Salidroside promotes the repair of spinal cord injury by inhibiting astrocyte polarization, promoting neural stem cell proliferation and neuronal differentiation.

Spinal cord injury (SCI) remains a formidable challenge, lacking effective treatments. Following SCI, neural stem cells (NSCs) migrate to SCI sites, offering a potential avenue for nerve regeneration, but the effectiveness of this intrinsic repair mechanism remains suboptimal. Salidroside has demonstrated pro-repair attributes in various pathological conditions, including arthritis and cerebral ischemia, and the ability to curtail early-stage inflammation following SCI. However, the specific role of salidroside in the late-stage repair processes of SCI remains less defined. In this investigation, we observed that continuous salidroside treatment in SCI mice improved motor function recovery. Immunofluorescence-staining corroborated salidroside's capacity to stimulate nerve regeneration and remyelination, suppress glial scar hyperplasia, reduce the activation of neurotoxic A1 astrocytes, and facilitate NSCs migration towards the injured region. Mechanistically, in vitro experiments elucidated salidroside's significant role in restraining astrocyte proliferation and A1 polarization. It was further established that A1 astrocytes hinder NSCs proliferation while inducing their differentiation into astrocytes. Salidroside effectively ameliorated this inhibition of NSCs proliferation through diminishing c-Jun N-terminal kinase (JNK) pathway phosphorylation and restored their differentiation into neurons by suppressing the signal transducer and activator of transcription 3 (STAT3) pathway. In summary, our findings suggest that salidroside holds promise as a therapeutic agent for traumatic SCI treatment.

Simplified Chinese version of the core outcome measures index (COMI) for patients with neck pain: cross-cultural adaptation and validation.

PURPOSE: The aim of this study was to translate and cross-culturally adapt the Core Outcome Measures Index for (COMI) into a Simplified Chinese version (COMI-SC) and to evaluate the reliability and validity of COMI-SC in patients with neck pain. METHODS: The COMI-neck was translated into Chinese according to established methods. The COMI-neck questionnaire was then completed by 122 patients with a hospital diagnosis of neck pain. Reliability was assessed by calculating Cronbach's alpha and intraclass correlation coefficient (ICC). Construct validity was assessed by correlating the COMI-neck with the Neck Pain and Disability Scale (NPDS), the Neck Disability Index (NDI), the VAS and the Short Form (36) Health Survey (SF-36). Using confirmatory factor analysis to validate the structural, convergent and discriminant validity of the questionnaire. RESULTS: The COMI-neck total scores were well distributed, with no floor or ceiling effects. Internal consistency was excellent (Cronbach's alpha = 0.861). Moderate to substantial correlations were found between COMI-neck and NPDS (r = 0.420/0.416/0.437, P < 0.001), NDI (r = 0.890, P < 0.001), VAS (r = 0.845, P < 0.001), as well as physical function (r = - 0.989, P < 0.001), physical role (r = - 0.597, P < 0.001), bodily pain (r = - 0. 639, P < 0.001), general health (r = - 0.563, P < 0.001), vitality (r = - 0.702, P < 0.001), social functioning (r = - 0.764, P < 0.001), role emotional (r = - 0.675, P < 0.001) and mental health (r = - 0.507, P < 0.001) subscales of the SF-36. An exploratory factor analysis revealed that the 3-factor loading explained 71.558% of the total variance [Kaiser-Mayer-Olkin (KMO) = 0.780, C2 = 502.82, P < 0.001]. CMIN/DF = 1.813, Tucker-Lewis index (TLI) = 0.966 (> 0.9), Comparative Fit Index (CFI) = 0.982 (> 0.9), Normed Fit Index (NFI) = 0.961 (> 0.9), RMSEA = 0.082 (< 0.5) indicating that the model fits well. CONCLUSION: COMI-neck was shown to have acceptable reliability and validity in patients with non-specific chronic neck pain and could be recommended for patients in mainland China. LEVEL OF EVIDENCE: Diagnostic: individual cross-sectional studies with consistently applied reference standard and blinding.

