Effects Of The Serum Containing Xinmaitong On Expression Of TNF-α And NF-kB During Oxidative Injury In Cardiomyocytes

Objective:People's quality of life is severely affected by myocardial ischemia. Myocardial ischemia is one of diseases with high morbidity and high mortality. Myocardial infarction resulting from acute myocardial ischemia is also a main reason for congestive heart failure,so it is very significant to protect ischemic heart disease(IHD).Xinmaitong is a compound recipe of traditional Chinese drug,its main functions include enhancing blood circulation and removing stasis, supplying Qi and nourishing blood.Clinical researches have shown that it has a good effect against ischemic heart disease(IHD).It can keep scathing things down,and increase the number of protective things.In this study,we detect the expressing of TNF-αand NF-kB in cardiocytes under the injury caused by H₂O₂ in order to relate an effect of Xinmaitong medicated mouse serum on heart injury by myocardial ischemic and possible mechanism at cellular and molecular level.Methods:Pour the traditional Chinese medicine compound recipe Xinmaitong solution into the mouse stomachs,withdrawal the blood from abdominal aorta of mouse,make herb-medicated mouse serum.Preparation of CMCs of neonate rats was done by the enzyme digestion and adherence of unequal velocity.CMC were identified by Immunocytochemical.Cells were seeded in 6,24 and 96 well culture plates,and were cultured in 37℃,5%CO₂,and 95%humidified condition.After 48h,Cells were randomly divided into four groups:Control group(15%non-medicated serum+DMEM),model group(15%non-medicated+H₂O₂+DMEM), Xinmaitong group(15%Xinmaitong-medicated serum +H₂O₂+DMEM),and DAng-I group(15%non-medicated+DAng-I+H₂O₂+DMEM).After 24h, CMCs were treated by H₂O₂(200μmol/L final concentration groups) for 6h. Observed the morphological changes of myocardial cell under inverted phase contrast microscope;detected cell viability by MTT chromatometry;Staining by ICC(Immunocytochemical),we block the cover slips and take pictures,20 pictures were taken for each sample and detect its nuclear translocation,extract 10 pictures randomly to compute their mean which is taken as the nuclear translocation of one sample.Compare different expressing of NF-kB in different groups;cells in the 6 well culture plates were immediately used for RNA extraction.Compare different expressing of NF-kB and TNF-a in different groups.Results:1.Detecting cell viability under the injury caused by H₂O₂ and the effect of Xinmaitong against the injury by MTT.â‘ .Injury effect of H₂O₂:Normal cardiomycytes were in fusiform or rhomubus shapes,with spurious leg interlaced like a net,cell beat simultaneously;when cardiomycytes were treated with H₂O₂ for 6h,their beating rate descend and even beating disappear, connection between cells break,with spurious leg shortened or disappeared, irregular rhythm,discordant frequency and poor refractive index.MTT show that the values of OD of control group and model group,are0.409±0.090,0.223±0.054 respectively.â‘¡.Protective effects of Xinmaitong-medicated serum against H₂O₂-induced CMCs' injury:Comparing with CMCs of model group,CMCs cultured in medium containing Xinmaitong-medicated serum are improved.Spurious leg is not retracted obviously.Cells show more regular rhythm of impulse,more coincident frequency and better refractive index.MTT show that the values of OD of Xinmaitong group,and DAng-I group are 0.35±0.012,0.37±0.013 respectively.2.Nuclear translocation of NF-kB:NF-kB was mainly expressed in cytoplasm in normal cardiomycytes,the translocation rate was 3.925±1.246.When cardiocytes were treated with H₂O₂ for 6h,the majority of NF-KB in cardiomycytes in model group translocated to the nuclear,with few NF-kB expressed in cytoplasm,the translocation rate was 46.546±5.163,which was significantly higher then the normal group(P＜0.05),in Xinmaitong group and DAng-I group,the majority of NF-kB expressed in cytoplasm,some expressed around the nuclear,the nuclear translocating rate were 31.124±5.582 and 33.977±3.925,it was significantly lower then model group(P＜0.05).It hints that protective effects of Xinmaitong-medicated serum and DAng-I against H₂O₂ induced CMCs' injury through reducing or inhibiting the nuclear translocating rate of NF-kB in the injured cardiomycytes.3.RT-PCR shows that:1)There is a certain amount of NF-kBmRNA in normal cardiomycytes;The expression of NF-kBmRNA in model group is significantly higher than control group(P＜0.05);and NF-kBmRNA in Xinnaitong and DAng-I group decreased significantly comparing with model group,(P＜0.05).There is no significance between Xinnaitong and DAng-I group in statistics.2)There is no expression of TNF-αmRNA in normal cardiomycytes;The expression of and TNF-αmRNA in model group is significantly higher than control group(P＜0.05);and TNF-αmRNA in Xinnaitong and DAng-I group decreased significantly comparing with model group(P＜0.05).There is no significance between Xinnaitong and DAng-I group in statistics.It hints that protective effects of Xinmaitong-medicated serum and DAng-I against H₂O₂ induced CMCs' injury through reducing or inhibiting the expressing of NF-kB and TNF-αin the injured cardiomycytes.Conclusions:(1)Peroxide damaged neonate rat cardiomycytes.(2)Xinmaitong serum could protect cardiomycytes by reducing the decline in cell viability and improving the condition of the cells that induced by peroxide. (3)The expression of NF-κB and TNF-αplayed an important role in H₂O₂-induced CMCs injury.(4)Xinmaitong serum could protect cardiocytes possibly by preventing or inhibiting the expression of NF-kB and TNF-αin the injured cardiomycytes.