| ObjectiveAn efficient and simple model for primary neuron oxysaccharide deprivation/reoxygenation was established.In vitro experiments,the effect of Scutellarin(Scu)on the survival rate of OGD neurons after injury was studied,and the effective range of its protective concentration was determined.Detection of the relevant apoptosis signal pathway to study the possible protective mechanism of Scutellarin against OGD injury of neurons;To investigate whether Scutellarin could regulate CX3CR1 after OGD injury in neurons.The expression of CX3CRI in primary neurons OGD injury model was studied,and the influence of CX3CR1 on neurons OGD injury and its regulatory mechanism were further discussedMethodsWithin 24 h of C57BL/6 mice were cortex,with shear,digestion,beat,filter to obtain cell suspension,vaccination in poly lysine package is more than 35 mm petri dish,complete medium into neurons after 4 h,48 h after purification using cytarabine,in liquid,after 72 h after every three days and an half amount in liquid,cultivate to 9 days after mature neurons for the experiment.Neurons were identified with neuron-specific marker map-2,so as to obtain neurons with reliable purity.MTT assay was used to detect the effect of Scu on the activity of neurons after OGD injury,and the protective effect of Scu on neurons and the optimal effective concentration were determined.Establishment of neuronal oxysaccharide deprivation method(OGD)model:primary neurons were cultured until day 9,and the neurons were observed in good shape under the microscope.The neurons were divided into 4 groups:Con,OGD,OGD+adjuvant,OGD+Scu.The basal cells were pretreated with Scu and Scu adjuvants for 30min,then the ogd-treated groups were replaced with sugar-free Neurobasal a-basal cells and placed into hypoxia cells for 15min,and cellular proteins were collected after hypoxia for subsequent Western Blotting experiments.Establishment of the pc-12 cell oxy glucose deprivation method(OGD)model:the pc-12 cells were inoculated in 6-well plates at an appropriate density and cultured in a high-glucose DMEM+10%FBS medium.After the cells matured,the cells requiring OGD treatment were replaced with sugar-free DMEM,and then put into a hypoxic chamber for 15min.After the hypoxia,proteins were collected for Western Blotting experiments.Western Blotting:neurons were cultured to mature and randomly divided into four groups:Con,OGD,OGD+adjuvant,and OGD+Scu.The optimal effective concentration of Scu was treated for 30min.Then,the OGD model was established,proteins were collected,and expressions of CX3CR1,cleaved PARP,cleaved caspase-3,ask-1,p-p38,and bcl-2 were detected by Western Blotting.Pc-12 cells were divided into four groups:CX3CR1siRNA,Con,Con+CX3CR1siRNA,OGD,OGD+CX3CR1siRNA were transfected into pc-12 cells by lip2000.After 24 hours,the OGD model was established to collect cell proteins.The expressions of CX3CR1,cleaved PARP,cleaved Caspase3,ask-1 and p-p38 were detected by Western Blotting,and the effects of silencing CX3CR1 gene on apoptosis were detected?ResultsMTT assay:compared with Con,the survival rate of neurons after OGD was significantly decreased(p<0.05).Compared with OGD group,pretreatment with 80nmol.L-1-160 nmol.L-1Scu for 30min can significantly improve the survival rate of neurons after OGD(p<0.05),while Scu 40nmol.L-1 has no obvious protective effect on neurons.Morphological observation:after OGD,the cell body expanded,the permeability decreased,and the cell fragments in the field increased.After Scu pretreatment,neuronal axons were relatively complete,cell fragments in the field of vision were significantly reduced,and disintegrating cells were not obvious,indicating that Scu could play a protective role on neurons.Western Blotting:compared with the Con group,the levels of cleaved caspase-3,cleaved PAR,ask-1,and p-p38 in the OGD group were significantly higher(*P<0.05),and bcl-2 were significantly lower(*P<0.05).Compared with OGD group,OGD+Scu group significantly reduced cleaved caspase-3,cleaved PARP,ask-1,and p-p38(*P<0.05),and significantly increased bcl-2(*P<0.05).In the pc-12 cell OGD model,CX3CR1 gene sequence siRNA was transfected into pc-12 cells for 24h and protein was collected.The results showed that compared with Con group,the expression of CX3CR1 in OGD+CX3CR1 siRNA group was significantly decreased,and the difference was statistically significant(p<0.05).Compared with Con group,CX3CR1,cleaved caspase-3,cleaved PARP,ask-1 and p-p38 were significantly increased after OGD(*P<0.05).Compared with OGD group,the CX3CR1,cleaved PARP,ask-1 and p-p38 of OGD+CX3CR1 siRNA group were significantly reduced,with statistically significant differences.(*p<0.05)(**p<0.01)Conclusion1:Scutellarin can significantly inhibit the apoptosis of neurons after cerebral ischemia in vitro.2:CX3CR1 is functionally expressed on neurons,and scutellarin can protect neurons by regulating the expression of CX3CR1.3,Scutellarin may protect neurons after cerebral ischemia through Ask-1/p-p38/Bcl-2 signaling pathway... |
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