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

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

Resveratrol Enhances Neurite Outgrowth and Synaptogenesis Via Sonic Hedgehog Signaling Following Oxygen-Glucose Deprivation/Reoxygenation Injury

Background/Aims: Neurite outgrowth and synaptogenesis are critical steps for functional recovery after stroke. Resveratrol promotes neurite outgrowth and synaptogenesis, but the underlying mechanism is not well understood, although the Sonic hedgehog (Shh) signaling pathway may be involved. Given that resveratrol activates sirtuin (Sirt)1, the present study examined whether this is mediated by Shh signaling. Methods: Primary cortical neuron cultures were pretreated with drugs before oxygen-glucose deprivation/reoxygenation (OGD/R). Cell viability and apoptosis were evaluated with Cell Counting Kit 8 and by terminal deoxynucleotidyl transferase dUTP nick end labeling, respectively. Neurite outgrowth and synaptogenesis were assessed by immunocytochemistry and western blotting, which was also used to examine the expression of Sirt1 and Shh signaling proteins. Results: Resveratrol and the Smoothened (Smo) agonist purmophamine, which activates Shh signaling, increased viability, reduced apoptosis, and stimulated neurite outgrowth after OGD/R injury. Moreover, the expression of growth-associated protein(GAP)-43, synaptophysin, Shh, Patched (Ptc)-1, Smo, glioma-associated oncogene homolog (Gli)-1, and Sirt1 were upregulated under these conditions. These effects were reversed by treatment with the Smo inhibitor cyclopamine, whereas the Sirt1 inhibitor sirtinol reduced the levels of Shh, Ptc-1, Smo, and Gli-1. Conclusions: Resveratrol reduces neuronal injury following OGD/R injury and enhances neurite outgrowth and synaptogenesis by activating Shh signaling, which in turn induces Sirt1

Resveratrol Enhances Neurite Outgrowth and Synaptogenesis Via Sonic Hedgehog Signaling Following Oxygen-Glucose Deprivation/Reoxygenation Injury

Background/Aims: Neurite outgrowth and synaptogenesis are critical steps for functional recovery after stroke. Resveratrol promotes neurite outgrowth and synaptogenesis, but the underlying mechanism is not well understood, although the Sonic hedgehog (Shh) signaling pathway may be involved. Given that resveratrol activates sirtuin (Sirt)1, the present study examined whether this is mediated by Shh signaling. Methods: Primary cortical neuron cultures were pretreated with drugs before oxygen-glucose deprivation/reoxygenation (OGD/R). Cell viability and apoptosis were evaluated with Cell Counting Kit 8 and by terminal deoxynucleotidyl transferase dUTP nick end labeling, respectively. Neurite outgrowth and synaptogenesis were assessed by immunocytochemistry and western blotting, which was also used to examine the expression of Sirt1 and Shh signaling proteins. Results: Resveratrol and the Smoothened (Smo) agonist purmophamine, which activates Shh signaling, increased viability, reduced apoptosis, and stimulated neurite outgrowth after OGD/R injury. Moreover, the expression of growth-associated protein(GAP)-43, synaptophysin, Shh, Patched (Ptc)-1, Smo, glioma-associated oncogene homolog (Gli)-1, and Sirt1 were upregulated under these conditions. These effects were reversed by treatment with the Smo inhibitor cyclopamine, whereas the Sirt1 inhibitor sirtinol reduced the levels of Shh, Ptc-1, Smo, and Gli-1. Conclusions: Resveratrol reduces neuronal injury following OGD/R injury and enhances neurite outgrowth and synaptogenesis by activating Shh signaling, which in turn induces Sirt1

Sonic Hedgehog Signaling Mediates Resveratrol to Increase Proliferation of Neural Stem Cells After Oxygen-Glucose Deprivation/Reoxygenation Injury in Vitro

Background/Aims: There is interest in drugs and rehabilitation methods to enhance neurogenesis and improve neurological function after brain injury or degeneration. Resveratrol may enhance hippocampal neurogenesis and improve hippocampal atrophy in chronic fatigue mice and prenatally stressed rats. However, its effect and mechanism of neurogenesis after stroke is less well understood. Sonic hedgehog (Shh) signaling is crucial for neurogenesis in the embryonic and adult brain, but relatively little is known about the role of Shh signaling in resveratrol-enhanced neurogenesis after stroke. Methods: Neural stem cells (NSCs) before oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro were pretreated with resveratrol with or without cyclopamine. Survival and proliferation of NSCs was assessed by the CCK8 assay and BrdU immunocytochemical staining. The expressions and activity of signaling proteins and mRNAs were detected by immunocytochemistry, Western blotting, and RT-PCR analysis. Results: Resveratrol significantly increased NSCs survival and proliferation in a concentration-dependent manner after OGD/R injury in vitro. At the same time, the expression of Patched-1, Smoothened (Smo), and Gli-1 proteins and mRNAs was upregulated, and Gli-1 entered the nucleus, which was inhibited by cyclopamine, a Smo inhibitor. Conclusion: Shh signaling mediates resveratrol to increase NSCs proliferation after OGD/R injury in vitro