Preliminary Study On The Regulation Of Microglia Inflammatory And Its Mechanism By Muse Cells

Spinal cord injury(SCI)mostly comes from damage to the human spine by external forces.When the spinal cord is damaged,it can affect the structure and function of the central nervous system(CNS)and is accompanied by complex and difficult-to-heal complications.It brings huge physiological pain and economic pressure to the patient,and also increases the country’s medical burden.The latest research shows that stem cells have the powerful paracrine function and differentiation ability.They can effectively nourish damaged nerves and promote nerve tissue regeneration in SCI.However,due to the survival rate and differentiation rate in vivo,the selection of seed cells is still a big problem.As neuroinflammation is an important pathological process that mediates various types of CNS diseases,the existence of inflammation increases the difficulty of the damage repair.Therefore,selecting suitable seed cells to improve the inflammatory microenvironment after SCI is a breakthrough in the treatment of SCI,providing a new idea for alleviating this major public health problem.Objective:To explore the effect and the possible mechanism of action of multilineage differentiating stress enduring cells(Muse cells)derived from human bone marrow on the neuroinflammatory microenvironment to provide evidence for Muse cells to treat SCI.Methods:Human bone marrow mesenchymal stem cells(BMSCs)were extracted from human bone marrow blood,and the cells were identified by microscopic observation combined with the staining of surface markers CD105 and CD45 using immunofluorescence cytochemistry technology.Muse cells were obtained by 8 h trypsin tolerance test,and Muse cells were identified.Muse cells or BMSCs were co-cultured with microglia stimulated by 1ug/ml LPS,and divided into Sham group,LPS group,BMSCs group and Muse group.Use q RT-PCR to semi-quantify the m RNA expression of TNF-αand IL-1βin microglia,ELISA to detect the release of TNF-α,IL-1βand IL-10,while CCK-8 was used to test the viability of microglia.The cell morphology was observed under the microscope,the number of i NOS~+cells was detected by Flow Cytometry,and the ratio of M1/M2 microglia was explored by q RT-PCR and Western Blot.Finally,we use Western Blot to explore the mechanism of Muse cells.Results:Both Muse cells and BMSCs could down-regulate the m RNA expression and secretion of pro-inflammatory factors TNF-αand IL-1β,and increase the level of IL-10.The expression levels of TNF-αand IL-1βin the Muse group were lower than those in the BMSCs group,while the secretion of IL-10 was higher,nearly twice as much as that in the LPS group.At the same time,Muse cells could increase the viability of microglia under the stimulation of LPS,and to a certain extent,restore the morphological changes of microglia after stimulation,promoting microglia to contact each other through a large number of protrusions.On the other hand,Muse cells could also reduce the ratio of i NOS~+M1 microglia and up-regulate the expression of CD206 and Arg-1,markers of M2 microglia.Western Blot results showed that Muse cells could significantly reduce the protein expression of TLR4 and My D88 in microglia in an inflammatory environment,and down-regulate the phosphorylation levels of p65,Ik Bαand p38.Conclusion:The results show that:(1)Muse cells could be isolated from bone marrow blood and obtained more efficiently through the adherence-suspension-adherent culture method.(2)Muse cells could be more effective than BMSCs in inducing the conversion of microglia to M2 type in the inflammatory environment,increasing the expression of M2microglia and reducing the proportion of M1 microglia,thereby improving the inflammatory microenvironment by regulating the phenotype of microglia.(3)Muse cells may exert anti-inflammatory effects by inhibiting TLR4/My D88/NF-κB and p38 MAPK signaling pathways in microglia.