DNA Repair Enzyme Poly ADP-ribose Research Articles

Sevoflurane Preconditioning Rescues PKMζ Gene Expression from Broad Hypoxia-Induced mRNA Downregulation Correlating with Improved Neuronal Recovery

Hypoxia due to stroke is a major cause of neuronal damage, leading to loss of cognition and other brain functions. Sevoflurane preconditioning improves recovery after hypoxia. Hypoxia interferes with protein expression at the translational level; however, its effect on mRNA levels for neuronal protein kinase and anti-apoptotic genes is unclear. To investigate the link between sevoflurane preconditioning and gene expression, hippocampal slices were treated with 4% sevoflurane for 15 min, a 5 min washout, 10 min of hypoxia, and 60 min of recovery. We used quantitative PCR to measure mRNA levels in the CA1 region of rat hippocampi. The mRNA levels for specific critical proteins were examined, as follows: Protein kinases, PKCγ (0.22), PKCε (0.38), and PKMζ (0.55) mRNAs, and anti-apoptotic, bcl-2 (0.44) and bcl-xl (0.41), were reduced 60 min after hypoxia relative to their expression in tissue not subjected to hypoxia (set to 1.0). Sevoflurane preconditioning prevented the reduction in PKMζ (0.88 vs. 1.0) mRNA levels after hypoxia. Pro-apoptotic BAD mRNA was not significantly changed after hypoxia, even with sevoflurane preconditioning (hypoxia 0.81, sevo hypoxia 0.84 vs. normoxia 1.0). However, BAD mRNA was increased by sevoflurane in non-hypoxic conditions (1.48 vs. 1.0), which may partially explain the deleterious effects of volatile anesthetics under certain conditions. The DNA repair enzyme poly ADP-ribose polymerase 1 (PARP-1) was increased by sevoflurane in tissue not subjected to hypoxia (1.23). PARP-1 mRNA was reduced in untreated tissue after hypoxia (0.21 vs. 1.0); sevoflurane did not improve PARP-1 after hypoxia (0.27). Interestingly, the mRNA level of the cognitive kinase PKMζ, a kinase essential for learning and memory, was the only one protected against hypoxic downregulation by sevoflurane preconditioning. These findings correlate with previous studies that found that sevoflurane-induced improvement of neuronal survival after hypoxia was dependent on PKMζ. Maintaining mRNA levels for critical proteins may provide an important mechanism for preserving neuronal function after stroke.

Dihydrokaempferol attenuates CCl4-induced hepatic fibrosis by inhibiting PARP-1 to affect multiple downstream pathways and cytokines

The pathophysiological mechanism of hepatic fibrosis (HF) is related to the excessive activation of the DNA repair enzyme poly ADP-ribose polymerase-1 (PARP-1). The drugs, targeting PARP-1, are scarce. Therefore, the lead compound, moderately inhibiting PARP-1, with anti-HF properties should be identified. This study screened dihydrokaempferol (DHK) from herbs based on preliminary studies to intervene in a CCl4-induced liver injury and HF model in mice. In vitro, the expression levels of PARP-1-regulated related proteins and phosphorylation were examined. The binding pattern of DHK and PARP-1 was analyzed using molecular docking and molecular dynamics platforms. The results showed that DHK could significantly attenuate CCl4-induced liver injury and HF in mice. Moreover, it could also attenuate the toxic effects of CCl4 on HepG2 and inhibit α-SMA and Collagen 1/3 synthesis of LX-2 cells in-vitro. Molecular docking revealed that DHK could competitively bind to the Glu-988 and His-862 residues of the upstream DNA repair enzyme PARP-1, moderately inhibiting its overactivation. This led to maintaining NAD+ levels and energy metabolism in hepatocytes and inhibiting the activation of PARP-1-regulated downstream signaling pathways (TGF-β1, etc.), related proteins (p-Smd2/3, etc.), and inflammatory mediators while acting indirectly. Thus, DHK could attenuate CCl4-induced liver injury and HF in mice in a different mechanism from those of the existing reported flavonoids. It was associated with inhibiting the expression of downstream pathways and related cytokines by competitively binding to PARP-1. This study might provide a basis and direction for the design and exploration of anti-HF lead compounds.

Effect of ethyl pyruvate on mitochondrial dynamics of lipopolysaccharide-induced human kidney-2 cells

