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Exogenous H2S contributes to recovery of ischemic post-conditioning-induced cardioprotection by decrease of ROS level via down-regulation of NF-κB and JAK2-STAT3 pathways in the aging cardiomyocytes.

Li L, Li M, Li Y, Sun W, Wang Y, Bai S, Li H, Wu B, Yang G, Wang R, Wu L, Li H, Xu C - Cell Biosci (2016)

Bottom Line: However, PC protection is ineffective in the aging cardiomyocytes.PC alone did not provide cardioprotection in H/R-treated aging cardiomyocytes, which was significantly restored by the addition of NaHS.Our results suggest that exogenous H2S contributes to recovery of PC-induced cardioprotection by decrease of ROS level via down-regulation of NF-κB and JAK2/STAT3 pathways in the aging cardiomyocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology, Harbin Medical University, Baojian Road, Harbin, 150081 China ; The Key Laboratory of Cardiovascular Medicine Research, Harbin Medical University, Ministry of Education, Harbin, China.

ABSTRACT

Background: Hydrogen sulfide (H2S), a third member of gasotransmitter family along with nitric oxide and carbon monoxide, generated from mainly catalyzed by cystathionine-lyase, possesses important functions in the cardiovascular system. Ischemic post-conditioning (PC) strongly protects against the hypoxia/reoxygenation (H/R)-induced injury and apoptosis of cardiomyocytes. However, PC protection is ineffective in the aging cardiomyocytes. Whether H2S restores PC-induced cardioprotection by decrease of reactive oxygen species (ROS) level in the aging cardiomyocytes is unknown.

Methods: The aging cardiomyocytes were induced by treatment of primary cultures of neonatal cardiomyocytes using d-galactose and were exposed to H/R and PC protocols. Cell viability was observed by CCK-8 kit. Apoptosis was detected by Hoechst 33342 staining and flow cytometry. ROS level was analyzed using spectrofluorimeter. Related protein expressions were detected through Western blot.

Results: Treatment of NaHS (a H2S donor) protected against H/R-induced apoptosis, cell damage, the expression of cleaved caspase-3 and cleaved caspase-9, the release of cytochrome c (Cyt c). The supplementation of NaHS also decreased the activity of LDH and CK, MDA contents, ROS levels and the phosphorylation of IκBα, NF-κB, JNK2 and STAT3, and increased cell viability, the expression of Bcl-2, the activity of SOD, CAT and GSH-PX. PC alone did not provide cardioprotection in H/R-treated aging cardiomyocytes, which was significantly restored by the addition of NaHS. The beneficial role of NaHS was similar to the supply of N-acetyl-cysteine (NAC, an inhibitor of ROS), Ammonium pyrrolidinedithiocarbamate (PDTC, an inhibitor of NF-κB) and AG 490 (an inhibitor of JNK2), respectively, during PC.

Conclusion: Our results suggest that exogenous H2S contributes to recovery of PC-induced cardioprotection by decrease of ROS level via down-regulation of NF-κB and JAK2/STAT3 pathways in the aging cardiomyocytes.

No MeSH data available.


Related in: MedlinePlus

Summary of experimental treatments protocol. The aging cardiomyocytes were exposed to hypoxic culture medium for 3 h and reoxygenated for 6 h by replacing the hypoxic culture medium with fresh DMEM with 10 % fetal bovine serum. For details of ischemic post-conditioning, NaHS, NAC, PDTC and AG 490 treatments see text
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Fig1: Summary of experimental treatments protocol. The aging cardiomyocytes were exposed to hypoxic culture medium for 3 h and reoxygenated for 6 h by replacing the hypoxic culture medium with fresh DMEM with 10 % fetal bovine serum. For details of ischemic post-conditioning, NaHS, NAC, PDTC and AG 490 treatments see text

Mentions: The aging cardiomyocytes were randomly divided into the following six groups. Each group included eight samples (n = 8) (Fig. 1): (1) Control group (Control): The aging cardiomyocytes were cultured for 9 h with 10 % fetal bovine serum-DMEM; (2) Hypoxia/reoxygenation group (H/R): The aging cardiomyocytes were exposed to hypoxic culture medium for 3 h and reoxygenated for 6 h by replacing the hypoxic culture medium with fresh DMEM with 10 % fetal bovine serum; (3) H/R + NaHS group: The procedure was similar to that for group 2, except that 100 μM NaHS were added in 6 h reoxygenation; (4) PC group: At the end of 3 h of hypoxia, the aging cardiomyocytes were exposed to normoxic culture solution for 5 min, after which cells were placed in hypoxic solution for 5 min. The PC cycle was repeated three times and followed by 6 h of reoxygenation; (5) PC + NaHS group: At the end of 3 h of hypoxia, initiated immediately at the onset of reoxygenation, 100 μM NaHS were given at the onset of reoxygenation for 5 min following with 5 min hypoxia. This protocol was repeated for another two times. The cells were then treated as those of group 3; (6) PC + NAC (or PDTC, or AG490) group: 5 mM NAC (or 100 μM PDTC or 100 μM AG490) were added to the medium 40 min before the end of hypoxia. The cells were then treated as those of group 4.Fig. 1


Exogenous H2S contributes to recovery of ischemic post-conditioning-induced cardioprotection by decrease of ROS level via down-regulation of NF-κB and JAK2-STAT3 pathways in the aging cardiomyocytes.

