Salvianolic acid A inhibits ferroptosis and protects against intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a common cerebral vascular disease with high incidence, disability, and mortality. Ferroptosis is a regulated type of iron-dependent, non-apoptotic programmed cell death. There is increasing evidence that ferroptosis may lead to neuronal damage mediated by hemorrhagic stroke mediated neuronal damage. Salvianolic acid A (SAA) is a natural bioactive polyphenol compound extracted from salvia miltiorrhiza, which has anti-inflammatory, antioxidant, and antifibrosis activities. SAA is reported to be an iron chelator that inhibits lipid peroxidation and provides neuroprotective effects. However, whether SAA improves neuronal ferroptosis mediated by hemorrhagic stroke remains unclear. The study aims to evaluate the therapeutic effect of SAA on Ferroptosis mediated by Intracerebral hemorrhage and explore its potential mechanisms. We constructed in vivo and in vitro models of intracerebral hemorrhage in rats. Multiple methods were used to analyze the inhibitory effect of SAA on ferroptosis in both in vivo and in vitro models of intracerebral hemorrhage in rats. Then, network pharmacology is used to identify potential targets and mechanisms for SAA treatment of ICH. The SAA target ICH network combines SAA and ICH targets with protein-protein interactions (PPIs). Find the specific mechanism of SAA acting on ferroptosis through molecular docking and functional enrichment analysis. In rats, SAA (10 mg/kg in vivo and 50 µM in vitro, p < 0.05) alleviated dyskinesia and brain injury in the ICH model by inhibiting ferroptosis (p < 0.05). The molecular docking results and functional enrichment analyses suggested that AKT (V-akt murine thymoma viral oncogene homolog) could mediate the effect of SAA. NRF2 (Nuclear factor erythroid 2-related factor 2) was a potential target of SAA. Our further experiments showed that salvianolic acid A enhanced the Akt /GSK-3ß/Nrf2 signaling pathway activation in vivo and in vitro. At the same time, SAA significantly expanded the expression of GPX4, XCT proteins, and the nuclear expression of Nrf2, while the AKT inhibitor SH-6 and the Nrf2 inhibitor ML385 could reduce them to some extent. Therefore, SAA effectively ameliorated ICH-mediated neuronal ferroptosis. Meanwhile, one of the critical mechanisms of SAA inhibiting ferroptosis was activating the Akt/GSK-3ß/Nrf2 signaling pathway.

Exosomes derived from human umbilical cord mesenchymal stem cells decrease neuroinflammation and facilitate the restoration of nerve function in rats suffering from intracerebral hemorrhage.

Exosomes derived from human umbilical cord mesenchymal stem cells (hUCMSC-ex) have become a hopeful substitute for whole-cell therapy due to their minimal immunogenicity and tumorigenicity. The present study aimed to investigate the hypothesis that hUCMSC-ex can alleviate excessive inflammation resulting from intracerebral hemorrhage (ICH) and facilitate the rehabilitation of the nervous system in rats. In vivo, hemorrhagic stroke was induced by injecting collagenase IV into the striatum of rats using stereotactic techniques. hUCMSC-ex were injected via the tail vein at 6 h after ICH model establishment at a dosage of 200 µg. In vitro, astrocytes were pretreated with hUCMSC-ex and then stimulated with hemin (20 µmol/mL) to establish an ICH cell model. The expression of TLR4/NF-κB signaling pathway proteins and inflammatory factors, including TNF-α, IL-1ß, and IL-10, was assessed both in vivo and in vitro to investigate the impact of hUCMSC-ex on inflammation. The neurological function of the ICH rats was evaluated using the corner turn test, forelimb placement test, Longa score, and Bederson score on the 1st, 3rd, and 5th day. Additionally, RT-PCR was employed to examine the mRNA expression of TLR4 following hUCMSC-ex treatment. The findings demonstrated that hUCMSC-ex downregulated the protein expression of TLR4, NF-κB/P65, and p-P65, reduced the levels of pro-inflammatory cytokines TNF-α and IL-1ß, and increased the expression of the anti-inflammatory cytokine IL-10. Ultimately, the administration of hUCMSC-ex improved the behavioral performance of the ICH rats. However, the results of PT-PCR indicated that hUCMSC-ex did not affect the expression of TLR4 mRNA induced by ICH, suggesting that hUCMSCs-ex may inhibit TLR4 translation rather than transcription, thereby suppressing the TLR4/NF-κB signaling pathway. We can conclude that hUCMSC-ex mitigates hyperinflammation following ICH by inhibiting the TLR4/NF-κB signaling pathway. This study provides preclinical evidence for the potential future application of hUCMSC-ex in the treatment of cerebral injury.

