Xinchun Ye

Intranasal administration of human umbilical cord mesenchymal stem cells-conditioned medium enhances vascular remodeling after stroke.

Stem cell-based treatments have been reported to be a potential strategy for stroke. However, tumorigenic potential and low survival rates of transplanted cells could attenuate the efficacy of the stem cell-based treatments. The application of stem cell-condition medium (CM) may be a practicable approach to conquer these limitations. In this study, we investigated whether intranasal administration of human umbilical cord mesenchymal stem cells (hUCMSCs)-CM has the therapeutic effects in rats after stroke. Adult male rats were subjected to middle cerebral artery occlusion (MCAo) and were treated by intranasal routine with or without hUCMSCs-CM (1 ml/kg/d), starting 24h after MCAo and daily for 14 days. Neurological functional tests, blood brain barrier (BBB) leakage, were measured. Angiogenesis and angiogenic factor expression were measured by immunohistochemistry, and Western blot, respectively. hUCMSCs-CM treatment of stroke by intranasal routine starting 24h after MCAo in rats significantly enhances BBB functional integrity and promotes functional outcome but does not decrease lesion volume compared to rats in DMEM/F12 medium control group and saline control group. Treatment of ischemic rats with hUCMSCs-CM by intranasal routine also significantly decreases the levels of Ang2 and increases the levels of both Ang1 and Tie2 in the ischemic brain. To take together, increased expression of Ang1 and Tie2 and decreased expression of Ang2, induced by hUCMSCs-CM treatment, contribute to vascular remodeling in the ischemic brain which plays an important role in functional outcome after stroke.

Purinergic 2X7 receptor/NLRP3 pathway triggers neuronal apoptosis after ischemic stroke in the mouse

ABSTRACT Previous research has shown that Purinergic 2X7 receptor (P2X7R) and NLRP3 inflammasome contribute to the inflammatory activation. In this study, we investigated whether P2X7R/NLRP3 pathway is involved in the caspase‐3 dependent neuronal apoptosis after ischemic stroke by using a focal cortex ischemic stroke model. The expressions of P2X7R, NLRP3 inflammsome components, and cleaved caspase‐3 were significantly enhanced in the ischemic brain tissue after stroke. However, the expression of cleaved caspase‐3 was significantly attenuated after treatment of stroke with P2X7R antagonist (BBG) or NLRP3 inhibitor (MCC950). The treatment also significantly reduced the infarction volume, neuronal apoptosis, and neurological impairment. In addition, in vitro data also support the hypothesis that P2X7R/NLRP3 pathway plays a vital role in caspase‐3 dependent neuronal apoptosis after ischemic stroke. Further investigation of effective regulation of P2X7R and NLRP3 in stroke is warranted. HIGHLIGHTSThe expressions of P2X7R, NLRP3 inflammsome components were increased after stroke.BBG treatment reduced neurological impairment, neuronal apoptosis.MCC950 treatment also reduced neurological impairment, neuronal apoptosis.NLRP3 mediated neuronal apoptosis could be ameliorated by a P2X7R antagonist.In vitro data also supported that P2X7R/NLRP3 pathway triggers neuronal apoptosis.

ROS/TXNIP pathway contributes to thrombin induced NLRP3 inflammasome activation and cell apoptosis in microglia

There is no effective therapy for intracerebral hemorrhage (ICH) because of poor understanding of the mechanisms of brain injury after hemorrhage. The NLRP3 inflammasome, as a vital component of innate immune system, which is associated with a wide range of human CNS disorders, including ICH. But its detailed mechanisms in ICH remain mainly unclear. In this study, BV2 cells with thrombin exposure were used to investigate the role of NLRP3 inflammasome in thrombin-induced brain injury. We used western blot to detect NLRP3 inflammasome activation and the expression of thioredoxin binding protein (TXNIP), DCFH-DA to investigate intracellular reactive oxygen species (ROS), flow cytometry to analyze apoptosis. Our results showed that ROS inhibitor N-acetyl-l-cysteine (NAC) suppressed the upregulation of intracellular ROS and TXNIP expression. Furthermore, the cell apoptosis and expression of apoptotic protein were significantly attenuated after treatment of thrombin with NAC or NLRP3 antagonist (MCC950). Thrombin activates ROS/TXNIP/NLRP3 signaling in BV2 cells, which may indicate a mechanism that pro-inflammatory and pro-apoptotic contributes to the development of ICH.

