scholarly journals Resveratrol Activated Sonic Hedgehog Signaling to Enhance Viability of NIH3T3 Cells in Vitro via Regulation of Sirt1

2018 ◽  
Vol 50 (4) ◽  
pp. 1346-1360 ◽  
Author(s):  
Shuang Guo ◽  
Hongyan Liao ◽  
Jie Liu ◽  
Jing Liu ◽  
Fanren Tang ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Hedgehog Signaling ◽  
Transcriptional Activity ◽  
Luciferase Reporter ◽  
Shh Signaling ◽  
Nih3t3 Cells ◽  
Cord Injury ◽  
Fibrotic Scar ◽  
Gli 1

Background/Aims: Injuries of the brain and spinal cord result in the formation of glial (reactive gliosis) and fibrotic (formed by fibroblasts) scars. Recent studies have shown that the fibrotic scar was much more important for hindering regeneration after brain or spinal cord injury than the astrocytic scar. However, it has been given much less attention for effects and mechanism of fibroblasts during formation of the fibrotic scar. Resveratrol may be a potential anti-scarring agent in burn-related scarring and keloid fibroblasts. However, it is unclear whether and how resveratrol affects formation of the fibrotic scar after brain or spinal cord injury. Earlier studies have shown that the activated Shh signaling has anti-apoptosis, anti-oxidation, anti-inflammation properties. Moreover, resveratrol can activate the Shh signaling. However, it is unclear how resveratrol activates the Shh signaling. Resveratrol is a activator of Sirt1. It is unknown whether resveratrol activates the Shh signaling via Sirt1. Methods: NIH3T3 cells, a fibroblast cell line, were used as model cells and treated with drugs. Cell viability was assessed by Cell Counting Kit 8. The expressions and activity of Shh signaling pathway proteins were evaluated by immunocytochemistry and Western blotting. Transcriptional activity of Gli-1 was detected with Dual-Luciferase Reporter Gene Assay Kit. Results: Resveratrol, Sirt1 agonist STR1720 and recombinant mouse Shh protein, an activator of hedgehog signaling, enhanced the viability of NIH3T3 cells, promoted Smo to translocated to the primary cilia and Gli-1 entered into the nuclei from cytoplasm, and upregulated expressions of Shh, Ptc-1, Smo, and Gli-1 proteins, which can be reversed by Smo antagonist cyclopamine and Sirt1 antagonist Sirtinol. Additionally, resveratrol increased transcriptional activity of Gli-1. Conclusion: We indicate in the first time that it may be mediated by Sirt1 for resveratrol activating the Shh signaling to enhance viability of NIH3T3 cells, and Sirt1 may be a regulator for upstream of the Shh signaling pathway.This study provides a basis for further investigating effects and mechanism of resveratrol during the formation of fibrous scar after brain or spinal cord injury.

scholarly journals miRNA-544a Regulates the Inflammation of Spinal Cord Injury by Inhibiting the Expression of NEUROD4

2018 ◽  
Vol 51 (4) ◽  
pp. 1921-1931 ◽  
Author(s):  
Lei Yang ◽  
Dawei Ge ◽  
Xi Chen ◽  
Chunzhi Jiang ◽  
Shengnai Zheng
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Grip Strength ◽  
Target Gene ◽  
Interleukin 1 ◽  
Luciferase Reporter ◽  
Interleukin 17 ◽  
Qrt Pcr ◽  
Gene Assay ◽  
Cord Injury

Background/Aims: To explore the potential role of miR-544a in spinal cord injury and the possible mechanism involved. Methods: We established a mouse model with spinal cord injury to examine the changes in grip force recovery of the forelimb or the posterior limb of the mouse. Microarray was performed to achieve differentiated miRNAs in the mice. The expressions of miR-544a, MCP-1, IL36B and IL17B after spinal cord injury were detected by qRT-PCR. Subsequently, miR-544a was overexpressed to observe changes in inflammation and grip strength after spinal cord injury. Target gene of miR-544a was then predicted using bioinformatics technology. Finally, dual luciferase reporter gene assay was used to verify the binding of miR-544a to its target gene. Results: Using mice models with spinal cord injury, we found that the strength of their four limbs began to recover 7 days after injury. The results of microarray and qRT-PCR confirmed that mir-544a level in mice with spinal cord injury decreased with increase of injury time, while the levels of inflammatory genes MCP-1 (monocyte chemoattractant protein-1), IL1 (interleukin-1) and TNF-α (tumor necrosis factor alpha) IL36B (interleukin-36 beta) and IL17B (interleukin-17 beta) were significantly increased. However, overexpression of miR-544a in the mice significantly reduced the level of inflammation and restored their grip strength in their four limbs. Finally, we found that miR-544a can bind to the NEUROD4 (Neurogenic differentiation 4) 3’UTR (Untranslated Region) region through bioinformatics website prediction, which was further confirmed by dual luciferase reporter assay. NEUROD4 level was significantly reduced following the overexpression of miR-544a. Conclusion: The expression of miR-544a was significantly decreased after spinal cord injury. High expression of miR-544a could alleviate the inflammation caused by spinal cord injury and promote the recovery of spinal cord via the inhibition of NEUROD4.

