Effect Of Iron On Iron-related Proteins Expression Of Primary Culture Rat Myocardial Cell

Iron is an essential element for all living organisms. It is a key functional component of oxygen transporting and storage molecules (eg, hemoglobin and myoglobin) and of many enzymes that catalyze the redox reactions required for the generation of energy (eg, cytochromes), the production of various metabolic intermediates, and for host defense (eg, nicotinamide adenine dinucleotide phosphate [NADPH] oxidase). And iron is also critical for cell proliferation, polarization, respiration and signal transduction. Excessive tissue iron causes widespread organ damage and induces many diseases including some cardiac and vascular diseases. Myocardial cells are highly polarization, so myocardial cells will not restore and regenerate with the irreversible pathological changes or damnifications. Myocardial cells in vitro possess some structural and functional characteristics as in vivo. Furthermore, we can directly observe the effects of some medicine on myocardial cells without the influence of nerves and body fluid. Therefore, myocardial cells are ideal experimental objects. Primary cultured myocardial cells can be easily controlled and repeated well, which provides an excellent model of studying the effect of single factor on myocardial cells.The primary cultured myocardial cells obtained from the ventricles of 1-day-old rats were made to do experiment. Different doses of FeSO4, ferric ammonium citrate (FAC) and desferrioxamine (DFO) were used to treat the primary cultured myocardial cells and then the activity, livability and the expression of iron-related proteins Ceruloplasmin (CP), Ferroportinl (FP1) and Hephaesin (HP) in myocardial cells were analyzed. The results of the experiments were analyzed with the software of STAT on computer. The results were as follows:1. Primary cultured myocardial cells:Cultured neonatal rat myocytes were prepared from 1-day-old Sprague-Dawley rats. Ventricular muscle was cut into pieces in lmm3 and dissociated with the use of 0.1% trypsin, then cells were suspended and cultured. According to the different adheret ability of different kinds of cells, the myocytes were purified. The chemical reagent was used to inhibit fibroblast proliferation to get purer cardiac myocytes. 24 hours later, the pseudopodium was stretched out to form network as a cluster or single layer of cells. The purity of myocardial cells is up to 96.94% with the analyses of immunohistochemical examination.2. Effect of DFO, FeSO4 and FAC on the myocardial cell livability:The livability of myocardial cells was observed with the method of trypan blue dyeing. Myocardial cells were seperatedly treated with DFO(0.5mmol/L, lmmol/L and 2mmol/L), FeSO4(0.2mmol/L, 0.4mmol/L and 0.8mmol/L) and FAC(20mg/L, 40mg/L and 80mg/L) for 24 hours, the change of livability of cells was not found.3. Effect of DFO, FeSO4 and FAC on the activity of myocardial cells:After incubation of the myocardial cells with DFO(0.5mmol/L, lmmol/L and 2mmol/L), FeSO4(0.2mmol/L, 0.4mmol/L and 0.8mmol/L) and FAC(20mg/L, 40mg/L and 80mg/L), the contractility of myocardial cells reduced and the myocardial cells degenerated in morphology (at least 80% of the cells developed hypercontractionthese).4. Effect of DFO, FeSO4 and FAC on CP, FP1 and Hp expression(1) Effect of DFO on CP, FP1 and Hp expression: The myocardial cells were separately incubated with 0.5mmol/L, lmmol/L and 2mmol/L DFO for 24 hours. CP expression obviously increases (P<0.05). FP1 expression did not decrease significantly (P>0.05) in 0.5mmol/L DFO, however, FP1 expression decreased significantly (P<0.05) in lmmol/L DFO and decreased slightly (P<0.01) in 2mmol/L DFO. There were no differences in the expression of FP1 between 0.5mmol/L and lmmol/L, lmmol/L and 2mmol/L DFO groups (P>0.05), whereas, there was obvious