| The cardiovascular disease is one of the three highest fatality of major human diseases nowadays, including ischemic heart disease, which is the major disease threatening people's health. Moreover, the acute myocardial ischemia is also found in the process of cardiac surgery. Therefore, the treatment, mechanism and prevention of acute ischemic myocardial injury are the hotspots of medical research at present.Endogenous hydrogen sulfide (H2S) is mainly produced from the substrate, such as L-cysteine and homocysteine, by the action of cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). It was firstly found that it could facilitate the induction of hippocampal long-term potentiation. Moreover, as the studies deepening, it was suggested that H2S may be involved in many physiological and pathophysiological processes. And now H2S is recognized as the third endogenous signaling gasotransmitter, following nitric oxide (NO) and carbon monoxide (CO). In the previous study of our laboratory, we observed the H2S/CSE system was down-regulated in acute myocardial ischemia in rats. In the present study, we investigated the effects of H2S on acute myocardial ischemia injury in rats from the aspects of oxidative stress, inflammatory factor and apoptosis, and explored the possible mechanisms.Part 1 Effects of hydrogen sulfide on oxidative stress in acute myocardial ischemia injury in ratsObjective: To investigate effects of H2S on myocardial ischemia injury in rats and explore the possible mechanism from oxidative stress.Methods: Fourty-eight male SD rats were randomly divided into sham operation group, ischemia group, ischemia + NaHS low, middle and high dose groups and ischemia + PPG group. The acute myocardial ischemia model was established by ligating the left anterior descending coronary artery (LAD) of the rats. Saline was intraperitoneally administrated in ischemia group. LADs were not ligated but only threaded in sham operation group in rats. In ischemia + NaHS low, middle and high dose groups or ischemia + PPG group, sodium hydrosulfide (NaHS) (0.78, 1.56, 3.12 mg/kg) or DL- propargylglycine (PPG) (30mg/kg) was intraperitoneally injected respectively at 3 hours after ischemia. All the rats were killed at 6 hours after the operation. The cardiac function indexes, such as the left ventricular developed pressure (LVDP), the left ventricular end diastolic pressure (LVEDP) and±dp/dtmax were respectively recorded. The content of H2S in plasma and the activity of cystathionine-γ-lyase (CSE) in myocardial tissue were respectively detected. The content of malondialdehyde (MDA), and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) in myocardial tissue were respectively measured. The pathological changes of myocardial tissue were observed with electron microscope.Results:1 Compared with those of the sham operation group, LVDP,±dp/dtmax were significantly decreased, but LVEDP was significantly increased in the ischemia group in rats (P<0.01). Compared with those of the ischemia group, LVDP was significantly increased in ischemia + NaHS low, middle and high dose groups in rats;±dp/dtmax was significantly increased, and LVEDP was significantly decreased in ischemia + NaHS middle and high dose groups in rats; LVDP,±dp/dtmax were significantly decreased, but LVEDP was significantly increased in ischemia + PPG group in rats (P<0.05 or P<0.01).2 Compared with those of the sham operation group, the content of H2S in plasma and the activity of CSE in myocardial tissue were significantly decreased in the ischemia group in rats (P<0.01). Compared with those of the ischemia group, the content of H2S in plasma and the activity of CSE in myocardial tissue were significantly increased in ischemia + NaHS low, middle and high dose groups in rats; the content of H2S in plasma and the activity of CSE in myocardial tissue were significantly decreased in ischemia + PPG group in rats (P<0.05 or P<0.01). 3 Compared with those of sham operation group, the content of MDA was significantly increased, and the activities of SOD and GSH-PX were significantly decreased in ischemia group in rats (P<0.01). Compared with those of ischemia group, the content of MDA was significantly decreased, and the activity of SOD was significantly increased in ischemia + NaHS low, middle and high dose groups in rats; the activity of GSH-PX was significantly increased in ischemia + NaHS middle and high dose groups in rats; the content of MDA was increased, the activities of SOD and GSH-PX were decreased in ischemia + PPG group in rats (P<0.05 or P<0.01).4 In the sham opearation group in rats, mitochondria showed regular cristae and intact membrane. Normal arrangement of sarcomeres was also evident. But the perinuclear gap slightly expanded. In ischemia group in rats, the cristae and membrane of mitochondria appeared to be