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Keywords:

  • atorvastatin;
  • fibrinogen-like protein 2;
  • myocardial ischemia/reperfusion;
  • no-reflow;
  • tumor necrosis factor-α

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Result
  6. Conclusion
  7. Declaration of Conflicting Interests
  8. Acknowledgement
  9. References

Atorvastatin is not only an antilipemic but also used as an anti-inflammatory medicine in heart disease. Our working hypothesis was that atorvastatin preconditioning could improve the forward blood flow in the no-reflow rats associated with inflammation. We investigated that two doses of atorvastatin preconditioning (20 and 5 mg/kg/day) could alleviate deterioration of early cardiac diastolic function in rats with inflammation detected by echocardiography and haemodynamics. This benefit was obtained from the effect of atorvastatin preconditioning on improving forward blood flow and preserving the infarct cardiomyocytes, which was estimated by Thioflavin S and TTC staining in rats with myocardial ischemia/reperfusion. Subsequently, the improving of forward blood flow was ascribed to reduction of microthrombus in microvascular and myocardial fibrosis observed by MSB and Masson's trichrome staining with atorvastatin preconditioning. Ultimately, we found that atorvastatin preconditioning could reduce inflammation factor, such as tumor necrosis factor-α and fibrinogen-like protein 2, both in myocardial and in mononuclear cells, which probably attribute to microcirculation dysfunction in no-reflow rats detected by immunohistochemistry staining, western blot, and ELISA detection, respectively. In conclusion, atorvastatin preconditioning could alleviate deterioration of early cardiac diastolic function and improve the forward blood flow in the no-reflow rats attributing to reduction of TNF-α and fgl-2 expression.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Result
  6. Conclusion
  7. Declaration of Conflicting Interests
  8. Acknowledgement
  9. References

A large number of acute myocardial infarction patients in clinical practice who received reperfusion therapy still suffered severe myocardial ischemia owing to various reasons, such as myocardial interstitial edema, microvascular spasm, endothelial cell blebbing, neutrophil infiltration, and micro-thromboembolism [1-3]. No matter which potential mechanism there was, it was clearly observed by coronary angiography that no or weak blood flow in distal vessels after the successful interventional operation of obstructive arteries in portion of patients graded with TIMI 0 or 1[4, 5]. This phenomenon was defined as no-reflow, which means inadequate myocardial perfusion though a given segment of the coronary circulation without angiographic evidence of mechanical vessel obstruction [6].

Many researches had indicated that no-reflow was closely related to inflammation effect [5, 7, 8]. It could induce myocardial interstitial edema, microvascular spasm, endothelial cell blebbing, micro-thromboembolism, and myocardial fibrosis. Subsequently, it would attribute to microvascular dysfunction in heart.

Atorvastatin is an inhibitor of the 3-hydroxy-3-methylglutaryl-CoA reductase. Its main effect is lowering the levels of cholesterol and triglyceride, meanwhile the effect of atorvastatin on resisting inflammation has also be generally manifested [9-11]. So we sought to determine whether atorvastatin improves the forward blood in the no-reflow rat associated with inflammation, and what is the effect of atorvastatin on cardiac function in early phase of myocardial ischemia/reperfusion.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Result
  6. Conclusion
  7. Declaration of Conflicting Interests
  8. Acknowledgement
  9. References

Animals and drug treatment

Adult male Sprague–Dawley rats (body weight: 240–260 g) were obtained from the animal center of Medical College of Xi'an Jiaotong University. Throughout the experiment, animals were given tap water and rodent diet ad libitum and treated according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health in 1996 and Laboratory Animals Regulation Administration published by China National Science and Technology Commission in 2004.

Rats were divided into the following groups: normal control without any treatment (group NC). Animals with thoracotomy without left anterior descending coronary artery (LAD) occlusion and treatment (group sham). LAD of animals subjected to 45 min of occlusion followed by reperfusion for 3 days (group I/R3d). Testing group was administered by different doses of atorvastatin for 7 days before cardiac operation and 3 days after cardiac operation (group H: high dose of atorvastatin, 20 mg/kg/day and group M: moderate dose of atorvastatin, 5 mg/kg/day). Atorvastatin (Pfizer Ireland Pharmaceuticals Ltd, Cork, Ireland) was administered orally by gavage once per day. A minimum of six rats were used in each groups per time period in a single method. There were totally 108 rats (90 total samples) in this research.