difference (P <0.05) between 0.5mmol/L and 2mmol/L.O.5mmol/L il H 1 mmol/L IS ffl it, 1 mmol/L& ^ 2mmol/Lï¿¡B ffi tfc 5E S (P>O.O5); 0.5mmol/Lï¿¡l %2mmol/Lilffi\t, ^tiJIIf (P<0.05)oHP expression did not increase significantly (P>0.05) in the incubation of 0.5mmol/L DFO for 24 hours. HP expression increased significantly after the incubation with 1 mmol/L DFO (P<0.05) and with 2mmol/L DFO(P<0.01). Nevertheless, there were no differences (P>0.05) in the expression of HP between 0.5mmol/L and 1 mmol/L, 1 mmol/L and 2mmol/L DFO groups. There were obvious differences (P <0.05) between 0.5mmol/L and(2) Effect of FeSC>4 on CP, FP1 and HP expression: Analyze the expression of CP, FP1 and HP in the myocardial cells with immunohistochemical method. There was apparent staining of CP, FP1 and HP on the myocardial cells. CP expression doesn't increase significantly (P>0.05) after incubating with 0.2mmol/L and 0.4mmol/L FeSO4 for 24 hours. CP expression increased significantly (P<0.05) after the incubation of 0.8mmol/L FeSO4. FP1 and HP expression increased dramatically (P<0.01) after the incubation of FeSO4. 0.4mmol/L ig.^ 0.8mmol/L iiffi tt, JcMM &M#(P>0.05); 0.2mmol/L M % 0.4mmol/L tiffitk, It ^'14 il if (P<0.05); 0.2mmol/L *i^ 0.8mmol/L Wfo bt. It#MM!:(P<0.01.).There was no difference in the expression of CP between 0.4mmol/L and 0.8mmol/L FeSO4 groups (P>0.05), but there were obvious differences between 0.2mmol/L and 0.4mmol/L(P<0.05), 0.2mmol/L and 0.8mmol/L(P<0.01). FeSO4 FP1 expression increased significantly incubation with 0.2mmol/L (P<0.05), 0.4mmol/L (P<0.01) and 0.8mmol/L (P<0.01) FeSO4. &%%tMWiWiffibk, 0.2mmol/L ï¿¡&-%' 0.4mmol/L iSMbt, ^EM^ttM^ (P>0.05); 0.4mmol/L tS^i 0.8mmol/L Sffitt. ^MMeM (P<0.05); 0.2mmol/L S -fe; 0.8mmol/LMffitL M^'fi^M^-CP^.Oljo While was no obvious difference (P>0,05) in the expression of FP1 between 0.2mmol/L and 0.4mmol/L group of FeSO4, there were obvious differences between 0.4mmol/L and 0.8mmol/L(P<0.05); 0.4mmol/L and 0.8mmol/L(P<0.01) FeSO4. HP expression increased significantly (P<0.05) after the incubation of 0.2mmol/L, 0.4mmol/L and 0.8mmol/L FeSO4 for 24hours. There were obvious differences (P<0.01) in the expression of HP between 0.2mmol/L and 0.4mmol/L, 0.2mmol/L and 0.8mmol/L FeSO4. However, there were no differences (P>0.05) between 0.4mmol/L and 0.8mmol/L. 0.2mmoI/L %LH 0.4mmol/L^ffitt, 0.2mmol/L m.^ 0.8mmol/L0.4mniol/L gl-% 0.8mmol/L(3) Effect of FAC on CP, FPl and HP expression: CP, FPl and HP staining in myocardial cells did not change obviously (P>0.05) after treated with 20mg/L FAC for 24 hours. However, CP, FPl and HP staining increased significantly in myocardial cells after the incubation with 40mg/L FAC (P<0.05) and 80mg/L FAC (P<0.0\) for 24 hours. It was found that FPl staining increased significantly between any two of these experimental groups.&^ftMMffi tfc. FPl&MFAC$c/f 51 -iSfConclusion1. The methodology of neonatal rat myocytes culture was established, we can use the primary myocardial cells to do experiment for our study.2. DFO (0.5mmol/L, lmmol/L and 2mmol/L), FeSO4 (0.2mmol/L, 0.4mmol/L and 0.8mmol/L) and FAC (20mg/L, 40mg/L and 80mg/L) have no obvious effect of dose-dependent relation with myocytes survival.3. The shrink frequency of myocardial cells reduced with DFO, FeSO4 and FAC treated notably and degenerate of myocardial cells in morphology. This result indicates the obvious drop of the myocardium cells' activation with the increasing density of these reagents.4. The primary myocardial cells were individually treated with DFO, FeSO4 and FAC. FPl expression decreased with the incubation of DFO, and the reverse with FeSO4 and FAC. This is compatible with documents reported about the expression of FPl in other organs. The FPl expression may be regulated by the mechanism of IRE-IRP.5. CP expression increases after the cells were treated with DFO. This is consistent...