Reduction in antioxidant enzyme expression and sustained inflammation enhance tissue damage in the subacute phase of spinal cord contusive injury

<p>Abstract</p> <p>Background</p> <p>Traumatic spinal cord injury (SCI) forms a disadvantageous microenvironment for tissue repair at the lesion site. To consider an appropriate time window for giving a promising therapeutic treatment for subacute and chronic SCI, global changes of proteins in the injured center at the longer survival time points after SCI remains to be elucidated.</p> <p>Methods</p> <p>Through two-dimensional electrophoresis (2DE)-based proteome analysis and western blotting, we examined the differential expression of the soluble proteins isolated from the lesion center (LC) at day 1 (acute) and day 14 (subacute) after a severe contusive injury to the thoracic spinal cord at segment 10. In situ apoptotic analysis was used to examine cell apoptosis in injured spinal cord after adenoviral gene transfer of antioxidant enzymes. In addition, administration of chondroitinase ABC (chABC) was performed to analyze hindlimb locomotor recovery in rats with SCI using Basso, Beattie and Bresnahan (BBB) locomotor rating scale.</p> <p>Results</p> <p>Our results showed a decline in catalase (CAT) and Mn-superoxide dismutase (MnSOD) found at day 14 after SCI. Accordingly, gene transfer of SOD was introduced in the injured spinal cord and found to attenuate cell apoptosis. Galectin-3, β-actin, actin regulatory protein (CAPG), and F-actin-capping protein subunit β (CAPZB) at day 14 were increased when compared to that detected at day 1 after SCI or in sham-operated control. Indeed, the accumulation of β-actin⁺immune cells was observed in the LC at day 14 post SCI, while most of reactive astrocytes were surrounding the lesion center. In addition, chondroitin sulfate proteoglycans (CSPG)-related proteins with 40-kDa was detected in the LC at day 3-14 post SCI. Delayed treatment with chondroitinase ABC (chABC) at day 3 post SCI improved the hindlimb locomotion in SCI rats.</p> <p>Conclusions</p> <p>Our findings demonstrate that the differential expression in proteins related to signal transduction, oxidoreduction and stress contribute to extensive inflammation, causing time-dependent spread of tissue damage after severe SCI. The interventions by supplement of anti-oxidant enzymes right after SCI or delayed administration with chABC can facilitate spinal neural cell survival and tissue repair.</p

Reduction in antioxidant enzyme expression and sustained inflammation enhance tissue damage in the subacute phase of spinal cord contusive injury

<p>Abstract</p> <p>Background</p> <p>Traumatic spinal cord injury (SCI) forms a disadvantageous microenvironment for tissue repair at the lesion site. To consider an appropriate time window for giving a promising therapeutic treatment for subacute and chronic SCI, global changes of proteins in the injured center at the longer survival time points after SCI remains to be elucidated.</p> <p>Methods</p> <p>Through two-dimensional electrophoresis (2DE)-based proteome analysis and western blotting, we examined the differential expression of the soluble proteins isolated from the lesion center (LC) at day 1 (acute) and day 14 (subacute) after a severe contusive injury to the thoracic spinal cord at segment 10. In situ apoptotic analysis was used to examine cell apoptosis in injured spinal cord after adenoviral gene transfer of antioxidant enzymes. In addition, administration of chondroitinase ABC (chABC) was performed to analyze hindlimb locomotor recovery in rats with SCI using Basso, Beattie and Bresnahan (BBB) locomotor rating scale.</p> <p>Results</p> <p>Our results showed a decline in catalase (CAT) and Mn-superoxide dismutase (MnSOD) found at day 14 after SCI. Accordingly, gene transfer of SOD was introduced in the injured spinal cord and found to attenuate cell apoptosis. Galectin-3, β-actin, actin regulatory protein (CAPG), and F-actin-capping protein subunit β (CAPZB) at day 14 were increased when compared to that detected at day 1 after SCI or in sham-operated control. Indeed, the accumulation of β-actin⁺immune cells was observed in the LC at day 14 post SCI, while most of reactive astrocytes were surrounding the lesion center. In addition, chondroitin sulfate proteoglycans (CSPG)-related proteins with 40-kDa was detected in the LC at day 3-14 post SCI. Delayed treatment with chondroitinase ABC (chABC) at day 3 post SCI improved the hindlimb locomotion in SCI rats.</p> <p>Conclusions</p> <p>Our findings demonstrate that the differential expression in proteins related to signal transduction, oxidoreduction and stress contribute to extensive inflammation, causing time-dependent spread of tissue damage after severe SCI. The interventions by supplement of anti-oxidant enzymes right after SCI or delayed administration with chABC can facilitate spinal neural cell survival and tissue repair.</p

Antroquinonol Targets FAK-Signaling Pathway Suppressed Cell Migration, Invasion, and Tumor Growth of C6 Glioma.

