12 research outputs found

    Lipoamide Acts as an Indirect Antioxidant by Simultaneously Stimulating Mitochondrial Biogenesis and Phase II Antioxidant Enzyme Systems in ARPE-19 Cells

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    <div><p>In our previous study, we found that pretreatment with lipoamide (LM) more effectively than alpha-lipoic acid (LA) protected retinal pigment epithelial (RPE) cells from the acrolein-induced damage. However, the reasons and mechanisms for the greater effect of LM than LA are unclear. We hypothesize that LM, rather than the more direct antioxidant LA, may act more as an indirect antioxidant. In the present study, we treated ARPE-19 cells with LA and LM and compared their effects on activation of mitochondrial biogenesis and induction of phase II enzyme systems. It is found that LM is more effective than LA on increasing mitochondrial biogenesis and inducing the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its translocation to the nucleus, leading to an increase in expression or activity of phase II antioxidant enzymes (NQO-1, GST, GCL, catalase and Cu/Zn SOD). Further study demonstrated that mitochondrial biogenesis and phase II enzyme induction are closely coupled via energy requirements. These results suggest that LM, compared with the direct antioxidant LA, plays its protective effect on oxidative damage more as an indirect antioxidant to simultaneously stimulate mitochondrial biogenesis and induction of phase II antioxidant enzymes.</p></div

    The effects of LM on oxygen consumption (A), mitochondrial membrane potential (MMP) (B), ROS production (C); cellular ATP level (D) and the expression of MnSOD,Trx2,Prx3,and Prx5 (E).

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    <p>ARPE-19 cells were treated with 40 ╬╝mol/L LM for 48 hours; then the following assays were carried out immediately. <b>(A)</b> LM promoted oxygen consumption. Results are expressed as the rate of oxygen consumption, with media without cells used as a blank. Values are means ┬▒ SEM from three independent experiments; three parallel measurements were used for each sample in every experiment. <b>(B)</b> LM treatment increased MMP as determined by JC-1 staining. Values are means ┬▒ SEM of the ratio of fluorescence at 590 nm to 530 nm from three independent experiments; 4 parallel wells for each group were used in each experiment. <b>(C)</b> LM treatment decreased ROS production examined by DCF-DA staining. Values are means ┬▒ SEM of 8 parallel wells of a representative experiment, from four independent experiments each showing similar trends. (D) LM treatment decreased cellular ATP level. Values are means ┬▒ SEM from 3 independent experiments. (E) Expression of MnSOD,Trx2,Prx3,and Prx5. ARPE-19 cells were treated with 40 ╬╝mol/L LM or LA for 48 h; then RNA was isolated and reverse-transcribed to cDNA. Real time PCR was employed to measure expression levels of the indicated genes. The results (from 5 independent experiments) are expression ratios of the target genes to 18SrRNA, and are normalized to control (control = 100). C stands for control, LM stands for 40 ╬╝mol/L LM treatment and LA stands for 40 ╬╝mol/L LA treatment. Statistical significance was established by one way ANOVA followed by the Tukey test (A, B, C, D) or LSD test (E). * p<0.05, ** p<0.01 vs. untreated control (0 ╬╝mol/L); <sup>#</sup>p<0.05, <sup>##</sup> p<0.01 vs. LA.</p

    The effects of LM and LA on expression and activity of GST, catalase, Cu/ZnSOD and G6PD.

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    <p>ARPE-19 cells were treated with 40 ╬╝mol/L LM or LA for 48 h. <b>(A)</b> A representative image of total GST expression detected by western blot. <b>(B)</b> Quantitative analysis of GST expression (4 independent experiments) and activity (5 independent experiments). <b>(C)</b> A representative image of catalase expression detected by western blot. <b>(D)</b> Quantitative analysis of catalase expression (4 independent experiments) and activity (3 independent experiments). The results are expressed in arbitrary units and each experiment was performed in duplicate. <b>(E)</b> Transcriptional expression of Cu/ZnSOD. Real time RT-PCR was employed to measure expression levels of Cu/ZnSOD. Results (from 5 independent experiments) are expressed as ratios of Cu/ZnSOD to 18SrRNA. <b>(F)</b> G6PD activity was measured as described in Methods. Values are means ┬▒ SEM of three independent experiments. All statistical significance were established by one way ANOVA followed by the Tukey test. * p<0.05, **p<0.01 vs. untreated controls (0 ╬╝mol/L), <sup>#</sup>p<0.05 vs. LA, and <sup>##</sup>p<0.01 vs. LA.</p

    LM increased ETC complex I, II, III, IV and V protein expression (A-E), mitochondrial DNA copy number (F) and viable mitochondria (G).

