Background: Acute spinal cord injury (ASCI) is a serious central nervous system condition that carries a high rate of disability and mortality, making it a primary focus of clinical research. The commonly used drugs for ASCI are ganglioside (GM-1) and methylprednisolone (MP). There are few studies available on the impact of GM-1 combined with MP pulse therapy on the clinical outcomes of patients with ASCI and the underlying mechanisms involved. Methods: In a randomized controlled trial, the 100 patients with ASCI who were treated at our hospital from 2021 to 2024 were selected for this study. They were arbitrarily separated into two equal cohorts, a control cohort and an observation cohort, each containing 50 patients. While the control cohort underwent treatment solely with GM-1, the observation cohort additionally received MP pulse therapy, both treatments administered over a span of six months. The general information of patients was recorded. The motor score, acupuncture pain score, light touch score, Barthel Index (BI) for evaluating daily living capabilities, American Spinal Injury Association (ASIA) neurological function score, and visual analogue scale (VAS) score were analyzed between the two groups before and after treatment to evaluate the recovery of neurological function. Additionally, quantitative real-time PCR (qRT-PCR) was used to assess the serum levels of miR-21-5p in both groups. Moreover, the concentrations of serum inflammatory markers, such as C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), along with nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), were quantified using ELISA. The Pearson method was employed to evaluate the correlation between miR-21-5p and the levels of CRP, TNF-alpha, IL-6, NGF, and BDNF. The receiver operating characteristic (ROC) curve was employed to evaluate the predictive ability of serum miR-21-5p regarding treatment effectiveness in patients with ASCI. Results: Three patients dropped out of the control group, and no patient dropped out of the observation group. There were no significant variations in the baseline data, index scores, or factor levels between the two groups prior to treatment (P > 0.05). After undergoing therapy, the observation group exhibited significantly higher BI index, ASIA motor and sensory scores, as well as increased in serum miR-21-5p levels, BDNF, and NGF compared to the reference group (P < 0.05). Furthermore, the observation group exhibited marked decreases in VAS ratings as well as serum CRP, TNF-alpha, and IL-6 levels, in stark contrast to the control group (P < 0.05). Furthermore, the level of miR-21-5p exhibited a negative correlation with the levels of CRP, TNF-alpha, and IL-6 (P < 0.05), while showing a positive correlation with the levels of BDNF and NGF (P < 0.05). The ROC curve analysis indicated that miR-21-5p possesses strong diagnostic potential for assessing ASCI efficacy, with an area under the curve (AUC) of 0.987, a sensitivity rate of 91.49%, and a specificity rate of 100.00%. Conclusions: The combination of GM-1 and MP pulse therapy can significantly enhance neurological symptoms and facilitate the restoration of neurological function in patients with ASCI. This effect may be ascribed to the increased levels of miR-21-5p, BDNF, and NGF, along with a reduction in serum inflammatory factors.

This article presents a method for using a conventional high-voltage cable as a transducer and exciting it with a pulse power supply to generate perfect cylindrical acoustic waves in water. The experimental results prove that the acoustic pulse strictly follows the propagation law of cylindrical waves. There is a slight fluctuation of sound pressure in the direction parallel to the cable and no directivity in the plane perpendicular to the cable. Under excitation energy of 1000 J, the duration of the acoustic pulse generated by a 12-m cable is about 500 μ s, the dominant frequency is around 3000 Hz, the effective source level reaches 195 dB, and the energy conversion efficiency is 0.012%. This method breaks through the traditional mode of forming a line source by combining point sources. The vibration and acoustic radiation along the cable are continuous, and the cylindrical waves are very stable. Based on the comparative analysis of marine single-channel seismic exploration, line source shows the potential to change the traditional method based on point source and spherical wave theory. It provides a new method for simplifying the survey system and improving exploration accuracy.

This work presents a case study about applying a homemade boomer source working with a well-designed field geometry in an offshore seismic survey. Some measurements were carried out to calibrate the boomer source acoustic characteristics in an anechoic tank. Then a catamaran, taking account of the boomer source signature, is designed to control the source depth and entirely use ghosts as part of the source wavelet. This way, the vertical resolution is sacrificed to signal-to-noise ratio (SNR), and the source wavelet meets the minimum phase characteristics. Furthermore, the detailed analytical results of the calibrated source wavelets also provide a reliable basis for determining post-processing parameters. Two surveys have been conducted on the same line using this source in combination with two different hydrophone streamers. Some valuable experiences in field data acquisition and post-processing are analyzed and summarized. The most important one is taking the ghost part of the source wavelet by controlling source depth. This work is a reliable reference to other research on improving the quality of seismic imaging of offshore shallow strata.

