Nrf2 Signaling Pathway: Focus on Oxidative Stress in Spinal Cord Injury.

Spinal cord injury (SCI) is a serious, disabling injury to the central nervous system that can lead to motor, sensory, and autonomic dysfunction below the injury plane. SCI can be divided into primary injury and secondary injury according to its pathophysiological process. Primary injury is irreversible in most cases, while secondary injury is a dynamic regulatory process. Secondary injury involves a series of pathological events, such as ischemia, oxidative stress, inflammatory events, apoptotic pathways, and motor dysfunction. Among them, oxidative stress is an important pathological event of secondary injury. Oxidative stress causes a series of destructive events such as lipid peroxidation, DNA damage, inflammation, and cell death, which further worsens the microenvironment of the injured site and leads to neurological dysfunction. The nuclear factor erythrocyte 2-associated factor 2 (Nrf2) is considered to be a key pathway of antioxidative stress and is closely related to the pathological process of SCI. Activation of this pathway can effectively inhibit the oxidative stress process and promote the recovery of nerve function after SCI. Therefore, the Nrf2 pathway may be a potential therapeutic target for SCI. This review deeply analyzed the generation of oxidative stress in SCI, the role and mechanism of Nrf2 as the main regulator of antioxidant stress in SCI, and the influence of cross-talk between Nrf2 and related pathways that may be involved in the pathological regulation of SCI on oxidative stress, and summarized the drugs and other treatment methods based on Nrf2 pathway regulation. The objective of this paper is to provide evidence for the role of Nrf2 activation in SCI and to highlight the important role of Nrf2 in alleviating SCI by elucidating the mechanism, so as to provide a theoretical basis for targeting Nrf2 pathway as a therapy for SCI.

Evaluation of Phenazine Derivatives from the Lichen-Associated Streptomyces flavidovirens as Potent Antineuroinflammatory Agents In Vitro and In Vivo.

Eighteen nitrogen-containing compounds (1-18) were isolated from cultures of the lichen-associated Streptomyces flavidovirens collected from the Qinghai-Tibet Plateau, including seven phenazine derivatives with three new ones, named subphenazines A-C (2-4), two new furan pyrrolidones (8-9), and nine known alkaloids. The structures were elucidated by spectroscopic data analysis, and absolute configurations were determined by single-crystal X-ray diffraction and ECD calculations. The phenazine-type derivatives, in particular compound 3, exhibited significantly better antineuroinflammatory activity than other isolated compounds (8-18). Compound 3 inhibited the release of proinflammatory cytokines including IL-6, TNF-α, and PGE2, and the nuclear translocation of NF-κB; it also reduced the oxidative stress and activated the Nrf2 signaling pathway in LPS-induced BV2 microglia cells. In vivo anti-inflammatory activity in zebrafish indicated that 3 inhibited LPS-stimulated ROS generation. These findings suggested that compound 3 might be a potent antineuroinflammatory agent through the regulation of the NF-κB/Nrf2 signaling pathways.

SPI1 exacerbates iron accumulation and promotes osteoclast formation through inhibiting the expression of Hepcidin.

