scholarly journals Quantitative Proteomic Analysis of Mouse Sciatic Nerve Reveals Post-injury Upregulation of ADP-Dependent Glucokinase Promoting Macrophage Phagocytosis

2021 ◽  
Vol 14 ◽  
Author(s):  
Kai Zhang ◽  
Qingyao Wang ◽  
Yiyao Liang ◽  
Yu Yan ◽  
Haiqiong Wang ◽  
...  
Keyword(s):  
Sciatic Nerve ◽  
Nerve Injury ◽  
Proteomic Analysis ◽  
Inflammatory Responses ◽  
Post Injury ◽  
Injured Nerve ◽  
Protein Levels ◽  
Macrophage Phagocytosis

Nerve injury induces profound and complex changes at molecular and cellular levels, leading to axonal self-destruction as well as immune and inflammatory responses that may further promote neurodegeneration. To better understand how neural injury changes the proteome within the injured nerve, we set up a mouse model of sciatic nerve injury (SNI) and conducted an unbiased, quantitative proteomic study followed by biochemical assays to confirm some of the changed proteins. Among them, the protein levels of ADP-dependent glucokinase (ADPGK) were significantly increased in the injured sciatic nerve. Further examination indicated that ADPGK was specifically expressed and upregulated in macrophages but not neurons or Schwann cells upon injury. Furthermore, culturing immortalized bone marrow-derived macrophages (iBMDMs) in vitro with the conditioned media from transected axons of mouse dorsal root ganglion (DRG) neurons induced ADPGK upregulation in iBMDMs, suggesting that injured axons could promote ADPGK expression in macrophages non-cell autonomously. Finally, we showed that overexpression of ADPGK per se did not activate macrophages but promoted the phagocytotic activity of lipopolysaccharides (LPS)-treated macrophages. Together, this proteomic analysis reveals interesting changes of many proteins within the injured nerve and our data identify ADPGK as an important in vivo booster of injury-induced macrophage phagocytosis.

Continuation of fast axonal transport in regenerating axons in vitro

1979 ◽  
Vol 57 (11) ◽  
pp. 1251-1255
Author(s):  
M. A. Bisby ◽  
C. E. Hilton
Keyword(s):  
Sciatic Nerve ◽  
Vagus Nerve ◽  
Axonal Transport ◽  
Nerve Injury ◽  
Axon Regeneration ◽  
Free Medium ◽  
Fast Axonal Transport ◽  
Sensory Axons

A previous study by McLean and co-workers reported that regenerating axons of the rabbit vagus nerve were unable to sustain axonal transport in vitro for several months after nerve injury. In contrast, we found that sensory axons of the rat sciatic nerve were able to transport 3H-labeled protein into their regenerating portions distal to the site of injury within a week after injury when placed in vitro. Transport in vitro was not significantly less than transport in axons maintained in vivo for the same period. Transport occurred in the medium that was used by the McLean group, but was significantly reduced in calcium-free medium. When axon regeneration was delared, only small amounts of activity were present in the nerve distal to the site of injury, showing that labeled protein normally present in that part of the nerve was associated with axons and was not a result of local precursor uptake by nonneural elements in the sciatic nerve. We were not able to explain the failure of McLean and co-workers to demonstrate transport in vitro in regenerating vagus nerve, but we conclude that there is no general peculiarity of growing axons that makes them unable to sustain transport in vitro.

scholarly journals Exosomal miR-25-3p from mesenchymal stem cells alleviates myocardial infarction by targeting pro-apoptotic proteins and EZH2

2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Yi Peng ◽  
Ji-Ling Zhao ◽  
Zhi-Yong Peng ◽  
Wei-Fang Xu ◽  
Guo-Long Yu
Keyword(s):  
Myocardial Infarction ◽  
Inflammatory Responses ◽  
Luciferase Assay ◽  
Protective Effects ◽  
Functional Studies ◽  
Protein Levels ◽  
Cardioprotective Effects ◽  
Apoptotic Proteins

