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Downregulation of Endogenous Hydrogen Sulfide Pathway Is Involved in Mitochondrion-Related Endothelial Cell Apoptosis Induced by High Salt.

Zong Y, Huang Y, Chen S, Zhu M, Chen Q, Feng S, Sun Y, Zhang Q, Tang C, Du J, Jin H - Oxid Med Cell Longev (2015)

Bottom Line: The cell apoptosis was detected by cell death ELISA and TdT-mediated dUTP nick end labeling (TUNEL) methods.However, supplementation of H2S donor markedly inhibited VEC oxidative stress and mitochondria-related VEC apoptosis induced by high salt.H2S/CSE pathway is an important endogenous defensive system in endothelial cells antagonizing high-salt insult.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Peking University First Hospital, Beijing 100034, China.

ABSTRACT

Background: The study aimed to investigate whether endogenous H2S pathway was involved in high-salt-stimulated mitochondria-related vascular endothelial cell (VEC) apoptosis.

Methods: Cultured human umbilical vein endothelial cells (HUVECs) were used in the study. H2S content in the supernatant was detected. Western blot was used to detect expression of cystathionine gamma-lyase (CSE), cleaved-caspase-3, and mitochondrial and cytosolic cytochrome c (cytc). Fluorescent probes were used to quantitatively detect superoxide anion generation and measure the in situ superoxide anion generation in HUVEC. Mitochondrial membrane pore opening, mitochondrial membrane potential, and caspase-9 activities were measured. The cell apoptosis was detected by cell death ELISA and TdT-mediated dUTP nick end labeling (TUNEL) methods.

Results: High-salt treatment downregulated the endogenous VEC H2S/CSE pathway, in association with increased generation of oxygen free radicals, decreased mitochondrial membrane potential, enhanced the opening of mitochondrial membrane permeability transition pore and leakage of mitochondrial cytc, activated cytoplasmic caspase-9 and caspase-3 and subsequently induced VEC apoptosis. However, supplementation of H2S donor markedly inhibited VEC oxidative stress and mitochondria-related VEC apoptosis induced by high salt.

Conclusion: H2S/CSE pathway is an important endogenous defensive system in endothelial cells antagonizing high-salt insult. The protective mechanisms for VEC damage might involve inhibiting oxidative stress and protecting mitochondrial injury.

No MeSH data available.


Related in: MedlinePlus

Changes in superoxide anion generation in human umbilical vein endothelial cells (HUVECs). (a) Fluorescent micrographs of superoxide anion generation in HUVEC detected by DHE probes. Red fluorescence indicates levels of superoxide anion generation in HUVEC; (b) quantification analysis of fluorescent intensity of superoxide anion generation in HUVEC detected by CellROX Green Reagent on Fluoroskan Ascent Fluorometer; (c) superoxide anion generation in HUVEC mitochondria detected by MitoSOX probes. Control: the cell treated with 137 mmol/L sodium. HS: high salt, the cell treated with 200 mmol/L sodium for 6 h. HS + NaHS: the cell pretreated with 200 μmol/L NaHS for 30 min following 200 mmol/L sodium for 6 h. ##P < 0.01 versus control group; ∗∗P < 0.01 versus HS group.
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fig2: Changes in superoxide anion generation in human umbilical vein endothelial cells (HUVECs). (a) Fluorescent micrographs of superoxide anion generation in HUVEC detected by DHE probes. Red fluorescence indicates levels of superoxide anion generation in HUVEC; (b) quantification analysis of fluorescent intensity of superoxide anion generation in HUVEC detected by CellROX Green Reagent on Fluoroskan Ascent Fluorometer; (c) superoxide anion generation in HUVEC mitochondria detected by MitoSOX probes. Control: the cell treated with 137 mmol/L sodium. HS: high salt, the cell treated with 200 mmol/L sodium for 6 h. HS + NaHS: the cell pretreated with 200 μmol/L NaHS for 30 min following 200 mmol/L sodium for 6 h. ##P < 0.01 versus control group; ∗∗P < 0.01 versus HS group.

Mentions: Compared with control group, the superoxide anion generation in HUVEC of HS group represented by both DHE staining image and fluorescence quantification with CellROX Green Reagent on fluorometer was significantly increased (Figures 2(a) and 2(b)). However, when pretreated with NaHS, the superoxide anion generation in high-salt-induced HUVEC was decreased markedly (Figures 2(a) and 2(b)). Furthermore, the change of superoxide anion generation in mitochondria of HUVEC detected by MitoSOX probe was in accordance with the change of superoxide anion generation in whole HUVEC (Figure 2(c)).