Exosomes derived from human umbilical cord mesenchymal stem cells pretreated by monosialoteterahexosyl ganglioside alleviate intracerebral hemorrhage by down-regulating autophagy.

PURPOSE: Intracerebral hemorrhage (ICH) results in substantial morbidity, mortality, and disability. Depleting neural cells in advanced stages of ICH poses a significant challenge to recovery. The objective of our research is to investigate the potential advantages and underlying mechanism of exosomes obtained from human umbilical cord mesenchymal stem cells (hUMSCs) pretreated with monosialoteterahexosyl ganglioside (GM1) in the prevention of secondary brain injury (SBI) resulting from ICH. PATIENTS AND METHODS: In vitro, hUMSCs were cultured and induced to differentiate into neuron-like cells after they were pretreated with 150 µg/mL GM1. The exosomes extracted from the culture medium following a 6-h pretreatment with 150 µg/mL GM1 were used as the treatment group. Striatal infusion of collagenase and hemoglobin (Hemin) was used to establish in vivo and in vitro models of ICH. RESULTS: After being exposed to 150 µg/mL GM1 for 6 h, specific cells displayed typical neuron-like cell morphology and expressed neuron-specific enolase (NSE). The rate of differentiation into neuron-like cells was up to (15.9 ± 5.8) %, and the synthesis of N-Acetylgalactosaminyltransferase (GalNAcT), which is upstream of GM1, was detected by Western blot. This study presented an increase in the synthesis of GalNAcT. Compared with the ICH group, apoptosis in the treatment group was remarkably reduced, as detected by TUNEL, and mitochondrial membrane potential was restored by JC-1. Additionally, Western blot revealed the restoration of up-regulated autophagy markers Beclin-1 and LC3 and the down-regulation of autophagy marker p62 after ICH. CONCLUSION: These findings suggest that GM1 is an effective agent to induce the differentiation of hUMSCs into neuron-like cells. GM1 can potentially increase GalNAcT production through "positive feedback", which generates more GM1 and promotes the differentiation of hUMSCs. After pretreatment with GM1, exosomes derived from hUMSCs (hUMSCs-Exos) demonstrate a neuroprotective effect by inhibiting autophagy in the ICH model. This study reveals the potential mechanism by which GM1 induces differentiation of hUMSCs into neuron-like cells and confirms the therapeutic effect of hUMSCs-Exos pretreated by GM1 (GM1-Exos) on an ICH model, potentially offering a new direction for stem cell therapy in ICH.

miR-122-5p Promotes Peripheral and Central Nervous System Inflammation in a Mouse Model of Intracerebral Hemorrhage via Disruption of the MLLT1/PI3K/AKT Signaling.

Intracerebral hemorrhage (ICH) is a recognized central nervous system inflammation complication. Several microRNAs (miRNAs or miRs) have been documented to be vital modulators in peripheral and central nervous system inflammation. Based on whole transcriptome sequencing and bioinformatics analysis, this study aims to reveal the possible molecular mechanisms by which miR-122-5p affects the inflammatory response in the peripheral and central nervous system in a mouse model of ICH. Differentially expressed ICH-related miRNAs were screened. Adeno-associated viral vectors were used to knock down miR-122-5p in mice to evaluate the effect of miR-122-5p on peripheral and central nervous system inflammation. The downstream target gene of miR-122-5p was analyzed. Neurons were isolated from mice and treated with hemin to construct an in vitro model of ICH, followed by transduction with miR-122-5p mimic or combined with oe-MLLT1. The neurons were then co-cultured with microglia BV2 to assess their activation. It was found that miR-122-5p was highly expressed in ICH, and MLLT1 was lowly expressed. In vivo experiments showed that miR-122-5p knockdown decreased neurological deficits, BBB permeability, and inflammation in the peripheral and central nervous system in ICH mice. It involved its binding to MLLT1 and downregulation of the activity of the PI3K/AKT pathway. In vitro data exhibited that miR-122-5p stimulated the generation of inflammatory factors and microglia activation by targeting MLLT1 and inhibiting the PI3K/AKT pathway. Collectively, our work reveals a novel miR-122-5p/MLLT1-mediated regulatory network in ICH that may be a viable target for neuroinflammation alleviation.