BACKGROUND: Osteoporosis (OP) can be caused by an overactive osteoclastic function. Anti-osteoporosis considerable therapeutic effects in tissue repair and regeneration because bone resorption is a unique osteoclast function. In this study, we mainly explored the underlying mechanisms of osteoclasts' effects on osteoporosis. METHODS: RAW264.7 cells were used and induced toward osteoclast and iron accumulation by M-CSF and RANKL administration. We investigated Hepcidin and divalent metal transporter 1 (DMT1) on iron accumulation and osteoclast formation in an ovariectomy (OVX)-induced osteoporosis. Osteoporosis was induced in mice by OVX, and treated with Hepcidin (10, 20, 40, 80 mg/kg, respectively) and overexpression of DMT1 by tail vein injection. Hepcidin, SPI1, and DMT1 were detected by immunohistochemical staining, western blot and RT-PCR. The bioinformatics assays, luciferase assays, and Chromatin Immunoprecipitation (ChIP) verified that Hepcidin was a direct SPI1 transcriptional target. Iron accumulation was detected by laser scanning confocal microscopy, Perl's iron staining and iron content assay. The formation of osteoclasts was assessed using tartrate-resistant acid phosphatase (TRAP) staining. RESULTS: We found that RAW264.7 cells differentiated into osteoclasts when exposed to M-CSF and RANKL, which increased the protein levels of osteoclastogenesis-related genes, including c-Fos, MMP9, and Acp5. We also observed higher concentration of iron accumulation when M-CSF and RANKL were administered. However, Hepcidin inhibited the osteoclast differentiation cells and decreased intracellular iron concentration primary osteoclasts derived from RAW264.7. Spi-1 proto-oncogene (SPI1) transcriptionally repressed the expression of Hepcidin, increased DMT1, facilitated the differentiation and iron accumulation of mouse osteoclasts. Overexpression of SPI1 significantly declined luciferase activity of HAMP promoter and increased the enrichment of HAMP promoter. Furthermore, our results showed that Hepcidin inhibited osteoclast differentiation and iron accumulation in mouse osteoclasts and OVX mice. CONCLUSION: Therefore, the study revealed that SPI1 could inhibit Hepcidin expression contribute to iron accumulation and osteoclast formation via DMT1 signaling activation in mouse with OVX.

Iron accumulation induced by hepcidin1 knockout accelerates the progression of aging osteoporosis.

OBJECTIVE: Iron accumulation is associated with osteoporosis. This study aims to explore the effect of chronic iron accumulation induced by hepcidin1 deficiency on aging osteoporosis. METHODS: Iron accumulation in hepcidin1 knockout aging mice was assessed by atomic absorption spectroscopy and Perl's staining. Bone microarchitecture was observed using Micro-CT. Hepcidin, ferritin, oxidative stress, and markers of bone turnover in serum were detected by enzyme-linked immunosorbent assay. Bone formation and resorption markers were measured by real-time quantitative PCR. Cell aging was induced by D-galactose treatment. CCK-8, flow cytometry, EdU assays, and Alizarin red staining were performed to reveal the role of hepcidin1 knockout in cell model. Iron Colorimetric Assay Kit and western blot were applied to detect iron and ferritin levels in cells, respectively. RESULTS: In hepcidin1-knockout mice, the ferritin and iron contents in liver and tibia were significantly increased. Iron accumulation induced by hepcidin1 knockout caused a phenotype of low bone mass and deteriorated bone microarchitecture. Osteogenic marker was decreased and osteoclast marker was increased in mice, accompanied by increased oxidative stress level. The mRNA expression levels of osteoclast differentiation markers (RANKL, Mmp9, OPG, Trap, and CTSK) were up-regulated, while bone formation markers (OCN, ALP, Runx2, SP7, and Col-1) were down-regulated in model group, compared to wild type mice. In vitro, hepcidin1 knockdown inhibited proliferation and osteogenic differentiation, while promoted apoptosis, with increased levels of iron and ferritin. CONCLUSION: Iron accumulation induced by hepcidin1 deficiency aggravates the progression of aging osteoporosis via inhibiting osteogenesis and promoting osteoclast genesis.

Reduction of the trans-cortical vessel was associated with bone loss, another underlying mechanism of osteoporosis.

RATIONALE: Numerous studies have established a robust association between bone morrow microvascular diseases and osteoporosis. This study sought to investigate the relationship between alterations in trans-cortical vessel (TCVs) and the onset of osteoporosis in various mouse models. METHODS: Aged mice, ovariectomized mice, and db/db mice, were utilized as osteoporosis models. TCVs in the tibia were detected using tissue clearing and light sheet fluorescence microscopy imaging. Femurs bone mass were analyzed using micro-CT scanning. Correlations between the number of TCVs and bone mass were analyzed using Pearson correlation analysis. RESULTS: All osteoporosis mouse models showed a significant reduction in the number of TCVs compared to the control group. Correlation analysis revealed a positive association between the number of TCVs and bone mass. TCVs were also expressed high levels of CD31 and EMCN proteins as type H vessels. CONCLUSIONS: This study underscores a consistent correlation between the number of TCVs and bone mass. Moreover, TCVs may serve as a potential biomarker for bone mass evaluation.

