratching. Then, injured cells were cultured in hydrogen-rich medium for 48 hours. Expression of miR-21, p-PI3K, p-Akt, p-GSK-3ß, Bax and Bcl-2 was measured using RT-qPCR, Western blot analysis and immunofluorescence staining. Rate of apoptosis was determined using TUNEL staining. Neuronal regeneration was assessed using immunofluorescence staining. The results showed that hydrogen-rich medium improved neurite regeneration and inhibited apoptosis in the injured cells. Scratch injury was accompanied by up-regulation of miR-21, p-PI3K, p-Akt and p-GSK-3ß. A miR-21 antagomir inhibited the expression of these four molecules, while a PI3K blocker only affected the three proteins and not miR-21. Both the miR-21 antagomir and PI3K blocker reversed the protective effect of hydrogen. In conclusion, hydrogen exerted a neuroprotective effect against neuronal apoptosis and impaired nerve regeneration through activation of miR-21/PI3K/AKT/GSK-3ß signalling in this in vitro model of traumatic brain injury.

Neuron-derived exosomes with high miR-21-5p expression promoted polarization of M1 microglia in culture.

BACKGROUND: Neuroinflammation is a characteristic pathological change of acute neurological deficit and chronic traumatic encephalopathy (CTE) after traumatic brain injury (TBI). Microglia are the key cell involved in neuroinflammation and neuronal injury. The type of microglia polarization determines the direction of neuroinflammation. MiR-21-5p elevated in neurons and microglia after TBI in our previous research. In this study, we explore the influence of miR-21-5p for neuroinflammation by regulating microglia polarization. METHODS: In this study, PC12 and BV2 used to instead of neuron and microglia respectively. The co-cultured transwell system used to simulate interaction of PC12 and BV2 cells in vivo environment. RESULTS: We found that PC12-derived exosomes with containing miR-21-5p were phagocytosed by microglia and induced microglia polarization, meanwhile, the expression of miR-21-5p was increased in M1 microglia cells. Polarization of M1 microglia aggravated the release of neuroinflammation factors, inhibited the neurite outgrowth, increased accumulation of P-tau and promoted the apoptosis of PC12 cells, which formed a model of cyclic cumulative damage. Simultaneously, we also got similar results in vivo experiments. CONCLUSIONS: PC12-derived exosomes with containing miR-21-5p is the essential of this cyclic cumulative damage model. Therefore, regulating the expression of miR-21-5p or the secretion of exosomes may be an important novel strategy for the treatment of neuroinflammation after TBI.

Increased miR-21-3p in Injured Brain Microvascular Endothelial Cells after Traumatic Brain Injury Aggravates Blood-Brain Barrier Damage by Promoting Cellular Apoptosis and Inflammation through Targeting MAT2B.

Our recent articles have reported that increased miR-21-5p in brain after traumatic brain injury (TBI) could improve the neurological outcome through alleviating blood-brain barrier (BBB) damage. miR-21-3p is another mature miRNA derived from pre-miR-21 after Dicer Procession other than miR-21-5p. Its roles in various diseases, such as tumors and myocardial disease, aroused great interest for research in recent years. To further explore the function and underlying mechanism of miR-21, especially miR-21-3p, in regulating the pathological development of BBB damage after TBI, we designed this research and focused on studying the impact of miR-21-3p on apoptosis and inflammation in brain microvascular endothelial cells (BMVECs), the major cellular component of BBB. We performed controlled cortical impact on mouse brain and employed the oxygen glucose deprivation/reoxygenation (OGD)-treated bEnd.3 cells injury model. We found that the miR-21-3p level in BMVECs from injured cerebral cortex of controlled cortical impact (CCI) mice and bEnd.3 cells with OGD treatment were both increased after injury. For in vitro experiments, downregulation on the miR-21-3p level by transfecting miR-21-3p antagomir in cultured cells alleviated OGD-induced BBB damage, characterized by decreased BBB leakage and increased expression of tight junction proteins. Besides, miR-21-3p antagomir could suppress cell death by anti-apoptosis and control inflammatory response by inhibiting the activity of NF-κB signaling. Using luciferase reporter assay and a MAT2B-silenced shRNA vector, we further proved that miR-21-3p exerted the above functions through targeting MAT2B. In addition, in vivo experiments also confirmed that intracerebroventricular infusion of miR-21-3p antagomir could alleviate BBB leakage after TBI. It reduced Evans Blue extravasation and promoted the expression of tight junction proteins, thus contributed to improve the neurological outcome of CCI mice. Taken together, increased miR-21-3p in BMVECs after TBI was bad for restoration of injured BBB. Downregulation on the miR-21-3p level in injured brain could be a promising therapeutic strategy for BBB damage after TBI.

