| Ischemia reperfusion (IR) injury can impair the systolic and diastolic functions of the heart. Apoptosis, necrosis and autophagy are reported to be the three different pathways of cell death in myocardial IR injury. Apoptosis is an active, programmed cell death, while necrosis is a passive pathway. Necrosis is the main pathway to death in ischemia myocardial cell, while apoptosis is in IR injury. Hydrogen sulfide (H2S) has been reported to be produced by special enzymes in several mammalian cells, which possesses comprehensive biological effects, and it also plays vital roles in many pathophysiological processes in cardiovascular system, nerves system and digestion system, et al. While many studies showed that endogenous H2S synthesized in cardiovascular tissue in various kinds of physiological and pathological states. H2S is involved in the relaxation of vascular smooth muscle, inhibition of vascular smooth muscle cell proliferation, regulations of the myocardial contractile force and other physiological acticities. It is also involved in hypertension, coronary heart disease and other pathological processes. It has been verified that H2S has an important endogenous protective role on myocardial IR injury. It not only reduces infarct size following IR injury, but significantly inhibits the incidence of myocardial apoptosis and improves cardiac function. H2S protection of myocardial cells on apoptosis, calcium overload and energy metabolism abnormality has been well studied. However, its exact mechanism remains unclear. Recently microRNA (miRNA., miR) has been proved to play an important role in the post-transcriptional gene regulation. It is well known that some cardiac-specific expression miRNA is involved in the signaling pathway of apoptosis through the regulation of expression of apoptosis-related target genes, so as to affect the apoptosis of myocardium. Therefore, we hypothesize that cardiac-specific expressed miRNA is involved in the H2S protection against myocardial IR injury, especially in post-transcriptional regulation of apoptosis-related target genes in anti-apoptotic process in cardiomyocytes.Part 1 H2S protects myocardium from IR injury in vivo1. ObjectiveWe aim to confirm the protective effects of H2S on IR myocardium and screen the obviously different expression of miRNAs between IR group and H2S protective group.2. MethodsMyocardial IR injury model in SD rats was established. SD rats were given 10,30,50, 100 μM/kg NaHS intraperitoneal injection to rats before suffered from IR injury. Evans blue and TTC staining method was used to detect the relative area of myocardial infarction rats (infarct area/risk area). TUNEL staining was used to detect the cell apoptotic rate in myocardial tissue. The RT-PCR and Western blot methods were used for quantitative detection of Bcl-2 and miR-1 expression. MiRNA chip was used to screen the obviously different expression of miRNAs between IR group and H2S protective group.3. Results3.1 Myocardial infarction area in IR group was significantly larger than that of Sham control group, (40.64 ± 11.26)% vs (0.85 ± 0.38)%. Meanwhile, H2S pretreatment could reduce the relative area of myocardial infarction. Compared with the IR group,30 μM/kg H2S group decreased most obviously.3.2 The LDH in IR group increased significantly compared with the Sham operation group. While, H2S pretreatment could reduce the LDH leakage in a concentration dependent manner, especially 30 μM/kg NaHS reducing the most obviously.3.3 Compared with Sham group, the apoptotic cells number increased significantly in IR group. After different concentrations of H2S pretreatment, the apoptosis rate decreased in different degree, especially in 30 μM/kg group reducing the most obviously.3.4 Compared with Con group, IR group Bcl-2 mRNA significantly decreased. Compared with IR group, in H2S groups Bcl-2 expression increased in a concentration dependent manner. When the H2S concentration was 30 μM/kg, compared with in IR group, the Bcl-2 mRNA was the most significant up-regulated (P<0.05). The expression of Bcl-2 protein was detected by Western blot method, and the change trend was consistent with mRNA expression.3.5 In H2S protection group, miR-1 was significantly down-regulated, which suggested that miR-1 may be involved in the inhibition of cardiomyocyte apoptosis.4. Conclusions4.1 H2S pretreatment could reduce myocardial infarct size, inhibit apoptosis, reduce the myocardial IR injury, protect myocardial cells, in a concentration dependent manner. And 30μM/kg was the most effective concentration.4.2 Compared with H2S protection group, miR-1 was significantly up-regulated in IR group.Part 2 H2S modulates miR-1 to inhibit myocardial cell apoptosis1. ObjectiveTo research the protective effects of H2S preconditioning on hypoxia reoxygenation (HR) cardiac cells in vitro. To study whether miR-1 was involved in the inhibition of H2S myocardial