scholarly journals Combined Treatment with Fasudil and Menthol Improves Functional Recovery in Rat Spinal Cord Injury Model

Biomedicines ◽  
2020 ◽  
Vol 8 (8) ◽  
pp. 258
Author(s):  
JeongHoon Kim ◽  
Hari Prasad Joshi ◽  
Kyoung-Tae Kim ◽  
Yi Young Kim ◽  
Keundong Yeo ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Functional Recovery ◽  
Combined Treatment ◽  
Scar Formation ◽  
Sprague Dawley ◽  
Static Compression ◽  
Cord Injury ◽  
Fibrotic Scar ◽  
Spinal Cord Dysfunction

Neuroprotective measures by preventing secondary spinal cord injury (SCI) are one of the main strategies for repairing an injured spinal cord. Fasudil and menthol may be potent neuroprotective agents, which act by inhibiting a rho-associated protein kinase (ROCK) and suppressing the inflammatory response, respectively. We hypothesized that combined treatment of fasudil and menthol could improve functional recovery by decreasing inflammation, apoptosis, and glial scar formation. We tested our hypothesis by administering fasudil and menthol intraperitoneally (i.p.) to female Sprague Dawley rats after moderate static compression (35 g of impounder for 5 min) of T10 spinal cord. The rats were randomly divided into five experimental groups: (i) sham animals received laminectomy alone, (ii) injured (SCI) and untreated (saline 0.2 mL/day, i.p.) rats, (iii) injured (SCI) rats treated with fasudil (10 mg/kg/day, i.p.) for two weeks, (iv) injured (SCI) rats treated with menthol (10 mg/kg/day, i.p.) for twoweeks, (v) injured (SCI) rats treated with fasudil (5 mg/kg/day, i.p.) and menthol (10 mg/kg/day, i.p.) for two weeks. Compared to single treatment groups, combined treatment of fasudil and menthol demonstrated significant functional recovery and pain amelioration, which, thereby, significantly reduced inflammation, apoptosis, and glial/fibrotic scar formation. Therefore, combined treatment of fasudil and menthol may provide effective amelioration of spinal cord dysfunction by a synergistic effect of fasudil and menthol.

scholarly journals Perivascular Fibroblasts Form the Fibrotic Scar after Contusive Spinal Cord Injury

2013 ◽  
Vol 33 (34) ◽  
pp. 13882-13887 ◽  
Author(s):  
C. Soderblom ◽  
X. Luo ◽  
E. Blumenthal ◽  
E. Bray ◽  
K. Lyapichev ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Cord Injury ◽  
Fibrotic Scar ◽  
Contusive Spinal Cord Injury

scholarly journals Transplantation of sh-miR-199a-5p-Modified Olfactory Ensheathing Cells Promotes the Functional Recovery in Rats with Contusive Spinal Cord Injury

2020 ◽  
Vol 29 ◽  
pp. 096368972091617 ◽  
Author(s):  
Zhengchao Gao ◽  
Yingjie Zhao ◽  
Xijing He ◽  
Zikuan Leng ◽  
Xiaoqian Zhou ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Functional Recovery ◽  
Diffusion Tensor ◽  
Olfactory Ensheathing Cells ◽  
Luciferase Reporter ◽  
Therapeutic Effects ◽  
Protein Levels ◽  
Cord Injury ◽  
Ensheathing Cells

