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Aim: Apoptosis of endothelial cells (ECs) is a crucial factor in blood-spinal cord barrier (BSCB) disruption post spinal cord injury (SCI). Insulin-like growth factor-1 (IGF-1) is a protective cytokine that plays an important role in multiple diseases, whereas the distinct role in SCI-induced remains critical questions to address. Here we designed to explore the role and underlying mechanism of IGF-1 in endothelial damage after SCI., Main Methods: In the current study, we established mouse microvascular endothelial cells (MVECs) injury model via LPS and cDNA of IGF-1 was transfected into MVECs. In vivo SCI mice, overexpression of IGF-1 (SCI-IGF-1) and its corresponding empty vehicle (SCI-NC) were conducted using lentivirus, then apoptosis degree, component of tight junction, and inflammatory damage were evaluated., Key Findings: IGF-1 treatment in MVECs displayed a milder apoptosis and cell damage under LPS insult. IGF-1 increased the level of PI3K/AKT pathway, which impeded the procedure of apoptosis. Blocking of PI3K/AKT pathway markedly neutralized the effect of IGF-1 treatment. Transfection of excess IGF-1 into SCI mice significantly corrected microenvironment of neural tissue repair, reduced area of injured core and improved functional recovery with greater activation of PI3K/AKT pathway., Significance: The results above argue that the promising roles played by IGF-1 is potentially vital for developing effective future therapies in SCI., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mitomycin C (MMC) is known to reduce epidural fibrosis, but the underlying mechanisms have not yet been elucidated. Aberrant miR-200b expressions have been reported in multiple types of fibrotic tissues from many diseases. The aim of this study was to clarify the mechanism by which MMC induces fibroblasts apoptosis and reduces epidural fibrosis. The expression of miR-200b in human fibroblasts was determined after MMC treatment, and the targeted association between miR-200b and RhoE was determined using the luciferase activity assay. The effects of MMC and miR-200b on human fibroblasts apoptosis were evaluated using flow cytometry and western blot analysis. The effects of MMC and miR-200b on epidural fibrosis were evaluated using the Rydell classification, hydroxyproline content, apoptotic cell count and histological analysis. The study revealed that MMC could significantly downregulate miR-200b expression and induce human fibroblasts apoptosis. The direct downregulation of miR-200b could induce human fibroblasts apoptosis. Furthermore, we identified the binding sequence for miR-200b within the 3' untranslated region of RhoE. RhoE was confirmed to be a direct target of miR-200b, and RhoE itself acted as a promoter of fibroblasts apoptosis. The inhibition of miR-200b increased fibroblasts apoptosis and reduced epidural fibrosis in rats, which was in accordance with the effect of MMC. This study suggests that MMC induces fibroblasts apoptosis and reduces epidural fibrosis by regulating miR-200b expression and its targeting of RhoE., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Mechanical stress has detrimental effects on cartilaginous endplate chondrocytes due to apoptosis in vivo and in vitro. In this study, we investigated the possible apoptosis signaling pathways induced by mechanical stress in cultured rat cervical endplate chondrocytes. Static mechanical load significantly reduced cell viability in a time- and load-dependent manner, as demonstrated by the Cell Counting Kit-8 (CCK-8) assay. Chondrocyte apoptosis induced by mechanical stress was confirmed by annexin V/propidium iodide (PI) staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Western blot analysis revealed that static load-induced chondrocyte apoptosis was accompanied by increased phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (MAPK). The loss of mitochondrial membrane potential (ΔΨm), increased Cytochrome c release, and activated Caspase-9 and Caspase-3, indicating that the mitochondrial pathway is involved in mechanical stress-induced chondrocyte apoptosis. Treatment with inhibitors of JNK (SP600125), p38 MAPK (SB203580), and ERK (PD98059) prior to mechanical stimulation reversed both the static load-induced chondrocyte apoptosis and the activation of JNK, p38 MAPK, and ERK. Taken together, the data presented in this study demonstrate that mechanical stress induces apoptosis in rat cervical endplate chondrocytes through the MAPK-mediated mitochondrial apoptotic pathway.