Myocardial ischemia/reperfusion model

The construction of model was as described previously [12]. After anesthetized by 2% pentobarbital (2 mL/kg) intraperitoneal injection, rats were given positive-pressure ventilation by small animal respirator (Harvard Apparatus, Millis, MA, USA) after tracheal intubation. Then, rats were opened left thorax for heart exposed. In group I/R3D, H, and M, LAD of rats was ligated for consecutive 45 min before reperfusion. Following chest closed layer by layer, benzylpenicillin sodium (400 000 U/kg) was injected immediately and in next 2 days. Successful myocardial ischemia/reperfusion (MI/R) was confirmed not only by visual inspection of left ventricle color but also change of electrocardiogram.

As we know, animal models of mechanical coronary artery ligation and reperfusion are suitable for researching the no-reflow phenomenon [12-14].

Echocardiography evaluation

From both the short- and the long-axis views, Simpson's method and M-mode Teichholz method of echocardiography were performed by a 10S probe connected with VIVID7 echocardiogram machine (GE medical, Milwaukee, WI, USA). Inherent analysis software captured digital images in three consecutive cardiac cycles, and average measurements were made at the same time on the 3rd day after operation.

Haemodynamics analysis

A sidearm of the aortic cannula was inserted into right carotid artery and connected with a pressure transducer (Millar Instruments, Houston, TX, USA), while BL-420F organism function experiment system was used to amplify the signal. Through the ascending aorta, the catheter was inserted into the left ventricle. Haemodynamics analysis was on the 3rd day after surgery.

Serum TNF-α measurement

One-millilitre endocardial blood samples were collected and centrifuged for 20 min at 3000 g per min. The serum was abstracted out and frozen at −80 °C until assayed. A TNF-α sandwich ELISA kit (NeoBiosciences, Shanghai, China) was used for quantifying the serum level of TNF-α.

Myocardial staining

Myocardial ischemic risk area and no-reflow area were, respectively, observed as previously described [14]. Thioflavin S is a fluorescent vial dye for endothelium with flow. By Thioflavin S staining, no-reflow phenomenon can be observed and the no-reflow size calculated in rats with acute MI/R. four per cent Thioflavin S (2 mL/kg) was injected into left auricle firstly. Then, 1% Evans Blue (1 mL) was injected at the same point after the coronary artery was re-ligated at the same point. Then, the whole heart was cut off and transected parallel to the atrioventricular groove at the center of the infarct area in 5 min. There were at least six rats in each group and three slices in each rat.

Histopathology staining and immunohistochemistry staining

Transverse sections were cut in each heart. Each sample was tested by three kinds of histopathology stainings and immunohistochemistry staining. Martius Scarlet Blue (MSB) staining and Masson's trichrome staining were used for identification of cardiac microthrombi. Myocardial collagenous fiber was detected by Masson's trichrome staining, and myocardial pathology was observed by hematoxylin and eosin (HE) staining. Cardiac fibrinogen-like protein 2(fgl-2) and TNF-α were detected and located by immunohistochemistry staining using with monoclonal mouse anti-rat fgl-2 (WH0010875M1, Sigma, St Louis, MO, USA) and polyclonal rabbit anti-rat TNF-α (500-P72; PeproTech, Rocky Hill, NJ, USA) following the instruction of the manufacturer.

Collagen volume fraction (CVF) and the ratio of perivascular collagen area to luminal area (lumen diameter < 150 μm) (PVCA/LA) were analyzed for collagen content by quantitative morphometry with automated image analysis (Media Cybernetics, Inc., Silver Spring, MD, USA) [15]. CVF was calculated in 4 fields of microscope in anterior wall of left heart in each Masson staining transverse slice. Those detected fields for CVF were chosen remote away from perivascular collagen, cicatricial tissue, and papillary muscles. And each group was detected 6 slices at least. PVCA/LA was calculated as previously [16, 17].

