| Apoptosis, also known as programmed cell death, is an active death form of cell by the gene's regulation. And the ischemia/reperfusion injury is an important pathophysiological process in cardiovascular disease; Cardiomyocyte apoptosis is also a major contributor to ischemia-reperfusion injury. Therefore, looking for a new way to inhibit cardiomyocyte apoptosis and save the damaged myocardial cells is important to treat a variety of myocardial cell apoptosis- related cardiovascular disease and protect normal heart function.In recent years, the studies found that the homeostasis of ion exchange between inside and outside of membrance is closely related to the apoptosis. In the nonage of apoptosis, cell shrinkage which named apoptotic volume decrease (AVD) is an important morphological characteristic of apoptosis. AVD and activation of ion channels in the process of apoptosis were demonstrated playing important roles in cell apoptosis as a perquisite of cell apoptosis. Chloride ion is the most important and abundant anion inorganisms, the apoptosis could be inhibited through blocking the ion channel and cell volume decrease in a variety of cells. So the purpose of this study is to establish the model of cardiomyocyte apoptosis induced by ischemia/reperfusion injury, to observe the effect of non-specific chloride channel blocker DIDS on cell survival and apoptosis, and to explore the effect of DIDS on intracellular signaling pathway of PI3K/Akt and its downstream molecular Bcl-2/Bax.Aims:1. To set up ischemia/reperfusion injury-induced cardiomyocyte apoptosis by reparing primary cultures of neonatal cardiomyocyte.2. To investigate the influence of non-specific chloride channel blocker DIDS on ischemia /reperfusion injury-induced apoptosis of myocardial.3. To explore the effects of chloride channel blocker DIDS on cell signaling pathway of PI3Kinase/Akt and its downdtream protein Bcl-2 and Bax.Methods:1. Primary cultured cardiomyocyte of SD neonatal rats were prepared and established the model of cardiomyocyte apoptosis by ischemia/reperfusion injury, and were divided into four groups: the normal control group (Con), ischemia/ reperfusion group (I/R), DIDS treatment group (I/R+DIDS) and LY294002 treatment group (I/R+DIDS+LY294002).2. The cell viability was measured by colorimetric method; the morphologic changes in cardiomyocyte was observed under Hoechst 33258 staining; caspase-3 activity was assayed using the caspase-3 Assay Kit/Fluorimetric; In addition, cell apoptosis was also confirmd by DNA fragmentation.3. The effects of chloride channel blocker DIDS on PI3K/Akt signaling pathway and its downstream molecular Bcl-2/Bax through Western blot.4. Bax distribution was assessed by double immunofluorescence staining. Results:1. There were significant differences (n=12, P<0.01 DIDS group vs. I/R group) between DIDS group and I/R group on cell survival rate and caspase-3 activity; Compare to I/R group, DIDS significantly inhibited DNA "ladder"-shaped formation of bands, Hoechst-33258 staining positive cells decreased significantly So it demonstrated that the chloride channel blocker DIDS could inhibit I/RI induced apoptosis.2. Compared with the DIDS group, it showed that cells survival rate reduced (58.4%), caspase-3 activity increased significantly (460%) and obvious characteristics of apoptosis: nuclear fragmentation, condensation and significant DNA "ladder"-shaped strip appeared in LY294002 treatment group.3. Western blot showed that the expression level of total Akt had no difference (n=12, P>0.05) among all groups, but the expression level of p-Akt in the DIDS group was significantly higher than I/R group (n=12, P<0.01 DIDS group vs. I/R group), even added DIDS to LY294002 treatment group, p-Akt levels had no difference (n=12, P>0.05 LY294002 group vs. I/R group) compared with I/R group; However, the level of p-Akt in LY294002 group was significantly lower than DIDS group(n=12, P<0.01 LY294002 group vs. DIDS group ).4. Western blot showed that the expression of Bcl-2, Bax had no significant difference (n=12, P>0.05) among all groups. Confocal laser scanning microscope showed that Bax was mainly distributed in the cytoplasm of normal cells, I/RI induced cardiomyocyte apoptosis accompanied by Bax protein translocation to the mitochondria; DIDS could inhibit the I/RI-induced Bax translocation from cytoplasm to mitochondria, but it showed that Bax translocation from cytoplasm to mitochondria in LY294002 group.Conclusions:1. DIDS prevented I/RI-induced cardiomyocyte apoptosis and enhanced cell viability.2. DIDS increased the level of p-Akt and inhibited I/RI-induced cardiomyocyte apoptosis through activating PI3K/Akt signaling pathway.3. In the I/RI-induced cardiomyocyte apoptosis modle, we first demonstrated DIDS involved in the mechanism of protecting cardiac myocytes by inhibiting Bax from the cytoplasm to the mitochondria.
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