Abstract Mesenchymal stem cell (MSC) therapy is a promising approach against myocardial infarction (MI). Studies have demonstrated that MSCs can communicate with other cells by secreting exosomes. In the present study, we aimed to identify exosomal microRNAs that might contribute to MSC-mediated cardioprotective effects. Primary cardiomyocytes were deprived of oxygen and glucose to mimic MI in vitro. For the animal model of MI, the left anterior descending artery was ligated for 1 h, followed by reperfusion for 12 h. MSC-derived exosomes were used to treat primary cardiomyocytes or mice. Cardioprotection-related microRNAs were determined, followed by target gene identification and functional studies with quantitative PCR, western blotting, MTT assay, flow cytometry assay, chromatin immunoprecipitation and dual-luciferase assay. We found that MSC co-culture reduced OGD-induced cardiomyocyte apoptosis and inflammatory responses. Cardioprotection was also observed upon treatment with MSC-derived exosomes in vitro and in vivo. In line with this, exosome uptake led to a significant increase in miR-25-3p in cardiomyocytes. Depletion of miR-25-3p in MSCs abolished the protective effects of exosomes. Mechanistically, miR-25-3p directly targeted the pro-apoptotic genes FASL and PTEN and reduced their protein levels. Moreover, miR-25-3p decreased the levels of EZH2 and H3K27me3, leading to derepression of the cardioprotective gene eNOS as well as the anti-inflammatory gene SOCS3. Inhibition of EZH2 or overexpression of miR-25-3p in cardiomyocytes was sufficient to confer cardioprotective effects in vitro and in vivo. We concluded that exosomal miR-25-3p from MSCs alleviated MI by targeting pro-apoptotic proteins and EZH2.

Regulation of angiopoietin expression by bacterial lipopolysaccharide

2008 ◽  
Vol 294 (5) ◽  
pp. L955-L963 ◽  
Author(s):  
Mahroo Mofarrahi ◽  
Thamir Nouh ◽  
Salman Qureshi ◽  
Loic Guillot ◽  
Dominique Mayaki ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Inflammatory Responses ◽  
Cell Types ◽  
Vascular Leakage ◽  
Saline Control ◽  
E Coli ◽  
Protein Levels ◽  
Angiopoietin 2

Angiopoietins are ligands for Tie-2 receptors and play important roles in angiogenesis and inflammation. While angiopoietin-1 (Ang-1) inhibits inflammatory responses, angiopoietin-2 (Ang-2) promotes cytokine production and vascular leakage. In this study, we evaluated in vivo and in vitro effects of Escherichia coli lipopolysaccharides (LPS) on angiopoietin expression. Wild-type C57/BL6 mice were injected with saline (control) or E. coli LPS (20 mg/ml ip) and killed 6, 12, and 24 h later. The diaphragm, lung, and liver were excised and assayed for mRNA and protein expression of Ang-1, Ang-2, and Tie-2 protein and tyrosine phosphorylation. LPS injection elicited a severalfold rise in Ang-2 mRNA and protein levels in the three organs. By comparison, both Ang-1 and Tie-2 levels in the diaphragm, liver, and lung were significantly attenuated by LPS administration. In addition, Tie-2 tyrosine phosphorylation in the lung was significantly reduced in response to LPS injection. In vitro exposure to E. coli LPS elicited cell-specific changes in Ang-1 expression, with significant induction in Ang-1 expression being observed in cultured human epithelial cells, whereas significant attenuation of Ang-1 expression was observed in response to E. coli LPS exposure in primary human skeletal myoblasts. In both cell types, E. coli LPS elicited substantial induction of Ang-2 mRNA, a response that was mediated in part through NF-κB. We conclude that in vivo endotoxemia triggers functional inhibition of the Ang-1/Tie-2 receptor pathway by reducing Ang-1 and Tie-2 expression and inducing Ang-2 levels and that this response may contribute to enhanced vascular leakage in sepsis.

ID: 109: PARKIN MEDIATES ENDOTHELIAL PRO-INFLAMMATORY RESPONSES IN ACUTE LUNG INJURY

2016 ◽  
Vol 64 (4) ◽  
pp. 963.1-963
Author(s):  
E Letsiou ◽  
H Wang ◽  
P Belvitch ◽  
S Dudek ◽  
S Sammani
Keyword(s):  
Acute Lung Injury ◽  
Endothelial Dysfunction ◽  
Lung Injury ◽  
Kinase Inhibitor ◽  
Inflammatory Responses ◽  
Neutrophil Adhesion ◽  
Protein Levels ◽  
Cell Counts

