Hydrogen Sulfide Protects H9c2 Cardiomyoblasts Against H2O2 ...

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Abstract

Reactive oxygen species (ROS) are highly reactive chemical species that may cause irreversible tissue damage, and play a critical role in cardiovascular diseases. Hydrogen sulfide (H2S) is a gasotransmitter that acts as a ROS scavenger with cardio-protective effects. In this study, we investigated the cytoprotective effect of H2S against H2O2-induced apoptosis in cardiomyocytes. H9c2 rat cardiomyoblasts were treated with H2S (100 μM) 24 h before challenging with H2O2 (100 μM). Apoptosis was then assessed by annexin V and PI, and mitochondrial membrane potential was measured using a fluorescent probe, JC-1. Our results revealed that H2S improved cell viability, reduced the apoptotic rate, and preserved mitochondrial membrane potential. An increased Bcl-2 to Bax ratio was also seen in myocytes treated with H2S after H2O2-induced stress. Our findings indicated a therapeutic potential for H2S in preventing myocyte death following ischemia/reperfusion.

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Figures

Figure 1.

Figure 1.. Hydrogen sulfide (H 2 S)…

Figure 1.. Hydrogen sulfide (H 2 S) reduced H 2 O 2 -induced H9c2 apoptosis. …

Figure 1.. Hydrogen sulfide (H2S) reduced H2O2-induced H9c2 apoptosis. A, Cell death analysis of treated cells was performed by flow cytometry with annexin V/PI double staining. Representative quantitative analysis is shown in B. Data are reported as means±SE (n=6). *P<0.05, **P<0.01 vs control; #P<0.05, ##P<0.01 vs NaHS; &p<0.01 vs H2O2 (ANOVA followed by Student-Newman-Keuls post hoc test).
Figure 2.

Figure 2.. Hydrogen sulfide (H 2 S)…

Figure 2.. Hydrogen sulfide (H 2 S) protected H9c2 cells against H 2 O 2 …

Figure 2.. Hydrogen sulfide (H2S) protected H9c2 cells against H2O2-induced oxidative stress (A). Intracellular superoxide anion production was detected with dihydroethidium and observed by fluorescent microscopy (B). The fluorescent signal was measured and quantified. Data are reported as means±SE (n=6). *P<0.01 vs control; #P<0.01 vs NaHS; &P<0.01 vs H2O2 (ANOVA followed by Student-Newman-Keuls post hoc test). ROS: reactive oxygen species.
Figure 3.

Figure 3.. Hydrogen sulfide (H 2 S)…

Figure 3.. Hydrogen sulfide (H 2 S) prevented the loss of mitochondrial membrane potential (ψ …

Figure 3.. Hydrogen sulfide (H2S) prevented the loss of mitochondrial membrane potential (ψm) (A). The ψm loss was determined by the lipophilic cationic probe JC-1. Red signal indicates JC-1 in mitochondria and green signal indicates cytosolic JC-1. Magnification ×400; bar: 50 μm. B, Quantitative analysis of membrane potential. Data are reported as means±SE (n=6). *P<0.01 vs control; #P<0.01 vs NaHS; &P<0.05 vs H2O2 (ANOVA followed by the Student-Newman-Keuls post hoc test).
Figure 4.

Figure 4.. Hydrogen sulfide (H 2 S)…

Figure 4.. Hydrogen sulfide (H 2 S) regulated the expression of apoptosis-related proteins. A ,…

Figure 4.. Hydrogen sulfide (H2S) regulated the expression of apoptosis-related proteins. A, Representative immunoblots showing the expression of Bax, Bcl-2, and activated caspase 3 p17 in the H9c2 cells. Bax (B) and Bcl-2 (C) expression normalized to GAPDH (n=6). D, Densitometric analysis of the ratio of Bcl-2 to Bax. E, Activated caspase 3 p17 expression normalized to GAPDH. Data are reported as means±SE (n=6) (ANOVA followed by Student-Newman-Keuls post hoc test). NS: not significant.
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References

    1. Lefer DJ, Granger DN. Oxidative stress and cardiac disease. Am J Med. 2000;109:315–323. doi: 10.1016/S0002-9343(00)00467-8. - DOI - PubMed
    1. McCord JM. Free radicals and myocardial ischemia: overview and outlook. Free Radic Biol Med. 1988;4:9–14. doi: 10.1016/0891-5849(88)90005-6. - DOI - PubMed
    1. Radomska-Lesniewska DM, Hevelke A, Skopinski P, Balan B, Jozwiak J, Rokicki D, et al. Reactive oxygen species and synthetic antioxidants as angiogenesis modulators: clinical implications. Pharmacol Rep. 2016;68:462–471. doi: 10.1016/j.pharep.2015.10.002. - DOI - PubMed
    1. Griendling KK, Touyz RM, Zweier JL, Dikalov S, Chilian W, Chen YR, et al. Measurement of reactive oxygen species, reactive nitrogen species, and redox-dependent signaling in the cardiovascular system: a scientific statement from the american heart association. Circ Res. 2016;119:e39–e75. doi: 10.1161/RES.0000000000000110. - DOI - PMC - PubMed
    1. Chen K, Keaney JF., Jr Evolving concepts of oxidative stress and reactive oxygen species in cardiovascular disease. Curr Atheroscler Rep. 2012;14:476–483. doi: 10.1007/s11883-012-0266-8. - DOI - PMC - PubMed
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