Transplantation of in vitro cultured endothelial progenitor cells repairs the blood-brain barrier and improves cognitive function of APP/PS1 transgenic AD mice.

To date, the pathogenesis of Alzheimer's disease (AD) remains unclear. It is well-known that excessive deposition of Aß in the brain is a crucial part of the pathogenesis of AD. In recent years, the AD neurovascular unit hypothesis has attracted much attention. Impairment of the blood-brain barrier (BBB) leads to abnormal amyloid-ß (Aß) transport, and chronic cerebral hypoperfusion causes Aß deposition throughout the onset and progression of AD. Endothelial progenitor cells (EPCs) are the universal cells for repairing blood vessels. Our previous studies have shown that a reduced number of EPCs in the peripheral blood results in cerebral vascular repair disorder, cerebral hypoperfusion and neurodegeneration, which might be related to the cognitive dysfunction of AD patients. This study was designed to confirm whether EPCs transplantation could repair the blood-brain barrier, stimulate angiogenesis and reduce Aß deposition in AD. The expression of ZO-1, Occludin and Claudin-5 was up-regulated in APP/PS1 transgenic mice after hippocampal transplantation of EPCs. Consistent with previous studies, EPC transplants also increased the microvessel density. We observed that Aß senile plaque deposition was decreased and hippocampal cell apoptosis was reduced after EPCs transplantation. The Morris water maze test showed that spatial learning and memory functions were significantly improved in mice transplanted with EPCs. Consequently, EPCs could up-regulate the expression of tight junction proteins, repair BBB tight junction function, stimulate angiogenesis, promote Aß clearance, and decrease neuronal loss, ultimately improve cognitive function. Taken together, these data demonstrate EPCs may play an important role in the therapeutic implications for vascular dysfunction in AD.

Long-Term Kinetics of Immunologic Components and Neurological Deficits in Rats Following Repetitive Mild Traumatic Brain Injury.

BACKGROUND Despite growing awareness of repetitive mild traumatic brain injury (rmTBI), understanding of the involvement of long-term kinetics of immunologic components in the central and peripheral immune system took part remains incomplete. The present study aimed to provide a quantitative assay for certain immune system parameters in rmTBI rats. MATERIAL AND METHODS Neurological functions were assessed by modified Neurological Severity Score (mNSS) and Morris Water Maze (MWM), immunologic components from brain and peripheral blood were analyzed by flow cytometry (FCM), and concentrations of inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 were measure by enzyme-linked immunosorbent assay (ELISA). RESULTS Neurological functions of rmTBI rats were seriously impaired. In the brain, T cells were up-regulated and peaked at week 1. The percentage of CD4+ T cells decreased from week 1 to week 4, while CD8+ T cells notably decreased at week 1, then increased until week 4. The infiltration proportion of Treg cells was reduced at week 1 and peaked at week 2. CD86+/CD11b+ M1 peaked at week 4 and CD206+/CD11b+ M2 rose at week 1. IL-6/IL-10 showed a similar pattern, whose rise corresponded to the decrease in TNF-α at week 2 after rmTBI. FCM demonstrated peripheral immune dysfunction after rmTBI. CONCLUSIONS mNSS and MWM demonstrated neuronal deficits in rmTBI rats, and central and peripheral immune systems were implicated in the pathophysiological processes of rmTBI. Long-term immune response may play dual roles in injury and repair of rmTBI.