Exosomes derived from platelet-rich plasma administration in site mediate cartilage protection in subtalar osteoarthritis.

Subtalar osteoarthritis (STOA) is often secondary to chronic ankle sprains, which seriously affects the quality of life of patients. Due to its etiology and pathogenesis was not studied equivocally yet, there is currently a lack of effective conservative treatments. Although they have been used for tissue repair, platelet-rich plasma-derived exosomes (PRP-Exo) have the disadvantage of low retention and short-lived therapeutic effects. This study aimed to determine whether incorporation of PRP-Exo in thermosensitive hydrogel (Gel) increased their retention in the joint and thereby playing a therapeutic role on STOA due to chronic mechanical instability established by transecting lateral ligaments (anterior talofibular ligament (ATFL)/calcaneal fibular ligament (CFL)). PRP-Exo incorporated Gel (Exo-Gel) system, composed of Poloxamer-407 and 188 mixture-based thermoresponsive hydrogel matrix in an optimal ratio, was determined by its release ability of Exo and rheology of Gel response to different temperature. The biological activity of Exo-Gel was evaluated in vitro, and the therapeutic effect of Exo-Gel on STOA was evaluated in vivo. Exo released from Exo-Gel continuously for 28 days could promote the proliferation and migration of mouse bone mesenchymal stem cells (mBMSCs) and chondrocytes, at the same time enhance the chondrogenic differentiation of mBMSCs, and inhibit inflammation-induced chondrocyte degeneration. In vivo experiments confirmed that Exo-Gel increased the local retention of Exo, inhibited the apoptosis and hypertrophy of chondrocytes, enhanced their proliferation, and potentially played the role in stem cell recruitment to delay the development of STOA. Thus, Delivery of PRP-Exo incorporated in thermosensitive Gel provides a novel approach of cell-free therapy and has therapeutic effect on STOA.

GIT1 protects traumatically injured spinal cord by prompting microvascular endothelial cells to clear myelin debris.

The clearance of myelin debris is a critical step in the functional recovery following spinal cord injury (SCI). As phagocytes do, microvascular endothelial cells (MECs) participate in myelin debris clearance at the injury site within one week. Our group has verified that G protein-coupled receptor kinase 2 interacting protein-1 (GIT1) is essential in autophagy and angiogenesis, both of which are tightly related to the uptake and degradation of myelin debris by MECs. Here, we analyzed the performance and mechanism of GIT1 in myelin debris clearance after SCI. The SCI contusion model was established and in vitro MECs were treated with myelin debris. Better recovery from traumatic SCI was observed in the GIT1 WT mice than in the GIT1 KO mice. More importantly, we found that GIT1 prompted MECs to clear myelin debris and further enhanced MECs angiogenesis in vivo and in vitro. Mechanistically, GIT1-mediated autophagy contributed to the clearance of myelin debris by MECs. In this study, we demonstrated that GIT1 may prompt MECs to clear myelin debris via autophagy and further stimulate MECs angiogenesis via upregulating VEGF. Our results indicate that GITI may serve as a promising target for accelerating myelin debris clearance and improving SCI recovery.

Salidroside Ameliorates Mitochondria-Dependent Neuronal Apoptosis after Spinal Cord Ischemia-Reperfusion Injury Partially through Inhibiting Oxidative Stress and Promoting Mitophagy.

