The Effect Of Fisetin-activated OECs On Neuronal Cell Growth Under The Damage Microenvironment And Underlying Molecular Mechanisms

ObjectiveThe purpose of this study was to investigate the promoting effects of fisetin-activated olfactory ensheathing(a OECs)on neuronal cells growth in the damage microenvironment and the underlying molecular mechanisms.MethodsPrimary OECs were isolated,cultured,purified from the olfactory bulb(OB)of adult SD rats,and treated with different concentrations of fisetin.CCK-8 assay and Brdu immunostaining were used to detect the cell viability and proliferative capacity.Concomitantly,the growth and proliferation of OECs were observed.Moreover,q RT-PCR was used to detect the expressions of CXCL1 and CXCL2,and western blot(WB)was used to identify the expression of transglutaminase-2(TG2)and phosphatidylserine receptor(PSR)in fisetin-treated OECs.Meanwhile,the activated or normal OECs were identified by immunofluorescence,and their culture supernatants were collected respectively.Subsequently,primary neural cell culture was prepared from the cerebral cortex of newborn SD rat pups of age postnatal 24 h.When neural cell processes formed a complicated net,an experimental model which mimics in vivo injury of central nervous system(CNS)was developed by in vitro mechanical scratch,and the cell debris was collected.In parallel,neuronal cells were isolated and cultured from the cerebral cortex of newborn SD rat pups of age postnatal 24 h.After 3-5d,neuronal cells were treated by various conditions as follows:fisetin-activated OECs supernatant,OECs supernatant,and normal cell medium(control group),and then the same dose of cell debris from mechanical scratch were delivered to the three different culture systems.Three groups without cell debris were set up respectively.Notably,the coverslips grown neuron were also prepared and transferred into the above-mentioned 6 different culture conditions for next experiments.At the end of the different treatment time points,immunofluorescence staining was performed to assess the growth and survival of neurons and compare the number of neurons among the different groups.In addition,WB was used to detect the expression level of neuron markers Tuj-1 and MAP2 following the different treatments.Eventually,the secretion of anti-inflammatory cytokines IL-4,IL-10,and TGF-βand brain-derived neurotrophic factor(BDNF),glial neurotrophic factor(GDNF)and nerve growth factor(NGF)in the supernatant of OECs treated with or without fisetin was detected by enzym-linked immunosorbent assay(ELISA).Meantime,WB were further carried out to test the activation PI3K/Akt/Cre B signaling pathway in neurons under the different treatment conditions.ResultsThe normal OECs in vitro displayed a spindle-shaped,bipolar morphology under phase-contrast microscopy,and there were considerable abundant thin and long process extension and arborizations,which formed a net.Immunofluorescence further showed that these cultured OECs were positive for p75.After treatment of fisetin,the processes extended from OECs became slightly thicker,and had richer arborizations than that of normal cultured cells,and the cell density was significantly increased.Strikingly,most of the cells were spindle-shaped and parallelly arranged,and there were many polygonal cells which extended a large number of thin neurites.CCK-8 and Brd U assays showed that fisetin can enhance the proliferative capacity of OECs and increase cell viability.Moreover,q RT-PCR results showed that fisetin drastically up-regulate the m RNA levels of CXCL1and CXCL2 in OECs.In addition,WB showed that fisetin could elevate the protein expression of TG2 and PSR in OECs,and the two selected molecules showed a time-dependent increase,which was significantly different from that of the control group(P<0.05).For mechanical injury,a variety of cell types,including astrocytes microglia neuronal cells and oligodendrocytes were seen in primary neural culture.After undergoing a mechanical scratch,most of the cell processes were broken,the soma of cell was swelling,and the normal cell structure was obviously destroyed,suggesting that a damage model was successfully established.When primary neurons were treated with a OECs and OECs supernatant,respectively.More Tuj-1 positive cells were seen in the a OECs supernatant group and OECs supernatant group than that in control.Moreover,cell count showed that the number of Tuj-1~+cells in a OECs supernatant group was significantly higher than that of the OECs supernatant group(P<0.05).When the cell debris was delivered into the cultures,the number of Tuj-1~+cells significantly decreased,and the a OECs supernatant can effectively mitigate the decline of Tuj-1~+cells,displaying more significant suppression of the number of Tuj-1~+cell decrease compared to OECs supernatant group and control group(P<0.05,P<0.01).As for the growth of neurons,OECs supernatant or OECs supernatant in the addition of cell debris also caused a significant increase of in the expression of Tuj-1 and MAP2 compared with in primary neurons,and the higher expression levels of the two selected markers were seen in a OECs groups(P<0.05).Moreover,the administration of a OECs or OECs supernatant to neuron culture in the presence of cell debris,markedly also elevated the expression of PI3K,Akt and Cre B.The highest levels were found in a OECs group.In addition,the antagonist LY94002 of PI3K/Akt signaling and antagonist U0126 of Cre B remarkably down-regulated the expression of neuronal marker Tuj-1.More importantly,ELISA assay showed the amount of anti-inflammatory factors IL-4,IL-10,TGF-βand neurotrophic factors BDNF,GDNF and NGF secreted from the fisetin-treated OECs were significantly elevated compared with that of OECs,and the difference was statistically significant(P<0.05).ConclusionFisetin can promote the activation of OECs.The activated OECs have an increased capacity for proliferation and cell viability,and effectively promote the growth of neurons under a damage condition.The a OECs promoting activity of growth of neurons in the damage environment may be closely related to the up-regulation of the secretion of anti-inflammatory cytokines IL-4,IL-10,TGF-βand neurotrophic factors BDNF,GDNF and NGF by a OECs,as well as the activation of PI3K/Akt/Cre B signaling pathway.