Hyperelatolides A-D, Antineuroinflammatory Constituents with Unusual Carbon Skeletons from Hypericum elatoides.

Four new δ- and γ-lactone derivatives, hyperelatolides A-D (1-4, respectively), were discovered from the aerial portions of Hypericum elatoides R. Keller. Their structures were elucidated by analysis of NMR spectra, HRESIMS, quantum chemical calculations of NMR and ECD spectra, and X-ray crystallographic data. Hyperelatolides A (1) and B (2) represent the first examples of δ-lactone derivatives characterized by a (Z)-(5,5-dimethyl-2-(2-oxopropyl)cyclohexylidene)methyl moiety and a benzoyloxy group attached to the ß- and γ-positions of the δ-lactone core, respectively, while hyperelatolides C (3) and D (4) are unprecedented γ-lactone derivatives featuring substituents similar to those of 1 and 2. All compounds were tested for their inhibitory effects on NO production in LPS-activated BV-2 cells. Lactones 1 and 2 exhibited considerable antineuroinflammatory activity, with IC50 values of 5.74 ± 0.27 and 7.35 ± 0.26 µM, respectively. Moreover, the mechanistic study revealed that lactone 1 significantly suppressed nuclear factor kappa B signaling and downregulated the expression of inducible nitric oxide synthase and cyclooxygenase-2 in LPS-induced cells, which may contribute to its antineuroinflammatory activity.

N-acetyl-L-cysteine attenuates oxidative stress-induced bone marrow endothelial cells apoptosis by inhibiting BAX/caspase 3 pathway.

Bone marrow endothelial cells (BMECs) play a crucial role in the maintenance of bone homeostasis. The decline in BMECs is associated with abnormal bone development and loss. At present, the mechanism of age-related oxidative stress enhancement in BMEC dysfunction remains unclear. Our experiment explored injury caused by oxidative stress enhancement in BMECs both in vivo and in vitro. The BMECs, indicators of oxidative stress, bone mass, and apoptosis-related proteins were analyzed in different age groups. We also evaluated the ability of N-Acetyl-L-cysteine (NAC) attenuate oxidative stress injury in BMECs. NAC treatment attenuated reactive oxygen species (ROS) overgeneration and apoptosis in BMECs in vitro and alleviated the loss of BMECs and bone mass in vivo. In conclusion, this study could improve our understanding of the mechanism of oxidative stress-induced BMECs injury and whether NAC has therapeutic potential in senile osteoporosis.

The role of PI3K/Akt signalling pathway in spinal cord injury.

Spinal cord injury (SCI) is a severely disabling central nervous system injury with complex pathological mechanisms that leads to sensory and motor dysfunction. The current treatment for SCI is aimed at symptomatic symptom relief rather than the pathological causes. Several studies have reported that signaling pathways play a key role in SCI pathological processes and neuronal recovery mechanisms. The PI3K/Akt signaling pathway is an important pathway closely related to the pathological process of SCI, and activation of this pathway can delay the inflammatory response, prevent glial scar formation, and promote neurological function recovery. Activation of this pathway can promote the recovery of neurological function after SCI by reducing cell apoptosis. Based on the role of the PI3K/Akt pathway in SCI, it may be a potential therapeutic target. This review highlights the role of activating or inhibiting the PI3K/Akt signaling pathway in SCI-induced inflammatory response, apoptosis, autophagy, and glial scar formation. We also summarize the latest evidence on treating SCI by targeting the PI3K/Akt pathway, discuss the shortcomings and deficiencies of PI3K/Akt research in the field of SCI, and identify potential challenges in developing these clinical therapeutic SCI strategies, and provide appropriate solutions.