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    <p>ARPE-19 cells were treated with the indicated concentrations of LM for 48 h, and then subunits expression of the complexes were detected by western blot. The subunits tested were 39 KD, 70 KD, 51.6 KD, 57 KD and 56.6 KD for complexes I to V (A to E), respectively. The images are representative; the quantitative results are from optical density analysis of images of three independent experiments. For complexes I, II and V, the loading control was ╬▓-actin; for complexes III and IV, it was ╬▒-tubulin instead. Results are the ratios of the complex densities to those of ╬▓-actin or ╬▒-tubulin. Values are means ┬▒ SEM. Differences were evaluated statistically with studentÔÇÖs t test. * p<0.05, and **p<0.01 vs. untreated control (0 ╬╝mol/L). <b>(F)</b> LM increased viable mitochondria and mitochondrial DNA copy numbers. ARPE-19 cells were treated with the indicated concentrations of LM for 48 h. For viable mitochondria measurement, cells were stained with Mitotracker Green. Fluorescence values read by flow cytometry were considered as estimates of viable mitochondria. Results are in arbitrary units normalized by setting the fluorescence of untreated (0 ╬╝mol/L LM) cells to 100. Values are means ┬▒ SEM from three independent experiments, each performed on three samples at each concentration. For mitochondrial DNA copy number measurement, real-time PCR was employed for assaying the D-LOOP region of mitochondrial DNA. The results shown are ratios of D-LOOP to 18S rDNA. Results are in arbitrary units normalized by setting the ratio of untreated (0 ╬╝mol/L LM) cells to 100. Values are means ┬▒ SEM from four independent experiments. Statistical significance of differences was established by studentÔÇÖs t test. * p<0.05, vs. 0 ╬╝mol/L treatment and **p<0.01 vs. untreated control (0 ╬╝mol/L) <b>(G)</b> A representative flow cytometry histogram was created with Flow Jo, Ver. 4.87 software. The fluorescence curves of 0, 10 and 40 ╬╝mol/L LM treatments were right-shifted with respect to the 0 ╬╝mol/L curve.</p

    LM treatment increased Nrf2 expression in both total and nuclear protein fraction, and increased expression and activity of NQO-1.

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    <p>ARPE-19 cells were treated with the indicated concentrations of LM for 48 h, or 40 ╬╝mol/L of LA or LM if concentrations not indicated. <b>(A)</b> A representative image of Nrf2 expression in total protein detected by western blot. Optical densities were analyzed with Quantity One software, and results are expressed as ratios of Nrf2 to ╬▓-actin in arbitrary units. <b>(B)</b> A representative image of Nrf2 expression in nuclear protein. Quantitative analysis of Nrf2 expression in nuclear protein was quantified in the same way as for Nrf2 expression in total protein. <b>(C)</b> A representative image of NQO-1 expression detected by western blot. Quantitative analysis of NQO-1 expression in total protein was performed in the same wa with Nrf2.. <b>(D)</b> A representative image of NQO-1 expression. <b>(E)</b> Quantitative analysis of NQO-1 expression and activity. All values are means ┬▒ SEM of four independent experiments. Statistical significance was established by one way ANOVA followed by the Tukey test. *p<0.05, and **p<0.01 vs. control, and <sup>#</sup>p<0.05, <sup>##</sup>p<0.01 vs. LA treatment.</p

    The Effects of Pharmacological Inhibition of Histone Deacetylase 3 (HDAC3) in HuntingtonÔÇÖs Disease Mice

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    <div><p>An important epigenetic modification in HuntingtonÔÇÖs disease (HD) research is histone acetylation, which is regulated by histone acetyltransferase and histone deacetylase (HDAC) enzymes. HDAC inhibitors have proven effective in HD model systems, and recent work is now focused on functional dissection of the individual HDAC enzymes in these effects. Histone deacetylase 3 (HDAC3), a member of the class I subfamily of HDACs, has previously been implicated in neuronal toxicity and huntingtin-induced cell death. Hence, we tested the effects of RGFP966 ((<i>E</i>)-N-(2-amino-4-fluorophenyl)-3-(1-cinnamyl-1<i>H</i>-pyrazol-4-yl)acrylamide), a benzamide-type HDAC inhibitor that selectively targets HDAC3, in the N171-82Q transgenic mouse model of HD. We found that RGFP966 at doses of 10 and 25 mg/kg improves motor deficits on rotarod and in open field exploration, accompanied by neuroprotective effects on striatal volume. In light of previous studies implicating HDAC3 in immune function, we measured gene expression changes for 84 immune-related genes elicited by RGFP966 using quantitative PCR arrays. RGFP966 treatment did not cause widespread changes in cytokine/chemokine gene expression patterns, but did significantly alter the striatal expression of macrophage migration inhibitory factor (<i>Mif)</i>, a hormone immune modulator associated with glial cell activation, in N171-82Q transgenic mice, but not WT mice. Accordingly, RGFP966-treated mice showed decreased glial fibrillary acidic protein (GFAP) immunoreactivity, a marker of astrocyte activation, in the striatum of N171-82Q transgenic mice compared to vehicle-treated mice. These findings suggest that the beneficial actions of HDAC3 inhibition could be related, in part, with lowered <i>Mif</i> levels and its associated downstream effects.</p></div

    Summary of cytokines array gene expression changes in striatum due to RGFP966 treatment.

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    <p>A. Heatmap of expression values for 84 cytokine/chemokine genes showing two-way clustering of expression levels and treatment groups. Red denotes increased relative gene expression levels for the indicated groups, with green denoting decreased expression levels. B. Volcano plots of expression changes due to RGFP966 treatment showing three different comparisons, as indicated. Dotted line on y-axis denotes the significance cut-off of p-value<0.05, using one-way ANOVA. Dotted lines on x-axis denote a fold-change cut-off of > +/- 2.</p

    Real-time qPCR results showing altered expression of <i>Mif</i> and <i>Il1b</i> in striatum and cortex of RGFP966 treated WT and N171-82Q mice.

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    <p>Groups of mice were treated with RGFP966 (25 mg/kg) for 12 weeks beginning at 8 weeks of age. Bar graphs shown the mean +/- S.E.M. expression value from n = 5ÔÇô6 mice per group normalized to the expression of <i>Hprt</i>. Significant differences of p<0.05 were measured by a two-tailed, unpaired StudentÔÇÖs <i>t</i> test and are indicated by an asterisk (*).</p
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