Drug delivery systems with high content of drug can minimize excipients administration, reduce side effects, improve therapeutic efficacy and/or promote patient compliance. However, engineering such systems is extremely challenging, as their loading capacity is inherently limited by the compatibility between drug molecules and carrier materials. To mitigate the drug-carrier compatibility limitation towards therapeutics encapsulation, we developed a sequential solidification strategy. In this strategy, the precisely controlled diffusion of solvents from droplets ensures the fast in-droplet precipitation of drug molecules prior to the solidification of polymer materials. After polymer solidification, a mass of drug nanoparticles is embedded in the polymer matrix, forming a nano-in-micro structured microsphere. All the obtained microspheres exhibit long-term storage stability, controlled release of drug molecules, and most importantly, high mass fraction of therapeutics (21.8–63.1 wt%). Benefiting from their high drug loading degree, the nano-in-micro structured acetalated dextran microspheres deliver a high dose of methylprednisolone (400 μg) within the limited administration volume (10 μL) by one single intrathecal injection. The amount of acetalated dextran used was 1/433 of that of low drug-loaded microspheres. Moreover, the controlled release of methylprednisolone from high drug-loaded microspheres contributes to improved therapeutic efficacy and reduced side effects than low drug-loaded microspheres and free drug in spinal cord injury therapy.

Spinal cord injury (SCI) can lead to severe motor and sensory dysfunction with high disability and mortality. In recent years, mesenchymal stem cell (MSC)-secreted nano-sized exosomes have shown great potential for promoting functional behavioral recovery following SCI. However, MSCs are usually exposed to normoxia in vitro, which differs greatly from the hypoxic micro-environment in vivo. Thus, the main purpose of this study was to determine whether exosomes derived from MSCs under hypoxia (HExos) exhibit greater effects on functional behavioral recovery than those under normoxia (Exos) following SCI in mice and to seek the underlying mechanism. Electron microscope, nanoparticle tracking analysis (NTA), and western blot were applied to characterize differences between Exos and HExos group. A SCI model in vivo and a series of in vitro experiments were performed to compare the therapeutic effects between the two groups. Next, a miRNA microarray analysis was performed and a series of rescue experiments were conducted to verify the role of hypoxic exosomal miRNA in SCI. Western blot, luciferase activity, and RNA-ChIP were used to investigate the underlying mechanisms. Our results indicate that HExos promote functional behavioral recovery by shifting microglial polarization from M1 to M2 phenotype in vivo and in vitro. A miRNA array showed miR-216a-5p to be the most enriched in HExos and potentially involved in HExos-mediated microglial polarization. TLR4 was identified as the target downstream gene of miR-216a-5p and the miR-216a-5p/TLR4 axis was confirmed by a series of gain- and loss-of-function experiments. Finally, we found that TLR4/NF-κB/PI3K/AKT signaling cascades may be involved in the modulation of microglial polarization by hypoxic exosomal miR-216a-5p. Hypoxia preconditioning represents a promising and effective approach to optimize the therapeutic actions of MSC-derived exosomes and a combination of MSC-derived exosomes and miRNAs may present a minimally invasive method for treating SCI.

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 viv o 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.

Spinal cord injury (SCI) is a severe neurological disease; however, there is no effective treatment for spinal cord injury. Neuroinflammation involves the activation of resident microglia and the infiltration of macrophages is the major pathogenesis of SCI secondary injury and considered to be the therapeutic target of SCI. Parthenolide (PN) has been reported to exert anti-inflammatory effects in fever, migraines, arthritis, and superficial inflammation; however, the role of PN in SCI therapeutics has not been clarified. In this study, we showed that PN could improve the functional recovery of spinal cord in mice as revealed by increased BMS scores and decreased cavity of spinal cord injury in vivo. Immunofluorescence staining experiments confirmed that PN could promote axonal regeneration, increase myelin reconstitution, reduce chondroitin sulfate formation, inhibit scar hyperplasia, suppress the activation of A1 neurotoxic reactive astrocytes and facilitate shift from M1 to M2 polarization of microglia/macrophages. To verify how PN exerts its effects on microglia/macrophages polarization, we performed the mechanism study in vitro in microglia cell line BV-2. PN could significantly reduce M1 polarization in BV2 cells and partially rescue the decrease in the expression of M2 phenotype markers of microglia/macrophage induced by LPS, but no significant effect on M2 polarization stimulated with IL-4 was observed. Further study demonstrated PN inhibited NF-κB signal pathway directly or indirectly, and suppressed activation of signal transducer and activator of transcription 1 or 3 (STAT1/3) via reducing the expression of HDAC1 and subsequently increasing the levels of STAT1/3 acetylation. Overall, our study illustrated that PN may be a promising strategy for traumatic SCI.