Endoplasmic reticulum (ER) stress-mediated cell apoptosis has been implicated in various cell types, including fibroblasts. Previous studies have shown that mitomycin C (MMC)-induced apoptosis occurs in fibroblasts, but the effects of MMC on ER stress-mediated apoptosis in fibroblasts have not been examined. Here, MMC-induced apoptosis in human primary fibroblasts was investigated by exposing cells to a single dose of MMC for 5 minutes. Significant inhibition of cell proliferation and increased apoptosis were observed using a cell viability assay, Annexin V/propidium iodide double staining, cell cycle analysis, and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining. Upregulation of proapoptotic factors, including cleaved caspase-3 and poly ADP-ribose polymerase (PARP), was detected by Western blotting. MMC-induced apoptosis was correlated with elevation of 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP), which are hallmarks of ER stress. Three unfolded protein response (UPR) sensors (inositol-requiring enzyme 1, IRE1; activating transcription factor 6, ATF6; and PKR-like ER kinase, PERK) and their downstream signaling pathways were also activated. Knockdown of CHOP attenuated MMC-induced apoptosis by increasing the ratio of BCL-2/BAX and decreasing BIM expression, suggesting that ER stress is involved in MMC-induced fibroblast apoptosis. Interestingly, knockdown of PERK significantly decreased ER stress-mediated apoptosis by reducing the expression of CHOP, BIM and cleaved caspase-3. Reactive oxygen species (ROS) scavenging also decreased the expression of GRP78, phospho-PERK, CHOP, and BIM. These results demonstrate that MMC-induced apoptosis is triggered by ROS generation and PERK activation.

Astrocytes play an important role in protecting neurons during ischemia and reperfusion in the central nervous system. Although many studies have shown that oxygen-glucose deprivation (OGD) can induce astrocyte apoptosis, the role of PERK/eIF2 alpha/ATF4 integrated stress response (ISR) in astrocyte apoptosis mediated by oxygen-glucose-serum deprivation (OGSD)/restoration remains uncertain. Astrocytes were subjected to a combination of oxygen, glucose, and serum deprivation for 8h followed by restoration. Hoechst 33342 staining was performed to quantify apoptotic astrocytes and cell viability was assessed with Cell Counting Kit-8 (CCK8). Immunocytochemical analysis and Western blotting for some related molecules, including pancreatic ER stress kinase (PERK), p-PERK, eukaryotic initiation factor 2 alpha (eIF2 alpha), p-eIF2 alpha, activating transcription factor 4 (ATF4), caspase-12, were examined. Caspase activation and apoptosis were detected in neonatal rat astrocytes from spinal cord subjected to OGSD/restoration. We also observed an increase in cytoplasmic staining of p-eIF2 alpha in astrocytes (8h OGSD/15 min restoration) compared with that of non-treated cells. In addition, we found the sequential activation of PERK, eIF2 alpha, and ATF4 during OGSD/restoration by Western blotting. These results indicate that both the PERK/eIF2 alpha/ATF4 ISR and activation of caspase-12 may be involved in apoptosis of spinal cord astrocytes induced by OGSD/restoration., ((c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Background Context: Recovery from spine surgery is usually accompanied with the development of epidural scar adhesions from the abnormal proliferation of fibroblast and extracellular matrix-related metabolism. Polyethylene glycol (PEG) could alleviate the postsurgical adhesion formation with still leaving a gap between the sheet and the dura. However, PEG film could not function as a three-dimensional barrier to prevent adhesion completely. Mitomycin C (MMC) could also reduce the scar formation after surgery, but cytotoxicity and the administrative pathway prevent its application., Purpose: Our purpose was to design and attest the role of new delivery