Casein kinase 2 attenuates brain injury induced by intracerebral hemorrhage via regulation of NR2B phosphorylation.

Objective: Intracerebral hemorrhage (ICH) is a common cerebrovascular disease with high incidence, disability, and mortality. Casein kinase 2 (CK2) is a serine/threonine kinase with hundreds of identified substrates and plays an important role in many diseases. This study aimed to explore whether CK2 plays protective roles in ICH-induced neuronal apoptosis, inflammation, and oxidative stress through regulation NR2B phosphorylation. Methods: CK2 expression level of brain tissues taken from ICH patients was determined by immunoblotting. Neurons from embryonic rat and astrocytes from newborn rats were cultured and treated by Hemoglobin chloride (Hemin). The proliferation of astrocytes, the apoptosis and oxidative stress of neurons and the inflammatory factors of astrocytes were detected. CK2 expression was determined in ICH model rats. The effects of CK2 overexpression plasmid (pc-CK2) on neurobehavioral defects and brain water content in ICH rats were observed. Results: CK2 expression in ICH patients was down-regulated. Overexpression of CK2 promoted the astrocyte proliferation, inhibited neuronal apoptosis, and reduced astrocyte-mediated inflammation. N-methyl-D-aspartate receptor 2B (NR2B) reversed the effects of pc-CK2 on neurons and astrocytes. CK2 phosphorylated NR2B at the S1480 site, down-regulated the expression of NR2B and interfered with the interaction between NR2B and postsynaptic density protein 95 (PSD95). In vivo experiments showed that the expression of CK2 decreased and the expression of NR2B increased in ICH rats. Furthermore, pc-CK2 attenuated neurobehavioral defects, brain water content and neuronal damage in ICH rats. Conclusion: CK2 phosphorylated NR2B, down-regulated the expression of NR2B, interfered with the interaction between NR2B and PSD95, alleviated inflammatory reactions, inhibited neuronal apoptosis and oxidative stress after ICH. CK2 and NR2B may be new potential therapeutic targets for the treatment of ICH. However, the limitation of this study is that we only investigated the regulation of NR2B by CK2.

Gabapentin Alleviates Brain Injury in Intracerebral Hemorrhage Through Suppressing Neuroinflammation and Apoptosis.

Neuroinflammation plays an important role in brain tissue injury during intracerebral hemorrhage. Gabapentin can reduce inflammation and oxidative stress through inhibiting nuclear factor κB (NFκB) signals. Here, we showed that gabapentin reduced brain tissue injury in ICH through suppressing NFκB-mediated neuroinflammation. ICH was induced by injecting collagenase IV into the right striatum of Sprague-Dawley rats. PC12 and BV2 cells injury induced by Hemin were used to simulate ICH in vitro. Inflammation and apoptosis were assessed in rat brain tissue and in vitro cells. The neurobehavioral scores were significantly decreased in ICH rats compared with sham rats. Phosphorylated IκB-α and cleaved caspase3, and apoptosis rate were significantly higher in tissue surrounding the hematoma than in brain tissues from rats subjected to sham surgery. Furthermore, serum IL-6 levels in ICH rats were higher than in sham rats. Gabapentin treatment significantly improved the behavioral scores, decreased levels of phosphorylated IκB-α and cleaved caspase3, apoptosis rate, and serum IL-6 level in ICH rats. Hemin-treated BV2 cells displayed higher levels of phosphorylated IκB-α, cleaved caspase3, and IL-6 in the supernatant compared with vehicle-treated cells. Hemin treatment induced a significantly lower level of peroxisome proliferator-activated receptor γ (PPARγ) in BV2 cells. BV2-PC12 co-culture cells treated by hemin displayed higher levels of cleaved caspase3 in PC12 cells. Furthermore, gabapentin treatment could reduce these effects induced by hemin and the protective effects of gabapentin were significantly attenuated by PPARγ inhibitor. Therefore, gabapentin may reduce inflammation and apoptosis induced by the ICH through PPARγ-NFκB pathway.

Intracerebral hemorrhage alters α2δ1 and thrombospondin expression in rats.