swollen, dissoluted or disappearanced. Loss of normal striations and disorganization of sarcomeres were also found, and the nuclear membrane partially disappearanced. The caryoplasm and perinuclear gap were in high degree of edema. However, the rats in the NaHS treated group had slightly swollen mitochondria, disorganized cristae and myofibrillar derangements, especially in ischemia + NaHS high dose group. The rats in ischemia + PPG group showed severe mitochondrial injury compared with those of ischemia group in rats.Summary: Administration of NaHS could ameliorate the injury after acute myocardial ischemia in rats. It could increase the synthesis of H2S and the activities of SOD and GSH-PX, and decrease the MDA synthesis. But administration of PPG could worsen the injury. It could be concluded that the protection of H2S on myocardial ischemian is related to diminishing oxidative stress.Part 2 Effects of hydrogen sulfide on inflammatory factors in acute myocardial ischemia injury in ratsObjective: To study effects of H2S on the activity of NF-κB in myocardial tissue and inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β(IL-1β), interleukin-6 (IL-6), and intercellular adhesion molecule-1 (ICAM-1) in acute myocardial ischemia in rats, and explore the possible mechanism.Methods: Fourty-eight male SD rats were randomly divided into sham operation group, ischemia group, ischemia + NaHS low, middle and high dose groups and ischemia + PPG group. The acute myocardial ischemia model was established by ligating the left anterior descending coronary artery (LAD) of the rats. Saline was intraperitoneally administrated in ischemia group. LADs were not ligated but only threaded in sham operation group in rats. In ischemia + NaHS low, middle and high dose groups or ischemia + PPG group, sodium hydrosulfide (NaHS) (0.78, 1.56, 3.12 mg/kg) or DL- propargylglycine (PPG) (30mg/kg) was intraperitoneally injected respectively at 3 hours after ischemia. All the rats were killed at 6 hours after the operation. The contents of IL-1β, IL-6 and TNF-αin serum and myocardial tissue were respectively measured by enzyme-linked immunosorbent assay (ELISA). The expression of intercellular adhesion molecule-1 (ICAM-1) mRNA and the nuclear factor-κB (NF-кB) in myocardial tissue were respectively detected by semi-quantitative PCR and Western- blotting.Results:1 Compared with those of the sham operation group, the contents of IL-1β, IL-6 and TNF-αin serum were significantly increased in ischemia group in rats (P<0.01). Compared with those of the ischemia group, the contents of IL-1βand IL-6 in serum were significantly deceased in ischemia + NaHS low, middle and high dose groups in rats; and the content of TNF-αin serum was significantly decreased in ischemia + NaHS middle and high dose groups in rats; the contents of IL-1β, IL-6 and TNF-αin serum were significantly increased in ischemia + PPG group in rats (P<0.05 or P<0.01).2 Compared with those of the sham operation group, the contents of IL-1β, IL-6 and TNF-αin myocardial tissue were significantly increased in ischemia group in rats (P<0.01). Compared with those of the ischemia group, the contents of IL-1βand TNF-αin myocardial tissue were significantly deceased in ischemia + NaHS low, middle and high dose groups in rats; and the content of IL-6 in myocardial tissue was significantly decreased in ischemia + NaHS middle and high dose groups in rats; the contents of IL-1β, IL-6 and TNF-αin myocardial tissue were significantly increased in ischemia + PPG group in rats (P <0.05 or P<0.01).3 The expression of ICAM-1 mRNA in myocardial tissue was significantly increased in ischemia group compared with that of the sham operation group in rats (P<0.01). Compared with that of the ischemia group, the expression of ICAM-1 mRNA in myocardial tissue was significantly decreased in ischemia + NaHS low, middle and high dose groups in rats (P<0.05 or P<0.01); the expression of ICAM-1 mRNA was not significantly altered in the ischemia + PPG group in rats.4 There was only trace of expression of NF-кB in myocardial tissue in the sham operation group in rats. Compared with that of the sham operation group, the expression of NF-кB was significantly increased in ischemia group in rats (P<0.01). Compared with that of the ischemia group, the expression of NF-кB in myocardial tissue was significantly decreased in ischemia + NaHS middle and high dose groups in rats; the expression of NF-кB in myocardial tissue was significantly increased in the ischemia + PPG group in rats (P<0.05 or P<0.01).Summary: The administration of NaHS after acute myocardial ischemia could inhibit the activation of NF-κB, reduce the expression of IL-1β, TNF-α, IL-6 and ICAM-1, thus ameliorate the myocardial injury. However, the administration of PPG could decrease the content of H2S, worsen the