Study Of Anti-Apoptotic Effect Of Compound Macrostem Onion Capsule (CMOC) On Endothelial Cell In Pulmonary Arterial Hypertension

Objective:The pathogenesis of chronic obstructive pulmonary disease(COPD) and pulmonary artery hypertension(PAH) are pulmonary endothelial dysfunction,hypoxic pulmonary vasoconstriction,vascular remodelling.Recent investigation proves that the damage of endothelial cell is the initiation of PAH.Compound Macrostem Onion Capsule (CMOC),which got patent right in 2004,NO.ZL02160139.9,is an effective formula for patients with COPD and PAH.This project study was designed to assess the protective effect of CMOC on hydrogen dioxided(H₂O₂) induced apoptosis in human umbilical vascular endothelial cell(HUVEC),apoptosis-related genes expression and signal transduction factors, to reveal the mechanism of anti-apoptosis of CMOC on Endothelial Cell.Methods:To establish the apoptotic models of HUVEC in vitro,using MTT assay to decide the best dose and the best time of H₂O₂.HUVEC were divided into five experimental groups:normal group,control group,CMOC group with three concentrations of 3μg/ml, 30μg/ml and 300μg/ml respectively MTT assay was used to measure the proliferation of the HUVEC.The apoptotic characteristics were detected by means of flow cytometry and Hoechst stain.Genes bcl-2 were detected by Western blotting.Using Rh123 dying to detect the expression of mitochondrial membrane potential.Using immunohistochemistry staining to detect the expression of caspase-3 and NF—KB.Results:1.The model of apoptotic were established by H₂O₂.The result showed that the best concentration of H₂O₂ was 200μmol/l,and the best duration to establish apoptotic model was 24 hours.2.MTT assay result:Cell optical density were lower than normal group and the proliferation rate of the cells was 9.29%in the control group(P＜0.01).When treated by CMOC,the OD value were increased,and the proliferation rate of the cells were14.77%, 31.98%and 41.45%in CMOC groups respectively which increased with the increase of concentration.There were apparent statistical significance in the middle and high concentration groups,as compared with the control group,P＜0.01.3.Hoechst dying result:Apoptosis cell were seldom found in the normal group the apoptosis rate was 2.0%.In the control group a mass of apoptosis were detected and the apoptosis rate was 32.17%.After treated with CMOC,apoptosis cells were reduced in number.The apoptosis rate were 28.33%,20.17%,13.33%in CMOC groups respectively,in direct proportion to concentration.There were apparent statistical significance in the middle and high concentration groups,as compared with the control group,P＜0.01.4.Flow cytometry assay result:The cellular apoptosis rate were 0.37%in the normal group,14.38%in the control group,9.1%,7.9%and 6.48%in CMOC groups respectively which were proportional to the increase of concentration.There were apparent statistical significance compared with the control group,P＜0.01.5.Western blot result:The value of Bcl-2 expression were 0.673 in normal group, 0.438 in control group,0.572,0.613 and 0.628 in CMOC groups respectively,in direct proportion to concentration.It shows that the anti-apoptosis gene expression was increased after CMOC treatment.6.Mitochondrial membrane potential result:In the normal group Rh123 dying emitted apparent green florescent light,while in the control group the florescent light were feeble. After treated with CMOC,the florescent light increased in intensity and were positively correlated with concentration,especially in the middle and high concentration groups.7.Caspase-3 immunohistochemistry result:The prunosus particle in the control group were the most apparent,after treated with CMOC,the prunosus particle reduced in number. The positive cells were 1.83%in normal group,20.67%in control group,19.33%,14.33% and 5.33%in CMOC groups respectively,in direct proportion to concentration.The most apparent statistical significance were found in the middle and high concentration groups,as compared with the control group,P＜0.01.8.NF—KB immunohistochemistry result:The prunosus particle in the control group were the most apparent,but after treated with CMOC,the prunosus particle reduced in number.The positive cells were 3.33%in normal group,18.5%in control group, 16.67%,12.33%and 6.67%in CMOC groups respectively,which were again in direct proportion to concentration.The most apparent statistical significance were found in the middle and high concentration groups,as compared with the control group,P＜0.01.Conclusions:These results indicate that Compound Macrostem Onion Capsule(CMOC) has the protective effect on the H₂O₂ induced cellular apoptosis in HUVEC,and has the function of anti-apoptosis.Its mechanism lies in its effect in increasing the expression of Bcl-2.The expression of this gene may change the permeability of mitochondrial,enhance the expression of membrane potential,reduced Caspae-3 and NF-KB signal pathway,and suppress caspase-3 induced cell apoptosis.Thus we think CMOC can protect HUVEC via many targets and can prevent apoptosis.