The heart had been washed out before further processing for ensuring that thrombi might not have formed during preparation.

Western blot analysis

The western blot analysis was as described previously [18]. Tissues of the anterior wall of left heart below the position of left anterior descending coronary artery ligated were preserved. The western blot analysis was as previously (mm). Equal amounts of protein of each sample (4-mL whole blood or 100-g tissue) were separated by 10% SDS-PAGE. Protein samples were homogenized in a buffer (Sigma, USA). Before blocked for 2 h in 5% defatting milk, the protein was transferred to nitrocellulose membranes. Then, they were responded with monoclonal mouse anti-rat fgl-2 (ab77642; Abcam, Cambridge, MA, USA) diluted 1 : 1000 and polyclonal rabbit anti-rat TNF-α (500-P72; PeproTech) diluted 1 : 600 overnight at 4 °C, and then reacting with the corresponding secondary antibody.

Statistical analyses

All values are expressed as mean ± SD and analyzed by SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). A value of P < 0.05 was considered as statistically significant differences. All statistics were analyzed by anova analysis.

Result

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Result
  6. Conclusion
  7. Declaration of Conflicting Interests
  8. Acknowledgement
  9. References

Atorvastatin improved the diastolic dysfunction in no-reflow rats

Echocardiography and haemodynamics were used to examine whether the atorvastatin improved cardiac function in a short period of rats with acute ischemia/reperfusion. There was no significant change about the parameter of LVIDs, EF, FS, left ventricular systolic pressure, left ventricular systolic pressure, or +dp/dtmax, which represented cardiac systolic function in the sham, the acute MI/R group on 3 day, group H, and group M (P > 0.05) (Figure 1). However, in equal heart rates, the −dp/dtmax of the acute MI/R group on 3d was significantly lower than that of the sham group (Group I/R3d: Group sham = (−1.56 ± 0.44): (−2.87 ± 0.66), n = 6, P < 0.05), while different doses of atorvastatin could benefit the diastolic function in no-reflow rats of MI/R (Group H: Group I/R3d = (−2.13 ± 0.93):(−1.56 ± 0.44), n = 6, P < 0.05; Group M: Group I/R3d = (−1.96 ± 0.88):(−1.56 ± 0.44), n = 6, P < 0.05) (Figure 2).

image

Figure 1. The effect of atorvastatin on the cardiac systolic function in no-reflow rats. There was no significant difference about cardiac systolic function, respectively, by echocardiography and by haemodynamic monitoring between each group (P > 0.05, n = 6). (a) Cardiac systolic function by echocardiography. LVIDs, systole left ventricular internal dimension. EF, left ventricular ejection fraction. FS, left ventricular fractional shortening. (b) Cardiac systolic function by haemodynamic monitoring. LVSP, left ventricular systolic pressure. LVEDP, left ventricular end-diastolic pressure. +dp/dtmax: the maximum change rate of left ventricular pressure rise. NC: normal control group; I/R3d, acute myocardial ischemia/reperfusion for 3 days; H, high dose of atorvastatin group; M, moderate dose of atorvastatin group.

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image

Figure 2. Atorvastatin improved the cardiac diastolic function in no-reflow rats. (a) Heart rates. There was no significant difference between each group (P > 0.05, n = 6). (b) −dp/dtmax: the maximum change rate of left ventricular pressure fall. The −dp/dtmax of the acute MI/R group on 3d was significantly lower than that of the sham group on 3d (*P < 0.05, n = 6), and different doses of atorvastatin could obviously improve the cardiac diastolic function in no-reflow rats (# vs. Group I/R3d P < 0.05, n = 6; & vs. Group I/R3d, P < 0.05, n = 6). And there was no significant different between the different doses of atorvastatin (P > 0.05, n = 6). NC: normal control group; I/R3d, acute myocardial ischemia/reperfusion for 3 days; H, high dose of atorvastatin group; M, moderate dose of atorvastatin group.