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine that is overexpressed in many types of tumors and plays a pivotal role in multiple cell signaling pathways involved in cell survival, migration, and proliferation. This study attempts to determine the effect of synthesized antroquinonol on the modulation of FAK signaling pathways and explore their underlying mechanisms. Antroquinonol significantly inhibits cell viability with an MTT assay in both N18 neuroblastoma and C6 glioma cell lines, which exhibits sub G1 phase cell cycle, and further induction of apoptosis is confirmed by a TUNEL assay. Antroquinonol decreases anti-apoptotic proteins, whereas it increases p53 and pro-apoptotic proteins. Alterations of cell morphology are observed after treatment by atomic force microscopy. Molecular docking results reveal that antroquinonol has an H-bond with the Arg 86 residue of FAK. The protein levels of Src, pSrc, FAK, pFAK, Rac1, and cdc42 are decreased after antroquinonol treatment. Additionally, antroquinonol also regulates the expression of epithelial to mesenchymal transition (EMT) proteins. Furthermore, antroquinonol suppresses the C6 glioma growth in xenograft studies. Together, these results suggest that antroquinonol is a potential anti-tumorigenesis and anti-metastasis inhibitor of FAK

Targeting the Y397 site of FAK with a structure-based molecular docking approach.

<p>(A) The structure of FAK (FERM domain, PDB code 2AL6), and (B) antroquinonol.</p

Effects of antroquinonol on FAK signaling pathway.

<p>Cells were treated with the indicated concentrations of antroquinonol (5 and 10 μM) for 24 h. Cells were harvested and lysed for western blot analysis. (A) Changes in the levels of FAK, pFAK, Src, pSrc, Cdc42, and Rac1 proteins after normalization to the levels of beta-actin are shown below each blot. (B) The <i>in vitro</i> assay was performed to detect the effect of antroquinonol on pFAK (Y397) (i) N18 and (ii) C6. (C) Cells were treated with PF 431396 (0.5 and 2 μM) for 24 h. Proteins were extracted and subjected to western blot analysis for FAK and pFAK. The results shown are the mean ±SEM of three independent experiments. *<i>p</i> < 0.05, **<i>p</i> < 0.01, and ***<i>p</i> < 0.001 when compared with the untreated controls. C—Control, A—antroquinonol and PF- PF 431396 (FAK inhibitor).</p

Effects of antroquinonol on EMT and MMP proteins in C6 and N18.

<p>Cells were treated with the indicated concentrations of antroquinonol (5 and 10 μM) for 24 h. (A) Cells were harvested and lysed for western blot analysis. Changes in the levels of E-cadherin, vimentin, NF-kB, beta-catenin, and Smad 2 and 3 proteins after being normalized to the levels of beta-actin are shown below each blot. (B) The supernatant of treated groups was collected for this assay. The MMP-2 and MMP-9 activities were measured using zymography and normalized with the control. These experiments were independently triplicated and similar results were obtained. C—Control and A—Antroquinonol.</p

Anti-tumor efficacy of antroquinonol on nude mice bearing glioma xenograft (n = 6).

<p>(A) Tumor size, (B) body weight of the mice, and (C) H&E staining of paraffin-embedded tissue sections of the liver, kidney, spleen, and tumor (magnification 100X). The values are represented as the mean ± SD of three individual experiments. **<i>p</i> < 0.01 when compared with the untreated controls.</p

AFM of C6 and N18 cells after antroquinonol treatment.

<p>Cells were grown on coverslips and treated with various concentrations (5 and 10 μM) of antroquinonol for 24 h, and then fixed with 1% glutaraldehyde for 5 min. An AFM probe was scanned across the cell surfaces at the rate of 0.5 to 2 Hz with an area of approximately 100 μm x 100 μm. Representative images were taken by the AFM, and the images were collected using JPK Software. C—Control and A—antroquinonol.</p