MicroRNAs (miRNAs) function as gene expression switches, and participate in diverse pathophysiological processes of spinal cord injury (SCI). Olfactory ensheathing cells (OECs) can alleviate pathological injury and facilitate functional recovery after SCI. However, the mechanisms by which OECs restore function are not well understood. This study aims to determine whether silencing miR-199a-5p would enhance the beneficial effects of the OECs. In this study, we measured miR-199a-5p levels in rat spinal cords with and without injury, with and without OEC transplants. Then, we transfected OECs with the sh-miR-199a-5p lentiviral vector to reduce miR-199a-5p expression and determined the effects of these OECs in SCI rats by Basso–Beattie–Bresnahan (BBB) locomotor scores, diffusion tensor imaging (DTI), and histological methods. We used western blotting to measure protein levels of Slit1, Robo2, and srGAP2. Finally, we used the dual-luciferase reporter assay to assess the relationship between miR-199-5p and Slit1, Robo2, and srGAP2 expression. We found that SCI significantly increased miR-199a-5p levels ( P < 0.05), and OEC transplants significantly reduced miR-199a-5p expression ( P < 0.05). Knockdown of miR-199a-5p in OECs had a better therapeutic effect on SCI rats, indicated by higher BBB scores and fractional anisotropy values on DTI, as well as histological findings. Reducing miR-199a-5p levels in transplanted OECs markedly increased spinal cord protein levels of Slit1, Robo2, and srGAP2. Our results demonstrated that transplantation of sh-miR-199a-5p-modified OECs promoted functional recovery in SCI rats, suggesting that miR-199a-5p knockdown was more beneficial to the therapeutic effects of OEC transplants. These findings provided new insights into miRNAs-mediated therapeutic mechanisms of OECs, which helps us to develop therapeutic strategies based on miRNAs and optimize cell therapy for SCI.

scholarly journals Zhenbao pill protects against acute spinal cord injury via miR-146a-5p regulating the expression of GPR17

2018 ◽  
Vol 38 (1) ◽  
Author(s):  
Yongxiong He ◽  
Bokang Lv ◽  
Yanqiang Huan ◽  
Bin Liu ◽  
Yutang Li ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Motor Function ◽  
Hind Limb ◽  
Neuronal Apoptosis ◽  
Acute Spinal Cord Injury ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Cord Injury

The aim of the present study was to observe the effect of zhenbao pill on the motor function of acute spinal cord injury (ASCI) rats and the molecular mechanisms involving miR-146a-5p and G-protein-coupled receptor 17 (GPR17). ASCI rat model was established by modified Allen method, and then the rats were divided into three groups. SH-SY5Y cells were cultured overnight in hypoxia condition and transfected with miR-146a-5p mimic or miR-146a-5p inhibitor. The hind limb motor function of the rats was evaluated by Basso, Beattie, Bresnahan (BBB) scoring system. Quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the expression of miR-146a-5p, GPR17, inducible nitric oxide synthase (iNOS), interleukin 1β (IL-1β), and tumor necrosis factor α (TNF-α). Neuronal apoptosis was measured using flow cytometry assay. Luciferase reporter assay was performed to determine the regulation of miR-146a-5p on GPR17. Zhenbao pill could enhance hind limb motor function and attenuate the inflammatory response caused by ASCI. Moreover, zhenbao pill increased the level of miR-146a-5p and decreased GPR17 expression in vivo and in vitro. Bioinformatics software predicted that GPR17 3′-UTR had a binding site with miR-146a-5p. Luciferase reporter assay showed that miR-146a-5p had a negative regulatory effect on GPR17 expression. Knockdown of miR-146a-5p could reverse the effect of zhenbao pill on the up-regulation of GPR17 induced by hypoxia, reversed the inhibitory effect of zhenbao pill on the cell apoptosis induced by hypoxia and the recovery of zhenbao pill on hind limb motor function in ASCI rats. Zhenbao pill could inhibit neuronal apoptosis by regulating miR-146a-5p/GPR17 expression, and then promoting the recovery of spinal cord function.

scholarly journals Fibronectin EDA forms the chronic fibrotic scar after contusive spinal cord injury

2018 ◽  
Vol 116 ◽  
pp. 60-68 ◽  
Author(s):  
John G. Cooper ◽  
Su Ji Jeong ◽  
Tammy L. McGuire ◽  
Sripadh Sharma ◽  
Wenxia Wang ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Cord Injury ◽  
Fibrotic Scar ◽  
Contusive Spinal Cord Injury

scholarly journals Comprehensive Effects of Suppression of MicroRNA-383 in Human Bone-Marrow-Derived Mesenchymal Stem Cells on Treating Spinal Cord Injury