Calcium voltage-gated channel auxiliary subunit (α2δ1) is a non-essential subunit of the voltage-gated calcium channel complex and is ubiquitously expressed in a number of tissues, including the brain. Thrombospondin (TSP)1 and TSP2 are extracellular matrix proteins and belong to the multi-domain glycoprotein family of macromolecular oligomers. TSP1/2 and α2δ1 promote synapse formation and functional recovery following cerebral infarction. However, to the best of our knowledge, the expression levels of α2δ1 and TSP1/2 in brain tissues, and the effects of intracerebral hemorrhage (ICH) on these levels have not yet been elucidated. The present study established a rat model of hemorrhage induced by injecting collagenase IV into the striatum to determine the changes in α2δ1 and TSP1/2 expression following ICH. The protein expression levels of α2δ1 and TSP1 in the striatum after hemorrhage were significantly increased on day 5 and returned to baseline levels on day 21; however, the protein expression levels of TSP2 were decreased on day 5, whereas they increased on day 14, subsequently returning to baseline levels. In addition, using proteomics analysis of tissues from the sham group (saline injection) and at 24 h post-ICH, it was found that both α2δ1 and TSP1 interacted with neural EGFL like 2. Taken together, these findings suggested that the expression levels of α2δ1 and TSP1/2 were altered in brain tissues in response to ICH.

Association of Single-Nucleotide Polymorphisms of Gab1 Gene with Susceptibility to Meningioma in a Northern Chinese Han Population.

BACKGROUND The Gab1 gene has an important role in cell proliferation in meningioma via various signaling pathways. However, the relationship between polymorphisms of the Gab1 gene and meningioma remains unknown. In this study, we aimed to investigate the plausible association of single-nucleotide polymorphisms (SNPs) of the Gab1 gene and meningioma risk in a northern Chinese Han population. MATERIAL AND METHODS This case-control study included 205 patients with meningioma and 297 healthy controls. Four loci of the Gab1 gene were genotyped using the multiplex snapshot technique. The odds ratio (OR) and 95% confidence interval (CI) were calculated by chi-squared and logistic regression analysis. The distributions of Gab1 SNP genotypes and allele frequencies were compared between patients with meningioma and healthy controls and among patients stratified by clinical phenotypes. RESULTS The allelic frequency distributions of G at rs3805236 and C at rs1397529 were significantly higher in patients with meningioma than in healthy controls. The frequency of the rs3805236-GG and rs1397529-AC genotypes were significantly higher in patients with meningioma than in controls. Furthermore, there was a statistically significant difference between the genotypes of patients versus healthy individuals at rs1397529, according to stratification by dural invasion. The allelic frequency distributions of alleles or genotypes at rs3805246 and rs3828512 were not different in patients with meningioma and healthy controls. CONCLUSIONS The Gab1 gene rs3805236A>G and rs1397529A>C SNPs increased the risk of meningioma in the northern Chinese Han population. Furthermore, rs1397529A>C may be related to enhanced dural invasion in patients with meningioma.

Outcomes of microsurgical clipping vs coil embolization for ruptured aneurysmal subarachnoid hemorrhage: A multicenter real-world analysis of 583 patients in China.

Aneurysmal subarachnoid hemorrhage (SAH) is a complex neurovascular syndrome with high disability and mortality. SAH patients may be managed with surgical clipping or coil embolization. In this study, we provided a real-world analysis of the outcome and prognostic factors of aneurysmal SAH in patients treated with coil embolization or microsurgical clipping.We retrospectively analyzed the medical records of aneurysmal SAH patients (n = 583) who underwent treatment at the First Hospital and the Second Hospital of Hebei Medical University, and Tangshan Worker's Hospital in China. All patients were evaluated by a combined neurosurgery and interventional neuroradiology team. Microsurgical aneurysmal clipping was performed using the skull base approach, while coil embolization was performed with bare platinum coils (with or without balloon assistance). The primary outcome was the Glasgow Outcome Scale (GOS) score at discharge.A total of 583 patients were included in this study, of which 397 (68.1%) of them underwent clipping and 186 (31.9%) received coil embolization. The patient cohort consisted of both poor grade and good grade aneurysmal SAH: 441 (75.6%) patients had good-grade (Hunt and Hess grade II or III) and 142 (24.4%) had poor grade (Hunt and Hess grade IV or V). Overall, 123 (21%) patients had unfavorable neurologic outcome (GOS score 1-3) and 460 (78.9%) patients had favorable neurologic outcome (GOS score 4 or 5). The mean GOS score at discharge was comparable for patients who underwent clipping and those received coil embolization (P > .05). Multivariate analysis showed that clipping only [OR (95%CI): 0.03 (0.01, 0.36); P = .000] and clipping with CSF drainage [OR (95%CI): 0.41 (0.18, 0.89); P = .001] were independent factors of a favorable outcome in patients with aneurysmal SAH. Coil embolization with hematoma removal [OR (95%CI): 0.03 (0.01, 0.36); P = .000] was also an independent determinant of a favorable outcome. High baseline Fisher grades were associated with significantly increased risk of an unfavorable outcome [OR (95%CI): 2.08 (1.30, 3.33); P = .002].Our findings suggested that both coil embolization and microsurgical clipping are viable treatment options for aneurysmal SAH patients. Procedures, such as CSF drainage and hematoma removal, performed in parallel with coil embolization and chipping should be considered when treating individual patients.

