| Objective: Spinal cord injury(SCI)is the most serious complication of both spinal cord injury and other serious neurological dysfunction diseases.Studies have shown that human umbilical cord mesenchymal stem cells(HUC-MSCs)used in the treatment of SCI rats can improve motor function,promote the expression of nerve growth factor,and inhibit IL-1? secretion.Low-dose ultrashort wave(USW),a wide-spread physical therapy,has been reported to achieved good results in the treatment of various acute inflammation diseases.It can assist in inhibiting inflammation,eliminating edema,improving blood circulation as well as promoting nerve regeneration.In inflammatory diseases,MAPK-activated protein kinase 2(MK2)can activate the RNA binding protein Tristetraprolin(TTP),a member of the zinc finger protein family,thereby regulating the translation of TNF-? and inflammatory factors,and promoting the expression of TNF-?and inflammatory factors.This study intends to use the improvement effect of USW on the inflammatory microenvironment,combined with HUC-MSCs to treat SCI,observe its therapeutic effect and influence on the inflammatory microenvironment,and explore the regulation mechanism of MK2/TTP signaling pathway and NLRP3 in order to improve HUC-MSCs treatment methods,and provide a new sight of scientific basis of stem cell therapy for the future.Methods: The spinal cord injury model of SD rats was established by Allen's striking method,to set up 5 groups,respectively:(1)the control group(sham): The SD rats in this group had back surgery without spinal cord striking;(2)spinal cord injury group(SCI):The SCI group did no treatment after spinal cord striking;(3)USW treatment group(USW): Spinal cord injury rats were treated with small doses of ultrashort waves;(4)HUC-MSCs treatment group(HUC-MSCs): Spinal cord injury rats were treated with HUC-MSCs cells injected into the back;(5)USW combined with HUC-MSCs treatment group(USW+HUC-MSCs):Rat spinal cord injury models were treated with small doses of ultrashort waves after injecting with HUC-MSCs.Basso Beatlie & Bresnahan(BBB)behavioral scoring was performed for each group of rats,pathological observation of spinal cord tissues of each group was performed using HE staining,neuronal cell activity was observed by Nissl staining,spinal cord neuronal injury was detected by immunofluorescence assay in each group of rats,neuronal apoptosis was detected by TUNEL method,and ELISA was used to detect The expression of inflammation-related factors M-CSF,IL-6,IL-10 and IL-1?,IL-18 and TNF-? in the spinal cord tissues of rats.The expression changes of MK2,p-MK2,TTP,NLPR3 and NLRP3-related proteins pro-IL-1?,pro-IL-18 and pro-casepase-1 were detected by Western-blot method,as well as the polarization markers i NOS and Arg-1 in spinal microglia by immunofluorescence double-labeling method,and observed the expression of MK2 and NLRP3 in microglia.The MK2 overexpression microglia injury model was also established,and microglia were divided into 6 groups:(1)Control group: normal cultured microglia without treatment;(2)LPS group: LPS-treated microglia and continued to be routinely cultured;(3)USW group: LPS-treated microglia,intervened with USW and continued to be cultured;(4)HUC-MSCs group: microglia treated with LPS,added to HUC-MSCs cells and co-cultured;(5)USW+HUC-MSCs group: microglia treated with LPS,intervened with USW and continued to be cultured with HUC-MSCs cells;(6)OV-MK2: MK2 overexpressing cell line treated with LPS,intervened with USW and then HUC-MSCs cells were added for co-culture.The change of protein expression levels of TTP,NLRP3,i NOS,Arg-1,pro-IL-18 and pro-IL-1? in each group of cells were detected by Western-blot,and the change of inflammatory factors such as IL-6,IL-10,IL-1? and IL-18 in the supernatant of each group of cells were detected by ELISA.Results: On the first day after spinal cord injury,the rat completely lost the hind limb motor function and was in a state of paresis.The BBB score was 0.The score gradually recovered 2 to 4 weeks after the operation.At 4 weeks,the BBB scores of the USW group and USW+MSCs group were both obvious It is better than the SCI group(P<0.05),while the MSCs group has no significant difference compared with SCI(P>0.05)(Figure1-A),indicating that within 4w after SCI,USW combined with MSCs treatment can improve the motor function of rats Mainly the role of USW.Histopathology showed that there were necrotic tissue and cell fragments on the dorsal side of the spinal cord during spinal cord injury,and inflammatory cell infiltration was obvious.Immunofluorescence detection of Neu N showed that the number of neurons at the head of the injury decreased However,the USW group,MSCs group and USW+MSCs group all improved spinal cord injury,the degree of inflammatory cell infiltration was reduced,and the number of damaged head neurons was significantly greater than that of the SCI group(P<0.05).It can be seen that in improving the behavioral function of rats,USW is the main role,and MSCs plays an auxiliary role.The combination of the two can improve spinal cord injury in SCI rats.The mechanism suggests that it is related to improving blood circulation,enhancing tissue nutrition,and reducing inflammation and edema..The occurrence of SCI can cause the activation of microglia and the infiltration of macrophages,forming a local inflammatory microenvironment.After the occurrence of SCI,the microglia eventually transformed into M1 and M2 types from the resting state.In this study,IBA-1 