The accumulation of brain injury leads to severe neuropathological and neurobehavioral changes after repetitive mild traumatic brain injury.

Traumatic brain injury (TBI) is a major public health problem with long-term neurobehavioral sequela. The evidences have revealed that TBI is a risk factor for later development of neurodegenerative disease and both the single and repetitive brain injury can lead to the neurodegeneration. But whether the effects of accumulation play an important role in the neurodegenerative disease is still unknown. We utilized the Sprague Dawley (SD) rats to develop the animal models of repetitive mild TBI and single mild TBI in order to detect the neurobehavioral changes. The results of neurobehavioral test revealed that the repetitive mild TBI led to more severe behavioral injuries than the single TBI. There were more activated microglia cells and astrocytes in the repetitive mild TBI group than the single TBI group. In consistent with this, the levels of TNF-α and IL-6 were higher and the expression of IL-10 was lower in the repetitive mild TBI group compared with the single TBI group. The expression of amyloid precursor protein (APP) increased in the repetitive TBI group detected by ELISA and western blot. But the levels of total tau (Tau-5) and P-tau (ser202) seem no different between the two groups in most time point. In conclusion, repetitive mild TBI could lead to more severe neurobehavioral impairments and the effects of accumulation may be associated with the increased inflammation in the brain.

miR-21 improves the neurological outcome after traumatic brain injury in rats.

The expression levels of microRNAs (miRNAs) including miR-21, have been reported to change in response to traumatic brain injury (TBI), suggesting that they may influence the pathophysiological process in brain injury. To analyze the potential effect of miR-21 on neurological function after TBI, we employed the fluid percussion injury rat model and manipulated the expression level of miR-21 in brain using intracerebroventricular infusion of miR-21 agomir or antagomir. We found that upregulation of miR-21 level in brain conferred a better neurological outcome after TBI by improving long-term neurological function, alleviating brain edema and decreasing lesion volume. To further investigate the mechanism underlying this protective effect, we evaluated the impact of miR-21 on apoptosis and angiogenesis in brain after TBI. We found that miR-21 inhibited apoptosis and promoted angiogenesis through regulating the expression of apoptosis- and angiogenesis-related molecules. In addition, the expression of PTEN, a miR-21 target gene, was inhibited and Akt signaling was activated in the procedure. Taken together, these data indicate that miR-21 could be a potential therapeutic target for interventions after TBI.

miR-21 alleviated apoptosis of cortical neurons through promoting PTEN-Akt signaling pathway in vitro after experimental traumatic brain injury.

Traumatic brain injury (TBI) is a major cause of chronic disability and death in young adults worldwide. Multiple cellular, molecular and biochemical changes impact the development and outcome of TBI. Neuronal cell apoptosis, which is an important pathological change in secondary brain damage, is crucial to determine the functional recovery after TBI. miR-21, a widely-reported oncogene, which can reduce cell apoptosis in cancer, has been confirmed to be a pronounced up-regulated miRNA after TBI in animal model. Our study is designed to investigate whether miR-21 has the function of antiapoptosis in experimental TBI model in vitro and to explore the possible regulatory mechanism of miR-21 on neuronal apoptosis. The scratch cell injury was performed to mimic TBI-induced apoptosis in neurons, and miR-21 agomir/antagomir was transfected to up-/down-regulate the miR-21 level. Our data suggests that miR-21 can reduce the number of TUNEL-positive neurons. Meanwhile, miR-21 decreased the expression level of PTEN, and increased the phosphorylation of Akt significantly. In neurons transfected with miR-21 agomir, the expression of Bcl-2 was promoted while the caspase-3, caspase-9 and Bax level were down-regulated, which are crucially the downstream apoptosis-related proteins of PTEN-Akt signaling pathway. In conclusion, miR-21 can exert the function of reducing neuronal apoptosis through activating the PTEN-Akt signaling pathway. Our research provides new insights into the molecular mechanisms of neuronal apoptosis following TBI, which reminds that miR-21may be a potential therapeutic target for TBI treatment.