IntroductionAcute lung injury (ALI) and its more severe form, the Acute Respiratory Distress Syndrome (ARDS), are serious conditions resulting from direct or indirect lung injury that occur in critically ill patients and are associated with an unacceptable mortality of up to 40%. A key biological event in the pathogenesis of ALI/ARDS is the dysfunction of the lung endothelium (EC), which is triggered by a variety of inflammatory insults leading to damaged EC, vascular leak, and excessive inflammation. Recently, we demonstrated that an Abl family tyrosine kinase inhibitor, imatinib, protects against LPS-induced endothelial dysfunction by inhibiting c-Abl kinase through mechanisms that remain largely unknown. In the present study, we identified parkin, a novel c-Abl substrate, as a critical mediator of endothelial dysfunction in ALI.MethodsIn vitro Human pulmonary artery endothelial cells (EC) were transfected with siRNA for parkin and then challenged with LPS (1 µg/ml, 3 hrs). Inflammatory mediators were determined in cell lysates and supernatants by Western blotting and ELISA respectively. In vivo C57BL/6 (WT) and parkin deficient (PARK2 KO) male mice (8–12 wks, n=5–8) were subjected to LPS (intratracheally, 1 mg/kg) or PBS (controls), and allowed to recover prior to harvest 18 hrs later. Leakage of proteins into the alveolar space was assessed by measuring the protein levels in the bronchoalveolar lavage (BAL). To assess lung inflammation, neutrophil cell counts, myeloperoxidase (MPO) activity, and IL-6 levels were determined in BAL.ResultsIn human lung EC, down-regulation of parkin by siRNA reduces LPS-induced VCAM-1 expression (adhesion molecule involved in neutrophil adhesion to EC) (by 35%, p<0.05), IL-8 (neutrophil chemoattractant) (by 59%, p<0.01), and IL-6 (inflammatory cytokine) release (by 79%, p<0.01). PARK2 KO mice exhibit less ALI after LPS compared to WT. In PARK2 KO, BAL protein levels were reduced by 27% (p=0.0024) compared to WT mice. LPS-induced neutrophil recruitment into the alveoli of PARK2 KO was attenuated by 47% compared to WT (p=0.0019). BAL MPO activity (marker of neutrophil activation) and BAL IL-6 levels were also significantly lower in PARK2 KO by 52% (p=0.03) and 28% (p=0.0061) respectively.ConclusionThese results suggest that endothelial parkin mediates EC activation and neutrophil adhesion/migration after LPS, and therefore it may represent a new potential therapeutic target in ALI/ARDS.

scholarly journals Macrophage-specific RhoA knockout delays Wallerian degeneration after peripheral nerve injury in mice

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Jiawei Xu ◽  
Jinkun Wen ◽  
Lanya Fu ◽  
Liqiang Liao ◽  
Ying Zou ◽  
...  
Keyword(s):  
Peripheral Nerve ◽  
Nerve Regeneration ◽  
Nerve Injury ◽  
Peripheral Nerve Injury ◽  
Axonal Regeneration ◽  
Wallerian Degeneration ◽  
Future Application ◽  
Injured Nerve

Abstract Background Plenty of macrophages are recruited to the injured nerve to play key roles in the immunoreaction and engulf the debris of degenerated axons and myelin during Wallerian degeneration, thus creating a conducive microenvironment for nerve regeneration. Recently, drugs targeting the RhoA pathway have been widely used to promote peripheral axonal regeneration. However, the role of RhoA in macrophage during Wallerian degeneration and nerve regeneration after peripheral nerve injury is still unknown. Herein, we come up with the hypothesis that RhoA might influence Wallerian degeneration and nerve regeneration by affecting the migration and phagocytosis of macrophages after peripheral nerve injury. Methods Immunohistochemistry, Western blotting, H&E staining, and electrophysiology were performed to access the Wallerian degeneration and axonal regeneration after sciatic nerve transection and crush injury in the LyzCre+/−; RhoAflox/flox (cKO) mice or Lyz2Cre+/− (Cre) mice, regardless of sex. Macrophages’ migration and phagocytosis were detected in the injured nerves and the cultured macrophages. Moreover, the expression and potential roles of ROCK and MLCK were also evaluated in the cultured macrophages. Results 1. RhoA was specifically knocked out in macrophages of the cKO mice; 2. The segmentation of axons and myelin, the axonal regeneration, and nerve conduction in the injured nerve were significantly impeded while the myoatrophy was more severe in the cKO mice compared with those in Cre mice; 3. RhoA knockout attenuated the migration and phagocytosis of macrophages in vivo and in vitro; 4. ROCK and MLCK were downregulated in the cKO macrophages while inhibition of ROCK and MLCK could weaken the migration and phagocytosis of macrophages. Conclusions Our findings suggest that RhoA depletion in macrophages exerts a detrimental effect on Wallerian degeneration and nerve regeneration, which is most likely due to the impaired migration and phagocytosis of macrophages resulted from disrupted RhoA/ROCK/MLCK pathway. Since previous research has proved RhoA inhibition in neurons was favoring for axonal regeneration, the present study reminds us of that the cellular specificity of RhoA-targeted drugs is needed to be considered in the future application for treating peripheral nerve injury.