Ischemia-reperfusion injury is the second most common injury of the spinal cord and has the risk of neurological dysfunction and paralysis, which can seriously affect patient quality of life. Salidroside (Sal) is an active ingredient extracted from Herba Cistanche with a variety of biological attributes such as antioxidant, antiapoptotic, and neuroprotective activities. Moreover, Sal has shown a protective effect in ischemia-reperfusion injury of the liver, heart, and brain, but its effect in ischemia-reperfusion injury of the spinal cord has not been elucidated. Here, we demonstrated for the first time that Sal pretreatment can significantly improve functional recovery in mice after spinal cord ischemia-reperfusion injury and significantly inhibit the apoptosis of neurons both in vivo and in vitro. Neurons have a high metabolic rate, and consequently, mitochondria, as the main energy-supplying suborganelles, become the main injury site of spinal cord ischemia-reperfusion injury. Mitochondrial pathway-dependent neuronal apoptosis is increasingly confirmed by researchers; therefore, Sal's effect on mitochondria naturally attracted our attention. By means of a range of experiments both in vivo and in vitro, we found that Sal can reduce reactive oxygen species production through antioxidant stress to reduce mitochondrial permeability and mitochondrial damage, and it can also enhance the PINK1-Parkin signaling pathway and promote mitophagy to eliminate damaged mitochondria. In conclusion, our results show that Sal is beneficial to the protection of spinal cord neurons after ischemia-reperfusion injury, mainly by reducing apoptosis associated with the mitochondrial-dependent pathway, among which Sal's antioxidant and autophagy-promoting properties play an important role.

Macrophage GIT1 Contributes to Bone Regeneration by Regulating Inflammatory Responses in an ERK/NRF2-Dependent Way.

Despite the best treatment, approximately 10% of fractures still face undesirable repair. Recently, many studies have focused on the importance of macrophages in bone repair; however, the cellular mechanisms by which they work are not yet fully understood. In this study, we explored the functions of macrophage G-protein-coupled receptor interacting protein 1 (GIT1) in healing a tibial monocortical defect model. Using GIT1flox/flox Lyz2-Cre (GIT1 CKO) mice, we observed that a GIT1 deficiency in the macrophages led to an exacerbation of interleukin 1ß (IL1ß) production, more M1-like macrophage infiltration, and impaired intramembranous ossification in vivo. The results of in vitro assays further indicated that the macrophage GIT1 plays a critical role in several cellular processes in response to lipopolysaccharide (LPS), such as anti-oxidation, IL1ß production alleviation, and glycolysis control. Although GIT1 has been recognized as a scaffold protein, our data clarified that GIT1-mediated extracellular-signal-regulated kinase (ERK) phosphorylation could activate nuclear factor (erythroid-derived 2)-like 2 (NRF2) in macrophages after LPS treatment. Moreover, we demonstrated that macrophage GIT1-activated ERK/NRF2 negatively regulates the 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3), facilitating the decrease of glycolysis. Our findings uncovered a previously unrecognized role of GIT1 in regulating ERK/NRF2 in macrophages to control the inflammatory response, suggesting that macrophage GIT1 could be a potential target to improve bone regeneration. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research..

Exosomal miRNA-128-3p from mesenchymal stem cells of aged rats regulates osteogenesis and bone fracture healing by targeting Smad5.

Transplantation of mesenchymal stem cells (MSCs) has been considered an effective therapeutic treatment for a variety of diseases including bone fracture. However, there are associated complications along with MSCs transplantation. There is evidence to show that exosomes (Exos) derived from MSCs exert a similar paracrine function. In addition, repair capabilities of MSCs decline with age. Hence, this study aims to confirm whether the Exos protective function on osteogenic differentiation and fracture healing from aged MSCs was attenuated. This information was used in order to investigate the underlying mechanism. MSCs were successfully isolated and identified from young and aged rats, and Exos were then obtained. Aged-Exos exhibited significantly attenuated effects on MSCs osteogenic differentiation in vitro and facture healing in vivo. Using miRNA array analysis, it was shown that miR-128-3p was markedly upregulated in Aged-Exos. In vitro experiments confirmed that Smad5 is a direct downstream target of miR-128-3p, and was inhibited by overexpressed miR-128-3p. A series gain- and loss- function experiment indicated that miR-128-3P serves a suppressor role in the process of fracture healing. Furthermore, effects caused by miR-128-3P mimic/inhibitor were reversed by the application of Smad5/siSmad5. Taken together, these results suggest that the therapeutic effects of MSCs-derived Exos may vary according to differential expression of miRNAs. Exosomal miR-128-3P antagomir may act as a promising therapeutic strategy for bone fracture healing, especially for the elderly.