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Atorvastatin improves the forward blood flow and preserves the infarct cardiomyocytes in no-reflow rats

We sought the reason of cardiac dysfunction by myocardial staining. By Evans Blue staining, myocardial ischemic risk area appeared red in contrast with blue-staining nonischemic risk myocardiums. Thioflavin S is a fluorescent vial dye for endothelium with flow. By Thioflavin S staining, no-reflow area appeared dark under ultraviolet ray, whereas fluorescence-staining area represented good blood supplied myocardiums. By TTC staining, infarct area appeared khaki, whereas the viable myocardium was red. Myocardial ischemic risk size was described by the percentage of myocardial ischemic area to the whole heart section area (AR/WH%). No-reflow size was described by the percentage of no-reflow area to the whole heart section area (AN/WH%). Infarct size was described by the percentage of infarct area to the whole heart section area (AI/WH%). The values of AR/WH% were no obvious difference in group I/R3D, group A, and group B (31.46 ± 2.28 (%), 33.75 ± 3.84 (%), 32.85 ± 3.76 (%)). Equal values of AR/WH% standardize our model and the size of each group's acute ischemia/reperfusion area. A step further, we found that atorvastatin could improve the forward blood flow in no-reflow rats significantly (Group H: Group I/R3d = (16.27 ± 4.31): (25.28 ±  2.01), n = 6, P < 0.05; Group M: Group I/R3d =  (18.85 ± 4.06): (25.28 ± 2.01), n = 6, P < 0.05). But high dose of atorvastatin had no significant effect on forward blood flow comparing to what low dose did (Group H: Group M = (16.27 ± 4.31): (18.85 ± 4.06), P > 0.05, n = 6) (Figure 3).

image

Figure 3. Atorvastatin improved the forward blood flow in no-reflow rats. The no-reflow phenomenon happened in MI/R rats. The similar values of AR/WH% (Evans Blue staining) excluded artificial error in model construction. By Thioflavin S staining and TTC staining, we detected that atorvastatin attuned the size of myocardial no-reflow and reduces the infarct size significantly (** vs. Group I/R3d, n = 6, P < 0.05; * vs. Group I/R3d n = 6, P < 0.05), and high dose of atorvastatin had no more significant effect on forward blood flow than what low dose did (P > 0.05, n = 6). I/R3d, acute myocardial ischemia/reperfusion for 3 days; H, high dose of atorvastatin group; M, moderate dose of atorvastatin group.

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Also, atorvastatin preconditioning could reduce the infarct size and preserve more myocardial cells after MI/R (Group H: Group I/R3d = (15.21 ± 0.78): (21.34 ± 3.97), n = 6, P < 0.05; Group M: Group I/R3d = (17.42 ± 0.26): (21.34 ± 3.97), n = 6, P < 0.0 5). However, there was no distinct evidence on dose-dependent effect in early time (Group H: Group M = (15.21 ± 0.78): (17.42 ± 0.26), n = 6, P < 0.05) (Figure 3).

The effect of atorvastatin on cardiac pathology in no-reflow rats

In cardiac microvessels of rats with acute MI/R, microthrombus-like substances were observed and further defined by MSB staining to be fibrin thrombi (Figure 4). HE staining slices of the ischemia/reperfusion section indicated myocardial pathological alterations. And myocardial collagenous fiber was identified by MSB and Masson's trichrome staining. Administrating different doses of atorvastatin previously, we detected that the cardial construction disorder had alleviated, the denatured and necrosis myocardial had decreased, and that the microthrombus-like substances had also reduced in no-reflow rats at the ischemia/reperfusion areas (Figure 4).

image

Figure 4. The change of cardiac pathology in rats. Cardiac microthrombosis was observed by Mason, MSB (Scarlet, Blue methocl) and hematoxylin and eosin (HE) staining (×400). Cardiac microthrombosis was identified by MSB staining. Both perivascular collagen and extracellular matrix collagen fiber were clearly showed by Masson staining. NC: normal control group; I/R3d, acute myocardial ischemia/reperfusion for 3 days; H, high dose of atorvastatin group; M, moderate dose of atorvastatin group.

