Involvement Of Necroptosis In The Injury-induced Activation Of Olfactory Neural Stem Cells

Olfactory epithelium is the only neural tissue capable of neuronal regeneration in adult mammals.Persistent neurogenesis of the olfactory epithelium provides a unique model for studying self-renewal of olfactory neural stem cells.The olfactory epithelium is a pseudostratified epithelium composed mainly of olfactory sensory neurons,sertoli cells,Bowman glands/ducts,microvillus cells,and olfactory neural stem cells.Horizontal basal cells(HBCs)and global basal cells(GBCs)are olfactory neural stem cells in the basal part of olfactory epithelium.Physiologically,GBCs are located in the basal olfactory epithelium between the HBCs and immature olfactory sensory neurons.HBCs is relatively stationery and acts as a reserved stem cell bank,which is activated after severe injury to the olfactory epithelium.Olfactory epithelium is exposed to air and is susceptible to external factors such as the chemicals,viruses,soot,etc.At mild injury,GBCs prolifically and rapidly regenerates into most olfactory epithelial cell types.In the case of severe injury,HBCs is rapidly activated,proliferating and differentiating into GBCs and other cell types of olfactory epithelium.After the olfactory epithelium is damaged,a large number of cells die,which also activates the proliferation and differentiation of stem cells.However,the specific types of cell death and the relationship between cell death and proliferation and differentiation of olfactory neural stem cells are still unclear and need further study.Key molecules of programmed cell necrosis were examined following olfactory epithelial impairment.Then interacting Serine/Threonine-protein Kinase 3(RIPK3)knockout mice and mixed lineage kinase domain like pseudokinase(MLKL)knockout mice were studied to investigate the effect of programmed cell necrosis on activation of olfactory neural stem cells after olfactory epithelial injury.Objective : Considering that necroptosis is a direct consequence of olfactory epithelial injury,we hypothesized that the olfactory nerve progenitor cells respond to the injury in response to cell death.However,it has not been studied whether olfactory cell death is involved in regulating reactive cell proliferation of olfactory epithelium.In this study,RIPK3 gene knockout mice and MLKL gene knockout mice,key molecules of programmed cell necrosis,were used to study the role of programmed cell necrosis in reactive cell proliferation after olfactory epithelial injury and the mechanism of necrosis on stem cell activation after olfactory epithelial injury.Methods : 1.C57 wild-type mice,receptor-interacting serine protein kinase 3(RIPK3)knockout mice,mixed lineage kinase domain-like pseudokinase(MLKL)knockout mice and methimazole were used to prepare olfactory epithelium injury models.2.Cell death was observed by terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling(TUNEL)staining and in vivo propidium iodide(PI)labeling.3.The levels of RIPK3,MLKL and phosphorylated MLKL(p-MLKL)were detected by Western Blotting.Changes in the expression levels of Wnt signaling-related protein molecules(Axin2,β-catenin,Wnt2,SFRP1);the changes of macrophage polarizationrelated protein molecules(i NOS,Argnase-1,CD206);NF-κB signaling pathway related protein molecules(P65,p-P65,IκB,p-IκB).4.And Brd U / SOX2 immunofluorescence double staining were used to observe the activation of stem cells after olfactory epithelial injury in mice.OMP / DCX double immunofluorescence staining was used to observe the proliferation of olfactory neurons after olfactory epithelium injury in mice.Cell proliferation was evalulated by Brd U incorporation.Results : 1.Three days after the olfactory epithelial injury,most of the olfactory epithelial cells underwent mainly developed necroptosis and a small number underwent apoptosis.2.Significantly increased activation and proliferation of olfactory epithelial stem cells in RIPK3 and MLKL knockout mice were observed 3 days after the olfactory epithelial injury,as compared with WT mice.3.Enhanced regeneration of olfactory neurons was observed 7 days after MLKL knockout mice,as compared with WT control.4.In comparison with the control group,there was a large number of macrophage/glial cell infiltration 3 days after the injury of olfactory epithelium in MLKL knockout mice.5.MLKL gene knockout had no significant effect on Wnt signaling and macrophage/ cell polarization after olfactory epithelial injury.6.MLKL gene knockout inhibits NF-κB signaling activation after olfactory epithelial injury.Conclusions :1.In the early stage of olfactory epithelial injury,olfactory epithelial cells mainly undergo necroptosis and olfactory neural stem cells are activated rapidly.2.MLKL and RIPK3 knockout can promote activation of olfactory epithelial stem cells and neuronal regeneration following injury.3.MLKL knockout has no significant effect on Wnt signal activation after olfactory epithelial injury.The knockout of MLKL and RIPK3 increases the invasion of macrophage in olfactory epithelium after injury.MLKL knockout inhibits NF-κB signaling after olfactory epithelial injury.