Li L, Li M, Li Y, Sun W, Wang Y, Bai S, Li H, Wu B, Yang G, Wang R, Wu L, Li H, Xu C - Cell Biosci (2016)

Summary of experimental treatments protocol. The aging cardiomyocytes were exposed to hypoxic culture medium for 3 h and reoxygenated for 6 h by replacing the hypoxic culture medium with fresh DMEM with 10 % fetal bovine serum. For details of ischemic post-conditioning, NaHS, NAC, PDTC and AG 490 treatments see text
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4836181&req=5

Fig1: Summary of experimental treatments protocol. The aging cardiomyocytes were exposed to hypoxic culture medium for 3 h and reoxygenated for 6 h by replacing the hypoxic culture medium with fresh DMEM with 10 % fetal bovine serum. For details of ischemic post-conditioning, NaHS, NAC, PDTC and AG 490 treatments see text
Mentions: The aging cardiomyocytes were randomly divided into the following six groups. Each group included eight samples (n = 8) (Fig. 1): (1) Control group (Control): The aging cardiomyocytes were cultured for 9 h with 10 % fetal bovine serum-DMEM; (2) Hypoxia/reoxygenation group (H/R): The aging cardiomyocytes were exposed to hypoxic culture medium for 3 h and reoxygenated for 6 h by replacing the hypoxic culture medium with fresh DMEM with 10 % fetal bovine serum; (3) H/R + NaHS group: The procedure was similar to that for group 2, except that 100 μM NaHS were added in 6 h reoxygenation; (4) PC group: At the end of 3 h of hypoxia, the aging cardiomyocytes were exposed to normoxic culture solution for 5 min, after which cells were placed in hypoxic solution for 5 min. The PC cycle was repeated three times and followed by 6 h of reoxygenation; (5) PC + NaHS group: At the end of 3 h of hypoxia, initiated immediately at the onset of reoxygenation, 100 μM NaHS were given at the onset of reoxygenation for 5 min following with 5 min hypoxia. This protocol was repeated for another two times. The cells were then treated as those of group 3; (6) PC + NAC (or PDTC, or AG490) group: 5 mM NAC (or 100 μM PDTC or 100 μM AG490) were added to the medium 40 min before the end of hypoxia. The cells were then treated as those of group 4.Fig. 1

Bottom Line: However, PC protection is ineffective in the aging cardiomyocytes.PC alone did not provide cardioprotection in H/R-treated aging cardiomyocytes, which was significantly restored by the addition of NaHS.Our results suggest that exogenous H2S contributes to recovery of PC-induced cardioprotection by decrease of ROS level via down-regulation of NF-κB and JAK2/STAT3 pathways in the aging cardiomyocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology, Harbin Medical University, Baojian Road, Harbin, 150081 China ; The Key Laboratory of Cardiovascular Medicine Research, Harbin Medical University, Ministry of Education, Harbin, China.

ABSTRACT

Background: Hydrogen sulfide (H2S), a third member of gasotransmitter family along with nitric oxide and carbon monoxide, generated from mainly catalyzed by cystathionine-lyase, possesses important functions in the cardiovascular system. Ischemic post-conditioning (PC) strongly protects against the hypoxia/reoxygenation (H/R)-induced injury and apoptosis of cardiomyocytes. However, PC protection is ineffective in the aging cardiomyocytes. Whether H2S restores PC-induced cardioprotection by decrease of reactive oxygen species (ROS) level in the aging cardiomyocytes is unknown.

Methods: The aging cardiomyocytes were induced by treatment of primary cultures of neonatal cardiomyocytes using d-galactose and were exposed to H/R and PC protocols. Cell viability was observed by CCK-8 kit. Apoptosis was detected by Hoechst 33342 staining and flow cytometry. ROS level was analyzed using spectrofluorimeter. Related protein expressions were detected through Western blot.

Results: Treatment of NaHS (a H2S donor) protected against H/R-induced apoptosis, cell damage, the expression of cleaved caspase-3 and cleaved caspase-9, the release of cytochrome c (Cyt c). The supplementation of NaHS also decreased the activity of LDH and CK, MDA contents, ROS levels and the phosphorylation of IκBα, NF-κB, JNK2 and STAT3, and increased cell viability, the expression of Bcl-2, the activity of SOD, CAT and GSH-PX. PC alone did not provide cardioprotection in H/R-treated aging cardiomyocytes, which was significantly restored by the addition of NaHS. The beneficial role of NaHS was similar to the supply of N-acetyl-cysteine (NAC, an inhibitor of ROS), Ammonium pyrrolidinedithiocarbamate (PDTC, an inhibitor of NF-κB) and AG 490 (an inhibitor of JNK2), respectively, during PC.

Conclusion: Our results suggest that exogenous H2S contributes to recovery of PC-induced cardioprotection by decrease of ROS level via down-regulation of NF-κB and JAK2/STAT3 pathways in the aging cardiomyocytes.

No MeSH data available.


Related in: MedlinePlus