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The CVF and the ratio of PVCA/LA were two excellent parameters to measure the extent of myocardial fibrosis (Figure 5). In sham group, there was less collagen fiber in extracellular matrix and lower PVCA/LA compared to I/R3d group. But when rats suffered ischemia/reperfusion, there were more aggregation of collagen in extracellular matrix, nearly four times to that of sham group (28.35 ± 3.12 to 6.18 ± 1.26, P < 0.05, n = 6). Similarly, the PVCA/LA showed significant change in no-reflow rats, nearly 3 times to that of sham group (2.15 ± 0.48 to 0.68 ± 0.26, P < 0.05, n = 6). Atorvastatin could reduce the CVF and the PVCA/LA in rats with no-reflow obviously. With atorvastatin precondition in high and moderate doses of atorvastatin, the CVF remarkably declined to 13.14 ± 2.54 (%) and 16.77 ± 2.88 (%), respectively, (P < 0.05, n = 6). And there was lower PVCA/LA in rats with precondition of atorvastatin (Group H: Group I/R3d = (1.24 ± 0.17): (2.15 ± 0.48), n = 6, P < 0.05; Group M: Group I/R3d = (1.43 ± 0.22): (2.15 ± 0.48), n = 4, P < 0.05).

image

Figure 5. Atorvastatin reduces the collagen volume fraction (CVF) and the ratio of perivascular collagen area to luminal area (PVCA/LA) in no-reflow rats. Both CVF and PVCA/LA were significantly higher in anterior wall of left heart in no-reflow rats (*P < 0.05, n = 6) compared to those of sham group. Different doses of atorvastatin could decline CVF and PVCA/LA obviously in rats with no-reflow compared to those of rats with acute ischemia/reperfusion for 3 days (#P < 0.05, n = 6; & P < 0.05, n = 6). But there was no clearly distinctive effect of two doses of atorvastatin on the CVF and PVCA/LA (P > 0.05, n = 6). NC: normal control group; I/R3d, acute myocardial ischemia/reperfusion for 3 days; H, high dose of atorvastatin group; M, moderate dose of atorvastatin group.

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Atorvastatin declined the expression of TNF-α in no-reflow rats

There was some expression of soluble TNF-α in normal tissue and serum (Figure 6). If rat suffers injury, even in sham group, the expression of TNF-α should increase, but not significantly. When rats underwent acute myocardial ischemia/reperfusion, the soluble TNF-α was significant higher expression than that of sham or normal rats both in myocardial tissue (Group I/R3d: Group Sham = 42.36 ± 4.43: 4.37 ± 1.34, n = 4, P < 0.05) and in serum (Group I/R3d: Group Sham = 66.32 ±  15.98 (pg/mL): 20.35 ± 5.19(pg/mL), n = 6, P < 0.05). With atorvastatin precondition, the expression of rat cardiac TNF-α was less at the third day after I/R (Group H/Group I = 28.74 ± 2.32/42.36 ± 4.43, n = 4, P < 0.05; Group M/Group I = 30.42 ± 2.78/42.36 ± 4.43, n = 4, P < 0.05). Also, we found different doses of atorvastatin can significantly reduce the serum concentrations of TNF-α (Group H: Group I/R3d = 46.38 ± 9.71(pg/mL): 66.32 ± 15.98(pg/mL), n = 6, P < 0.05; Group M: Group I/R3d = 48.42 ±  10.19(pg/mL): 66.32 ± 15.98(pg/mL), n = 6, P < 0.05 ) (Figure 6).

image

Figure 6. Atorvastatin reduced both cardiac expression and serum level of tumor necrosis factor-α (TNF-α) in no-reflow rats. (a) Cardiac expression of TNF-α was detected in rats with no-reflow by immunohistochemistry (×400). (b) Atorvastatin reduced cardiac expression of TNF-α was detected in rats with no-reflow by western blot. (c) Atorvastatin declined serum level of TNF-α in rats with no-reflow. Cardiac expression and serum level of TNF-α were significantly higher in no-reflow rats (*P < 0.05, n = 6) compared to those of sham group. Two doses of atorvastatin could decline both of them significantly in rats with no-reflow compared to those of rats with acute ischemia/reperfusion for 3 days (#P < 0.05, n = 6; & P < 0.05, n = 6). But there was no discernible change of different doses of atorvastatin on the expression of TNF-α both in cardiomyocytes and serum (P > 0.05, n = 6). NC: normal control group; I/R3d, acute myocardial ischemia/reperfusion for 3 days; H, high dose of atorvastatin group; M, moderate dose of atorvastatin group.

