Protective Effect Of Tetramethylpyrazine Diphenylmethyl Piperazidine On Endothelial Cell Damage Induced By Hydrogen Peroxide

The cardiac and cerebral vessels diseases have strong relationships with dysfunction of endothelial cells (EC). The EC cells injury, sush as, swell, shed, apoptosis and necrosis are induced by many factors, such as acute ischemia, intoxication, oxidative stress, homocysteic acid emia.Tetramethylpyrazine(TMP) is the major efficient component of the Chuanxiong, and is used widely in clinic with the effect of scavenging oxygen free radical, antagonizing calcium, dilating blood vessel, antiplatelet aggregation and anti — thrombsis.Because the methyls group in TMP molecule can be easily oxygenized and metabolized to products with high water solubility which can be eliminated quickly, has short half-life and low bioavailability. According to the structure-activity relationship of TMP and its derivates, we synthesize TMPDP by keeping its pharmacophore and replaced the second methyl of TMP with the (4,4'-fiuorine)diphenyl-methyl-1-piperazidine. The Flunarizine is an important drug for cardiac and cerebral vessels disease, the (4,4'-fluorine)diphenyl-methy-l-piperazidine-methylium is its main active group. We presume that the new compound will have longer half-life and higher bioavailability compared with TMPDP.At the meamtime, pharmacodynamic action of TMPDP can be increased with the synergistic effect of TMP with Flunarizine. Thereby, we supposed TMPDP have stronger effects on cardiac and cerebral vessels disease.In this paper, the detailed characterization of the blood vessel endothelial cells response to the presence of hydrogen peroxide was studied, and the effects of TMPDP on the cardiac and cerebral vessels disease were evaluated1 Effect of TMPDP on the viability in ECV-304 cells injured by H₂O₂The viability of ECV-304 cells was detected by MTT assay. The results indicated that the viability of ECV-304 cells viability decreased significantly by oxidative damage (P<0.01). After the administration of TMPDP at the dosage of 10, 50, 100 μmol/L, the cells viability was increased significantly(P<0.01).2 Effect of TMPDP on the release of LDH in ECV-304 cells injured by H₂O₂ We collected the supernate and lysate of ECV-304 cells injured by H₂O₂ anddetermined the activity of LDH by kit. The results showed that after oxidative damage, the permeability of cellular membrane was raised and LDH efflux increased, so activity of LDH in extra-cellular markedly ascended (P<0.01). After the treatment of TMPDP at the dosage of 10, 50, 100 μmol/L, the leaking of LDH efflux induced by H₂O₂ was attenuated and LDH level in extra-cellular was decreased (P<0.01).3 Effect of TMPDP on MDA contents and SOD, GSH-P_X activities in ECV-304 cells injured by H₂O₂We collected the supernate of ECV-304 cells injured by H₂O₂ and detected the MDA content and SOD, GSH-P_X activity by kit. There were marked increase of MDA content and decrease of SOD and GSH-P_X activity when cultures were incubated with H₂O₂ (P<0.01). 10, 50, 100μmol/L TMPDP significantly inhibited the generation of MDA(P<0.01) and the reduction of SOD and GSH-P_X activity (P<0.01).4 Effect of TMPDP on ROS in ECV-304 cells injured by H₂O₂The cells were collected. After DCFH-DA was added to cell cultures. The cells were divided into 6 groups randomly: the normal group; the injured group; the protected groups with different contents of TMPDP, and added to 96-well plate. Then we detected the fluorescence of cells (λ_ex=488nm ,λ_em=525nm). The content of ROS increased by the injury of H₂O₂ (P<0.01). 10, 50, 100 μmol/L TMPDP protected the injured cells by decreasing the concentration of ROS.5 Effect of TMPDP on nuclear morphology in ECV-304 cells injured by H₂O₂Chromosomal condensation and morphological change in the nuclear were observed using the chromatin dye Hoechst 33258 and photoed by the fluorescence microscope. The cells induced by H₂O₂ obviously exhibited apoptotic nuclear condensation. The morphous of cells with the protection of TMPDP were tend to be normal.6 Effect of TMPDP on apoptosis in ECV-304 cells injured by H₂O₂The percentage of apoptosis were monitored by flow cytometry analysis after Annexin V-FITC/PI staining. The result suggested that after oxidative damage, apoptosis in injured group cells was emerged. TMPDP inhibited apoptosis by inhibiting oxidative damage.7 Effect of TMPDP on the concentration of free calcium in ECV-304 cells injured by H₂O₂Cells were cultured with the calcium dye Fura-2/AM, and divided into 6 groups: the normal group; the injured group; the protected groups with different contents of TMPDP, then added to 96-well plate. Average calcium concentration were expressed as a ratio of the fluorescence emissions obtained using two different exicitation wavelengths at λ_ex=340 nm/λ_em=510 nm and λ_ex=380 nm/λ_em=510 nm. The fluorescence intensities were then calibrated to provide an estimation of the relative change in the intracellular calcium concentration following equation to calculate [Ca²⁺]_i. The result showed that after oxidative damage, the intracellular calcium concentration increased significantly. The 50, 100μmol/L TMPDP can antagonized the up-regulation of concentration of free calcium induced by H₂O₂(P<0.01).8 Effect of TMPDP on mitochondrial membrane potential in ECV-304 cells injured by H₂O₂Cells were collected, and cultured with fluorescent dye Rh123. The MMP was monitored using flow cytometry. The result showed when cells were exposed to H₂O₂, the MMP was rapidly reduced. After administration of TMPDP, the MMP increased with dose-dependent.9 Effect of TMPDP on the expression of Bcl-2, P-53, Caspase-3 in ECV-304cells injured by H₂O₂After treatment with H₂O₂, ECV-304 cells cultured on coverslips were washed and fixed. Then the expression of Bcl-2, P-53, Caspase-3 protein was detected using S ABC immunohistochemistry. The results showed that there was a decrease of Bcl-2 level and an increase of P-53 and Caspase-3 in ECV-304 cells after oxidative damage (P<0.05), administration of 50, 100μmol/L TMPDP antagonized the down-regulation of Bcl-2 level and the up-regulation induced by H₂O₂(P<0.05).