Study Of The Myocardic Cell Apoptosis And The Expression Of Nuclear Factor-κb In Heart Failure Rat Induced By Homoeysteine

BackgroundHomoeysteine(Hcy) is a thiols-containing amino acid formed during the metabolism of methionine.Elevated level of serum homoeysteine are an independent risk factor for coronay heart disease. however,the precise mechanism of homoeysteine-associated cardiovascular disease is still unclear.To day,There is no research report that HCY induced chronic heart failure(CHF) of coronary heart disease.The pathogenesis of homoeysteine-associated heart failure is uncertain. Nuclear factor-κB is inflammatory transcription factor.The novol evidence suggests that nuclear factor-KB may play a important role in the development and progression of heart failure. The relationship between nuclear factor-KB and CHF is becoming research focus.The purpose is to establish Wistar rat model of chronic heart failure (CHF) by giving it different dose homocysteine food. The changes of myocardial ultramicrostructure by light microscope.Adopted terminal deoxynucleotidy transferase_mediated dUTP nick end labeling law, detected myocardial cell's apoptosis and detected nuclear factor-κB(NF-κB)by using electrophoretic mobility shift assay (EMSA).The purpose is to study the correlation between Hcy and heart failure, NF-κB and heart failure in rat.Materials and MethodsMajority healthy Wistar rats divided into three groups.Rat model of diversity level HCY by giving low,middle and high dose of HCY induced food to the groups for 12 weeks.1.The spirits,activities,eatings and color pattern were observed during the lab procedure.2.The Index of myocardium organization's morphology:fetched right amount of size of cardiac muscle of left ventricle,and fixed, wraped up and buried,HE dyed regularly,then were observed under light microscope.3. apoptosis of myocardial cell:cut the heart completely,the appropriate amount of the whole layer of cardiac muscle was fethed in the fixed position of the left ventricle's apex to prepare myocardium wax sample. Apoptotic cell in myocardium were determined by in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end Labeling(TUNEL), according to the directions of the kit of testing-apoptosis in situ.The nucleus submitting buffy grains or brown were judged for apoptotic cell. According to thedistribution situation of apoptosis,each slice shooted 5 positive visual fields. Take the proportion of apoptosis's cell with normal myocardial cell as apoptosis rate.Myocardial cell apoptotic index(AI)=(the count of myocardial apoptosis cell/the count of normal myocardial cell)×100%.Quantification of myocardical cell apototic index,the data were assayed by spss17.0 statistic software.4. Nuclear proteins are extracted from myocardial tissue, according to the directions of the kit of Nuclear and Cytoplasmic Extraction Reagents.NF-κB binding activity in the nuclears was measured by electrophoretic mobility shift assay (EMSA). Procedure for Electrophoretic mobility shift assay,according to the direction of the kit.①Prepare and pre-run gel.②Prepare and perform binding reactions.③Electrophorese binding reaction④Electrophoretic transfer of binding reactions to nylon membrane.⑤Cross-link transferred DNA to membrane.⑥Detect biotin-labeled DNA by chemiluminescence.⑦Expose membrane to X-ray film.Quantification the gray scale of strap, the data were assayed by spss statistic software.Result1. After 12 weeks, Wistar rat model of chronic heart failure was established.2. The Index of myocardium organization's morphology:After 12 weeks, myocardial ultrastructure of heart failure groups were seriously damaged compared with control group. Compared with high doze group, myocardial ultrastructure of the low doze group were mildly damaged. There was no difference in the Index of myocardial ultrastructure between ow doze group and middle doze group. 3. After 12 weeks, apoptotic cells were rarely found in the control group with the TUNEL assay,but the myocardic apototic index in heart failure rats was high compared with control group (2.90±0.89%) (P<0.01). Compared with high doze group (14.17±2.73%), the myocardic apototic index reduced significantly in the middle doze group (8.70±1.92%) (P<0.01), There was no difference in the myocardic apototic index between low doze group (7.35±1.26%) and middle doze group (8.70±1.92%) (P=0.139).4. The activation of NF-κB increased signifieantly in heart failure groups compared with control group (the gray scale of strap:272.64±26.51)(P<0.01). Compared with high doze group (the gray scale of strap:780.07±45.34),the activation of NF-κB decreased significantly in the middle doze group (the gray scale of strap:617.61±45.63) (P<0.01).Compared with middle doze group (the gray scale of strap:617.61±45.63), the activation of NF-κB decreased significantly in the low doze group (the gray scale of strap:485.00±36.07) (P<0.01)Conclusions1. Hyperhomocysteinemia can induce the generate and development of CHF in rats. There was close relationship between the severity degree of the cardiac function and the dose of Hcy,which were gave to Wistar rats.2. The activation of NF-κB increased signifieantly in rats of CHF. There was close relationship between the severity degree of the cardiac function and the activation of NF-κB.NF-κB might play an important role in the process of CHF.Decreased the activation of NF-κB might play an important myocardial preservation role in CHF.