Comparison of repeated human umbilical cord mesenchymal stem cells administration and tetramethylpyrazine combination with single injection of human umbilical cord mesenchymal stem cells in cuprizone-induced demyelinating mice

Introduction : Multiple sclerosis (MS) is an autoimmune disease characterized by inflammation and demyelination in the central nervous system, which can lead to neurological dysfunction. Our previous studies have showed tetramethylpyrazine (TMP) treated human umbilical cord mesenchymal stem cells (hUC-MSCs) showed better therapy effects in experimental autoimmune encephalomyelitis (EAE) mice, compared to hUC-MSCs alone. Recently, studies showed that repeated injection of MSCs in MS patients improved cognition and neurological functional tests of patients. Therefore, we wanted to explore the comparison therapy effects among repeated hUC-MSCs injection and TMP combination of single injection of hUC-MSCs in demyelinating mice. Methods : Neurological function, microglial polarization, myelin regeneration, and blood brain barrier (BBB) integrity were evaluated in cuprizone (CPZ)-induced demyelinating mice in vivo . In vitro , the supernatants were collected from co-cultureof BV2 microglia and hUC-MSCs. The supernatants were used to culture endothelial cells or oligodendrocyte precursor cells (OPCs). Microglial polarization was detected by flow cytometry; BBB integrity was assessed via fluorescein sodium leakage assay, and OPCs differentiation was examined by immunofluorescence staining. Results : Both therapeutic regimens improved neurological function, regulated microglial polarization, protected BBB integrity, and promoted myelin regeneration. However, repeated hUC-MSCs injection showed superior effects on neurological function, up-regulating the expression of CD206, IL-10, myelin basic protein, claudin-5, down-regulating the expression of glial fibrillary acidic protein, TNF-α, and IL-1β, as well as decreasing evans blue leakage in vivo . In vitro experiments further verified that both therapies could induce the polarization of LPS-stimulated BV2 microglia from M1 to M2 phenotype, thereby protecting BBB integrity and promoting the differentiation of OPCs into oligodendrocytes. Nevertheless, repeated hUC-MSCs therapy exhibited better efficacy in up-regulating the expression of Claudin-5 and CNPase, and down-regulating the permeability of sodium fluorescein sodium in vitro . Conclusion : In conclusion, repeated hUC-MSCs therapy exerts superior efficacy in promoting myelin regeneration and protecting BBB integrity by regulating microglial polarization, this study provides additional results for optimizing clinical treatment regimens for MS.