[Mechanism of inhibition of apoptosis of human umbilical vein endothelial cells induced by oxidized-low density lipoprotein by erythropoietin].

OBJECTIVE: To explore the protective effect of erythropoietin (EPO) against oxidized-low density lipoprotein (ox-LDL)-induced apoptosis in human umbilical vein endothelial cells (HUVECs) in an ox-LDL induced apoptosis model. METHODS: Third-sixth passage of cultured HUVECs were used, and they were divided into two groups. The cells were pretreated with different concentrations (6.25, 50, 100 kU/L) of recombinant human erythropoietin (rhEPO) for 24 hours, then they were exposed to ox-LDL (100 mg/L) for 48 hours; another group of cells were pretreated with antisense to 0.5 micromol/L LOX-1 mRNA or 0.5 micromol/L sense for 24 hours, and then HUVECs were exposed to ox-LDL (100 mg/L) for 12 hours. Apoptosis was assessed by the apoptosis ratio, cell viability, and Bcl-2/Bax ratio. RESULTS: As compared to untreated controls, pretreatment with rhEPO led to increased cell survival of HUVECs and decreased cell apoptosis in a dose-dependent manner (all P<0.05). Consistently, the Bcl-2/Bax ratios were also increased in a similar fashion. The ratio of apoptosis protein Bcl-2/Bax was increased in the antisense LOX-1 mRNA group than that of ox-LDL group (P<0.05), but the one in the sense LOX-1 mRNA group was not significantly different from that of ox-LDL group. CONCLUSION: The ox-LDL can induce apoptosis in HUVECs by regulating LOX-1 mRNA, and rhEPO can increase Bcl-2/Bax ratio and inhibit ox-LDL-induced apoptosis of HUVECs.

[Effect of erythropoietin on the caspase-3 subfamily in preventing apoptosis of endothelial cell].

OBJECTIVE: To explore effect of erythropoietin on the caspase-3 subfamily in preventing apoptosis of human umbilical vein endothelial cells (HUVECs) induced by oxidized-low density lipoprotein (ox-LDL). METHODS: Third-sixth passages of HUVECs were used. Two experiments were conducted. In the first experiment, there was a blank control group, ox-LDL control group (100 mg/L, incubated for 48 hours), and low, medium, and high recombinant human erythropoietin (rhEPO) groups (6.25, 25.00, 100.00 kU/L rhEPO incubated for 24 hours+100 mg/L ox-LDL incubated for 48 hours). Another experimental protocol consisted of groups of the cells pretreated with either caspase-3 inhibitor DEVD-CHO, or caspase-8 inhibitor z-IETD-fmk, or caspase-9 inhibitor z-LEHD-fmk of 25 micromol/L for 24 hours, then HUVECs were exposed ox-LDL (100 mg/L) incubated for 48 hours. The activity of caspase-3, caspase-8, or caspase 9 was determined by caspase colorimetric assay. The cell survival rate was assessed with methyl thiazolyl tetrazolium (MTT) method. The positive expression rate of caspase-3 and apoptotic rate were measured by flow cytometer. RESULTS: The activity of caspase-3 was significantly decreased and cell survival rate was increased in the caspase-3 inhibitor group (both P<0.05). The activity of caspase-8 was decreased in the caspase-8 inhibitor group (P<0.05), but the cell survival rate was not significantly different from that of ox-LDL group (P>0.05). The activity of caspase-3 or caspase-9 was lower and cell survival rate was higher in the caspase-9 inhibitor group than that of ox-LDL group (all P<0.05). The pretreatment with rhEPO led to decreased activity of caspase-3, caspase-9, positive expression rate of caspase-3 and apoptotic rate in a dose-dependent manner compared with ox-LDL group (all P<0.05), but the activity of caspase-8 showed no significant difference from rhEPO pretreatment groups (all P>0.05). CONCLUSION: These results demonstrate that rhEPO can significantly inhibit the activity of caspase-3 or caspase-9 in endothelial cell apoptosis in a dose-dependent manner. Activation of caspase-3 or caspase-9 is involved in ox-LDL-induced HUVECs apoptotic signaling pathway, but caspase-8 is not involved.