2018 ◽  
Vol 47 (1) ◽  
pp. 129-139 ◽  
Author(s):  
Guo-Jun Wei ◽  
Ke-wen Zheng ◽  
Gang An ◽  
Zuo-Wei Shi ◽  
Kai-Fu Wang ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Stem Cells ◽  
Bone Marrow ◽  
Spinal Cord Injury ◽  
Mesenchymal Stem Cells ◽  
Therapeutic Potential ◽  
Luciferase Reporter ◽  
Therapeutic Effects ◽  
Cord Injury

Background/Aims: Transplantation of bone-marrow-derived mesenchymal stem cells (MSCs) promotes neural cell regeneration after spinal cord injury (SCI). Recently, we showed that suppression of microRNA-383 (miR-383) in MSCs increased the protein levels of glial cell line derived neurotrophic factor (GDNF), resulting in improved therapeutic effects on SCI. However, the overall effects of miR-383 suppression in MSCs on SCI therapy were not determined yet. Here, we addressed this question. Methods: We used bioinformatics tools to predict all miR-383-targeting genes, confirmed the functional bindings in a dual luciferase reporter assay. The effects of alteration of candidate genes in MSCs on cell proliferation were analyzed by MTT assay and by Western blotting for PCNA. The effects on angiogenesis were assessed by HUVEC assay. The effects on SCI in vivo were analyzed by transplantation of the modified MSCs into nude rats that underwent SCI. Results: Suppression of miR-383 in MSCs not only upregulated GDNF protein, but also increased vascular endothelial growth factor A (VEGF-A) and cyclin-dependent kinase 19 (CDK19), two other miR-383 targets. MiR-383-suppression-induced increases in CDK19 resulted in a slight but significant increase in MSC proliferation, while miR-383-suppression-induced increases in VEGF-A resulted in a slight but significant increase in MSC-mediated angiogenesis. Conclusions: Upregulation of CDK19 and VEGF-A by miR-383 suppression in MSCs further improve the therapeutic potential of MSCs in treating SCI in rats.

Ethamsylate attenuates mutilated secondary pathogenesis and exhibits a neuroprotective role in experimental model of spinal cord injury

2021 ◽  
Author(s):  
Sonam Dolma ◽  
Kirti Adhikari ◽  
Teena Mamidi ◽  
Abhishek Roy ◽  
Zarna Pathak ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Motor Neurons ◽  
Proteolytic Enzymes ◽  
Motor Impairment ◽  
Scar Formation ◽  
Secondary Damage ◽  
Cord Injury ◽  
Fibrotic Scar ◽  
Blood Spinal Cord Barrier

Abstract Deficits in the neuronal connection that succumbs to the impairment of sensory and motor neurons are the hallmarks of spinal cord injury (SCI). Secondary pathogenesis, which initiates after the primary mechanical insult to the spinal cord, depicts a pivotal role in producing inflammation, lesion formation and ultimately causes fibrotic scar formation in the chronic period. This fibrotic scar formed acts as a major hindrance in facilitating axonal regeneration and is one of the root causes of motor impairment. Cascade of secondary events in SCI begins with injury-induced blood spinal cord barrier rupture that promotes increased migration of neutrophils, macrophages, and other inflammatory cells at the injury site to initiate the secondary damages. This phenomenon leads to the release of matrix metalloproteinase, cytokines and chemokines, reactive oxygen species, and other proteolytic enzymes at the lesion site. These factors assist in the activation of TGF-β1 signalling pathway, which further leads to excessive proliferation of perivascular fibroblast, followed by deposition of collagen and fibronectin matrix, which are the main components of the fibrotic scar. Subsequently, this scar formed inhibits the propagation of action potential from one neuron to adjacent neurons. Ethamsylate, an anti-hemorrhagic drug, has the potential to maintain early hemostasis as well as restores capillary resistance. Therefore, we hypothesized that ethamsylate, by virtue of its anti-hemorrhagic activity, reduces hemorrhagic ischemia-induced neuronal apoptosis, maintains the blood spinal cord barrier integrity, and decreases secondary damage severity, thereby reduce the extent of fibrotic scar formation, and demonstrates a neuroprotective role in SCI.

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