Reactive Oxygen Species Mediating The Up-regulation Of Monocyte Chemoattractant Protein-1 In Rat Cardiac Remodeling As Well As Its Signal Pathway And Simvastatin Intervention

Backgroud Myocardial fibrosis is one of the important pathological changes in hypertensive left ventricular hypertrophy (LVH) and its pathogenesy is complicated. Recent study suggested inflammatory cell and fibroblast concomitance in cardiac fibrosis area in hypertensive rat, which indicates inflammation participates in the pathology process. Infiltration of inflammatory cells, particularly monocyte and macrophage, into cardiac interstitium is the key step in initiation of inflammation reaction, and chemokines were found to play a pivotal role in migration of inflammatory cells. Monocyte chemoattractant protein-1 (MCP-1) is one of CC chemoattractant protein and it has a potent chemoattractant for monocytes. Up-regulation of MCP-1 may be one of the mechanisms in pressure-overload rat Cardiac Remodeling. Recent study indicates reactive oxygen species (ROS) may play multiple roles in LVH and congestive heart failure, including regulation of cell growth and apoptosis, modulation of extracellular matrix, and activation of various protein kinases. But whether ROS participates in myocardial fibrosis and its relation with MCP-1 expression still remain unclear. ROS may active multiple mitogen-activated protein kinases (MAPKs),including P38-MAPK,JNK1/2 and ERK1/2, but through which ROS induces MCP-1 expression remains unidentified. Recently it has been identified that 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase inhibitor(statins) have multiple effects of anti-inflammatory, anti-oxidation stress, anti-fibrosis. But whether statins can inhibit oxidation stress and MCP-1 is still unidentified. Aim The study was designed to observe oxidative stress and MCP-1 expression and their associativity ,aim to investigate the role of P38-MAPK,JNK1/2 and ERK1/2 pathway in the modulation effects of ROS inducing MCP-1 expression, to study the possible mechanism of anti-inflammatory and antioxidation of simvastatin, elucidate the effects of oxidative stress and inflammation in hypertensive myocardial fibrosis, and provide new theoretical evidences and treatment approaches for hypertension and myocardial fibrosis.Methods and contents In this study, abdominal aortic coarctation(AC) rats were used as experiment models. On the other hand, spectrophotometer, enzyme linked immunosorbent assay(ELISA), reverse transcription polymerase chain reaction(RT-PCR),Western blot, immunohistochemistry and computerized image analysis were applied to identified : (1) the effects of AC on myocardial fibrosis;(2) the effects of AC on ROS, erythrocuprein(SOD), malonaldehyde(MDA), catalase(CAT)in pressure-overload induced cardiac remodeling;(3) the effects of AC on MCP-1 expression and macrophage infiltration;(4) the intervention effects of N-acetylcysteine(NAC) on oxidation stress indices and MCP-1 expression; (5) the activation of P38-MAPK,JNK1/2 and ERK1/2 during the modulation effects on MCP-1 expression induced by ROS;(6) the effects of simvastatin on ROS and MCP-1 expression in myocardium and reverse effects on cardiac remodeling. Results (1) After AC, mean arterial blood pressure (MBP) and LVW/BW ratio were increased gradually and were significantly greater than those in sham operation group(P<0.05).(2)AC caused significant interstitial fibrosis and perivascular fibrosis. Collagen volume fraction(CVF) of 2～4 week group were 3.28%±0.22%, 5.38%±0.17% and 7.09%±0.33%, which were significantly different compared to sham group(0.87%±0.19%)(P<0.01);CVF of 1-week group was 1.06%±0.17%, which was no significantly different compared to sham group(P>0.05). Perivascular collagen area(PVCA) of 2～4 week group were 15.03%±0.88%, 20.12%±1.01%, 24.99%±0.91%, which were significantly different compared to sham group(6.19%±0.41%)(P<0.01);PVCA of 1-week group was 9.02%±0.6%, which was no significantly different compared to sham group(P>0.05).(3) ROS increased as early as 1 week after AC(30.43±1.50 U/mg pro), peaked at 2 week(42.27±1.33 U/mg pro), returned slightly at 3 and 4 week(34.91±1.60,32.33±1.36 U/mg pro),and sustained at a higher level compared to the sham group(17.29±1.78 U/mg pro)(P<0.01). NAC treatment for 1～4 week significantly decreased ROS in myocardium(12.12±0.75, 11.76±0.67, 12.08±0.78 and 14.0±0.7 U/mg pro), compared to every corresponding AC group (P<0.01). (4) After AC, SOD of 1～4 week group in myocardium were 261.19±6.69, 185.86±3.59, 192.93±5.44 and 217.37±3.10 U/mg pro, which were obviously higher than every corresponding sham group(P<0.01). NAC treatment for 1～4 week significantly decreased SOD in myocardium compared to every corresponding AC group (P<0.01).(5) After AC ,MDA of 1～4 week group in myocardium were 3.90±0.19, 4.26±0.30, 4.12±0.15 and 4.07±0.18 nmol/mg pro, which were obviously higher than every corresponding sham group(P<0.01). MDA of NAC treatment for 1～4 week group were 1.79±0.20, 1.93±0.11, 1.88±0.18 and 1.80±0.16 nmol/mg pro, which were obviously lower than every corresponding AC group(P<0.01).