Hydrogen Sulfide Protects HUVECs against Hydrogen Peroxide Induced Mitochondrial Dysfunction and Oxidative Stress

10.1371/journal.pone.0053147PLoS ONE82

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Effects of NaHS on protein expressions of antioxidant enzymes.

<p>(<b>A</b>) Western-blot analysis showing the intensities of Catalase, SOD-1, SOD-2, GST and GPx in each group, (<b>B</b>)–(<b>F</b>) bar charts indicating the different intensities of antioxidant proteins between groups. Values were normalized against the control values. The data shown are mean ± SEM (n = 6). * <i>p</i><0.05, ** <i>p</i><0.01 vs control. # <i>p</i><0.05, ## <i>p</i><0.01 vs vehicle + H₂O₂ group.</p

Conceptualization of the way in which H₂S may influence on H₂O₂-induced cell damage by preserving mitochondrial functions and displaying antioxidative abilities though CSE/H₂S pathway.

<p>Conceptualization of the way in which H₂S may influence on H₂O₂-induced cell damage by preserving mitochondrial functions and displaying antioxidative abilities though CSE/H₂S pathway.</p

Antioxidant enzyme activities in each study groups.

<p>The data shown are mean ± SEM (n = 6). ** <i>p</i><0.01 vs control. # <i>p</i><0.05, ## <i>p</i><0.01 vs vehicle + H₂O₂ group.</p

Effects of NaHS on H₂S levels and H₂S synthesizing enzyme activities.

<p>(<b>A</b>) The changes of H₂S levels in medium for each treatment group (expressed in μM). (<b>B</b>) CSE activities in HUVECs lysate of each group, presented as μmol/h/g. (<b>C</b>) CSE protein expressions levels as determined using western blot analysis. (<b>D</b>) CSE mRNA expression levels as determined by real-time PCR. (<b>E</b>) CBS protein expressions levels and (<b>F</b>) The CBS mRNA expression tested by real-time PCR. The values in (C)–(F) were normalized against the control values. The data shown are mean ± SEM (n = 6). * <i>p</i><0.05, ** <i>p</i><0.01 vs control. # <i>p</i><0.05 vs vehicle + H₂O₂ group.</p

Ultrastructural changes in HUVECs induced by H₂O₂ using transmission electron microscopy.

<p>(<b>A</b>)–(<b>D</b>) showed HUVECs with legible nucleus. Scale bar is shown at 1 μm. (<b>E</b>)–(<b>H</b>) showed mitochondria. Scale bar is shown at 0.2 μm. (<b>A</b>) and (<b>E</b>) cell and mitochondria in the control group; (<b>B</b>) and (<b>F</b>) cell and mitochondria in vehicle + H₂O₂ group; (<b>C</b>) and (<b>G</b>) cell and mitochondria in NaHS + H₂O₂ group; (<b>D</b>) and (<b>H</b>) cell and mitochondria in PAG + H₂O₂ group.</p

Effects of NaHS on mitochondrial function.

<p>(<b>A</b>) ATP synthesis. After pretreatment with vehicle, 300 μM NaHS or 10 mM PAG for 6 hours and followed by exposure to 600 μM H₂O₂ for another 4 hours, HUVECs were harvested to collect mitochondria. The rate of ATP synthesis was expressed by µmol ATP/min/g of mitochondrial protein. (<b>B</b>) Release of cytochrome c from mitochondria. After treatments as previous description, HUVECs were harvested to collect mitochondria and cytosol. The protein expression was tested by western blot. The bar chart showed the ratio of cytochrome c in cytosol to that in mitochondria, indicating the intensity of release of cytochrome c. (<b>C</b>) MDA changes in HUVECs mediated by H₂O₂. The data are expressed at nmol/mg. (<b>D</b>) Fluorescent intensity of DPPP in HUVECs mediated by H₂O₂. (<b>E</b>) ROS production was stained by 10 μM H₂DCFDA for 20 min, whose oxidation product (DCF) fluorescence indicated ROS formation. (<b>F</b>) ROS production was stained by 5 μM DHE for 30 min, which fluorescence indicated ROS formation. The absorbance values in (D)–(F) of HUVECs were normalized against the values for normal controls and expressed as a percentage of control. (<b>G</b>)–(<b>L</b>) JC-1 staining. Red fluorescence represents the mitochondrial aggregate form of JC-1, indicating intact mitochondrial membrane potential. Green fluorescence represents the monomeric form of JC-1, indicating dissipation of Δ<i>Ψ</i>_m. (G)–(J) HUVECs were stained with JC-1. (K) CCCP was the positive control. Cells were observed under ×200 microscopy. Scale bar is shown at 100 μm. (L) Ratio of red to green fluorescence, indicating ratio of JC-1 polymer/monomer. The data shown are mean ± SEM (n = 6). * <i>p</i><0.05, ** <i>p</i><0.01vs control. # <i>p</i><0.05, ## <i>p</i><0.01 vs vehicle + H₂O₂ group.</p

Cell viability and death assay of HUVECs subjected to different concentrations of NaHS with or without H₂O₂.

<p>(<b>A</b>)–(<b>C</b>) MTT assay. (<b>A</b>) HUVECs were treated with 10–500 μM NaHS for 6 hours. (<b>B</b>) HUVECs were treated with 0.2–1.5 mM H₂O₂ for 4 hours. (<b>C</b>) HUVECs were pretreated with vehicle or 30–500 μM NaHS for 6 hours, followed by exposure to 600 μM H₂O₂ for another 4 hours. (<b>D</b>) LDH Release. HUVECs were pretreated with vehicle or 300 μM NaHS and 10 mM PAG for 6 hours, followed by exposure to 600 μM H₂O₂ for another 4 hours. Cell viability in each treatment group is expressed as a percentage of control. (<b>E</b>)–(<b>H</b>) Hoechst staining. HUVECs were pretreated with vehicle, 300 μM NaHS or 10 mM PAG for 6 hours, followed by exposure to 600 μM H₂O₂ for another 4 hours. Cells were observed under ×200 microscopy. Scale bar is shown at 100 μm. (<b>I</b>)–(<b>M</b>) Annexin V/PI staining detected by flow cytometry. HUVECs were pretreated with vehicle or 300 μM NaHS and 10 mM PAG for 6 hours, followed by exposure to 600 μM H₂O₂ for another 4 hours. The data shown are mean ± SEM (n = 9). ** <i>p</i><0.01 vs control. ## <i>p</i><0.01 vs vehicle + H₂O₂.</p