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

<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

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

<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 β-actin; for complexes III and IV, it was α-tubulin instead. Results are the ratios of the complex densities to those of β-actin or α-tubulin. Values are means ± SEM. Differences were evaluated statistically with student’s t test. * p<0.05, and **p<0.01 vs. untreated control (0 μmol/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 μmol/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 μmol/L LM) cells to 100. Values are means ± SEM from four independent experiments. Statistical significance of differences was established by student’s t test. * p<0.05, vs. 0 μmol/L treatment and **p<0.01 vs. untreated control (0 μmol/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 μmol/L LM treatments were right-shifted with respect to the 0 μmol/L curve.</p

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

<p>ARPE-19 cells were treated with the indicated concentrations of LM for 48 h, or 40 μmol/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 β-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 ^#p<0.05, ^##p<0.01 vs. LA treatment.</p

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).

<p>ARPE-19 cells were treated with 40 μmol/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 μmol/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 μmol/L LM treatment and LA stands for 40 μmol/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 μmol/L); ^#p<0.05, ^## p<0.01 vs. LA.</p

Inhibition of Prostaglandin Transporter (PGT) Promotes Perfusion and Vascularization and Accelerates Wound Healing in Non-Diabetic and Diabetic Rats

<div><p>Peripheral ischemia, resulting from diminished arterial flow and defective local vascularization, is one of the main causes of impaired wound healing in diabetes. Vasodilatory prostaglandins (PGs), including PGE₂ and PGI₂, regulate blood flow in peripheral tissues. PGs also stimulate angiogenesis by inducing vascular endothelial growth factor. However, PG levels are reduced in diabetes mainly due to enhanced degradation. We hypothesized that inhibition of the prostaglandin transporter (PGT) (SLCO2A1), which mediates the degradation of PGs, would increase blood flow and stimulate vascularization, thereby mitigating peripheral ischemia and accelerating wound healing in diabetes. Here we report that inhibiting PGT with intravenously injected PGT inhibitor, T26A, increased blood flow in ischemic hind limbs created in non-diabetic rats and streptozotocin induced diabetic rats. Systemic, or combined with topical, T26A accelerated closure of cutaneous wounds. Immunohistochemical examination revealed that inhibition of PGT enhanced vascularization (marked by larger numbers of vessels formed by CD34+ cells), and accelerated re-epithelialization of cutaneous wounds. In cultured primary human bone marrow CD34+ cells and human epidermal keratinocytes (HEKs) either inhibiting or silencing PGT increased migration in both cell lines. Thus PGT directly regulates mobilization of endothelial progenitor cells (EPCs) and HEKs, which could contribute to PGT-mediated vascularization and re-epithelialization. At the molecular level, systemic inhibition of PGT raised circulating PGE₂. Taken together, our data demonstrate that PGT modulates arterial blood flow, mobilization of EPCs and HEKs, and vascularization and epithelialization in wound healing by regulating vasodilatory and pro-angiogenic PGs.</p></div

Primers and annealing temperature.

<p>Primers and annealing temperature.</p

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

<p>ARPE-19 cells were treated with 40 μmol/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 μmol/L), ^#p<0.05 vs. LA, and ^##p<0.01 vs. LA.</p

Inhibition of PGT Stimulates Vascularization.

<p>Representative images of CD34 (A) staining of cutaneous wounds of non-diabetic (ND) Sprague Dawley rats or STZ diabetic (D) rats. Rats were treated with both i.p. injected 500 μL of vehicle (2% DMSO + 2% cremophor in water) or 1.2 mM T26A, once daily, and topically applied 15 μL of vehicle or 2 mM T26A, once every other day. (B) Analysis of CD34+ cells. Numbers of CD34+ cells were counted in 5 random high power fields for each rat tissue. Five rats (per treatment) were used. Values are average ± SEM. *p < 0.05 or **p < 0.01, p values were obtained by ANOVA test.</p

Monitoring and Evaluating the Quality Consistency of Compound Bismuth Aluminate Tablets by a Simple Quantified Ratio Fingerprint Method Combined with Simultaneous Determination of Five Compounds and Correlated with Antioxidant Activities

<div><p>A combination method of multi-wavelength fingerprinting and multi-component quantification by high performance liquid chromatography (HPLC) coupled with diode array detector (DAD) was developed and validated to monitor and evaluate the quality consistency of herbal medicines (HM) in the classical preparation Compound Bismuth Aluminate tablets (CBAT). The validation results demonstrated that our method met the requirements of fingerprint analysis and quantification analysis with suitable linearity, precision, accuracy, limits of detection (LOD) and limits of quantification (LOQ). In the fingerprint assessments, rather than using conventional qualitative “Similarity” as a criterion, the simple quantified ratio fingerprint method (SQRFM) was recommended, which has an important quantified fingerprint advantage over the “Similarity” approach. SQRFM qualitatively and quantitatively offers the scientific criteria for traditional Chinese medicines (TCM)/HM quality pyramid and warning gate in terms of three parameters. In order to combine the comprehensive characterization of multi-wavelength fingerprints, an integrated fingerprint assessment strategy based on information entropy was set up involving a super-information characteristic digitized parameter of fingerprints, which reveals the total entropy value and absolute information amount about the fingerprints and, thus, offers an excellent method for fingerprint integration. The correlation results between quantified fingerprints and quantitative determination of 5 marker compounds, including glycyrrhizic acid (GLY), liquiritin (LQ), isoliquiritigenin (ILG), isoliquiritin (ILQ) and isoliquiritin apioside (ILA), indicated that multi-component quantification could be replaced by quantified fingerprints. The Fenton reaction was employed to determine the antioxidant activities of CBAT samples <i>in vitro</i>, and they were correlated with HPLC fingerprint components using the partial least squares regression (PLSR) method. In summary, the method of multi-wavelength fingerprints combined with antioxidant activities has been proved to be a feasible and scientific procedure for monitoring and evaluating the quality consistency of CBAT.</p></div

Systemic Inhibition of PGT Increases Perfusion to Distal Limb.

<p>(A) Images of representative blood flow in hind limbs of non-diabetic Sprague Dawley rats before and after various treatments. Left hind limb of each rat was partially occluded. 500 μL of vehicle (2% DMSO + 2% cremophor in water), or 10 μM PGE₂, or 1.2 mM T26A was injected via jugular vein. (B) Statistical analysis of blood flow as percentage of blood flow before occlusion (BO). AO, after occlusion. (C) Representative pharmacodynamics of the effects of PGE₂ and T26A on blood flow presented as percentage of blood flow before occlusion. (D) Images of representative blood flow in hind limbs of STZ diabetic rats before and after various treatments. Left hind limb of each rat was partially occluded. 500 μL of vehicle (2% DMSO + 2% cremophor in water), or 10 μM PGE₂, or 1.2 mM T26A was injected via jugular vein. (E) Statistical analysis of blood flow as percentage of blood flow before occlusion (BO). Blood flow was measured using a PeriScan PIM 3. For all analyses of laser Doppler measurements the color scale was set at 0–800, and the intensity was set at 0.34. (F) Plasma PGE₂. Values are average ± SEM (n = 5). *p < 0.05, p values were obtained by t-test for E and by ANOVA test for the rest.</p