The Relationship Between The Expression Of NF-κB In Hypothalamic Paraventricular Nucleus And The Plasma Aldosterone Myocardial Cell Apoptosis In Chronic Heart Failure Rats

Background and purposeChronic heart failure (CHF) is the clinical syndrome, participated by a variety ofcytokines, which was characterized by excessive activation of neuroendocrine hormonesand ventricular remodeling, and the end stage of various heart diseases.With agingpopulations and increasing in incidence of cardiovascular disease, incidence rate of heartfailure is increasing year by year. Hypothalamic paraventricular nucleus (PVN) as animportant nuclei of visceral and neuroendocrine regulation is the most important centralsites and integate parts in regulation of sympathetic efferent activity. The studiesconfirmed that the changes of neurochemical factors of PVN in heart failure such as ROS(reactive oxygen species ROS), inflammatory cytokines (proinflammatory cytokines byPIC), may be important factors which spured the increasement of the sympatheticnervous system outflow in heart failure.The nuclear factor κB (NF-κB) widely distributein body tissues and cells, involved in the transcriptional regulation of gene of numerousfactors in the process of inflammation and immune response. Recent studies found that NF-κB in the hypothalamic paraventricular nucleus,an important cardiovascular regulationcenter in rats with heart failurer was significantly increased.NF-κB mediates the pivotalrole of circulating cytokines in chronic heart failure through a series of signal transductionpathways,and induces inflammatory response and apoptosis. NF-κB plays an importantrole in heart failure pathogenesis.Rise of aldosterone is an independent risk factor ofheart failure, mainly through myocardial aldosterone receptor-mediated myocardialremodeling and cardiac hypertrophy.In chronic heart failure high levels of aldosterone inthe cycle can also effct ventricular reconstruction through the central effect, and thusinvolved in the occurrence and development.In this study,CHF was induced by myocardial infarction through ligation of left anterior descending coronary artery, The rats weretreatedwith PDTC or EPL in drinking water,and then we observed the changes of plasmaaldosterone levels and expression of NF-κB in the hypothalamic paraventricular nucleusInfluenced on process of the heart failure, explored the interaction and the regulation ofmyocardial cell apoptosis, We can get a new idea and new way in heart failure central cureMaterials and Methods1. Model preparationAdult male Sprague-Dawley (SD) rats,250-300g, were purchased from animalexperimental center, Shandong University of Traditional Chinese Medicine. We randomlydivided the rats into2groups: CHF group and SHAM group.CHF was induced bymyocardial infarction through ligation of left anterior descending coronary artery. The leftanterior descending coronary artery was only exposed without ligation in SHAM rats.Thenwe randomly divided the CHF rats into3groups: untreated rats (CHF group, n=20), ratstreated with PDTC (PDTC group, n=20), and rats treated with EPL (EPL group, n=20).SHAM group was used as experimental control(SHAM group, n=10). In treatmentgroups,the rats were treated with NF-κB inhibitor pyrrolidine dithiocarbamate(PDTC)150mg/kg or mineralocorticoid receptor inhibitor eplerenone (EPL)30mg/kg in drinkingwater per day for6weeks.2. The judgment of the degree in heart failureLeft ventricular weight/body weight ratio (LVW/BW (mg/g)), right ventricleweight/body weight ratio (RVW/BW (mg/g)) and lung weight/body weight ratio(LW/BW (mg/g,)) were measured to evaluate cardiac function and degree of heartfailure after6weeks.3. The determination of plasma aldosterone(ALD) level by radioimmunoassayThe blood samples from artery in each group of rats after completing the pressurecurve recording, were taken5ml, mixed into the EDTA-tube bathing in the ice-water, wellmix. Separate the plasma by3000rpm/min centrifuge for15min in4℃, place thesupernatant in the tube and messure the plasma aldosterone level by radioimmunoassayaccordancing with the instructions.4. The determination the cardiac cell apoptosis by TUNEL stainingTake full-thickness myocardial tissue in left ventricular apical, prepare myocardialparaffin-embedded specimens according to the routine pathological