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

The cardia and cerebral vessels disease may be due to the disfunction of endothelial cells.The cells swell,shed,apoptosis and necrosis are induced by many factors,such as acute ischemia,intoxication,oxidative stress,homocysteic acid emia.Ligustrazine is one major efficient component from Chinese traditional medicine herb Chuanxiong,which is currently widely used in China for the treatment of coronary atherosclerotic cardiovascular disease and ischemic cerebrocardiac vascular disease.Ligustrazine has been reported to inhibit the platelet aggregation,to cause negative chronotropic and inotropic responses on isolated atria,to inhibit vasoconstriction in isolated vascular strips,and to act as a vasodilator,a free-radical scavenger,and anti-thrombosis and antihypertension agent.More recently,it has been found to be more effective in protection against injured vascular endothelial cells. However,pharmacokinetics studies found that Ligustrazine presented low bioavailability and to be metabolized fast in vivo with short half-life of t_1/2=2.89 h, so accumulated toxicity often appeared in the patients for keeping an effective plasma concentration by the frequent administration.Therefore,it is necessary to develop a new generation of the cerebrocardiac vascular drugs from molecular modification of Ligustrazine.According to the principles of bioisosteric replacement in medicinal chemistry,a series of novel Ligustrazine derivatives was designed,among which,OH was replaced by C1,CSTMP was synthesized.Therefore,the aim of the present study was to examine the protective effect of CSTMP in HUVECs cell injured by H₂O₂ by measuring cell viability,NO level,NOS activity,LDH leakage,the change of nuclear morphology,and apoptotic protein expression.1.Effect of CSTMP on viability in HUVECs cells injured by H₂O₂To evaluate whether CSTMP protected HUVECs against oxidative stress,cells were treated with 150umol/L H₂O₂ and CSTMP for 12h.The cell viability was measured by MTT assay.H₂O₂ decreased the viability of HUVECs markedly,while CSTMP relieved the cell damage induced by H₂O₂.The result suggested that CSTMP protected HUVECs from oxidative damage.2.Effect of CSTMP on the release of LDH in HUVECs cells injured by H₂O₂To investigate the effect of CSTMP on the LDH leakage of HUVECs cells,cells were treated with 150umol/L H₂O₂ and CSTMP for 12h.Then,the level of LDH in cell lysate and cultured supernatant was measured by kit.Exposure to H₂O₂ for 12h markedly increased LDH leakage from cells into medium,while treatment of HUVECs cells with various concentrations of CSTMP inhibited H₂O₂-induced LDH leakage.The same protective effect was observed in TMP treated-cell group.3.Effect of CSTMP on MDA,GSH contents and SOD activities in HUVECs cells injured by H₂O₂To determine the effect of CSTMP on the MDA,GSH contents and SOD activities of H₂O₂-damaged HUVECs cells,cells were treated with 150umol/L H₂O₂ and CSTMP for 12h.Cells were incubated with H₂O₂ accompanied with the decrease of GSH level and SOD activity,while CSTMP attenuated the H₂O₂-induced decrease of GSH and SOD activity.The same protective effect was observed in TMP group.The MDA content of HUVECs was significantly increased after incubated with H₂O₂, which could be attenuated by the treatment of CSTMP.4.Effect of CSTMP on nuclear morphology in HUVECs 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 and the morphous of cells with the protection of CSTMP has little difference with the normal group. 5.Effect of CSTMP on the apoptosis in HUVECs cells injured by H₂O₂The percentage of apoptosis was monitored with flow cytometry analysis after Annexin V-FITC/PI staining.The result showed that after the oxidative damage, apoptosis cells in injured group cells were increased,while that in CSTMP groups were fewer,which indicate a strong cell protection of CSTMP from apoptosis.6.Effect of CSTMP on HUVECS cells NO production and NOS activityThe NOS activity and NO level of H₂O₂-damaged HUVECs cells were determined by kits.Cells were treated with 150umol/L H₂O₂ and CSTMP for 12h. The results showed that after the oxidative damage,NO contents were decreased and NOS activity was inhibited,while CSTMP increase the release of NO directly by increasing the activity of NOS on HUVECs cells in a concentration-dependent manner.7.Effect of CSTMP on the expression of ERK1/2,P-ERK1/2 in HUVECs cells injured by H₂O₂HUVECs cells were incubated with H₂O₂ in the presence or absence of CSTMP (100umol/L) for 15-min,30-min,1-hr,4-hr,8-hr,and 12-hr,and the lysates were probed with specific anti-phospho-ERK1/2 antibodies.Beta-tubulin was used as the intrinsic reference of phospho-ERK1/2.A significant increase in the expression of phospho-ERK1/2 was observed after cells were injured by H₂O₂.The expression of p-ERK1/2 increased since 30min.At the time of 12hr,CSTMP significantly enhanced the phosphorylation level of ERK1/2,which is much higher than H₂O₂ group. however,no change in the total ERK1/2.These results suggest that the pro-survival effect of CSTMP was mediated through activation of the ERK1/2 pathway.8.Effect of CSTMP on the expression of caspase-3 in HUVECs cells injured by H₂O₂H₂O₂ can induce apoptosis of cultured endothelial cells.To observe the effect of CSTMP on apoptotic HUVEC_s,we investigate the expression of caspase-3 using Western blotting method.The result showed that CSTMP significantly decrease the level of caspase-3,which is markedly different with that of H₂O₂ group(P＜0.01).The results showed that H₂O₂ up-regulated the expression of caspase-3,while CSTMP can inhibit the expression of caspase-3,which suggested that the anti-apoptosis effect of CSTMP was exerted by inhibition of caspase-3.