Synaptic Plasticity of Human Umbilical Cord Mesenchymal Stem Cell Differentiating into Neuron-like Cells In Vitro Induced by Edaravone.

OBJECTIVE: The human umbilical cord mesenchymal stem cells (hUMSCs) are characterized with the potential ability to differentiate to several types of cells. Edaravone has been demonstrated to prevent the hUMSCs from the oxidative damage, especially its ability in antioxidative stress. We hypothesized that Edaravone induces the hUMSCs into the neuron-like cells. METHODS: The hUMSCs were obtained from the human umbilical cord tissue. The differentiation of hUMSCs was induced by Edaravone with three different doses: 0.65 mg/ml, 1.31 mg/ml, and 2.62 mg/ml. Flow cytometry was used to detect the cell markers. Protein and mRNA levels of nestin, neuron-specific enolase (NSE), and glial fibrillary acidic protein (GFAP) were detected by Western blot and RT-PCR. The expression of synaptophysin (SYN), growth-associated protein 43 (GAP43), and postsynaptic density 95 (PSD95) was detected by Real-Time PCR. RESULTS: As long as the prolongation of the culture, the hUMSCs displayed with the long strips or long fusiform to fat and then characterized with the radial helix growth. By using flow cytometry, the cultured hUMSCs at the 3rd, 5th, and 10th passages were expressed with CD73, CD90, and CD105 but not CD11b, CD19, CD34, CD45, and HLA-DR. Most of the hUMSCs cultured with Edaravone exhibited typical nerve-immediately characters including the cell body contraction, increased refraction, and protruding one or more elongated protrusions, which were not found in the control group without addition of Edaravone. NSE, nestin, and GFAP were positive in these neuron-like cells. Edaravone dose-dependently increased expression levels of NSE, nestin, and GFAP. After replacement of maintenance fluid, neuron-like cells continued to be cultured for five days. These neuron-like cells were positive for SYN, PSD95, and GAP43. CONCLUSION: Edaravone can dose-dependently induce hUMSCs to differentiate into neuron-like cells that expressed the neuronal markers including NSE, nestin, and GFAP and synaptic makers such as SYN, PSD95, and GAP43.

Minimally effective concentration of zoledronic acid to suppress osteoclasts in vitro.

Zoledronic acid is regarded as the most potent bisphosphonate and is widely used in patients with osteoporosis; however, its side effects, including acute-phase reactions, gastrointestinal complaints, renal dysfunction and bisphosphonate-associated osteonecrosis impair the safety and quality of life of patients. The present study was designed to determine the minimal effective concentration of zoledronic acid through testing the dose-dependent effects of zoledronic acid on osteoclast suppression. A primary culture of bone marrow mononuclear cells obtained from C57 mice (age, 6 weeks) was established and induced to form osteoclasts. The number of multinuclear cells was determined by tartrate-resistant acid phosphatase staining and compared among cultured marrow cells treated with different concentrations of zoledronic acid. Furthermore, the cellular properties, including adhesion, migration and bone resorption, were compared at the minimal effective concentration. At a concentration of 1×10-6 mol/l, zoledronic acid significantly inhibited the formation of osteoclasts. This inhibitory effect was further enhanced at the concentration of 1×10-5 mol/l. However, the inhibitory effect of zoledronic acid tapered at the concentration of 1×10-4 mol/l and there was no further dose-dependent increase. In addition, the concentration of 1×10-6 mol/l was sufficient to alter cellular functions, including cell adhesion, migration and bone resorption. In conclusion, zoledronic acid was effective in reducing osteoclast formation and suppressing cellular functions. The minimal effective concentration of zoledronic acid in vitro was 1 µmol/l. Based on these results, a comparable dosage should be explored in clinical applications.

