| Objective1. To observe the effects of folate on proliferation and differentiation of neural stem cells (NSCs) in the hypoxic-ischemic model by establishing rat model of middle cerebral artery occlusion (MCAO) and hypoxic NSCs model in vitro and detecting proliferation and differentiation of NSCs by double immunofiuorescence techniques. To confirm if folate can show neuroprotective effects on rats with cerebral infarction by stimulating NSCs proliferation and differentiation.2. To explore the mechanisms of folate on regulating proliferation and differentiation of NSCs in hypoxic-ischemic model by detecting mRNA and protein expression of genes involved in Notch and ERK signal pathways. Further observe cell proliferation and differentiation after inhibiting ERK pathway.3. To observe the effect of folate on apoptosis of NSCs in hypoxic-ischemic model and explore the possible mechanism by detecting cell apoptosis rate, detecting different genes expression with PCR array and testing expression levels of proteins involved in mitochondrial pathway. To provide science basis for researching the therapeutic and preventive effects of folate on hypoxic-ischemic brain damage.Methods1. Sprague-Dawley rats were randomly assigned to four groups. The groups were: sham operation plus vehicle (Sham OP), middle cerebral artery occlusion plus vehicle (MCAO), MCAO plus low dose folate (4 mg/kg·d) (MCAO+LFA) and MCAO plus high dose folate (12 mg/kg-d) (MCAO+HFA). MCAO was induced by the intraluminal filament technique. The vehicle (saline,10 ml/kg-d) and folate were administered by gastric lavage for 28 d prior to sham or MCAO operation. The sensorimotor integrity was conducted to assess the neurobehavior. Brain water content was measured. Infarct volume were measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Serum folate and plasma homocysteine were both detected. Apoptosis rate of neural cells were tested by TUNEL method. The histological observation used the HE stain. Learning and memory abilities were tested by Y-maze. Endogenous NSCs proliferation and differentiation were detected by double immunofluorescence techniques.?-Tubulin-III protein expression level in brain tissues were detected by western blot analysis. Notchl, Hesl/5, Mashl, Ngnl/2 mRNA expression levels in brain tissues were analyzed by situ hybridization. Notchl, Hesl/5 and pERKl/2 protein expression level were also detected by western blot. After blocking enzymatic activity of MEK1 in ERK pathway by U0126, NSCs proliferation and pERKl/2 protein expression were detected again.2. NSCs from newborn SD rats were culture in vitro. Cells were divided into Control, Hypoxia, Hypoxia plus low folate (Hypoxia+FA-L), Hypoxia plus high folate (Hypoxia+FA-H) and Hypoixa with folate deficiency (Hypoxia+FA-D) groups. The folate concentrations in 5 groups were 4?g/ml,4?g/ml,8?g/ml,44?g/ml and 0.65?g/ml respectively. Cells were exposed to 1%O2 for 8 hours. Cell viability was measured by MTT assay. On the 6th day in proliferation period, NSCs proliferation was detected by double immunofluorescence. Flow cytometry was performed to measure the cell apoptosis. On the 6th day in differentiation period, GFAP and?-Tubulin-III proteins expression were measured by western blot. Expression levels of Notchl, Hesl/5, Mashl, Ngnl/2 and ERK1/2 mRNA were detected by real-time PCR. Notchl, Hesl/5 and pERK proteins were detected by western blot. After blocking ERK pathway, NSCs proliferation and pERK protein expression were detected again. Different gene expressions in Hypoxia and Hypoxia+FA-H groups were detected by apoptosis PCR array. Cytochrome C (Cyt C), Bcl-2, Bax, Cleaved Caspase 3/9 protein expressions in 5 groups were further measured by western blot. Homocysteine concentration in all groups was also detected.Results1. Effects of folate on proliferation and differentiation of endogenous NSCs in rats with cerebral infarction and NSCs under hypoxic injury in vitro:Folate supplementation could raise serum folate concentration and reduce plasma homocysteine level. Neural deficit scores and infarct volume in MCAO+LFA and MCAO+HFA were lower than those in MCAO group. Furthermore, the learning or memory abilities in those 2 groups were better than that in MCAO group. Brain water content and apoptosis rate of neural cells in MCAO+HFA