Macrophage MSR1 promotes the formation of foamy macrophage and neuronal apoptosis after spinal cord injury.

BACKGROUND: A sustained inflammatory response following spinal cord injury (SCI) contributes to neuronal damage, inhibiting functional recovery. Macrophages, the major participants in the inflammatory response, transform into foamy macrophages after phagocytosing myelin debris, subsequently releasing inflammatory factors and amplifying the secondary injury. Here, we assessed the effect of macrophage scavenger receptor 1 (MSR1) in phagocytosis of myelin debris after SCI and explained its possible mechanism. METHODS: The SCI model was employed to determine the critical role of MSR1 in phagocytosis of myelin debris in vivo. The potential functions and mechanisms of MSR1 were explored using qPCR, western blotting, and immunofluorescence after treating macrophages and RAW264.7 with myelin debris in vitro. RESULTS: In this study, we found improved recovery from traumatic SCI in MSR1-knockout mice over that in MSR1 wild-type mice. Furthermore, MSR1 promoted the phagocytosis of myelin debris and the formation of foamy macrophage, leading to pro-inflammatory polarization in vitro and in vivo. Mechanistically, in the presence of myelin debris, MSR1-mediated NF-κB signaling pathway contributed to the release of inflammatory mediators and subsequently the apoptosis of neurons. CONCLUSIONS: Our study elucidates a previously unrecognized role of MSR1 in the pathophysiology of SCI and suggests that its inhibition may be a new treatment strategy for this traumatic condition.

The protective effort of GPCR kinase 2-interacting protein-1 in neurons via promoting Beclin1-Parkin induced mitophagy at the early stage of spinal cord ischemia-reperfusion injury.

In spinal cord ischemia-reperfusion (I/R) injury, large amounts of reactive oxygen species can cause mitochondrial damage. Therefore, mitophagy acts as the main mechanism for removing damaged mitochondria and protects nerve cells. This study aimed to illustrate the important role of GPCR kinase 2-interacting protein-1 (GIT1) in mitophagy in vivo and in vitro. The level of mitophagy in the neurons of Git1 knockout mice was significantly reduced after ischemia-reperfusion. However, the overexpression of adeno-associated virus with Git1 promoted mitophagy and inhibited the apoptosis of neurons. GIT1 regulated the phosphorylation of Beclin-1 in Thr119, which could promote the translocation of Parkin to the mitochondrial outer membrane. This process was independent of PTEN-induced kinase 1 (PINK1), but it could not rescue the role in the absence of PINK1. Overall, GIT1 enhanced mitophagy and protected neurons against ischemia-reperfusion injury and, hence, might serve as a new research site for the protection of ischemia-reperfusion injury.

Hypoxic mesenchymal stem cell-derived exosomes promote bone fracture healing by the transfer of miR-126.

Increasing evidence has suggested that paracrine mechanisms might be involved in the underlying mechanism of mesenchymal stem cells (MSCs) transplantation, and exosomes are an important component of this paracrine role. However, MSCs are usually exposed to normoxia (21% O2) in vitro but experience large differences in oxygen concentration in the body under hypoxia. Indeed, hypoxic precondition of MSCs can enhance their paracrine effects. The main purpose of this study was to determine whether exosomes derived from MSCs under hypoxia (Hypo-Exos) exhibit greater effects on bone fracture healing than those under normoxia (Exos). Using in vivo bone fracture model and in vitro experiments including cell proliferation assay, cell migration assay and so on, we confirmed that Hypo-Exos administration promoted angiogenesis, proliferation and migration to a greater extent when compared to Exos. Furthermore, utilizing a series in vitro and in vivo gain and loss of function experiments, we confirmed a functional role for exosomal miR-126 in the process of bone fracture healing. Meanwhile, we found that knockdown of hypoxia inducible factor 1 (HIF-1α) resulted in a significant decrease of miR-126 in MSCs and exosomes, thereby abolishing the effects of Hypo-Exos. In conclusion, our results demonstrated a mechanism by which Hypo-Exos promote bone fracture healing through exosomal miR-126. Moreover, hypoxia preconditioning mediated enhanced production of exosomal miR-126 through the activation of HIF-1α. Hypoxia preconditioning represents an effective and promising method for the optimization of the therapeutic actions of MSC-derived exosomes for bone fracture healing. STATEMENT OF SIGNIFICANCE: Studies have confirmed that transplantation of exosomes exhibit similar therapeutic effects and functional properties to directly-transplanted stem cells but have less significant adverse effects. However, during in vitro culture conditions, MSCs are usually exposed to normoxia (21% O2) which is very different to the oxygen concentrations found in the body under natural physiological conditions. Our results demonstrated a mechanism by which Hypo-Exos promote bone fracture healing through exosomal miR-126 and the SPRED1/Ras/Erk signaling pathway. Moreover, hypoxia preconditioning mediated enhanced production of exosomal miR-126 through the activation of HIF-1α. Hypoxia preconditioning represents an effective and promising method for the optimization of the therapeutic actions of MSC-derived exosomes for bone fracture healing.

