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OBJECTIVES@#Nerve growth factor (NGF) induces neuron transdifferentiation of adrenal medulla chromaffin cells (AMCCs) and consequently downregulates the secretion of epinephrine (EPI), which may be involved in the pathogenesis of bronchial asthma. Mammalian achaete scute-homologous 1 (MASH1), a key regulator of neurogenesis in the nervous system, has been proved to be elevated in AMCCs with neuron transdifferentiation in vivo. This study aims to explore the role of MASH1 in the process of neuron transdifferentiation of AMCCs and the mechanisms.@*METHODS@#Rat AMCCs were isolated and cultured. AMCCs were transfected with siMASH1 or MASH1 overexpression plasmid, then were stimulated with NGF and/or dexamethasone, PD98059 (a MAPK kinase-1 inhibitor) for 48 hours. Morphological changes were observed using light and electron microscope. Phenylethanolamine-N-methyltransferase (PNMT, the key enzyme for epinephrine synthesis) and tyrosine hydroxylase were detected by immunofluorescence. Western blotting was used to test the protein levels of PNMT, MASH1, peripherin (neuronal markers), extracellular regulated protein kinases (ERK), phosphorylated extracellular regulated protein kinases (pERK), and JMJD3. Real-time RT-PCR was applied to analyze the mRNA levels of MASH1 and JMJD3. EPI levels in the cellular supernatant were measured using ELISA.@*RESULTS@#Cells with both tyrosine hydroxylase and PNMT positive by immunofluorescence were proved to be AMCCs. Exposure to NGF, AMCCs exhibited neurite-like processes concomitant with increases in pERK/ERK, peripherin, and MASH1 levels (all P<0.05). Additionally, impairment of endocrine phenotype was proved by a signifcant decrease in the PNMT level and the secretion of EPI from AMCCs (all P<0.01). MASH1 interference reversed the effect of NGF, causing increases in the levels of PNMT and EPI, conversely reduced the peripherin level and cell processes (all P<0.01). MASH1 overexpression significantly increased the number of cell processes and peripherin level, while decreased the levels of PNMT and EPI (all P<0.01). Compared with the NGF group, the levels of MASH1, JMJD3 protein and mRNA in AMCCs in the NGF+PD98059 group were decreased (all P<0.05). After treatment with PD98059 and dexamethasone, the effect of NGF on promoting the transdifferentiation of AMCCs was inhibited, and the number of cell processes and EPI levels were decreased (both P<0.05). In addition, the activity of the pERK/MASH1 pathway activated by NGF was also inhibited.@*CONCLUSIONS@#MASH1 is the key factor in neuron transdifferentiation of AMCCs. NGF-induced neuron transdifferentiation is probably mediated via pERK/MASH1 signaling.
Subject(s)
Animals , Rats , Adrenal Medulla , Cell Transdifferentiation , Chromaffin Cells , Dexamethasone , Epinephrine/pharmacology , Mammals , Nerve Growth Factor , Neurons , Peripherins , Protein Kinases , Tyrosine 3-MonooxygenaseABSTRACT
Objective To investigate the effects of Mash-1 gene overexpression on neural cell proliferation, differentiation and learning and memory ability in C57BL/6 adult male mice after brain trauma. Methods One hundred and sixty healthy adult male C57BL/6 mice were randomly divided into sham operation group, simple trauma group, negative control group and overexpression group. Gene transfection using recombinant adenovirus Ad5-mMash-1. Detection of Mash-1 mRNA level by RT-PCR at 1 d before TBI and 1 d, 3 d, 7 d, 14 d after traumatic brain injury (TBI). Western blotting was used to detect the expression of Mash-1 protein. The learning and memory ability was evaluated by means of water maze. The proliferation of nerve cells in dentate gyrus and cerebral cortex of hippocampus at 3 d and 7 d after TBI were detected by immunofluorescence. Results Compared with those in sham operation group, the relative expression of Mash-1 mRNA in simple trauma group and negative control group at 1 d, 3 d, 7 d, 14 d after TBI were significantly lower (P<0. 05-0. 01), and the relative expression of Mash-1 mRNA in overexpression group at 1 d, 3 d, 7 d, 14 d after TBI were significantly higher ( all P<0. 01 ). The relative expression of Mash-1 mRNA in overexpression group at 1 d , 3 d , 7 d , 1 4 d after TBI were significantly higher than those in simple trauma group and negative control group (all P<0. 