CREB3L1 and PTN expressions correlate with prognosis of brain glioma patients.

The present study was conducted to investigate the clinical significance of cAMP responsive element binding protein 3 like 1 (CREB3L1) and pleiotrophin (PTN) expression in prognosis of patients with brain gliomas. Human brain tissue samples were collected from normal glial tissues (control), low- and high-grade glioma tissues. CREB3L1 and PTN expression levels in cells were assessed by immunohistochemistry (IHC), and population distribution of the CREB3L1- and PTN-presenting patients was examined. The CREB3L1 and PTN mRNA expression levels in three types of the brain cells was determined by RT-PCR. Survival rates for population of the CREB3L1- and PTN-presenting patients were examined. CREB3L1+ cell counts were decreased with increased PTN+ cells in the low-grade and high-grade glioma tissues as compared with the control. Population proportion of the CREB3L1+-presenting patients decreased from the control to the high-grade glioma and the population of the PTN+-presenting patients increased in low- and high-grade gliomas as compared with the control (both P<0.05). The decrease in the CREB3L1 mRNA expression was associated with the increase in the PTN mRNA expression in the low- and high-grade gliomas (P<0.05). Survival time for patients with CREB3L1- and PTN+ gliomas was shorter than patients with CREB3L1+ and PTN- gliomas in the investigated cohorts (both P<0.05). There was a relationship between the expression levels of both proteins and survival time. CREB3L1 and PTN expression levels serve as biomarkers with utility in grading gliomas. Absence of CREB3L1 and presence of PTN in brain glioma cells correlate with survival time of the glioma patients.

MicroRNA-182 targets protein phosphatase 1 regulatory inhibitor subunit 1C in glioblastoma.

Glioblastoma (GBM) is an incurable cancer, with mean post-diagnosis survival time of 14-16 months. Metagenomic analysis by The Cancer Genome Atlas (TCGA) program has identified microRNA-182-5p (miR-182-5p or miR-182) as the only miRNA associated with favorable disease prognosis and temozolomide (TMZ) susceptibility. Previous reports have indicated that miR-182 down regulates expression of BCL2L12, c-MET, and HIF2A. However, other messenger RNA (mRNA) targets of miR-182 have not been validated which would explain its association with a favorable disease prognosis. In situ analysis revealed that protein phosphatase 1 regulatory inhibitor subunit 1C (PPP1R1C) is a putative target of miR-182. PPP1R1C protein and RNA expression as assessed by tissue microarray and quantitative real time PCR, respectively, was inversely correlated to miR-182 expression in glioblastoma patients and in the metastatic glioblastoma cell line U87-MG. Reporter assays using PPP1R1C 3' untranslated region (UTR) showed that miR-182 can interact with the wild-type but not a miR-182-5-seed mutant. Ectopic expression of miR-182 mimic in the U87-MG cell line significantly decreased proliferation as well as suppressed in vitro migration and invasion. Opposite observations were made when the non-malignant neuronal cell line HCN-2 was transfected with anti-miR-182 antagomir. The miR-182 mimic or siRNA targeting PPP1R1C induced TMZ susceptibility indicating that decreased susceptibility to TMZ in GBM patients might be attributed to high expression of PPP1R1C. Inverse correlation of PPP1R1C mRNA and miR-182 levels in 20 GBM patients confirmed the same. Cumulatively, our results indicate that loss of miR-182 leads to increased expression of PPP1R1C which in part explain disease progression and resistance to TMZ therapy.

Tetramethylpyrazine induces differentiation of human umbilical cord-derived mesenchymal stem cells into neuron-like cells in vitro.