system MMC-PEG controlled-release film in preventing the epidural scar adhesions after laminectomy in the rat model., Study Design/setting: A total laminectomy of L1 in the rat model was used to assess epidural fibrosis between and around the spinal nerves using a histochemistry assessment along with flow cytometry analysis., Patient Sample: The sample comprises 64 adult male Sprague-Dawley rats., Outcome Measures: The outcome measures are macroscopic evaluation, histological analysis, and flow cytometry analysis., Methods: Lumbar laminectomies at L1 and L2 with a L1-L2 disc injury were performed on 64 adult male Sprague-Dawley rats. The rats were then randomized into four groups. In Group I, 25 mg PEG film was applied on the dura mater in the laminectomy area before the layers were sutured. In Group II, a cotton pad soaked with 0.05% MMC solution was kept on the laminectomy area for 5 minutes. In Group III, 25 mg PEG film containing 0.01% MMC was implanted on the laminectomy area. In Group IV, the laminectomy area was flushed with saline before wound closure. The rats were sacrificed 4 weeks after the operation. Macroscopic evaluation and histological analysis of epidural scar adhesion with the hematoxylin and eosin stain and Masson stain were used followed by the quantification of hydroxyproline (Hyp) and flow cytometry analysis of the apoptosis of fibroblasts in the scar tissues., Results: The recovery of all rats was uneventful after the operations. In the laminectomy sites of rats treated with MMC or MMC-PEG, the dura mater was clean without any evident adhesion or membrane. Collagen tissue hyperplasia significantly decreased in the MMC- or MMC-PEG-treated models. Accordingly, Hyp concentration was significantly reduced in these two groups compared with saline-control group. In addition, the apoptosis of fibroblasts, however, was significantly elevated in the MMC or MMC-PEG group compared with the saline-control group., Conclusions: These results demonstrate that the treatment of postlaminectomy wounds with MMC-PEG film reduces the severity of adhesion by decreasing the concentration of Hyp and increasing the apoptosis of fibroblasts., (Copyright 2010 Elsevier Inc. All rights reserved.)

Objective: To explore the molecular mechanism of the protective effect of nerve growth factor (NGF) on injured spinal cord., Methods: The posterior T(8) (the 8th thoracic segment) spinal cords of 60 Wistar rats were injured by impacts caused by objects (weighing 10 g) falling from a height of 2.5 cm with Allen's way. Solution with nerve growth factors (NGF) was given to 30 rats (the NGF group) through a microtubule inserted into the subarachnoid cavity immediately, and at 2, 4, 8, 12 and 24 hours after spinal cord injury (SCI) respectively. Normal saline (NS) with same volume was given to the other 30 rats (the NS group) with the same method. And 5 normal rats were taken as the normal controls. The expression of bcl-2 and bax proteins in spinal cord was detected with immunohistochemistry. The apoptotic neurons in spinal cord were measured with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling of DNA fragments (TUNEL) staining., Results: The positive expression of bcl-2 protein was strong in the normal controls, but decreased in the NS group, and increased significantly in the NGF group as compared with that of the NS group (P<0.01). The positive expression of bax protein was also strong in the normal controls, but increased in the NS group, and decreased significantly in the NGF group as compared with that of the NS group (P<0.01). Apoptotic neurons were found in the NS group, and they decreased significantly in the NGF group as compared with that of the NS group (P<0.01)., Conclusions: NGF can protect the injured nerve tissues through stimulating the expression of bcl-2 protein, inhibiting the expression of bax protein and inhibiting the neuronal apoptosis after SCI.