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Atorvastatin reduced the expression of fgl-2 in no-reflow rats

In the normal rat, we had not found the obvious expression of fibrinogen-like protein 2 (fgl-2) by immunohistochemistry and western blot analysis. But in the MI/R rats, the fgl-2 was at microvascular walls of the ischemia/reflow areas (Group I/R3d: Group Sham = (5.09 ± 0.35): (0.11 ± 0.0068), n = 4, P < 0.05) (Figure 7). The expression of fgl-2 was lower when atorvastatin was prior administrated (Group H: Group I/R3d = (3.87 ± 0.64): (5.09 ± 0.35), n = 4, P < 0.05; Group M: Group I/R3d = (4.26 ± 0.38): (5.09 ± 0.35), n = 4, P < 0.05). Meanwhile, we also detected the abundant fgl-2 in mononuclear cells of no-reflow rats (Group I/R3D: Group Sham = (115.7 ± 3.97):(1.15 ± 0.83), n = 4, P < 0.05). The expression of fgl-2 was significantly declined after atorvastatin administration (Group H: Group I/R3d = (64.2 ±  3.54):(115.7 ± 3.97), n = 4, P < 0.05; Group M: Group I/R3d = (78.3 ± 4.76): (115.7 ± 3.97), n = 4, P < 0.05) (Figure 7). The high dose of atorvastatin had not obviously decreased the expression of fgl-2 both in cardial and mononuclear cells (P > 0.05).

image

Figure 7. Atorvastatin reduced the expression of fgl2 in cardiomyocytes and mononuclear cells of no-reflow rats. (a) Cardiac expression of fgl2 was detected in rats with no-reflow by immunohistochemistry (×400). It was located at cardiac microvessels (diameter < 150 μm). (b) Atorvastatin reduced cardiac expression of fgl2 in rats with no-reflow by western blot. (c) Atorvastatin declined the expression of fgl2 in mononuclear cells of rats with no-reflow. The expression of fgl2 in cardiomyocytes and mononuclear cells was significantly higher in no-reflow rats (*P < 0.05, n = 6) compared to those of sham group. High and moderate doses of atorvastatin could decline both of them significantly in rats with no-reflow compared to those of rats with acute ischemia/reperfusion for 3 days (#P < 0.05, n = 6; & P < 0.05, n = 6). But there was no clearly change effect of different doses of atorvastatin on the expression of fgl2 in cardiomyocytes and peripheral mononuclear cells (P > 0.05, n = 6). NC: normal control group; I/R3d, acute myocardial ischemia/reperfusion for 3 days; H, high dose of atorvastatin group; M, moderate dose of atorvastatin group.

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Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Result
  6. Conclusion
  7. Declaration of Conflicting Interests
  8. Acknowledgement
  9. References

Our major point was the effect of atorvastatin precondition on the microcirculation of no-reflow rat in this test. In our prior study, we had demonstrated that microthrombosis was involved in cardiac microvascular dysfunction or disturbance of rats with acute myocardial ischemia/reperfusion and no-reflow size was markedly or significantly larger in the acute MI/R group on 3 days [19]. A growing number of researches indicated that an increased myocardial TNF-α concentration leading to cardiac microembolization and cardiac diseases, although there is low expression of TNF-α in tissue and circulation of health individual [20, 21, 8]. Subsequent studies showed that preischemic with TNF-α antibody could reduce infarct size in animals after ischemia/reperfusion [22, 23]. Also, preadministration with atorvastatin improved the forward blood flow in rat with acute MI/R owing to the effect of atorvastatin on reducing the expression of TNF-α in our study.