miR-624-5p promoted tumorigenesis and metastasis by suppressing hippo signaling through targeting PTPRB in osteosarcoma cells.

BACKGROUND: Accumulating evidence indicates that aberrant microRNA (miRNA) expression contributes to osteosarcoma progression. This study aimed to elucidate the association between miR-624-5p expression and osteosarcoma (OS) development and to investigate its underlying mechanism. METHODS: We analyzed GSE65071 from the GEO database and found miR-624-5p was the most upregulated miRNA. The expression of miR-624-5p and its specific target gene were determined in human OS specimens and cell lines by RT-PCR and western blot. The effects of miR-624-5p depletion or ectopic expression on OS proliferation, migration and invasion were evaluated in vitro using CCK-8 proliferation assay, colony formation assay, transwell assay, would-healing assay and 3D spheroid BME cell invasion assay respectively. We investigated in vivo effects of miR-624-5p using a mouse tumorigenicity model. Besides, luciferase reporter assays were employed to identify interactions between miR-624-5p and its specific target gene. RESULTS: miR-624-5p expression was upregulated in OS cells and tissues, and overexpressing miR-624-5p led to a higher malignant level of OS, including cell proliferation, migration and invasion in vitro and in vivo. Protein tyrosine phosphatase receptor type B (PTPRB) was negatively correlated with miR-624-5p expression in OS tissues. Using the luciferase reporter assay and Western blotting, PTPRB was confirmed as a downstream target of miR-624-5p. PTPRB restored the effects of miR-624-5p on OS migration and invasion. The Hippo signaling pathway was identified as being involved in the miR-624-5p/PTPRB axis. CONCLUSIONS: In conclusion, our results suggest that miR-624-5p is a negative regulator of PTPRB and a risk factor for tumor metastasis in OS progression.

Blocking Notch signal pathway suppresses the activation of neurotoxic A1 astrocytes after spinal cord injury.

Spinal cord injury (SCI) is a catastrophic disease which has complicated pathogenesis including inflammation, oxidative stress and glial scar formation. Astrocytes are the most abundant cells in central nervous system and fulfill homeostatic functions. Recent studies have described a new reactive phenotype of astrocytes, A1, induced by inflammation, which may have negative effects in SCI. As the Notch signaling pathway has been linked to cell differentiation and inflammation, we aimed to investigate its potential role in the differentiation of astrocytes in SCI. Contusive SCI rat model showed elevated A1 astrocyte numbers at the damage site 28 days after SCI and the expression levels of Notch signaling and its downstream genes were upregulated parallelly. Western blotting, RT-qPCR and immunofluorescence revealed that blocking of Notch pathway using γ-secretase blocker (DAPT) suppressed the differentiation of A1 astrocytes. Flow cytometry, and TUNEL staining indicated that DAPT alleviated neuronal apoptosis and axonal damage caused by A1 astrocytes likely through the Notch-dependent release of pro-inflammatory factors. CO-IP and western blotting revealed an interaction between Notch pathway and signal transducer and activator of transcription 3 (Stat3), which played a vital role in differentiation of A1 astrocytes. We conclude that phenotypic transition of A1 astrocytes and their neurotoxity were controlled by the Notch-Stat3 axis and that Notch pathway in astrocytes may serve as a promising therapeutic target for SCI.