ATF1/miR-214-5p/ITGA7 axis promotes osteoclastogenesis to alter OVX-induced bone absorption.

BACKGROUND: The dynamic balance of osteoblast and osteoclast is critical for bone homeostasis and overactive osteoclastic function may lead to osteoporosis. Activating transcription factor 1 (ATF1) is involved in osteoclastogenesis. However, the detailed mechanisms remain to be explored. METHODS: RAW264.7 cells were used and induced toward osteoclast by RANKL administration. We performed flow cytometry, CCK-8 assay and tartrate-resistant acid phosphatase (TRAP) staining to examine cell apoptosis, proliferation and differentiation of RAW264.7 cells, respectively. Mice were subjected to ovariectomy to induce osteoporosis. Micro CT, HE staining and TRAP staining were performed to evaluate bone loss in the OVX mouse model. Bioinformatics methods, luciferase assays and Chromatin Immunoprecipitation (ChIP) were used to predict and validate the interaction among ATF1, miR-214-5p, and ITGA7. RESULTS: ATF1 and miR-214-5p were up-regulated while ITGA7 was inhibited in RANKL-induced osteoclasts. MiR-214-5p was transcriptionally activated by ATF1. ATF1 knockdown suppressed osteoclast formation by miR-214-5p inhibition. ITGA7 was the direct target of miR-214-5p. Knockdown of miR-214-5p abolished osteoclastogenesis, which was reversed by ITGA7 knockdown. In OVX model, miR-214-5p knockdown suppressed osteoclast differentiation and prevented bone loss. CONCLUSION: ATF1/miR-214-5p/ITGA7 axis regulated osteoclast formation both in vivo and in vitro, thereby affecting OVX-induced bone resorption in mice. Knockdown of ATF1 might be a promising strategy to manage osteoporosis.

Ferric Ion Induction of Triggering Receptor Expressed in Myeloid Cells-2 Expression and PI3K/Akt Signaling Pathway in Preosteoclast Cells to Promote Osteoclast Differentiation.

OBJECTIVE: Iron plays a significant role in multiple biological processes. The purpose of this study was to measure whether iron mediated osteoclast differentiation through regulation of triggering receptor expressed in myeloid cells-2 (Trem-2) expression and the PI3K/Akt signaling pathway. METHODS: The effects of six different concentrations of ferric ammonium citrate (FAC) (100, 80, 40, 20, 10 and 0 µmol/L) on RAW 264.7 cells proliferation were assessed by Cell Counting Kit-8 (CCK-8) gassay. Tartrate resistant acid phosphatase (TRAP) assay was performed to detect the effects of FAC on osteoclast formation. The expression of osteoclast differentiation-related (TRAP, NFATc-1, and c-Fos) and Trem-2 mRNA and proteins was analyzed by reverse transcription-polymerase chain reaction and western blot, respectively. Si-Trem-2 was constructed and transfected to RAW264.7 to measure the effects of Trem-2 on FAC-mediated osteoclast formation. TRAP assay and osteoclast differentiation-related gene analyses were further performed to identify the role of Trem-2 in osteoclastogenesis. The Search Tool for the Retrieval of Interacting Genes (STRING) was used to explore the target genes of Trem-2. Trem-2-related gene ontology and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were used for further in-depth analysis. PI3K/Akt pathway-related proteins were detected by immunofluorescence and western blot. RESULTS: In groups with FAC concentration of 10 (102.5 ± 3.1), 20 (100.5 ± 1.5), and 40 µmol/L (98.7 ± 3.1), compared with the control group (100.1 ± 2.2), cell viability was not significantly different from the control (P > 0.05). When the concentration of FAC exceeded 80 µmol/L, cell viability was significantly decreased (87.5 ± 2.8 vs 100.1 ± 2.2, P < 0.05). FAC promotes Trem-2 expression and osteoclast differentiation in a dose-response manner (P < 0.05). The number of osteoclast-like cells was found to be reduced following transfection with the siRNA of Trem-2 (42 ± 3 vs 30 ± 5, P < 0.05). We observed that most of Trem-2 target genes are primarily involved in response to organic substance, regulation of reactive oxygen species metabolic process, and regulation of protein phosphorylation. The STRING database revealed that Trem-2 directly target two gene nodes (Pik3ca and Pik3r1), which are key transcriptional cofactors of the PI3K/Akt signaling pathway. KEGG pathways include the "PI3K-Akt signaling pathway," the "thyroid hormone signaling pathway", "prostate cancer," the "longevity regulating pathway," and "insulin resistance." Expression of p-PI3K and p-Akt protein, measured by immunofluorescence and western blotting, was markedly increased in the FAC groups. Trem-2 siRNA caused partial reduction of these two proteins (p-PI3K and p-Akt) compared to the FAC alone group. CONCLUSION: The FAC promoted osteoclast differentiation through the Trem-2-mediated PI3K/Akt signaling pathway. However, its regulation osteoclastogenesis should be verified through further in vivo studies.