Downregulation of Endogenous Hydrogen Sulfide Pathway Is Involved in Mitochondrion-Related Endothelial Cell Apoptosis Induced by High Salt.

Zong Y, Huang Y, Chen S, Zhu M, Chen Q, Feng S, Sun Y, Zhang Q, Tang C, Du J, Jin H - Oxid Med Cell Longev (2015)

Changes in superoxide anion generation in human umbilical vein endothelial cells (HUVECs). (a) Fluorescent micrographs of superoxide anion generation in HUVEC detected by DHE probes. Red fluorescence indicates levels of superoxide anion generation in HUVEC; (b) quantification analysis of fluorescent intensity of superoxide anion generation in HUVEC detected by CellROX Green Reagent on Fluoroskan Ascent Fluorometer; (c) superoxide anion generation in HUVEC mitochondria detected by MitoSOX probes. Control: the cell treated with 137 mmol/L sodium. HS: high salt, the cell treated with 200 mmol/L sodium for 6 h. HS + NaHS: the cell pretreated with 200 μmol/L NaHS for 30 min following 200 mmol/L sodium for 6 h. ##P < 0.01 versus control group; ∗∗P < 0.01 versus HS group.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4442413&req=5

fig2: Changes in superoxide anion generation in human umbilical vein endothelial cells (HUVECs). (a) Fluorescent micrographs of superoxide anion generation in HUVEC detected by DHE probes. Red fluorescence indicates levels of superoxide anion generation in HUVEC; (b) quantification analysis of fluorescent intensity of superoxide anion generation in HUVEC detected by CellROX Green Reagent on Fluoroskan Ascent Fluorometer; (c) superoxide anion generation in HUVEC mitochondria detected by MitoSOX probes. Control: the cell treated with 137 mmol/L sodium. HS: high salt, the cell treated with 200 mmol/L sodium for 6 h. HS + NaHS: the cell pretreated with 200 μmol/L NaHS for 30 min following 200 mmol/L sodium for 6 h. ##P < 0.01 versus control group; ∗∗P < 0.01 versus HS group.
Mentions: Compared with control group, the superoxide anion generation in HUVEC of HS group represented by both DHE staining image and fluorescence quantification with CellROX Green Reagent on fluorometer was significantly increased (Figures 2(a) and 2(b)). However, when pretreated with NaHS, the superoxide anion generation in high-salt-induced HUVEC was decreased markedly (Figures 2(a) and 2(b)). Furthermore, the change of superoxide anion generation in mitochondria of HUVEC detected by MitoSOX probe was in accordance with the change of superoxide anion generation in whole HUVEC (Figure 2(c)).

Bottom Line: The cell apoptosis was detected by cell death ELISA and TdT-mediated dUTP nick end labeling (TUNEL) methods.However, supplementation of H2S donor markedly inhibited VEC oxidative stress and mitochondria-related VEC apoptosis induced by high salt.H2S/CSE pathway is an important endogenous defensive system in endothelial cells antagonizing high-salt insult.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Peking University First Hospital, Beijing 100034, China.

ABSTRACT

Background: The study aimed to investigate whether endogenous H2S pathway was involved in high-salt-stimulated mitochondria-related vascular endothelial cell (VEC) apoptosis.

Methods: Cultured human umbilical vein endothelial cells (HUVECs) were used in the study. H2S content in the supernatant was detected. Western blot was used to detect expression of cystathionine gamma-lyase (CSE), cleaved-caspase-3, and mitochondrial and cytosolic cytochrome c (cytc). Fluorescent probes were used to quantitatively detect superoxide anion generation and measure the in situ superoxide anion generation in HUVEC. Mitochondrial membrane pore opening, mitochondrial membrane potential, and caspase-9 activities were measured. The cell apoptosis was detected by cell death ELISA and TdT-mediated dUTP nick end labeling (TUNEL) methods.

Results: High-salt treatment downregulated the endogenous VEC H2S/CSE pathway, in association with increased generation of oxygen free radicals, decreased mitochondrial membrane potential, enhanced the opening of mitochondrial membrane permeability transition pore and leakage of mitochondrial cytc, activated cytoplasmic caspase-9 and caspase-3 and subsequently induced VEC apoptosis. However, supplementation of H2S donor markedly inhibited VEC oxidative stress and mitochondria-related VEC apoptosis induced by high salt.

Conclusion: H2S/CSE pathway is an important endogenous defensive system in endothelial cells antagonizing high-salt insult. The protective mechanisms for VEC damage might involve inhibiting oxidative stress and protecting mitochondrial injury.

No MeSH data available.


Related in: MedlinePlus