Background/Aims: Hypertrophic scars (HS) formation results from reduced apoptosis and increased proliferation of fibroblasts. Therefore, apoptosis of fibroblasts is a key target for the development of novel therapeutic strategies for HS. Previous reports demonstrated that FK506 could attenuate scar formation in vivo and FK506 could also induce endoplasmic reticulum stress (ER stress). However, the effects of FK506 on ER stress-mediated apoptosis in fibroblasts remain unclear. Methods: Rat skin fibroblasts were used in the study. Cell viability was examined using cell counting Kit-8. Apoptosis was detected by Annexin V/Propidium Iodide Double Staining. Gene silencing was performed using Small Interfering RNAs (siRNAs) or via lentiviral infection. The expression of apoptosis-related proteins was determined via Western blot. Interaction between proteins was explored by co-immunoprecipitation. Results: FK506 significantly reduced cell viability and induced apoptosis in fibroblasts. Interestingly, ER stress was also activated after FK506 treatment. We further demonstrated that FK506-induced apoptosis was mediated by ER stress via activating CHOP, evidenced by decreased apoptosis after inhibition of ER stress using TUDCA or silencing expression of CHOP. Furthermore, Co-immunoprecipitation results indicated that treatment of FK506 induced disassociation of FKBP12.6 from RyR2 and its translocation from ER membrane to cytosol, consequently promoting ER stress-mediated apoptosis. Conclusion: FK506-induced fibroblasts apoptosis was mediated by ER stress via CHOP signaling pathway. [ABSTRACT FROM AUTHOR]

Background/Aims: Rheumatoid arthritis (RA) is a systemic chronic inflammatory disease characterised by prominent synoviocyte hyperplasia and a potential imbalance between the growth and death of fibroblast-like synoviocytes (FLS). Mitomycin C (MMC) has previously been demonstrated to inhibit fibroblast proliferation and to induce fibroblast apoptosis. However, the effects of MMC on the proliferation and apoptosis of human RA FLS and the potential mechanisms underlying its effects remain unknown. Methods: Cell viability was determined using the Cell Counting Kit-8 assay. Apoptotic cell death was analysed via Annexin V-FITC/PI double staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling. The production of intracellular reactive oxygen species (ROS) was assessed via flow cytometry, and the changes in mitochondrial membrane potential (ΔΨm) were visualized based on JC-1 staining via fluorescence microscopy. The expression of apoptosis-related proteins was determined via Western blot. Results: Treatment with MMC significantly reduced cell viability and induced apoptosis in RA FLS. Furthermore, MMC exposure was found to stimulate the production of ROS and to disrupt the ΔΨm compared to the control treatment. Moreover, MMC increased the release of mitochondrial cytochrome c, the ratio of Bax/Bcl-2, the activation of caspase-9 and caspase-3, and the subsequent cleavage of poly(ADP-ribose) polymerase. Conclusion: Our findings suggest that MMC inhibits cell proliferation and induces apoptosis in RA FLS, and the mechanism underlying this MMC-induced apoptosis may involve a mitochondrial signalling pathway. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress-mediated cell apoptosis has been implicated in various cell types, including fibroblasts. Previous studies have shown that mitomycin C (MMC)-induced apoptosis occurs in fibroblasts, but the effects of MMC on ER stress-mediated apoptosis in fibroblasts have not been examined. Here, MMC-induced apoptosis in human primary fibroblasts was investigated by exposing cells to a single dose of MMC for 5 minutes. Significant inhibition of cell proliferation and increased apoptosis were observed using a cell viability assay, Annexin V/propidium iodide double staining, cell cycle analysis, and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining. Upregulation of proapoptotic factors, including cleaved caspase-3 and poly ADP-ribose polymerase (PARP), was detected by Western blotting. MMC-induced apoptosis was correlated with elevation of 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP), which are hallmarks of ER stress. Three unfolded protein response (UPR) sensors (inositol-requiring enzyme 1, IRE1; activating transcription factor 6, ATF6; and PKR-like ER kinase, PERK) and their downstream signaling pathways were also activated. Knockdown of CHOP attenuated MMC-induced apoptosis by increasing the ratio of BCL-2/BAX and decreasing BIM expression, suggesting that ER stress is involved in MMC-induced fibroblast apoptosis. Interestingly, knockdown of PERK significantly decreased ER stress-mediated apoptosis by reducing the expression of CHOP, BIM and cleaved caspase-3. Reactive oxygen species (ROS) scavenging also decreased the expression of GRP78, phospho-PERK, CHOP, and BIM. These results demonstrate that MMC-induced apoptosis is triggered by ROS generation and PERK activation. [ABSTRACT FROM AUTHOR]