apoptosis.2. MethodsCariomyocytes HR injury model was established in vitro. Cariomyocytes were given 10,30,50 or 100 μM NaHS before suffered from HR injury. MTT method was used for detecting myocardial viability. LDH leakage of cell culture supernatant was detected by Automatic biochemical analyzer. Flow cytometry was used to detect the cell apoptotic rate. The RT-PCR and Western blot methods were used for quantitative detection of Bcl-2 and miR-1 expression. Cariomyocytes were transfected with positive fragments miR-1 mimic, then suffered from HR injury.3. Results3.1 Compared with Con group, cell viability in HR group decreased. While, in pretreatment of H2S groups the cell viability was significantly higher than the HR group. Of which in 30 μM group the cell viability was the most significantly increased compared with in HR group.3.2 Compared with Con, LDH concentration was significantly increased in HR group. NaHS pretreatment could decrease the LDH leakage compared with HR group, and a concentration gradient change. Of which in 30 μM group the LDH was the most significantly decreased compared with in HR group.3.3 HR group apoptosis rate was significantly higher than that in Con group, while NaHS pretreatment could decrease cell apoptosis rates compared with the HR group, with a concentration gradient change. Compared with in HR, in 30 μM group the apoptosis rate decreased the most significantly.3.4 Compared with Con group, in HR group Bcl-2 mRNA significantly decreased (P<0.05). Compared with HR group, expression of Bcl-2 in NaHSpretreatment groups was increased, and with a concentration gradient. When the H2S concentration was 30μM, the Bcl-2 mRNA was the most significantly up-regulated. The expression of Bcl-2 protein was detected by Western blot method, and the change trend was consistent with mRNA expression.3.5 Compared with Con group, miR-1 increased significantly in HR group. Compared with HR group, expression of miR-1 in NaHS pretreatment groups was decreased with a concentration gradient. When the NaHS concentration was 30μM, compared with HR group, miR-1 was the most significantly down-regulated.3.6 Compared with NC group, in NC+H2S group the cell apoptosis rate had no significant change. Compared with in NC group, cell apoptosis rate in mimic group increased significantly, suggesting that transfection of miR-1 mimic can worsen the apoptosis. Compared with in HR+NC group, in HR+mimic group cell apoptosis rate increased. Compared with in HR+NC+H2S group, in HR+mimic+H2S group apoptosis rate was significantly increased. Ater HR injury and up-regulation of miR-1, NaHS pretreatment did not significantly reduced myocardial apoptosis rate. The results suggested that up-regulation the expression of miR-1 decreased the cardioprotective effect of NaHS on apoptosis.4. Conclusions4.1 H2S exhibited a clear protective effect of myocardial cells, and 30 μM was the most effective concentration.4.2 MiR-1 was involved in the signaling pathway of H2S inhibition the apoptosis of myocardial cells.Part 3 H2S regulates miR-1-Bcl-2 pathway to inhibit cardiomyoctes apoptosis1. ObjectiveTo study the possible mechanism of miR-1 participate in myocardial protection.2. MethodsUsing bioinformatics method (websites such as Targetscan, miR-base, et al) to query and predict the potential target genes for miRNA. Bcl-2 was one of the potential target genes of miR-1.Cardiomyocytes were transfected with positive fragments miR-1 mimic or antagonist AMO-1, then Bcl-2 protein expression was detected. Cardiomyocytes were transfected with positive fragments Bcl-2 siRNA, then suffered from HR injury.3. Results3.1 The expression of miR-1 in mimic group was higher than that in NC group, while the expression of Bcl-2 were lower than that in negative groupin a concentration gradient. In contrast, miR-1 in AMO group was lower than that in NC group; and the expression of Bcl-2 was higher than that in NC group.3.2 Compared with NC group, in NC+H2S group the cell apoptosis rate had no significant change. Compared with NC group, cell apoptosis rate in siRNA group increased significantly, suggesting that transfection of Bcl-2 siRNA could increase the apoptosis of myocardial cells. Compared with HR+NC group, in HR+siRNA group cell apoptosis rate increased. Compared with HR+NC+H2S group, in HR+siRNA+H2S group apoptosis rate was significantly increased. After HR injury and down-regulation of Bcl-2, NaHS pretreatment did not significantly reduced myocardial apoptosis rate. The results suggested that down-regulation the expression of Bcl-2 decreased the cardioprotective effect of H2S on apoptosis.4. Conclusions4.1 Bcl-2 is one downstream target gene of miR-1.4.2 Bcl-2 was involved in the signaling pathway of H2S inhibiting the apoptosis of myocardial cells.In summary, miR-1-Bcl-2 signaling pathway was involved in the protective effect of H2S on myocardium. |
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