inflammation and myocardial damage.Part 3 Effects of hydrogen sulfide on apoptosis in acute myocardial ischemia injury in ratsObjective: To study effects of H2S on cardiomyocytes apoptosis in acute myocardial ischemia injury in rats and explore the possible mechanism.Methods: Fourty-eight male SD rats were randomly divided into sham operation group, ischemia group, ischemia + NaHS low, middle and high dose groups and ischemia + PPG group. The acute myocardial ischemia model was established by ligating the left anterior descending coronary artery (LAD) of the rats. Saline was intraperitoneally administrated in ischemia group. LADs were not ligated but only threaded in sham operation group in rats. In ischemia + NaHS low, middle and high dose groups or ischemia + PPG group, sodium hydrosulfide (NaHS) (0.78, 1.56, 3.12 mg/kg) or DL- propargylglycine (PPG) (30mg/kg) was intraperitoneally injected respectively at 3 hours after ischemia. All the rats were killed at 6 hours after the operation. The apoptotic rate of cardiomyocytes was detected by flow cytometry (FCM). The expression of Bcl-2 and Bax in myocardial tissue were respectively detected by immuno- histochemisty. The expression of Caspase-3 and Cyt-c in myocardial tissue were respectively analysised by Westernblotting.Results:1 The apoptotic rate of cardiomyocytes was significantly increased in ischemia group compared with that of the sham operation group in rats (P<0.01). Compared with that of the ischemia group, the apoptotic rate of cardiomyocytes was significantly decreased in ischemia + NaHS low, middle and high dose groups in rats, and it was significantly increased in the ischemia + PPG group in rats (P<0.05 or P<0.01).2 In the ischemia group, the expression of Bcl-2 was significantly decreased, and the expression of Bax was significantly increased (P<0.05 or P<0.01). Compared with that of the ischemia group, the expression of Bcl-2 was significantly increased, and the expression of Bax was significantly decreased in ischemia+NaHS low, middle and high dose groups in rats (P<0.05 or P<0.01). Compared with that of the ischemia group, the expression of Bcl-2 was significantly decreased in ischemia + PPG group in rats (P<0.05), but the expression of Bax was not significantly altered.3 There was only a small amount of expression of Caspase-3 and Cyt-c in the sham operation group in rats. The expression of Caspase-3 and Cyt-c were significantly increased in ischemia group compared with those of the sham operation group in rats (P<0.01). Compared with that of the ischemia group, the expression of Caspase-3 was significantly decreased in ischemia + NaHS middle and high dose groups in rats; the expression of Cyt-c was significantly decreased in ischemia + NaHS low, middle and high dose groups in rats; the expression of Caspase-3 and Cyt-c were significantly increased in ischemia + PPG group in rats (P<0.05 or P<0.01).Summary: The administration of NaHS could decrease the apoptotic rate of cardiomyocytes, attenuate the expression of Bax, and increase the expression of Bcl-2 in myocardial tissue in rats. The administration of PPG could worsen the injury of myocardium. The results showed that H2S plays a protective role in acute myocardial ischemia by inhibiting cardiomyocytes apoptosis.Conclusion:1 After the acute myocardial ischemia, myocardial ultrastructure and cardiac function were damaged, the H2S/CSE system was down-regulated, and the oxidative stress was increased in rats. The administration of NaHS could increase the production of H2S, reduce the damage of myocardial structure and cardiac function, and reduce the lipid oxidation reaction, thus reducing the myocardial injury.2 The contents of TNF-α, IL-1β, IL-6 in serum and myocardial tissue were increased, and the expressions of ICAM-1 mRNA and NF-κB protein were also significantly increased after acute myocardial ischemia in rats. The administration of NaHS could decrease the contents of TNF-α, IL-1β, IL-6 in serum and myocardial tissue, and reduce the expressions of ICAM-1mRNA and NF-κB protein in myocardium, thus reducing the myocardial injury, and playing the role in myocardial protection. However, the administration of PPG could increase the contents of TNF-α, IL-1β, IL-6 in serum and myocardial tissue, increase the expression of NF-κB protein in myocardial tissue, and increase the myocardial damage.3 The cardiocyte apoptosis was increased acute myocardial ischemia in rats. The administration of NaHS could decrease the apoptotic rate of cardiomyocytes, attenuate the expressions of Bax, Cyt-c, and Caspase-3, and increase the expression of Bcl-2 in myocardial tissue in rats. The administration of PPG could worsen the injury of myocardium. It could be concluded that H2S inhibition of apoptosis is one of the mechanisms of myocardial protection. |
PDF Full Text Request