The Molecular Mechanism On Inducing Endothelial Cell Apoptosis By Myeloperoxidase

ObjectiveTo investigate the molecular mechanism on inducing human umbilical vein endothelial cell apoptosis by myeloperoxidase.Methods1. Human umbilical vein endothelial cells (HUVEC-12) were treated with different concentrations (0.1u/ml,0.2u/ml,0.4u/ml,0.6u/ml)of myeloperoxidase, the growth inhibitory rate of endothelial cell was determined by MTT assay, and cell apoptosis rate by Flow cytometry.2. Reverse transcription-PCR (RT-PCR) and real-time PCR were used to assess relative expression level of caspase-3and bax mRNA expression. Each gray value was measured by gray level analyzer to calculate the ratio of the gray value of the target gene and the internal reference gene (GAPDH) in RT-PCR. Besides, the comparative threshold method was used to calculate the ratio of different treatment groups with the negative control group to compare the relative expression level in real-time PCR.3. Western-blotting was used to detect caspase-3and bax protein expression, the measurement of each gray value by the gray scale analyzer to calculate the ratio of the gray value of the target protein and the internal reference one(β-actin).Results1. The changes of the growth inhibitory rate and cell apoptosis rate in HUVEC-12:Compared with the negative control group, growth inhibitory rate of HUVEC-12cells was significantly higher (P<0.05) in0.4u/ml MPO treatment group,0.6u/ml MPO treatment group and the positive control group, and cell apoptosis rate was significantly higher (P<0.05) in0.2u/ml MPO treatment group,0.4u/ml MPO treatment group,0.6u/ml MPO treatment group, and the positive control group. Besides, the effects of MPO was dose-dependent (P<0.05).2. The expression of caspase-3mRNA and protein in HUVEC-12: Compared with the negative control group, the expression of caspase-3 mRNA in HUVEC-12cells was significantly increased (P<0.05) when they were treated by higher concentration (RT-PCR0.4u/ml、0.6u/ml, Real-time PCR0.2u/ml、0.4u/ml and0.6u/ml) of MPO and peroxide(positive control group). Compared with the negative control group, caspase-3precursor fragment of protein expression was significantly lower(P<0.05) but activation fragment of caspase-3protein expression was significantly higher (P<0.05) in0.4u/ml MPO treatment group,0.6u/ml MPO treatment group and positive control group. Besides, the effects of MPO was dose-dependent (P<0.05).3. The expression of bax mRNA and protein in HUVEC-12: Compared with the negative control group, the expression of bax mRNA in HUVEC-12cell was significantly increased (P<0.05) when they were treated by higher concentration(RT-PCR0.4u/ml、0.6u/ml, Real-time PCR0.2u/ml、0.4u/ml and0.6u/ml) of MPO and peroxide(positive control group). Compared with the negative control group, bax protein expression was significantly higher (P<0.05) in0.4u/ml MPO treatment group,0.6u/ml MPO treatment group and positive control group. Besides, the effects of MPO was dose-dependent (P<0.05).Conclusion1. Myeloperoxidase induces cell apoptosis in HUVEC-12endothelial cells within certain concentration range, and the effect of MPO is dose-dependent.2. MPO induces cell apoptosis may be by activating of caspase-3pathway in HUVEC-12endothelial cells3. Bax may also be involved in the process of HUVEC-12endothelial cell apoptosis caused by myeloperoxidase.