histology,using theterminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL method) thecardiac cell apoptosis was detected. Steps accord with the instructions of in situ apoptosis detection kit. Positive staining showed that the nuclei are in brown color. Five positivevisual fields were photographed in each section. The apoptosis index (AI) was calculatedas ratio of apoptosis cell number over total number of cells. The myocardial cell apoptosisindex（AI%）=(the count of myocardial apoptosis cell/the count of myocardial normal cell)×100%.Quantification of the myocardial cell apoptosis index,the data was assayed bySPSS13.0statistic software.5. The expression of NF-κB protein in the PVN was determined by western blotProtein was extracted from the PVN tissue and separated by denaturing polyacryla-mide gel (sample volume:20μg total protein). Then protein was transferred to membraneby using electric transfer membrane method. The membrane was incubated with mouseanti-rabbit NF-κB p65(1:200) overnight incubation at4℃and then conjugated antibody(horseradish peroxidase-labeled goat anti-rabbit IgG,1:2000) for2h at37℃. Protein bandswere observed by using the UVP condensate gel image scanning system and wereexpressed as the ratio of the absorbance value in each group to the value of the controlgroup. Protein levels were expressed as its ratio to β-actin optical density. Quantification ofthe gray scale of strap,the data was assayed by SPSS13.0statistic software.Result1. The ratios of right ventricular weight/body weight、left ventricular weight/bodyweight and lung wet weight/body weight were significantly higher in CHF rats than insham rats, there were statistical differences(P<0.05). These two ratios were significantlyreduced in rats treated with PTDC or EPL.The reduction of these two ratios in EPL-treatedrats were greater than that in PDTC-treated rats, there were statistical differences (P<0.05).The heart failure model of experimental rat was successfully created.2. Positive apoptotic cells showed that the nuclei are in brown color. The myocardialcell apoptosis index in CHF group(36.82±6.34%）was significantly higher than SHAMgruop （9.68±8.40%）(P <0.01). Compared with CHF group(36.82±6.34%）,the apoptosisindex in EPL gruop (24.21±6.44%）was significantly lower (P <0.01), and PDTC gruopalso had lower the myocardial cell apoptosis index (P<0.05). However the apoptosis indexin EPL group was significantly lower than in PDTC group (P<0.05).Those results promptthe inhibition role of EPL in cardiomyocyte apoptosis is larger than PDTC, EPL has betterefficacy than PDTC in the inhibition of cardiomyocyte apoptosis and improving thesymptoms of heart failure.3. The plasma aldosterone level in CHF group (0.30±0.07) was significantly higherthan SHAM group (0.14±0.07)(P<0.01). EPL group (0.20±0.12) significantly reduced plasma aldosterone level compared with CHF group (P<0.01), while PTDC group(0.28±0.12) failed to change aldosterone level (P>0.05). EPL group was significantlylower than in PTDC group (P <0.01), but did not differ from that in SHAM group (P>0.05).4. Westem blot results show group EPL(0.54±0.14), group PDTC(0.70±0.11), groupCHF (0.83±0.31)within expression levels of NF-κB in PVN were increased in turncompared with group SHAM(0.27±0.10), the differences is highly significant (P <0.01);the difference was highly significant between group CHF and group EPL,(P <0.01), andthe difference is significant between group CHF and groupPDTC (P <0.05);, the differenceis significant between group EPL and group PDTC (P <0.05).Conclusion1.The plasma aldosterone level in CHF rats was significantly higher than sham rats2. The expression level of NF-κBp65in PVN was significantly higher in CHF ratsthan in sham rats,which is closely related to the severity of heart failure.NF-κB may playan important role in the process of heart failure, inhibiting its activity may become a newtarget of treatment in heart failure.3. There are correlation between the plasma aldosterone level and expression level ofNF-κBp65in PVN in CHF rats.In heart failure a high level plasma aldosterone can activatethe NF-κB in PVN, and activation of NF-κB in PVN can mediate myocardial cellapoptosis, impact the development of heart failure, so inhibition of plasma aldosterone orcentral NF-κB levels can delay the development of heart failure.