The Role And Mechanism Of VPA In The Coversation Of Satellite Glial Cells Into Neurons

Satellite glial cells are developed from neural crest cells and are a type of glial cells with multiple differentiation potential that surround sensory neurons in the dorsal root ganglion.Study finds that under the influence of neural cell injury microenvironment SGCs can differentiate into sensory neurons to promote the repair of nervous system injury,but the mechanisms that affect and control this transformation process have not been established.In this study,we investigate the molecular mechanism of SGCs differentiation by inducing SGCs differentiated into neurons in vitro.Studies have shown that Wnt,FGF,Notch signaling pathways and epigenetic modifications are involved in the differentiation of neural crest cells into sensory neurons.DRG-SGCs were incubated with VCRS——the combination of four small molecules:histone deacetylase inhibitor VPA(V),Wnt pathway activator CHIR99021(C),Notch pathway inhibitor RO4929097(R)and FGF pathway inhibitor SU5402(S)for 7 days,significant changes in cell morphology and expression of neuronal markers Tuj1 and Map2 were observed.After continuous treatment in 28 days,the cells expressed mature neuron marker NeuN,indicating that DRG-SGCs can transform into neuron-like cells after combined treatment with VCRS.In order to explore which small molecule plays a more important role in the differentiation of DRG-SGCs into neuron-like cells,we removed a single small molecule from the VCRS combination and treated DRG-SGCs for 7 days.Only the removal of VPA can significantly reduce the number of cells expressing Tuj1,suggesting that VPA made the most significant contribution to the differentiation of DRG-SGCs into neuron-like cells in the treatment with VCRS for 7 days.To further verify the effectiveness of VPA,we treated DRG-SGCS with VCRS for 7 days,and then removed S,SR or VSR and continued to treat for 7 days.Immunofluorescence staining reveals the removal of S or SR did not affect the number of cells expressing Tuj1,while the removal of VSR significantly reduced the number of cells.It shows that DRG-SGCs could transform into neuron-like cells after 7 days of VCRS combination treatment,while the neuron-like characteristics were no longer appeared after 7 days of incubation without VPA in the combination,thus proving that VPA played a key role in the differentiation of DRG-SGCs into neuron-like cellsNext,we explored the effect of VPA treatment alone on DRG-SGCs,and found that after 7 days of VPA stimulation,the cell morphology of DRG-SGCs significantly changed and expressed the neuronal marker Tuj1,indicating that VPA alone can induce DRG-SGCs to differentiate into neuron-like cells.As a histone deacetylase inhibitor,VPA can widely activate the transcription of neuronal development-related genes,we observed that after VPA treatment of DRG-SGCs for 0,1,3,5,and 7 days,the expression of the neural system development-related gene Tescalcin(TESC)is up-regulated,suggesting that TESC may participate in the VPA-mediated differentiation of DRG-SGCs into neuron-like cells.Interfering with the expression of TESC in DRG-SGCs reduced the number of cells expressing Tuj 1 obtained from DRG-SGCS treated with VPA for 7 days,indicating that TESC interference could reduce the differentiation efficiency of DRG-SGCS induced by VPA.To further verify that VPA induces the differentiation of DRG-SGCs into neuron-like cells by up-regulating the expression of TESCs,we used lentivirus to make the TESC highly expressed in DRG-SGCs.After 7 days,the cells expressed the neuron marker Tuj 1,indicating that TESC with high expression could also transform DRG-SGCs into neuron-like cellsDuring the development of peripheral nerves,transcription factors such as Brn3a,Isl1,Ngn1,and Ngn2 regulate the differentiation of neural crest cells to sensory neurons,and the differential expression of Runx1,Runx3 and Shox2 may transform them into different types of sensory neurons.We speculated that these transcription factors also play roles in the transformation of SGCs to neurons.After 7 days of treatment with DRG-SGCs by VPA,the expressions of neuron-related transcription factors Brn3a,Isl1,Ngn2,Runx1 and Runx3 were significantly up-regulated,and Lentiviral interference-mediated downregulation of TESC caused the downregulation of Brn3a,Isl1,Ngn2 and Runx1 expression,indicating that TESC is involved in the process of VPA up-regulating the expression of neuron-related transcription factors.In order to further verify that VPA up-regulates the expression of neural-related transcription factors through TESC,we used lentivirus to increase the expression of TESC in DRG-SGCs.After 7 days,the expression of Brn3a and Isl1 in the cells were up-regulated,further indicating that TESC can regulate the expression of nerve-related transcription factors that are up-regulated by VPA.In summary,we found that VPA induced the differentiation of DRG-SGCs into neuron-like cells and made neuron-related transcription factors upregulated,lentiviral interference-mediated downregulation of TESC caused neuron-related transcription factors down-regulated and reduced the differentiation efficiency of DRG-SGCs induced by VPA.High expression of TESC up-regulated the expression of neuron-related transcription factors and transforms DRG-SGCs into neuron-like cells,indicating TESC participates in the VPA-mediated differentiation of DRG-SGCs into neuron-like cells by regulating the expression of neuron-related transcription factors.This experiment investigated the mechanism of DRG-SGCs differentiate into neuron-like cells by VPA,which provides a theoretical basis for the clinical treatment of peripheral nervous system injury.