scholarly journals A Brief Review of In Vitro Models for Injury and Regeneration in the Peripheral Nervous System

2022 ◽  
Vol 23 (2) ◽  
pp. 816
Author(s):  
Parvathi Varier ◽  
Gayathri Raju ◽  
Pallavi Madhusudanan ◽  
Chinnu Jerard ◽  
Sahadev A. Shankarappa
Keyword(s):  
Nervous System ◽  
Peripheral Nervous System ◽  
Nerve Injury ◽  
Axonal Injury ◽  
In Vitro Models ◽  
Model Systems ◽  
Neural Regeneration ◽  
Post Injury

Nerve axonal injury and associated cellular mechanisms leading to peripheral nerve damage are important topics of research necessary for reducing disability and enhancing quality of life. Model systems that mimic the biological changes that occur during human nerve injury are crucial for the identification of cellular responses, screening of novel therapeutic molecules, and design of neural regeneration strategies. In addition to in vivo and mathematical models, in vitro axonal injury models provide a simple, robust, and reductionist platform to partially understand nerve injury pathogenesis and regeneration. In recent years, there have been several advances related to in vitro techniques that focus on the utilization of custom-fabricated cell culture chambers, microfluidic chamber systems, and injury techniques such as laser ablation and axonal stretching. These developments seem to reflect a gradual and natural progression towards understanding molecular and signaling events at an individual axon and neuronal-soma level. In this review, we attempt to categorize and discuss various in vitro models of injury relevant to the peripheral nervous system and highlight their strengths, weaknesses, and opportunities. Such models will help to recreate the post-injury microenvironment and aid in the development of therapeutic strategies that can accelerate nerve repair.

scholarly journals Long Non-coding RNA MSTRG.24008.1 Regulates the Regeneration of the Sciatic Nerve via the miR-331-3p–NLRP3/MAL Axis

2021 ◽  
Vol 9 ◽  
Author(s):  
Gang Yin ◽  
Ying Peng ◽  
Yaofa Lin ◽  
Peilin Wang ◽  
Zhuoxuan Li ◽  
...  
Keyword(s):  
Sciatic Nerve ◽  
Peripheral Nerve ◽  
Nerve Injury ◽  
Peripheral Nerve Regeneration ◽  
Clinical Problem ◽  
Neural Regeneration ◽  
Luciferase Reporter ◽  
Complex Biological Process

Peripheral nerve injury (PNI) is a common clinical problem, which can cause severe disability and dramatically affect a patient’s quality of life. Neural regeneration after PNI is a complex biological process that involves a variety of signaling pathways and genes. Emerging studies demonstrated that long non-coding RNAs (lncRNAs) were abnormally expressed after PNI and played pivotal roles in peripheral nerve regeneration. Based on the rat sciatic nerve injury model, we found that the expression levels of several lncRNAs were increased significantly in the sciatic nerve after injury. Software prediction prompted us to focus on one up-regulated lncRNA, MSTRG.24008.1. Dual-luciferase reporter assay, RNA pull-down assay and RNA interference approach verified that MSTRG.24008.1 regulated neuroregeneration via the miR-331-3p/nucleotide-binding oligomerization domain-like pyrin domain containing 3 (NLRP3)/myelin and lymphocyte protein (MAL) axis in vitro. Subsequently, we performed gastrocnemius muscle gravity and sciatic functional index experiments to evaluate the recovery of injured sciatic nerves after MSTRG.24008.1 siRNA interference in vivo. In conclusion, knockdown of MSTRG.24008.1 promotes the regeneration of the sciatic nerve via the miR-331-3p/NLRP3/MAL axis, which may provide a new strategy to evaluate and repair injured peripheral nerves clinically.

scholarly journals The GDF11 Promotes Nerve Regeneration After Sciatic Nerve Injury in Adult Rats by Promoting Axon Growth and Inhibiting Neuronal Apoptosis

2022 ◽  
Vol 9 ◽  
Author(s):  
Junhao Lin ◽  
Jie Shi ◽  
Xiang Min ◽  
Si Chen ◽  
Yunpeng Zhao ◽  
...  
Keyword(s):  
Sciatic Nerve ◽  
Nerve Injury ◽  
Cell Apoptosis ◽  
Neuronal Apoptosis ◽  
Axonal Growth ◽  
Sciatic Nerve Injury ◽  
Smad Pathway ◽  
Sciatic Nerves