was used to label microglia,and immunofluorescence double labeling was used to observe the M1 type marker i NOS and the M2 type marker arg-1 As a result,after 4 weeks,the microglia in the net interest state(M0)were polarized into M1 and M2,and the expression of i NOS and arg-1 were significantly increased,and the over-activated M1 Type microglia secrete a large number of inflammatory factors,and M-CSF and IL-6 were significantly increased;USW group,MSCs group and USW+MSCs group M2 type microglia Cells gradually became dominant,arg-1expression increased,IL-10 content increased,while i NOS expression decreased,M-CSF and IL-6 content decreased.Among them,the USW+MSCs group had the most significant performance and decreased inflammatory factors,while the USW group There is no significant difference between the MSCs group and the MSCs group,suggesting that the combination of USW and MSCs can provide a more suitable microenvironment for neurons.The initiation of the inflammatory response requires the involvement of the protein complex of the inflammasome.It has been confirmed that the expression level of NLRP3 inflammasome after spinal cord injury is significantly up-regulated,and inhibition of NLRP3 inflammasome can improve motor function after SCI.In this study,immunofluorescence double labeling was used to observe the expression of microglial inflammasomes.The results showed that the expressions of spinal microglial markers IBA-1 and NLRP3 increased simultaneously in the SCI group,and NLRP3 catalyzed pro-caspase-l The expression is up-regulated,thereby promoting the expression of pro-IL-l? and pro-IL-18,and at the same time transforming them into biologically active IL-l? and IL-18,participating in the follow-up The inflammatory response caused by the apoptosis of neuronal cells in the spinal cord.The USW group,MSCs group and USW+MSCs group all showed different degrees of inhibition of NLRP3.Among them,the MSCs group had a weak inhibitory effect on the immune microenvironment,while the combined application of USW and MSCs had a significant effect,showing inhibition of pro-caspase-1.The role of pro-IL-l? and pro-IL-18,while inhibiting the release of IL-l? and IL-18,and reducing the apoptosis of spinal cord neurons,suggesting that the combined application of USW and MSCs can inhibit inflammasome and reduce Spinal cord neuron injury.Our previous results show that the expression of MK2 in the serum of SCI patients is higher than that of non-SCI patients.The up-regulation of MK2 gene expression may be related to the local inflammatory microenvironment.Our previous results show that the expression of MK2 in the serum of SCI patients is higher than that of non-SCI patients.The up-regulation of MK2 gene expression may be related to the local inflammatory microenvironment.This experiment explored the effects of HUC-MSCs combined with USW on MK2 in SCI rats.The results showed that the occurrence of SCI can promote the phosphorylation of MK2,inhibit the activity of TTP,and promote the release of downstream factor TNF-?.Interestingly,we also found that the expression of MK2 increased in microglia,suggesting that MK2 is involved in the formation of inflammatory microenvironment of SCI rats.The USW group,MSCs group and USW+MSCs group all showed different levels of inhibition of MK2.The combined application of USW and MSCs had a significant effect,the phosphorylation level of MK2 was inhibited,the expression of TTP was up-regulated,and the content of TNF-?decreased.It is speculated that when SCI occurs,the expression of MK2 is up-regulated,inhibiting the activity of TTP,and at the same time promoting the expression of NLRP3,increasing the secretion of inflammatory factors,thereby promoting the formation of local inflammatory microenvironment.To further investigate the effective mechanisms of MK2/TTP pathway and NLRP3 in combination therapy-relieved SCI in rats,We selected rat spinal cord microglia,used LPS to simulate spinal cord injury cell models,and used USW,co-culture with HUC-MSCs,and USW combined with HUC-MSCs to intervene the cells.Results showed USW combined with HUC-MSCs can regulate the polarization state of microglia,inhibit M1 polarization of microglia,and promote their M2 polarization for repair and regeneration.At the same time,the content of IL-6 is reduced and the content of IL-10 is increased;When the expression of MK2 was up-regulated in vitro study,the polarization state regulated by the combination therapy was destroyed,M1 type dominated,and the content of inflammatory factors increased;Meanwhile,Overexpression of MK2 relieved the inhibition of NLRP3 expression by TTP,and increased the expression of NLRP3,pro-IL-1? and pro-IL-18.It is suggested that HUC-MSCs combined with USW can down-regulate the expression of NLRP3 by inhibiting MK2 phosphorylation,reduce inflammation,improve the immune microenvironment,and alleviate spinal cord injury.Conclusion: 1.Ultrashort wave combined with human umbilical cord mesenchymal stem cells can repair nerve cells and reduce inflammatory factors,which can effectively reduce spinal cord injury.2.Ultrashort wave combined with human umbilical cord mesenchymal stem cells can inhibit the secretion of NLRP3 inflammasome and regulate the polarization of microglia.3.Ultrashort wave combined with human umbilical cord mesenchymal stem cells can improve the inflammatory microenvironment and relieve spinal cord injury through the MK2/TTP/NLRP3 signaling pathway. |
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