The Regulatory Effect Of UMSCs Combined With GM1 On The Immune-inflammatory Microenvironment In Rats With Traumatic Brain Injury

Part I Effects of UMSCs combined with GM1 on neural network reconstruction in rats with traumatic brain injuryObjective: To explore the effect of human umbilical cord mesenchymal stem cells(UMSCs)combined with monosialotetrahexosy1 ganglioside(GM1)on neural network reconstruction in rats with traumatic brain injury.Method: A total of 70 healthy adult male SD rats were used to construct a TBI model with a rat hydraulic brain injury device.56 survived 7 days after modeling.They were randomly divided into GM1+UMSCs groups(A7d,A14d),UMSCs group(B7d,B14d),GM1 group(C7d,C14d)and NS group(D7d,D14d)according to the random number table method,all 8 groups,7 in each group,were treated respectively with GM1+UMSCs,UMSCs,GM1 and NS on the 7th day after TBI,neurological severity score(m NSS)was performed on 7d and 14 d after treatment,and then the rats were sacrificed by decapitation to obtain brain tissue samples from the injured area.The expression of neuron-like cell-related markers NE,β-tublinⅢ,NF-200 and MAP-2 were detectted by Immunofluorescence and western blot,HE and LFB staining were used to analyze the damage cavity,myelin protection and regeneration,to understand the effect of UMSCs combined with GM1 on neural network reconstruction in rats with traumatic brain injury.Result: 7d and 14 d after UMSCs transplantation,Western Blot and immunofluorescence results showed that the protein expressions of NE,NF-200,MAP-2 and β-tubulin Ⅲ increased in the GM1+UMSCs group,GM1 group and UMSCs group compared with the NS group.The protein expression was most significant around 7 days after UMSCs transplantation in the GM1+UMSCs group(P<0.05),and the expression was significantly decreased at 14 days.There was no significant difference between NF-200 andβ-tubulin III(P>0.05),and the GM1+UMSCs group had the lowest m NSS score,and The neurological functional recovery was the most obvious(P>0.05).HE and LFB staining results showed that GM1+UMSCs group had the least cavities,dense myelin sheaths,and small gaps the 7d after UMSCs transplantation.There was no significant difference in the other groups.While in the 14 d,NS group had the most cavities,loose myelin sheaths,and the largest gaps,the other groups showed no significant difference.Conclusion:1.UMSCs combined with GM1 may synergistically promote UMSCs differentiation or secrete related cytokines to activate endogenous neural stem cells to differentiate into neuron-like or glial-like cells,reduce cavitation and myelin damage,and promote neural network reconstruction to restoration of the area;2.7d may be the best time window for the intervention effect of GM1 combined with UMSCs on TBI rats.Part II The regulatory effect of UMSCs combined with GM1 on local microglial polarization in rats with traumatic brain injuryObjective: To investigate the regulatory effect of human umbilical cord mesenchymal stem cells(UMSCs)combined with monosialotetrahexosy1ganglioside(GM1)on the polarization of microglia in rats with traumatic brain injury.Method: A total of 70 healthy adult male SD rats were used to construct a TBI model with a rat hydraulic brain injury device.56 survived 7 days after modeling.They were randomly divided into GM1+UMSCs groups(A7d,A14d),UMSCs group(B7d,B14d),GM1 group(C7d,C14d)and NS group(D7d,D14d)according to the random number table method,all 8 groups,7 in each group,were treated respectively with GM1+UMSCs,UMSCs,GM1 and NS on the 7th day after TBI,neurological severity score(m NSS)was performed on 7d and 14 d after treatment,and then the rats were sacrificed by decapitation to obtain brain tissue samples from the injured area.Immunofluorescence,PCR and western blot were used to detect the microglia polarization markers CD163 and i NOS.The expression of key proteins TLR4 and My D88 in TLR4 signaling pathway was detected by western blot,to understand the regulation effect and possible mechanism