The present study evaluated the ability and optimal concentration of tetramethylpyrazine (TMP) to induce human umbilical cord-derived mesenchymal stem cells (hUMSCs) to differentiate into neuron­like cells in vitro. Human umbilical cords from full-term caesarean section patients were used to obtain hUMSCs by collagenase digestion after removal of the umbilical artery and vein. The surface antigen expression profile of cultured hUMSCs was monitored by flow cytometry. After amplification, cells of the 5th passage were divided into experimental groups A­C treated with TMP at 4.67, 2.34 and 1.17 mg/ml, respectively, in low glucose­Dulbecco's Modified Eagle's Medium (L­DMEM) (induction medium), while group D (control) was exposed to L­DMEM culture medium only. Differentiation of hUMSCs into neuron­like cells and morphological changes were observed every 0.5 h with an inverted phase contrast microscope for 6 h. After the 6­h induction period, proportions of cells expressing neuronal markers neuron­specific enolase (NSE), neurofilament protein (NF­H) and glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry. The optimal concentration of TMP was selected on the basis of neuron­like cell positive rate. Western blotting and RT­polymerase chain reaction were applied to detect the expression of NSE, NF­H, and GFAP of the group of optimal concentration in each point­in­time. Results showed that most primary cells were adherent 12 h after seeding and first appeared as diamond or polygon shapes. Thereafter, they gradually grew into long spindle­shaped cells and finally in a radiating or swirling pattern. The cells maintained a strong proliferative capacity after continuous passage. Flow cytometry analysis of cultured hUMSCs at the 3rd, 5th and 10th passages expressed CD73, CD90 and CD105, but not CD11b, CD19, CD34, CD45 or human leukocyte antigen­DR. After 6 h of TMP treatment, typical neuron­like cells with many protrusions connected into a net­like pattern were observed in all experimental groups. These neuron­like cells were positive for NSE and NF­H, but negative for GFAP. Among the tested treatment groups, group A with TMP at 4.67 mg/ml had the highest expression of NSE and NF­H. By contrast, no change was found after induction in the control group. The mRNA expression of cells expressing neuronal markers as well as GAPDH was observed, with the relative NSE transcript levels of 0, 1.303±0.031, 1.558±0.025, 1.927±0.019 and 2.415±0.033 after 0, 1, 2, 4 and 6 h of treatment, respectively; the mRNA expression of NH­F was 0, 1.429±0.025, 1.551±0.024, 1.930±0.042 and 1.398±0.014 after 0, 1, 2, 4 and 6 h of treatment, respectively. There was no expression of GFAP before or after induction and all the groups showed high expression of GAPDH at each time point. Protein expression was also observed on cells expressing neuronal markers as well as GAPDH at each time point. The protein expression of NSE was 0, 0.717±0.097, 0.919±0.056, 1.097±0.143 and 1.157±0.055 in proper order; the protein expression of NH­F was 0, 0.780±0.103, 0.973±0.150, 1.053±0.107 and 0.753±0.094 in proper order. There was no expression of GFAP before or after induction, and all the groups showed high expression of GAPDH at each tested time point. Our results demonstrated that TMP can induce hUMSCs to differentiate into neuron­like cells effectively with the optimal concentration of 4.67 mg/ml. After induction, the NSE and NF-H of the neuron-like cells were positive, but the GFAP-2 was negative.

Monosialoteterahexosyl ganglioside induces the differentiation of human umbilical cord-derived mesenchymal stem cells into neuron-like cells.

In the present study, human umbilical cord-derived mesenchymal stem cells (hUMSCs) were investigated for their potential to be induced to differentiate in vitro into neuron-like cells by monosialoteterahexosyl ganglioside (GM1). Mononuclear cells obtained from umbilical cords from women with full-term pregnancies whose babies were delivered by cesarean section were cultivated in vitro and their surface antigen expression profiles were monitored. Following amplification, the cells were divided into 5 groups, of which 4 (groups A-D) were treated with GM1 at doses of 50, 100, 150 and 200 µg/ml, respectively. The control (group E) was treated with the vehicle only. The ability of GM1 to induce the differentiation of the hUMSCs into neuron-like cells was monitored for 6 h. The expression levels of microtubule-associated protein-2 (MAP-2), neurofilament protein (NF-H) and glial fibrillary acidic protein (GFAP) were measured by immunohistochemistry. Following exposure to GM1, the hUMSCs first appeared to have a diamond or polygonal shape and gradually grew into long spindle-shaped cells, finally exhibiting a radiating or swirling pattern. The cells maintained a strong proliferative capacity after continuous passage. Flow cytometry revealed that the hUMSCs expressed CD73, CD90 and CD105 up to passage 10, but not CD11b, CD19, CD34, CD45 or HLA-DR. Treatment with GM1 for 6 h led to the appearance of neuron-like cells with oval-shaped cell bodies and protruding neurites. These neuron-like cells were positive for MAP-2 and NF-H, but negative for GFAP expression. No changes in the expression of these markers were observed in the control group. Thus, the findings of the present study demonstrate that GM1 effectively induces hUMSCs to differentiate into neuron-like cells.