Abstract: Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK), which plays a pivotal role in cell apoptosis. To determine the mechanism of ASK1 induction during reperfusion of ischemic spinal tissue, we used a model of rabbit spinal cord ischemia and reperfusion. To assess the role of ASK1 in spinal cord ischemia-reperfusion injuries, we examined alterations in spinal tissue morphology, protein–protein interactions, and activation of key members of the ASK1-mediated signaling pathway. Changes in spinal cord morphology were observed with hematoxylin and eosin (H&E) staining and electron microscopy. The phosphorylation levels of ASK1, JNK, and p38 were assessed by immunoblotting proteins from animals that received 30min of ischemia followed by 1 or 24h of reperfusion. We observed increased phosphorylation of ASK1, JNK, and p38 after reperfusing ischemic spinal cords. Immunohistochemical studies were performed to determine the cellular localization of phosphorylated ASK1 (pASK1) and 14-3-3. Following reperfusion for 24h, we observed increased cytoplasmic localization of pASK1 and decreased cytoplasmic localization of 14-3-3. Immunoprecipitation analyses suggested that 14-3-3 dissociates from ASK1 during reperfusion of ischemic spinal cords. These results indicate that activation of ASK1 may play an important role in the apoptotic signaling mechanisms that occur in reperfused spinal cord injuries. [Copyright &y& Elsevier]

• P38 serves as a direct target of miR-340-5p. • miR-340-5p overexpression attenuates neuroinflammation both in vitro and in vivo SCI models. • miR-340-5p mimics prevent inflammation-mediated oxidative stress and apoptosis via reducing P38 level. • miR-340-5p mimics improve locomotor recovery by alleviating P38/MAPK pathway-induced neuroinflammation. Spinal cord injury (SCI) is a destructive polyneuropathy that can result in loss of sensorimotor function and sphincter dysfunction, and even death in critical situations. MicroRNAs (miRs) are a series of non-coding RNA molecules that are involved in transcriptional regulation. Previous studies have demonstrated that modulation of multiple miRs is involved in neurological recovery after SCI. However, the functions of miR-340-5p in SCI remain uncertain. Therefore, we probed the therapeutic effect and mechanism of miR-340-5p in microglia in vitro and in vivo in SCI rats. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were employed to examine the alterations in miR-340-5p and P38 levels in SCI rats. miR-340-5p targets in microglia were ascertained using luciferase reporter assays, immunofluorescence analyses, and western blotting. We also established an SCI model and administered miR-340-5p. The effects of miR-340-5p on the amelioration of inflammation, oxidative stress, and apoptosis following SCI were assessed using immunofluorescence, immunohistochemistry, and histological analyses. Finally, locomotor function recovery was determined using the Basso, Beattie, Bresnahan rating scale. In our study, the expression profiles and luciferase assay results clarified that P38 was a target of miR-340-5p, which was associated with activation of the P38-MAPK signaling pathway. Elevation of miR-340-5p decreased P38 expression, subsequently inhibiting the inflammatory reaction. SCI-induced secondary neuroinflammation was relieved under miR-340-5p treatment. Moreover, by controlling neuroinflammation, the increased levels of miR-340-5p might counter oxidative stress and reduce the degree of apoptosis. We also observed decreasing gliosis and glial scar formation and increasing neurotrophin expression at the chronic stage of SCI. Together, these potential effects of miR-340-5p treatment ultimately improved locomotor function recovery in SCI rats. [ABSTRACT FROM AUTHOR]