Bone marrow stromal cells inhibits HMGB1-mediated inflammation after stroke in type 2 diabetic rats

High-mobility group box 1 (HMGB1), a ligand of receptor for advanced glycation endproducts (RAGE), functions as a proinflammatory factor. It is mainly involved in inflammatory activation and contributes to the initiation and progression of stroke. By using a model of transient middle cerebral artery occlusion (MCAo) in type 2 diabetic rats, we investigated the changes of pro-inflammation mediators, blood-brain barrier (BBB) leakage and functional outcome after stroke. Type 2 diabetic rats did not show an increased lesion volume, but exhibited significantly increased expression of HMGB1 and RAGE, BBB leakage, as well as decreased functional outcome after stroke compared with control rats. Injection of bone marrow stromal cells (BMSCs) into type 2 diabetic rats significantly reduced the expression of HMGB1 and RAGE, attenuated BBB leakage, and improved functional outcome after stroke. BMSCs-treated type 2 diabetic rats inhibited inflammation and improved functional outcome after stroke. Furthermore, in vitro data support the hypothesis that BMSCs-induced reduction of HMGB1 and RAGE in T2DM-MCAo rats contributed to attenuated inflammatory response in the ischemic brain, which may lead to the beneficial effects of BMSCs treatment. Further investigation of BMSCs treatment in type 2 diabetic stroke is warranted.

The protective role of (-)-epigallocatechin-3-gallate in thrombin-induced neuronal cell apoptosis and JNK-MAPK activation.

(−)-Epigallocatechin-3-gallate (EGCG), the major polyphenolic component of green tea, has anti-inflammatory and antioxidant properties and provides neuroprotection against central nervous system diseases. Yet, it is not known whether EGCG may be neuroprotective against intracerebral hemorrhage. In this study, we used a simplified in-vitro model of thrombin neurotoxicity to test whether EGCG provides neuroprotection against thrombin-associated toxicity. Exposure of primary cortical neurons to thrombin (100 U/ml) caused dose-dependent and time-dependent cytotoxicity. Cell Counting Kit 8 and lactate dehydrogenase were used to monitor cell viability after exposure of neurons to thrombin or EGCG and after EGCG pretreatment. Flow cytometric analysis and western blotting demonstrated that thrombin-induced neuron degeneration occurs through apoptosis. A concentration of 25 &mgr;M EGCG significantly abolished thrombin-induced toxicity and prevented apoptosis by suppressing c-Jun-N-terminal kinase (JNK) phosphorylation, and the JNK inhibitor SP600125 reduced thrombin-induced caspase 3 activation and apoptosis. These data suggest that EGCG may have protective effects against thrombin-induced neuroapoptosis by inhibiting the activation of JNK, leading to caspase 3 cleavage. EGCG is a novel candidate neuroprotective agent against intracerebral hemorrhage-induced neurotoxicity.

Nrf2/ARE pathway inhibits ROS-induced NLRP3 inflammasome activation in BV2 cells after cerebral ischemia reperfusion.

ObjectiveCurrent therapies for ischemia/reperfusion are insufficient because of our poor understanding of the mechanisms of brain injury after ischemic stroke. As a vital component of the innate immune system, NLRP3 inflammasome contributes to ischemic brain injury; however, a detailed understanding of their molecular mechanisms is unknown. This study was designed to investigate the effect of nuclear factor E2-related factor-2 (Nrf2) on NLRP3 inflammasome.Materials and methodsBV2 microglial cells were pretreated with tert-butylhydroquinone or Nrf2 CRISPR plasmid before oxygen–glucose deprivation/reoxygenation (OGDR) exposure. Then we observed the effect of Nrf2 on NLRP3 inflammasome.ResultsWe identified that Nrf2 activation inhibited NLRP3 inflammasome expression and subsequent IL-1β generation. Furthermore, the activation of NLRP3 inflammasome was sensitive to the reactive oxygen species (ROS) level and Nrf2 could decrease the production of ROS. Additionally, as a Nrf2-targeted ARE gene, NADPH quinone oxidoreductase 1 was involved in the inhibition of the NLRP3 inflammasome.ConclusionWe elucidated an inhibitory regulation of Nrf2/ARE pathway on ROS-induced NLRP3 inflammasome activation in BV2 microglial cells after OGDR exposure.