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.

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.

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 (CD31(hi)Emcn(hi)) 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 CD31(hi)Emcn(hi) vessel subtype. In the current study, GIT1 knockout (GIT1 KO) mice displayed a significant decrease in trabecular bone mass and CD31(hi)Emcn(hi) vessel number, compared to their wild-type counterparts. In the fracture healing mouse model, GIT1 KO mice contained a lower number of CD31(hi)Emcn(hi) 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 CD31(hi)Emcn(hi) 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 CD31(hi)Emcn(hi) vessel subtype, highlighting a novel biologic function of this scaffold protein in preosteoclasts.

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.

The neuronal apoptosis program associated with spinal cord injury (SCI) has a severe impact on spinal cord function, which leads to further secondary and permanent neuronal damage that may cause irreparable damage to the central nervous system. Activation of the Wnt/β-catenin signaling pathway is effective in reducing apoptosis and preventing SCI. Harpagide is one of the main active constituents of the iridoid class of molecules, which have neuroprotective effects after SCI. In this study, we demonstrated that harpagide attenuated neuronal apoptosis via activation of the Wnt/β-catenin signaling pathway. This resulted in a promotion of axonal regeneration and an inhibition of glial scar formation, which ultimately improved functional behavioral recovery after SCI in rats. Specifically, the administration of harpagide after SCI increased the expression levels of β-catenin, c-myc and cyclin D1 proteins in spinal cord neurons, as well as increased the number of motor neurons and reduced the size of the SCI lesion area. In addition, the administration of harpagide after SCI also decreased the protein expression levels as well as the number of cells immuno-stained for the pro-apoptotic proteins Bax and cleaved-caspase 3. The expression level of the anti-apoptotic protein Bcl-2 was also increased. When the Wnt /β-catenin signaling pathway was inhibited, a weakened anti-apoptotic effect of harpagide was observed. Additionally, the application of harpagide led to an increase in NF200 staining and a reduction in GFAP staining in the SCI injury site. In summary, our study suggested that harpagide may be a promising drug for the treatment of SCI.

Spinal cord injury (SCI) can cause severe irreversible motor dysfunction and even death. Neural stem cell (NSC) transplantation can promote functional recovery after acute SCI in experimental animals, but numerous issues, including low-transplanted cell survival rate, cell de-differentiation, and tumor formation need to be resolved before routine clinical application is feasible. Recent studies have shown that transplanted stem cells facilitate regeneration through release of paracrine factors. Small extracellular vesicles (sEVs), the smallest known membrane-bound nanovesicles, are involved in complex intercellular communication systems and are an important vehicle for paracrine delivery of therapeutic agents. However, the application of NSC-derived small extracellular vesicles (NSC-sEVs) to SCI treatment has not been reported. We demonstrate that NSC-sEVs can significantly reduce the extent of SCI, improve functional recovery, and reduce neuronal apoptosis, microglia activation, and neuroinflammation in rats. Furthermore, our study suggests that NSC-sEVs can regulate apoptosis and inflammatory processes by inducing autophagy. In brief, NSC-sEVs increased the expression of the autophagy marker proteins LC3B and beclin-1, and promoted autophagosome formation. Following NSC-sEV infusion, the SCI area was significantly reduced, and the expression levels of the proapoptotic protein Bax, the apoptosis effector cleaved caspase-3, and the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were significantly reduced, whereas the expression level of the anti-apoptotic protein Bcl-2 was upregulated. In the presence of the autophagy inhibitor 3MA, however, these inhibitory effects of NSC-sEVs on apoptosis and neuroinflammation were significantly reversed. Our results show for the first time that NSC-sEV treatment has the potential to reduce neuronal apoptosis, inhibit neuroinflammation, and promote functional recovery in SCI model rats at an early stage by promoting autophagy.

Neural stem cell-derived small extracellular vesicles (NSC-sEVs) play an important role in the repair of tissue damage. Our previous in vitro and in vivo studies found that preconditioning with NSC-sEVs promoted the recovery of functional behaviors following spinal cord injury by activating autophagy. However, the underlying mechanisms for such observations remain unclear. In this study, we further explored the mechanisms by which NSC-sEVs repair spinal cord injury via autophagy. We found that NSC-sEVs contain 14-3-3t protein, of which the overexpression or knockdown enhanced and decreased autophagy, respectively. In addition, 14-3-3t overexpression enhanced the anti-apoptotic and anti-inflammatory effects of NSC-sEVs, further promoting functional behavior recovery following spinal cord injury. The overexpression of 14-3-3t was used to further validate the in vivo results through a series of in vitro experiments. Conversely, knockdown of 14-3-3t attenuated the anti-apoptotic and anti-inflammatory effects of NSC-sEVs. Further studies also confirmed that NSC-sEVs increased Beclin-1 expression, with which 14-3-3t interacted and promoted its localization to autophagosome precursors. In this study, we found that NSC-sEVs deliver 14-3-3t, which interacts with Beclin-1 to activate autophagy. Our results indicate that 14-3-3t acts via a newly-discovered mechanism for the activation of autophagy by NSC-sEVs.