of UMSCs combined with GM1 on the polarization of microglia in rats with traumatic brain injury were investigated.Result: 7d and 14 d after UMSCs transplantation,PCR,Western Blot and immunofluorescence results showed that the protein expression of CD163 in the GM1+UMSCs group,the GM1 group and the UMSCs group was up-regulated compared with the NS group,and the protein expression of i NOS was down-regulated compared with the NS group.The expression levels of the proteins TLR4 and My D88 were significantly decreased,and the protein expression of the GM1+UMSCs group had the most significant changes in at 7 d after transplantation,and the GM1+UMSCs group had the lowest m NSS score and the most obvious neurological recovery,P<0.05.Conclusion:1.UMSCs combined with GM1 may reduce the secondary neurodegeneration and neuroinflammation of TBI by synergistically regulating the polarization state of microglia;2.7d may be the best time window for the intervention effect of GM1 combined with UMSCs on TBI rats;3.TLR4 signaling pathway is a possible mechanism for UMSCs combined with GM1 to coordinately regulate the polarization state of microglia.Part III The regulatory effect of UMSCs combined with GM1 on the local inflammatory microenvironment in rats with traumatic brain injuryObjective: To explore the effect of human umbilical cord mesenchymal stem cells(UMSCs)combined with monosialotetrahexosy1 ganglioside(GM1)on the local immune inflammatory microenvironment in rats with traumatic brain injury.Method: A total of 70 healthy adult male SD rats were used to construct a TBI model with a rat hydraulic brain injury device.56 survived 7 days after modeling.They were randomly divided into GM1+UMSCs groups(A7d,A14d),UMSCs group(B7d,B14d),GM1 group(C7d,C14d)and NS group(D7d,D14d)according to the random number table method,all 8 groups,7 in each group,were treated respectively with GM1+UMSCs,UMSCs,GM1 and NS on the 7th day after TBI,neurological severity score(m NSS)was performed on 7d and 14 d after treatment,and then the rats were sacrificed by decapitation to obtain brain tissue samples from the injured area.Immunohistochemistry,RT-PCR and ELISA were used to detect local inflammatory reaction and immune regulation.The expression of related proteins was detected by western blot in NF-κB/IκBα signaling pathway,to understand the effect and possible mechanism of UMSCs combined with GM1 on the local immune inflammatory microenvironment in traumatic brain injury rats.Result: 7d and 14 d after UMSCs transplantation,the results of immunohistochemistry,RT-PCR and ELISA showed that the expressions of local inflammatory factors MPO,ICAM-1 and ED-1 decreased in the GM1+UMSCs group,There was no significant difference in the GM1 group,UMSCs group and NS group.The expression of the injured local immunoregulatory factor IL-6 decreased and the expression of TGF-βincreased in GM1+UMSCs group,but there was no significant difference in the other groups.Compared with 7d,the Expression amplitude of IL-6 and TGF-β decreased on 14 d,but the trend was basically same.The expressions of p65 and IκBα were up-regulated in the TBI rat model,the expressions of p-p65 and p-IκBα were significantly down-regulated in the GM1+UMSCs group,The expression of factor such as MPO,ICAM-1,ED-1,IL-6,TGF-β,COX-2,etc.had the most significant change in GM1+UMSCs group,and the change was the most obvious at 7d after UMSCs transplantation,the m NSS score of the GM1+UMSCs group was the lowest,and the neurological function recovery was the most obvious,P<0.05.Conclusion:1.UMSCs combined with GM1 may synergistically regulate the local immune inflammatory microenvironment in traumatic brain injury rats and reduce the secondary neuroinflammation and neurodegeneration of TBI;2.7d may be the best time window for the intervention effect of UMSCs combined with GM1 on TBI rats;3.NF-κB/IκBα signaling pathway is a possible mechanism for synergistically regulating the local immune inflammatory microenvironment in traumatic brain injury rats.