Abstract
Objective To investigate the curative effects of HUC-MSCs combined with USW on spinal cord injury (SCI) and the effects on inflammatory microenvironment and to explore the regulatory mechanisms of MK2/TTP signalling pathway and NLRP3 inflammasome. Methods The SCI rat model was established using the modified Allen method; rats were administered with USW, HUC-MSCs, and combination therapy of USW and HUC-MSCs; the therapeutic efficacies in each group of rats were monitored and represented in BBB score. SCI levels were observed using HE staining and IF. The microglia polarisation state and released contents of inflammatory factors were detected. IF and Western Blotting were performed on to detect the expression levels of MK2/TTP signalling pathway and NLRP3 inflammasome-related proteins. Furthermore, the regulatory mechanisms of MK2/TTP pathway and NLRP3 were explored by performing on the in vitro study. Results Combination therapy of USW and HUC-MSCs was found of significant efficacy on improving motor functions of SCI rats, and it was further proved that this combination therapy can reduce spinal cord injury in SCI rats, of which USW plays a more important role. While transplantation of HUC-MSCs can promote microglial cells developing to SCI repair, and M2 microglial cells were taking advantages gradually. The combination therapy can inhibit the expression of MK2; downregulate NLRP3 inflammasome; suppress the expression levels of pro-caspase-1, pro-IL-1β, and pro-IL-18; and simultaneously suppress the release of IL-1β and IL-18, thereby reducing spinal cord neurons apoptosis. It was found that the steady state of microglial polarisation maintained by combined treatment of USW and HUC-MSCs was destroyed with the upregulation of MK2 expression in cells, of which, M1 type microglial cell was dominant and the increased contents of inflammatory factors were detected. However, overexpressed MK2 relieved the inhibition of NLRP3 expression by TTP. Conclusions Combination therapy of USW and HUC-MSCs can downregulate NLRP3 expression, relieve inflammatory responses, improve immune microenvironment, and rescue spinal cord injury via suppressing phosphorylation level of MK2.
Highlights
Spinal cord injury (SCI) usually leads to the patient’s loss of feeling below the injury level and motor dysfunction, which greatly reduces the patient’s quality of life [1]
Immunofluorescence staining showed that HUC-mesenchymal stem cells (MSCs) were positive for the surface markers CD105, CD29, CD44, and CD73; they are negative for hematopoietic stem cell marker CD45 (Figure S1B)
NeuN was detected using IF, illustrating that the number of neurons was decreased with SCI occurrence (Figure 1(c)), while monotherapy with ultrashort wave (USW) and combination therapy were both found of improvement efficacy on SCI
Summary
Spinal cord injury (SCI) usually leads to the patient’s loss of feeling below the injury level and motor dysfunction, which greatly reduces the patient’s quality of life [1]. Studies have suggested that human umbilical cord mesenchymal stem cells (HUC-MSCs) used in the treatment of SCI. The severe microenvironment of the injury site is a primary reason for limiting the clinical application of HUC-MSCs, as the occurrence of SCI can break the original balance of the spinal cord microenvironment, and the inflammatory cells and inflammatory factors in the microenvironment can cause the apoptosis of the implanted mesenchymal stem cells (MSCs), inhibiting the posttransplanted survival rate in the body [9, 10]. IFN-γ, TNF-α, and IL-1β can inhibit MSC proliferation and affect the differentiation trend of MSCs [12], suggesting that the SCI-caused immune microenvironment has limited the survival rate and therapeutic effects of HUC-MSCs after transplantation
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