Therapy Effects of Bone Marrow Stromal Cells on Ischemic Stroke.

Stroke is the second most common cause of death and major cause of disability worldwide. Recently, bone marrow stromal cells (BMSCs) have been shown to improve functional outcome after stroke. In this review, we will focus on the protective effects of BMSCs on ischemic brain and the relative molecular mechanisms underlying the protective effects of BMSCs on stroke.

Oxygen-glucose deprivation of neurons transfected with toll-like receptor 3-siRNA: Determination of an optimal transfection sequence.

Toll-like receptor 3 protein expression has been shown to be upregulated during cerebral ischemia/reperfusion injury in rats. In this study, rat primary cortical neurons were subjected to oxygen-glucose deprivation to simulate cerebral ischemia/reperfusion injury. Chemically synthesized small interfering RNA (siRNA)-1280, -1724 and -418 specific to toll-like receptor 3 were transfected into oxygen-glucose deprived cortical neurons to suppress the upregulation of toll-like receptor 3 protein expression. Western blotting demonstrated that after transfection with siRNA, toll-like receptor 3 protein expression reduced, especially in the toll-like receptor 3-1724 group. These results suggested that siRNA-1724 is an optimal sequence for inhibiting toll-like receptor 3 expression in cortical neurons following oxygen-glucose deprivation.

Dual effects of heme oxygenase-1 on astrocyte injury induced by hemin in vitro

Abstract Primary objective: The purpose of this study was to investigate the effects of heme oxygenase-1 (HO-1) on astrocyte injury induced by hemin. Research design: Primary astrocytes were isolated from Sprague Dawley rat pups and cultured in vitro. The expression of HO-1 was induced by hemin in a quantitative fashion and the effects of HO-1 on hemin-induced astrocyte injury were estimated by cell viability, cell membrane permeability and apoptosis. Methods and procedures: Astrocytes were divided into control group, hemin 5 μM group, hemin 5 μM + Zn-PPIX group, hemin 30 μM group and hemin 30 μM + Zn-PPIX group. Survival quality of astrocyte was measured by WST-8 assay, LDH assay, Hoechst 33258 Staining and annexin V-FITC/PI assay and apoptotic-related proteins were measured using Western blotting. Main outcome and results: Hemin could dose-dependently up-regulate the expression of HO-1. HO-1 exerted a protective role on astrocyte damage induced by 5 μM hemin, including increased cell survival rate and anti-apoptotic proteins expression (Bcl-2 and p-AKT), as well as decreased LDH release, apoptosis ratio and apoptotic protein expression (Bax, p-ERK and cleaved-caspase3). However, the effect of HO-1 on astrocyte injury between 30 μM hemin-treated groups was opposite of the protective role in 5 μM hemin-treated groups. Conclusions: There were dual effects of HO-1 in 5 μM and 30 μM hemin-induced astrocyte injuries.

Neuroprotective Effects of 17β-Estradiol against Thrombin-Induced Apoptosis in Primary Cultured Cortical Neurons

Aims: 17β-estradiol (E2) is a powerful neuroprotective agent in the central nervous system; however, little is known about its effects on intracerebral hemorrhage. This study examined the effects of E2 on thrombin-induced apoptosis in vitro and investigated the potential mechanisms. Methods: Primary cultured cortical neurons were treated with E2 or vehicle and then the cells were exposed to thrombin. Neuronal apoptosis was assessed by flow cytometry. The phosphorylated c-Jun-N-terminal kinase (p-JNK), phosphorylated extracellular signal-regulated kinases 1/2 (p-ERK1/2), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and caspase-3 were assayed by western blot. Results: Consequently, we found that E2 has significantly reduced the apoptosis in thrombin-treated neurons. E2 also exhibited a downregulation in the ratio of Bax/Bcl-2, caspase-3 and p-JNK. However, E2 had little effect on p-ERK1/2 proteins activation. Conclusion: Taken together, E2 has shown neuroprotective effects on thrombin-induced neuronal apoptosis, and the molecular mechanisms may correlate with the inhibition of the JNK signaling pathway.