(6) After AC ,CAT of 1～4 week group in myocardium were 7.36±0.86, 5.80±0.91, 6.49±0.63 and 6.70±0.78 U/mg pro, which were obviously lower than every corresponding sham group(P<0.01). CAT of NAC treatment for 1～4 week group were 13.42±1.12, 11.06±1.24, 11.83±0.67 and 12.22±0.90 U/mg pro, which were obviously higher than every corresponding AC group(P<0.01). (7) In AC rats, the number of ED-1 positive cells per section in myocardium increased significantly at 1 week after surgery (246±24), peaked at 2 week (582±33), and then declined slightly at 3 and 4 weeks (512±30,386±26),which were significantly different higher than that of every corresponding sham group(P<0.01). NAC treatment for 1～4 week significantly decreased ED-1 positive cells per section (95±9, 144±39, 126±15 and 106±26) compared to every corresponding AC group (P<0.01). (8) After AC, MCP-1 protein of 1～4 week group in myocardium were 66.95±4.53, 84.79±5.92, 43.10±3.11 and 36.23±3.18 pg/mg pro, which were obviously higher than every corresponding sham group(P<0.01). MCP-1 protein of NAC treatment for 1～4 week group were 21.34±3.02, 33.73±2.38, 22.3±2.76 and 19.35±2.82 pg/mg pro, which were obviously lower than every corresponding AC group(P<0.01). (9)The MCP-1mRNA levels of 1～4 week group (0.83±0.08, 0.57±0.07, 0.45±0.06 and 0.3±0.07) were significantly increased compared to every corresponding sham group (P<0.01). NAC treatment for 1～4 week significantly decreased MCP-1mRNA levels(0.34±0.08, 0.26±0.04, 0.22±0.04 and 0.21±0.04), compared to every corresponding AC group (P<0.01). (10) In AC rats, the phosphorylation of P38-MAPK was no significantly different compared to sham group. After NAC treatment, it was also no significantly different compared to AC group.(11) In AC rats, the phosphorylation of ERK1/2 was enhanced significantly by 53%compared to sham group(P<0.01); NAC treatment slightly decreased the phosphorylation of ERK1/2(150%±10% vs 153%±11%), but has no statistical significance( P>0.05). (12) In AC rats, the phosphorylation of JNK1/2 was enhanced significantly by 40%compared to sham group(P<0.01); NAC treatment significantly decreased the phosphorylation of JNK1/2(140%±10% vs 117%±11%) (P<0.01).(13) MBP of AC group(179.00±6.78 mmHg) was obviously higher than that of sham group (95.83±5.30 mmHg)(P<0.05); MBP of simvastatin group(176.33±5.74 mmHg) was obviously lower than that of AC group (P<0.05). LVW/BW of AC group(3.27±0.04 mg/g) was obviously higher than that of sham group(2.24±0.05 mg/g)(P<0.05); LVW/BW of simvastatin group(2.74±0.07 mg/g) was obviously lower than that of AC group (P<0.01).(14) In AC rats, CVF and PVCA were 7.09%±0.33%, 24.99%±0.91%, respectively, which were obviously higher than that of sham group(0.87%±0.19%,6.19%±0.41%) (P<0.01); simvastatin treatment significantly decreased CVF and PVCA(4.32%±0.23%,16.42%±0.58%) (P<0.01). (15) In AC rats, ROS(32.33±1.36 U/mg pro)was significantly increased compared to sham group(17.44±1.92 U/mg pro)(P<0.01); simvastatin treatment significantly decreased ROS(13.96±0.74 U/mg pro)(P<0.01).(16) In AC rats, The number of ED-1 positive cells (386±26) was obviously higher than that of sham group(39±11) (P<0.01); simvastatin treatment significantly decreased the number of ED-1 positive cells(196±16)(P<0.01).(17) In AC rats, MCP-1 protein(36.23±3.18 pg/mg pro) was significantly increased compared to sham group(13.34±1.88 pg/mg pro)(P<0.01); simvastatin treatment significantly decreased the level of MCP-1 protein (23.68±2.64 pg/mg pro)(P<0.01).(18)The mRNA expression levels of MCP-1 in sham group, AC group and simvastatin group were 0.18±0.05,0.3±0.07 and 0.21±0.04. It was much higher in AC group than that of sham group and it was much lower in simvastatin group than that of AC group(P<0.01).Conclusion (1) Hypertensive myocardial fibrosis model in rats can be established by AC. (2) In AC rats, up-regulation of ROS, MDA and down-regulation of SOD, CAT in myocardium were involved in myocardial fibrosis. (3) In AC rats, up-regulation of MCP-1mRNA and protein level in myocardium were relevant with myocardial fibrosis. (4)The intervention of NAC could significantly decreased oxidative stress and MCP-1mRNA and protein expression, which indicates MCP-1 was mediated by ROS. (5) JNK1/2 and ERK1/2 were involved in myocardial fibrosis, but ROS could up-regulate JNK1/2 activation, could not influence P38-MAPK and ERK1/2 activation.(6) Simvastatin could reverse cardiac remodeling by down-regulation of oxidative stress and MCP-1 expression.To summarise above all, oxidative stress and inflammation were involved in the process of myocardial fibrosis. JNK1/2 pathway may play a critical role in MCP-1 expression mediated by ROS. Simvastatin could reverse cardiac remodeling by down-regulation of ROS and MCP-1 expression.