NBP Cytoprotective Effects Promoting Neuronal Differentiation in BMSCs by Inhibiting the p65/Hes1 Pathway.

Background: Bone marrow-derived mesenchymal stem cell (BMSC) transplantation has become an effective method for treating neurodegenerative diseases. Objectives: This study investigated the effect of 3-N-butylphthalide (NBP) on the neuronal differentiation of BMSCs and its potential mechanism. Methods: In this study, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to detect cell proliferation and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining was conducted to detect the apoptosis of BMSCs. Quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analysis were performed to detect the messenger RNA (mRNA) and protein expression levels, respectively. An enzyme-linked immunosorbent serologic assay assessed the levels of interleukin-1ß, tumor necrosis factor-α, and cyclic adenosine monophosphate (cAMP). Moreover, a flow cytometry assay was used to detect the proportion of active ß-tubulin III (TUJ-1) cells, and TUJ-1 expression was observed by immunofluorescence assay. Results: The results showed that a low concentration of NBP promoted the proliferation and induction of BMSC neuronal differentiation while inhibiting apoptosis, the production of inflammatory factors, and p65 expression. Compared with differentiation induction alone, combined NBP treatment increased the levels of nestin, neuron-specific enolase (NSE), TUJ-1, and microtubule-associated protein 2 (MAP2) protein, as well as the ratio of TUJ-1-positive cells and cAMP expression. Furthermore, p65 overexpression weakened the effect of NBP, and the overexpression of hairy and enhancer of split homolog-1 (HES1) reversed the effect of NBP in the induction of BMSC neuronal differentiation in vitro. Conclusions: We confirmed that NBP exhibited potential therapeutic properties in the stem cell transplantation treatment of neurodegenerative diseases by protecting cells and promoting BMSC neuronal differentiation by inhibiting the p65/HES 1 pathway.

MicroRNA-124a regulates the differentiation of bone marrow mesenchymal stem cells into neurons.

OBJECTIVE: This study investigated the effects of microRNA-124a on the differentiation of bone marrow mesenchymal stem cells (BMSCs) and its underlying mechanism. METHODS: Flow cytometry was used for isolation and identification of BMSCs. Real-time polymerase chain reaction (RT-PCR) was used to detect gene mRNA expression. Apoptosis was detected using Annexin V-FITC/PI Apoptosis Detection Kit. Cell proliferation ability was tested using Cell Counting Kit-8 (CCK-8). The differentiation of BMSCs into neuron inducers ß-thiol ethanol or baicalin formed the basis of the study. RESULTS: ß-thiol ethanol markedly suppressed the microRNA-124a expression of BMSCs, baicalin markedly induced the microRNA-124a expression of BMSCs and ß-thiol ethanol or baicalin promoted apoptosis and reduced the growth of BMSCs. Only the microRNA-124a inhibitor did not affect apoptosis or the differentiation of BMSCs, and it increased the effects of ß-thiol ethanol or baicalin on the apoptosis of BMSCs. CONCLUSION: ß-thiol ethanol and baicalin treatment could affect microRNA-124a expression in BMSCs. We demonstrated that microRNA-124a promoted the differentiation of BMSCs into neurons.

The value of next-generation sequencing for the diagnosis of Streptococcus suis meningitis

SUMMARY OBJECTIVE: The aim of this study was to investigate the value of next-generation sequencing for the diagnosis of Streptococcus suis meningitis. METHODS: Patients with meningitis in the Department of Neurology of the Hainan General Hospital were recruited and divided into a next-generation sequencing group and a control group. In the next-generation sequencing group, we used the next-generation sequencing method to detect the specific pathogenic bacteria in the patients. In the control group, we used the cerebrospinal fluid bacterial culture method to detect the specific pathogenic bacteria in the patients. RESULTS: A total of 28 participants were recruited for this study, with 14 participants in each group. The results showed similarities in both the average age and average course of the disease between the two groups (p>0.05). The white blood cell count, percentage of neutrophils, and level of C-reactive protein in the next-generation sequencing group were significantly higher than those in the control group (p<0.05). There were similarities in both the temperature and intracranial pressure between the two groups (p>0.05). In the next-generation sequencing group, all patients (100%) were detected as having had the S. suis meningitis infection by next-generation sequencing, while only 6 (43%) patients in the control group had been detected as having the S. suis meningitis infection by cerebrospinal fluid bacterial culture. CONCLUSIONS: The positive detection rate of S. suis by the next-generation sequencing method was significantly higher compared with using a cerebrospinal fluid bacterial culture. Therefore, the next-generation sequencing method is valuable for the diagnosis of S. suis meningitis and is worthy of clinical application.

[Effect of p65 gene inhibited by siRNA on differention of rat marrow mesenchymal stem cells into neurons].

OBJECTIVE: To investigate the effect of p65 gene inhibited by siRNA on neuronic differentiation in the marrow mesenchymal stem cells (MSCs). METHODS: The MSCs were transfected with Rn-p65-siRNA. Fasudil hydrochloride induced MSCs differentiating into neurons. The non-transfected group and negative control group (transfected with negative control siRNA marked by Cy3) were used as controls. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope at 24 h,48 h and 72 h after transfected with negative control siRNA. The viability of MSCs was detected by MTT at 24 h, 48 h and 72 h after transfected with Rn-p65-siRNA. The expressions of p65 mRNA and protein in MSCs were detected by RT-PCR and Western blot respectively. The expressions of p65 protein, NSE, MAP-2 and glial fibrillary acidic protein (GFAP) were detected by immunocytochemical method after transfection for 6 h. RESULTS: The fluorescence of MSCs was mostly displayed after transfection of 72 hours and the efficiency of transfection was up to 83.3% ± 3.8%. Meanwhile, the p65 mRNA and p65 protein expressed by MSCs of transfected group were significantly decreased (P < 0.05); MTT displayed that the viability of MSCs was also significantly reduced (P < 0.05). The best efficiency of induction was observed in the transfected group. There were higher expressions of NSE and MAP-2 than the other group (P < 0.05). CONCLUSION: The p65 gene inhibited by siRNA can promote the marrow mesenchymal stem cells to differentiate into neurons.