Reliability of Atmosphere Pressure-Plasma Enhanced Chemical Vapor Deposition Deposited Indium Gallium Zinc Oxide Resistive Random Access Memory Device with Microwave Annealing.

Recently resistive random access memory (RRAM) is considered to be the most promising one to become the next generation memory since its simple Metal/Insulator/Metal (MIM) structure, lower power consumption and fabrication cost (Meena, J.S., et al., 2014. Overview of emerging nonvolatile memory technologies. Nanoscale Research Letters, 9(1), p.526). Due to some bottlenecks for current flash memory, such as high operation voltage, low operation speed, poor retention time and endurance, RRAM device is regarded as an alternative solution (Fuh, C.S., et al., 2011. Role of environmental and annealing conditions on the passivation-free In-Ga-Zn-O TFT. Thin Solid Films, 520, pp.1489-1494). In this investigation, the memory layer of RRAM device is IGZO, and it is deposited with AP-PECVD technique which can operate under atmosphere, reduce cost of the process. Microwave annealing (MWA) is used to enhance the RRAM device reliability (Fuh, C.S., et al., 2011. Role of environmental and annealing conditions on the passivation-free In-Ga-Zn-O TFT. Thin Solid Films, 520, pp.1489-1494). Experiment shows that with appropriate MWA treatment, the IGZO RRAM device exhibits better electrical characteristics, reliability issues such as numbers of switching cycle and data retention time are also improved (Teng, L.F., et al., 2012. Effects of microwave annealing on electrical enhancement of amorphous oxide semiconductor thin film transistor. Applied Physics Letters, 101, p.132901).

Investigation of Microwave Annealing on Resistive Random Access Memory Device with Atmospheric Pressure Plasma Enhanced Chemical Vapor Deposition Deposited IGZO Layer.

Non-volatile memory (NVM) is essential in almost every consumer electronic products. The most prevalent NVM used nowadays is flash memory (Meena, J.S., et al., 2014. Overview of emerging nonvolatile memory technologies. Nanoscale Res. Letters, 9(1), p.526). However, some bottlenecks of flash memory have been identified, such as high operation voltage, low operation speed, and poor retention time. Resistive random access memory (RRAM) is considered to be the most promising one to become the next generation NVM device since its simple structure, fast program/erase speed, and low power consumption. In this experiment, the RRAM device is fabricated, and its IGZO (memory) layer is deposited with AP-PECVD technique which can reduce cost of the process. Microwave annealing (MWA) is used to enhance electrical characteristics of the RRAM device (Fuh, C.S., et al., 2011. Role of environmental and annealing conditions on the passivation-free In-Ga- Zn-O TFT. Thin Solid Films, 520, pp.1489-1494). Experiment results show that with appropriate MWA treatment, the IGZO RRAM device exhibits better electrical characteristics under bipolar operation, all forming/set/reset voltage for RRAM device is simultaneously lowered.