GIT1 regulates angiogenic factor secretion in bone marrow mesenchymal stem cells via NF-κB/Notch signalling to promote angiogenesis.

OBJECTIVES: Osteogenesis is coupled with angiogenesis during bone remodelling. G-protein-coupled receptor (GPCR) kinase 2-interacting protein-1 (GIT1) is an important protein that participates in fracture healing by regulating angiogenesis. This study investigated whether GIT1 could affect bone mesenchymal stem cells (BMSCs) to secrete angiogenic factors to enhance fracture healing by promoting angiogenesis and its possible mechanism. MATERIALS AND METHODS: The angiogenesis of mice post-fracture was detected by micro-CT and immunofluorescence. Subsequently, vascular endothelial growth factor (VEGF) level in mouse and human BMSCs (hBMSCs) under TNF-α stimulation was detected. The hBMSCs were transfected with GIT1 shRNAs to further explore the relationship between GIT1 and VEGF and angiogenesis in vitro. Furthermore, based on previous research on GIT1, possible signal pathways were investigated. RESULTS: GIT1 knockout mice exhibited impaired angiogenesis and delayed fracture healing. And GIT1 deficiency remarkably reduced the expression of VEGF mRNA in BMSCs, which affected the proliferation and migration of human umbilical vein endothelial cells. GIT1 knockdown inhibited the activation of Notch and NF-κB signals by decreasing nuclear transportation of NICD and P65/P50, respectively. Overexpression of the canonical NF-κB subunits P65 and P50 markedly increased NICD-dependent activation of recombination signal-binding protein-jκ reporter. Finally, GIT1 enhanced the affinity of NF-κB essential modulator (NEMO) for K63-linked ubiquitin chains via interaction with NEMO coiled-coil 2 domains. CONCLUSION: These data revealed a positive role for GIT1 by modulating the Notch/NF-κB signals which promoting paracrine of BMSCs to enhance angiogenesis and fracture healing.

GIT1 is critical for formation of the CD31hiEmcnhi vessel subtype in coupling osteogenesis with angiogenesis via modulating preosteoclasts secretion of PDGF-BB.

G protein-coupled receptor kinase 2 interacting protein-1 (GIT1) is a scaffold protein that plays a vital role in bone modeling and remodeling during osteogenesis coupled with angiogenesis. Recent studies have shown that a specialized subset of vascular endothelium strongly positive for CD31 and Endomucin (CD31hiEmcnhi) is coupled with anabolic bone formation. Based on our previous finding that GIT1 knockout (GIT1 KO) mice have impaired angiogenesis and bone formation, we hypothesized that GIT1 affects formation of the CD31hiEmcnhi vessel subtype. In the current study, GIT1 knockout (GIT1 KO) mice displayed a significant decrease in trabecular bone mass and CD31hiEmcnhi vessel number, compared to their wild-type counterparts. In the fracture healing mouse model, GIT1 KO mice contained a lower number of CD31hiEmcnhi vessels in fracture callus at days 7 and 14. However, no significant differences in the number of preosteoclasts in bone marrow, trabecular bone and callus in GIT1 KO mice were observed, compared with wild-type mice. Notably, concentrations of serum platelet-derived growth factor-BB(PDGF-BB) secreted by preosteoclasts associated with CD31hiEmcnhi vessel formation were lower in GIT1 KO mice. In addition, PDGF-BB-associated expression of phosphorylated extracellular signal-regulated kinase- 1/2 (ERK1/2) and specificity protein 1 (SP1) was significantly decreased in preosteoclasts of GIT1 KO mice. These results collectively suggest that GIT1 is a critical participant in formation of the CD31hiEmcnhi vessel subtype, highlighting a novel biologic function of this scaffold protein in preosteoclasts.