Introduction: Sciatic nerve injury is a common injury of the nervous system. Stem cell-based therapies, drug-based therapies and rehabilitation physiotherapy therapies are currently available, but their limited therapeutic efficacy limits their use. Here, we aimed to explore a novel lentiviral-based gene therapeutic strategy and to elaborate its mechanism.Materials and Methods: Recombinant GDF11 protein was used for the in vitro treatment of dorsal root ganglion (DRG) cells. Lentivirus was used to construct a vector system for the in vivo expression of GDF11. The nerve conduction function was detected using action-evoked potentials at different time periods, and the regulatory effect of nerves on target organs was detected by weighing the gastrocnemius muscle. Immunofluorescence of NF200 and S100 was used to show the regeneration of the sciatic nerve, and myelin and Nissl staining were performed to observe the pathological features of the tissue. Western was used to validate signaling pathways. The expression of related genes was observed by qPCR and Western blotting, and cell apoptosis was detected by flow cytometry.Result: GDF11 promotes the axonal growth of DRG cells and inhibits DGR cell apoptosis in vitro. GDF11 acts by activating the Smad pathway. GDF11 promotes the recovery of damaged sciatic nerve function in rats, the regeneration of damaged sciatic nerves in rats, and myelin regeneration of damaged sciatic nerves in rats. GDF11 also exerts a protective effect on neuronal cells in rats.Conclusion: Based on the present study, we conclude that GDF11 promotes axonal growth and inhibits DRG cell apoptosis in vitro through the Smad pathway, and lentivirus-mediated GDF11 overexpression in vivo can promote the recovery of sciatic nerves after transection by promoting axonal growth and inhibiting neuronal apoptosis in the spinal cord.

scholarly journals Recovery from rat sciatic nerve injury in vivo through the use of differentiated MDSCs in vitro

2012 ◽  
Vol 5 (1) ◽  
pp. 193-196 ◽  
Author(s):  
XIANGYI ZENG ◽  
LI ZHANG ◽  
LIANG SUN ◽  
DAI ZHANG ◽  
HENGWU ZHAO ◽  
...  
Keyword(s):  
Sciatic Nerve ◽  
Nerve Injury ◽  
Sciatic Nerve Injury ◽  
Rat Sciatic Nerve

scholarly journals Exosomes from Human Gingiva-Derived Mesenchymal Stem Cells Combined with Biodegradable Chitin Conduits Promote Rat Sciatic Nerve Regeneration

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Feng Rao ◽  
Dianying Zhang ◽  
Tengjiaozi Fang ◽  
Changfeng Lu ◽  
Bo Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Sciatic Nerve ◽  
Peripheral Nerve ◽  
Nerve Regeneration ◽  
Nerve Injury ◽  
Peripheral Nerve Injury ◽  
Peripheral Nerve Regeneration

At present, repair methods for peripheral nerve injury often fail to get satisfactory result. Although various strategies have been adopted to investigate the microenvironment after peripheral nerve injury, the underlying molecular mechanisms of neurite outgrowth remain unclear. In this study, we evaluate the effects of exosomes from gingival mesenchymal stem cells (GMSCs) combined with biodegradable chitin conduits on peripheral nerve regeneration. GMSCs were isolated from human gingival tissue and characterized by surface antigen analysis and in vitro multipotent differentiation. The cell supernatant was collected to isolate the exosomes. The exosomes were characterized by transmission electron microscopy, Western blot, and size distribution analysis. The effects of exosomes on peripheral nerve regeneration in vitro were evaluated by coculture with Schwann cells and DRGs. The chitin conduit was prepared and combined with the exosomes to repair rat sciatic nerve defect. Histology, electrophysiology, and gait analysis were used to test the effects of exosomes on sciatic nerve function recovery in vivo. We have successfully cultured GMSCs and isolated exosomes. The exosomes from GMSCs could significantly promote Schwann cell proliferation and DRG axon growth. The in vivo studies showed that chitin conduit combined with exosomes from GMSCs could significantly increase the number and diameter of nerve fibers and promote myelin formation. In addition, muscle function, nerve conduction function, and motor function were also obviously recovered. In summary, this study suggests that GMSC-derived exosomes combined with biodegradable chitin conduits are a useful and novel therapeutic intervention in peripheral nerve repair.

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