To examine the effects of ethyl pyruvate (EP) on mitochondrial dynamics and cell apoptosis in lipopolysaccharide (LPS)-induced human kidney-2 (HK-2) cells. HK-2 cells were divided into three groups: HK-2 cells were challenged with LPS (800 μg/L) for 24 hours as LPS group, or LPS mixed with EP (0.25 mmol/L) for 24 hours as EP group. Cells were incubated with normal saline for 24 hours as control group. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and intracellular adenosine triphosphate (ATP) were detected by enzyme linked immunosorbent assay (ELISA). JC-1 staining and Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) assays were used to evaluate mitochondrial membrane potential and cell apoptosis, respectively. Western Blot was used to evaluate the protein expressions of mitochondrial dynamics, including death-associated protein kinase 2 (DAPK-2), mitofusin (Mfn-1 and Mfn-2), and apoptotic associated biomarkers, including caspase-3, caspase-9, Bcl-2, Bcl-xL, cytochrome C (Cyt C), and DNA repair enzyme poly ADP-ribose polymerase (PARP). Compared with the NC group, MDA, IL-6, TNF-α of LPS group were significantly increased, the expression of SOD, mitochondrial membrane potential and ATP level were significantly decreased, the expression of mitochondrial fission protein DAPK-2 was significantly increased, and mitochondrial fusion proteins Mfn-1 and Mfn-2 were significantly decreased, cell apoptosis and apoptotic protein caspase-3, caspase-9 and Cyt C were increased, and anti-apoptotic protein Bcl-2, Bcl-xL, PARP were significantly decreased. Compared with the LPS group, the oxidative activities and inflammatory factors above were inhibited in EP group [MDA (μmol/L): 12.35±2.21 vs. 45.95±1.76, SOD (kU/L): 54.68±1.42 vs. 40.73±1.60, IL-6 (ng/L): 67.87±2.61 vs. 338.92±20.91, TNF-α (ng/L): 19.23±1.80 vs. 180.69±6.51], mitochondrial membrane potential and ATP level were significantly increased [mitochondrial membrane potential: (99.43±0.25)% vs. (69.40±0.75)%, ATP (×106 RLU): 0.19±0.01 vs. 0.12±0.05], the expression of mitochondrial fission protein was significantly decreased (DAPK-2/β-actin: 0.03±0.01 vs. 0.61±0.02), mitochondrial fusion proteins were significantly increased (Mfn-1/β-actin: 0.43±0.04 vs. 0.17±0.01, Mfn-2/β-actin: 0.201±0.004 vs. 0.001±0.001), percentage of cell apoptosis was significantly decreased [(5.25±0.17)% vs. (34.42±0.64)%], the expressions of apoptotic proteins were significantly decreased (caspase-3/β-actin: 0.25±0.15 vs. 1.76±0.01, caspase-9/β-actin: 0.09±0.02 vs. 1.52±0.12, Cyt C/β-actin: 0.001±0.001 vs. 0.350±0.030), and the expressions of anti-apoptotic proteins and PARP were significantly increased (Bcl-2/β-actin: 0.500±0.010 vs. 0.009±0.004, Bcl-xL/β-actin: 0.550±0.010 vs. 0.009±0.001, PARP/β-actin: 0.94±0.01 vs. 0.16±0.13), with statistically significant differences (all P < 0.05). There are enhanced mitochondrial fission and diminished mitochondrial fusion in LPS-induced HK-2 cells. EP can protect mitochondria functions by regulate mitochondrial dynamics, and reducethe apoptosis of LPS-induced HK-2 cells.

Delta 9-tetrahydrocannabinol induces the apoptotic pathway in cultured cortical neurones via activation of the CB1 receptor.

Delta 9-tetrahydrocannabinol, the principal psychoactive component of marijuana, exerts a variety of effects on the CNS, including impaired cognitive function and neurobehavioural deficits. The mechanisms underlying these neuronal responses to tetrahydrocannabinol are unclear but may involve alterations in neuronal viability. Tetrahydrocannabinol has been shown to influence neuronal survival but the role of the cannabinoid receptors in the regulation of neuronal viability has not been fully clarified. In this study we demonstrate that tetrahydrocannabinol promotes the release of cytochrome c, activates caspase-3, promotes cleavage of the DNA repair enzyme poly-ADP ribose polymerase and induces DNA fragmentation in cultured cortical neurones. These effects of tetrahydrocannabinol were completely abrogated by the CB(1) receptor antagonist AM-251. The findings of this study demonstrate that tetrahydrocannabinol induces apoptosis in cortical neurones in a manner involving the CB1 subtype of cannabinoid receptor.

Effects of free radicals on ciliary movement in the human nasal epithelial cells

Objective: There have been few reports on the effects of free oxygen radicals on ciliary mobility of nasal respiratory epithelial cells. The aim of this study was to determine the effects of free radicals and antioxidants on human nasal epithelial cells (HNECs) using video-computerized analysis. Methods: Human nasal epithelial cells were obtained from the nasal cavity of normal volunteers. Ciliary beat frequency (CBF) was calculated as the mean value of ten randomly selected cells. The proportion of the area with normal CBF (above 8 Hz) was calculated from 10 randomly selected sites per specimen. Free radicals were produced by xanthine–xanthine oxidase enzymatic system. The generation of free radicals was confirmed by chemoilluminometer. CBF and the proportion of the area with normal CBF were measured at every 5 min for 30 min after the addition of enzyme. For the evaluation of the antioxidant effects on free radical-mediated ciliary slowing in HNECs, cells were incubated in superoxide dismutase solution (300 unit/ml) for 30 min and 3-aminobenzamide (5 mM). Results: Superoxide produced by 0.4 mM xanthine and 400 miliunit/ml xanthine oxidase decreased CBF (7.71±1.91 Hz). A total of 2 min later, ciliary slowing was evident (3.87±1.10 Hz). Regarding the changes in proportion of epithelial area that showed normal CBF experimental group showed a significant decrease in percentage of epithelial area with normal CBF over time. Superoxide dismutase prevented ciliary slowing (8.76±0.99 Hz). Moreover, 3-aminobenzamide, an inhibitor of the DNA repair enzyme poly-ADP ribose polymerase, prevented inhibition of CBF (8.32±0.61 Hz). Conclusions: These results suggest that oxygen-mediated damage to DNA may be the mechanism of the deterioration effects of oxygen radicals on the ciliated respiratory nasal epithelium.