Furthermore, we found the atorvastatin could reduce the CVF and the PVCA/LA in no-reflow rats, both of which were index responding the myocardial fibrosis. It had been given evidences by many researchers that the perivascular collagen had been accounted for abnormal coronary vasodilator reserve and supply decrease of oxygen and nutrients to the myocardium. Meanwhile, an increasing accumulation of collagen fibrosis in extracellular matrix of the impaired left ventricle had a response for abnormal myocardial stiffness and cardiac diastole dysfunction [17, 24]. As far as known, TNF-α over-expression resulted to matrix metalloproteinase activation, collagen degradation intramyocardial inflammation, and fibrosis in rats [25-27]. Thus, atorvastatin as an anti-TNF-α agent should attenuate myocardial fibrosis, increase the supply of blood flow, and reverse cardiac diastole dysfunction in rats after acute myocardial ischemia/reperfusion.

No matter how complex was the mechanism of no-reflow phenomenon, but its root was that myocardial hypoperfusion resulted of microcirculation dysfunction results to myocardial infarct and cardial dysfunction [28]. In this research, we approved that atorvastatin reduced the expression of TNF-α preserving the no-reflow area of acute MI/R in no-reflow rats, both from anti-microthrombosis and from anti-myocardial-fibrosis. However, there was no significant difference between two doses of atorvastatin on each parameter. Patients with coronary heart disease are usually treated with statins after admission of ESC Guidelines. The dose to achieve maximal benefit appears high (e.g., 80 mg of atorvastatin) [29]. Furthermore, according to the result of the Atorvastatin for Reduction of Myocardial Damage during Angioplasty-Acute Coronary Syndromes (ARMYDA-ACS) randomized trial, even short-term pretreatment with atorvastatin (80 mg 12 h before PCI, with a further 40-mg preprocedure dose) may improve outcomes in patients with ACS undergoing early invasive strategy [30]. These data suggest that higher doses of atorvastatin may be able to improve clinical outcomes of CHD patients, even in a portion of patients receiving percutaneous coronary intervention (PCI). According to the previous reports of atorvastatin on rats [9-11], 20 or 5 mg/kg/day of atorvastatin on rats is preferred in researches of myocardial ischemia–reperfusion injury. Following the method of dosage conversion between animals and humans [31], 20 mg/kg/day of atorvastatin on rats is equal to 120–140 mg/kg/day of atorvastatin on humans. In our speculations, no significant difference between two doses of atorvastatin on anti-microthrombosis and anti-myocardial fibrosis might be owing to different pharmacokinetics between rats and humans.

Finally, we detected that atorvastatin preconditioning could reduce the expression of fgl-2 both in cardial tissue and mononuclear cells. Fgl-2 was a novel inducible endothelial cell procoagulant and an important immune regulator of both innate and adaptive responses on the surface of macrophages and endothelial cells of impaired tissues or organs of microvascular dysfunction diseases such as viral hepatitis [32], severe acute respiratory syndrome (SARS) [33] and so on [34, 35]. It was demonstrated that Fgl2 existed as both a type II transmembrane protein and can be constitutively secreted by both CD4+ and CD8+ T cells [36]. And researches suggested expression of Fgl-2 obviously increased in rats of 3 days during acute vascular xenograft rejection and induction of fgl2 expression by cultured vascular endothelial cell activated by TNF-α [35, 37]. In this study, the expression of fgl-2 in cardial tissue and mononuclear cells was declined when the no-reflow size was decreased with atorvastatin preconditioning. That may be a potential novel mechanism about atorvastatin improving forward blood flow in no-reflow rats.

In conclusion, this research indicated that atorvastatin preconditioning could alleviate deterioration of early cardiac diastolic function and improve forward blood flow in no-reflow rats by declining the expression of TNF-α and fgl-2.

Declaration of Conflicting Interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Result
  6. Conclusion
  7. Declaration of Conflicting Interests
  8. Acknowledgement
  9. References

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Result
  6. Conclusion
  7. Declaration of Conflicting Interests
  8. Acknowledgement
  9. References

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: supported by Natural Science Foundation of Hubei Province (No. 201051299454) and Scientific and Technological Project of Wuhan city of China (No. 201161038340-08).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Result
  6. Conclusion
  7. Declaration of Conflicting Interests
  8. Acknowledgement
  9. References
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