group were lower than those in MCAO group (P<0.05). Proliferation peak of NSCs appeared 7 days after ischemia in each group. BrdU+Nestin+cell amount in rat brains in MCAO+HFA was greater than those in other groups (P<0.05). BrdU+GFAP+and BrdU+NeuN+cell amounts in rat brains reached highest level 14 days after ischemia in all groups, those cell amounts in MCAO+HFA were greater than other groups (P<0.05). Expression levels of (3-Tubulin-?proteins in brain tissue in MCAO+LFA and MCAO+HFA groups were higher than those in other 2 vehicles (P<0.05). In vitro, after hypoxia, cell viabilitis in Hypoxia+FA-L group and Hypoxia+FA-H group were higher than that in Hypoxia group. Cell viabilitis in Hypoxia+FA-D group were lower than that in Hypoxia (P<0.05). On the 6th day in proliferation period, Nestin+BrdU+cell percentages in Hypoxia+FA-L and Hypoxia+FA-H group were both higher than that in Hypoxia group. This percentage in Hypoxia+FA-D group was lower than that in Hypoxia group (P<0.05). On the 6th day in differentiation period, GFAP protein expression in Hypoxia+FA-H group was higher than that in Hypoxia group. P-tubulin-III protein expression in Hypoxia+FA-H group was lower than that in Hypoxia group. Those 2 proteins expressions in Hypoxia+FA-D group were both lower than those in other groups (P<0.05).2. Effect of folate on expression of Notch and ERK signal pathway in brain tissue of rats with cerebral infarction and NSCs under hypoxic injury:7 days after infarction, Notch1 and Hesl/5 mRNA and protein expressions in MCAO+HFA group were higher than those in other groups (P<0.05). Mashl mRNA expressions in two folate administration groups were lower than that in MCAO group (P<0.05). Ngn1/2 mRNA expression levels were both lower in MCAO+HFA than those in other groups (P<0.05). In vitro, both in proliferation and differentiation periods, Notch1 and Hesl/5 mRNA expression levels were highest in Hypoxia+FA-H group, were lowest in Hypoxia+FA-D group (P<0.05). Mashl and Ngnl/2 mRNA expression levels were lowest in Hypoxia+FA-H group (P<0.05).7 days after infarction, pERKl/2 protein expressions in MCAO+LFA and MCAO+HFA groups were higher than that in MCAO group (P<0.05). After inhibiting ERK pathway by U0126, we found that U0126 could reduce pERKl/2 protein expression as well as BrdU+Nestin+cell amount in folate administration goup. In vitro, on the 6th day in proliferation period, pERKl/2 protein expressions in Hypoxia+FA-L and Hypoxia+FA-H groups were higher than those in Hypoxia and Hypoxia+FA-D group. Inhibiting ERK pathway by U0126 could reduce cell viability, BrdU+Nestin+ cell percentage and pERK1/2 expression in folate administration group.3. Effects of folate on apoptosis and expression of gene involved in mitochondrial pathway in NSCs after hypoxia in vitro:On the 6th day in proliferation period, Cell apoptosis rate in Hypoxia was higher than those in Control, Hypoxia+ FA-L and Hypoxia+FA-H group, but lower than that in Hypoxia group (P<0.05). Compared to Hypoxia group, expressions of bcl2 and Hprtl mRNA were higher, expressions of p53, apaf-1, bax and caspase-3/9 mRNA were lower in Hypoxia+FA-H group. Western blot detection showed that Cyt C, Bax, Cleaved Caspase-3/9 protein expressions in Hypoxia+FA-L and Hypoxia+FA-H groups were lower than those in Hypoxia group. Those protein expressions were higher in Hypoxia+FA-D group compared to Hypoxia group (P<0.05). However, Bcl-2 protein had higher expressions in 2 folate administration groups than that in Hypoxia group. It was lower in Hypoxia+FA-D group compared to Hypoxia group (P<0.05).ConclusionNeuroprotective effect of folate on rats with cerebral infarction may relate to its ability for stimulating proliferation of endogenous NSCs. These can be proved by experiments in vivo and vitro. Folate can promote proliferation of NSCs after hypoxia injury in vitro and help cells differentiating to astrocytes and inhibit NSCs differentiating to neurons. Folate deficiency then inhibits NSCs proliferation and differentiation. Notch and ERK signal pathways play important roles in mechanism of folate regulating NSCs proliferation and differentiation. Apoptosis inhibitory effect of folate on NSCs after hypoxic injury in vitro may relate to mitochondrial pathway. |
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