05). Compared with those in sham operation group, expression of Mash-1 protein in simple trauma group at 1 d, 7 d, 14 d after TBI and negative control group and overexpression group at 1 d, 3 d, 7 d, 14 d after TBI (P<0. 05 -0. 01), expression of Mash-1 protein in simple trauma group at 3 d after TBI (P<0. 05). The expression of Mash-1 protein in overexpression group at 1 d, 3 d, 7 d, 14 d after TBI were significantly higher than those in simple trauma group and negative control group (all P<0. 05). Compared with those in sham operation group, the number of BrdU positive cells in simple trauma group at 3 d, 7 d after TBI and the number of DCX positive cells at 3 d after TBI were significantly decreased (P<0. 05-0. 01), and they were significantly increased in overexpression group (all P<0. 05). The number of BrdU positive cells at 3 d, 7 d after TBI and the number of DCX positive cells at 3 d after TBI in overexpression group were significantly increased than those in simple trauma group (all P<0. 05). There was no statistical difference of escape latency between simple trauma group, negative control group and overexpression group at 1 d, 3 d, 7 d, 14 d after TBI (all P>0. 05). Compared with those in sham operation group, escape latency in simple trauma group, negative control group and overexpression group at 1 d, 3 d, 7 d, 14 d after TBI were significantly increased (all P<0. 05). Conclusion Overexpression of Mash-1 gene increases neuronal proliferation and differentiation in dentate gyrus and cortex of adult C57BL/6 mice after traumatic brain injury, but it has no effect on learning and memory ability.
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Objective To investigate the effects of overexpression of Mash-1 gene on functional recovery and neural differentiation of embryonic stem cells in spinal cord injury mice. Methods CE3 cell line with overexpression of Mash-1 gene was generated with murine stem cell virus. Spinal cord injury model was established with forceps compression in 4-week-old KM mice. Normal saline (model group, n=12), CE3 cells with or without overexpression Mash-1 gene (CE3-Mash-1 and CE3 groups, n=12 respectively) were transplanted into the ar-eas of injury 3 days after injury. They were assessed with the Basso Mouse Scale (BMS) 1, 7, 14, 21, and 28 days after injury. 6 mice from each group were sacrificed 14 and 28 days after injury respectively. The spinal cord area remained were observed with HE stained, and the expression of Oct3/4, nestin,β-tubulin III and glial fibrillary acidic protein (GFAP) were detected with immunofluorescence in the injured spinal cord in the CE3 and CE3-Mash-1 groups. Results The score of BMS significantly improved in CE3 and CE3-Mash-1 groups com-pared with that of the model group (F>84.471, P49.990, P0.05). Conclusion Overexpression of Mash-1 gene promotes CE3 cells to differentiate into neurons in spinal cord injury mice, and improve the motor function recovery.
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Objective To investigate the effect and mechanism of Shouwu Yizhi Capsule (Capsule of Radix Polygoni Multiflori and Semen Coicis for dementia) on the Notch/Delta signal pathway in experimental rats with vascular dementia (VD). Methods The models were established with the modified Pulsinelli 4 vessel occlusion method. They were randomized into Shouwu Yizhi Capsule group,piracetam group,model group,and sham operation group with 14 rats in each group. Seven days after modeling,the Shouwu Yizhi Capsule group was administered intragastrically Shouwu Yizhi Capsule solution 0.08g/ml,the piracetam group was prescribed intragastrically piracetam solution 0.02g/ml,and the model and sham operation groups were given intragastrically normal saline. Twenty days later,the mRNA expression of HES1,Mash1,and ?-APP on Notch/Delta singal pathway was determined by real-time quantitative fluorescent PCR. Results Compared with the model and piracetam groups,the mRNA expression of HES1 the in Shouwu Yizhi Capsule group was significantly increased (P0.05). Conclusion Shouwu Yizhi Capsule could effectively activate the interaction of HES1,Mash1,and ?-APP on the Notch/Delta signal pathway,being the possible mechanism in the treatment of VD.