ABSTRACT 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.

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.

Osteosarcoma (OS) has emerged as the most common primary musculoskeletal malignant tumor which affects children and adolescents. A growing number of relevant studies have shown that many microRNAs (miRNAs) play a vital regulatory role in the etiology of various types of cancer. miR-1258 has been widely studied in various cancers, but there have been few studies of its role in OS. In this present study, miR-1258 expression was dramatically decreased in OS tissues as well as OS cell lines. In addition, decreased expression of miR-1258 was significantly associated with malignant clinical manifestations and poor clinical prognosis of patients with OS. Moreover, upregulation of miR-1258 significantly inhibited cell proliferation as well as promoting cell cycle arrest at G0/G1. AKT3 was identified as a direct target of miR-1258 by binding to its 3′-UTR, and miR-1258 was negatively correlated with AKT3 expression in clinical OS tissues. AKT3 was evidently upregulated in OS tissues and cells and upregulation of AKT3 accelerated the progression of OS. Moreover, through a series of rescue experiments, we demonstrated that AKT3 can abolish the role of miR-1258 in suppressing proliferation as well as regulating the cell cycle in OS cells. In conclusion, our results suggest that the miR-1258–AKT3 axis may be a promising prognostic marker and therapeutic target for human OS.

GPCR kinase 2-interacting protein-1 (GIT1) is a scaffold protein that plays an important role in cell adaptation, proliferation, migration, and differentiation; however, the role of GIT1 in the regulation of neuronal death after spinal cord injury remains obscure. Here, we demonstrate that GIT1 deficiency remarkably increased neuronal apoptosis and enhanced JNK/p38 signaling, which resulted in stronger motor deficits by ischemia-reperfusion in vivo, consistent with the finding of oxygen-glucose deprivation/reoxygenation-induced neuronal injury in vitro. After treatment with JNK and p38 inhibitors, abnormally necroptotic cell death caused by GIT1 knockdown could be partially rescued, with the recovery of neuronal viability, which was still poorer than that in control neurons. Meanwhile, overactivation of JNK/p38 after GIT1 depletion was concomitant with excessive activity of apoptosis signal-regulating kinase-1 (ASK1) that could be abolished by ASK1 silencing in HEK293T cells. Finally, GIT1 could disrupt the oligomerization of ASK1 via interaction between the synaptic localization domain that contains the coiled-coil (CC)-2 domain of GIT1 and the C-terminal CC domain of ASK1. It suppressed the autophosphorylation of ASK1 and led to decreasing activity of the ASK1/JNK/p38 pathway. These data reveal a protective role for GIT1 in neuronal damage by modulating ASK1/JNK/p38 signaling.-Chen, J., Wang, Q., Zhou, W., Zhou, Z., Tang, P.-Y., Xu, T., Liu, W., Li, L.-W., Cheng, L., Zhou, Z.-M., Fan, J., Yin, G.-Y. GPCR kinase 2-interacting protein-1 protects against ischemia-reperfusion injury of the spinal cord by modulating ASK1/JNK/p38 signaling.

Approximately 10-15% of all bone fractures do not heal properly, causing patient morbidity and additional medical care expenses. Therefore, better mechanism-based fracture repair approaches are needed. In this study, a reduced number of osteoclasts (OCs) and autophagosomes/autolysosomes in OC can be observed in GPCR kinase 2-interacting protein 1 (GIT1) knockout (KO) mice on days 21 and 28 post-fracture, compared with GIT1 wild-type (GIT1 WT) mice. Furthermore, in vitro experiments revealed that GIT1 contributes to OC autophagy under starvation conditions. Mechanistically, GIT1 interacted with Beclin1 and promoted Beclin1 phosphorylation at Thr119, which induced the disruption of Beclin1 and Bcl2 binding under starvation conditions, thereby, positively regulating autophagy. Taken together, the findings suggest a previously unappreciated role of GIT1 in autophagy of OCs during fracture repair. Targeting GIT1 may be a potential therapeutic approach for bone fractures.