| More and more patients of coronary artery disease have benefit from percutaneous coronary intervention (PCI). About 30 000 cases of PCI were performed in 2002 in China. It rises to 75 000 in 2005. By the end of 2009, the PCI cases has been over 240 000. Now, the number is still increasing.No/slow reflow phenomenon and contrast induced nephropathy are the main complications that limited the short and long-term efficacy of PCI. Although the exact mechanism remains uncertain, it was accepted that dysfunction of microcirculation, inflamation and oxidant stress were among the common pathophysiological basis.Both statins and anisodamine possess these characteristics such as anti-inflammation, antioxidation, improving the function of the endothelium. It has been confirmed that common dose statins improve myocardial perfusion and prevent the incidence of CIN. Data in our laboratory also support the use of anisodamine in the setting of NRP. So far, the effect of intensive statin dose on myocardial perfusion, the preventive effect of anisodamine on CIN and the effect of their combination have not been carried out. Based on our previous work, this study was designed to evaluate the effect of intensive simvastatin, anisodamine and their combination on myocardila perfusion and CIN. The possible mechanisms were also probed.Part I Intensive cholesterol lowering with simvastatin improves the outcomes of percutaneous coronary intervention in patients with acute coronary syndromeObjective: The incidence of no reflow penomenon limits the clinical outcomes of percutaneous coronary intervention (PCI). We designed a randomized controlled study to evaluate the immediate protective effects of intensive statin pretreatment on myocardial perfusion and myocardial ischemic injury during PCI.Methods: A total of 228 acute coronary syndrome (ACS) patients were randomly divided into standard statin group (SSG, n=115) and intensive statin group (ISG, n=113). Patients in SSG received 20 mg simvastatin and patients in ISG received 80 mg simvastatin for 7 days before PCI. TIMI grade flow (TGF), corrected TIMI frame count (CTFC) and TIMI myocardial perfusion grade (TMPG) of the intervened vessel were recorded before and after stent deployment. CPK isoenzyme MB, troponin I and plasma level of hs-CRP, P-selectin and ICAM were measured before and 24 hours after the procedure.Results: The TFG after stent deployment was significantly improved with less TIMI 0-1 patients and more TIMI 3 blood flow in ISG than in SSG (all p<0.05). Patients with no reflow phenomenon were less in ISG (P<0.001). The CTFC was lower in ISG than SSG (P<0.001). TMPG was also improved in ISG than SSG (P=0.001). Twenty-four hours after the procedure, although PCI caused significantly increase in CK-MB, the elevated CK-MB value was lower in ISG than SSG (18.74±8.41 vs 21.78±10.64 P=0.018). Similar changes were also found with regard to Troponin I (0.99±1.07 in ISG vs 1.47±1.54 in SSG P=0.006). CK-MB elevation occurred in 27.8% (32/115) in SSG versus 15.9% (18/113) in ISG (P=0.030). Among them, myocardial necrosis was detected in 4.4% (5/115) of the patients in SSG, whereas 0.9% (1/113) in ISG (P=0.341). No myocardial infarction was found. Similarly, the patients with increased troponin I were much more in SSG (36.5% 42/115) than ISG (19.5% 22/113) (P=0.04). Among them, myocardial necrosis was detected in 13% (15/115) of the patients in SSG, while 4.4% (5/113) in ISG (P=0.021). Myocardial infarction was found in 4.4% (5/115) in the patients in SSG and 0.9% (1/113) in ISG (P=0.213).Conclusion: Intensive statin pretreatment for 7 days before PCI can further improve myocardial blood perfusion, protect myocardium from ischemic injury. This benefit is associated with the lowering of hs-CRP, P-selectin and ICAM level.Part II Protective effect of simvastatin in combined with anisodamine on myocardial perfusion in swine no reflow model. Objectives: Slow reflow or no reflow phenomenon not only increase the risk during percutaneous coronary intervention, but also limit the long prognosis of this therapy. Study of its mechanism and find out the prevention measures remain the main focus of this field. Anisodamine can improve the state of mircrocirculation, antioxidant and protect the myocardium from ischemic injury, while statins posses the ability of improving the function of endothelium, anticoagulation, antiplatelet, antioxidation, antiinflammation and improving blood hemodynamics. So far, data on the combination of the two drugs on the prevention of SRP/NRP is absent. So, based on our previous study of experimental SRP/NRP model on minipig, we evaluated the preventive effect of their combination. We also probed the possible mechanism by measuring the level of hs-CRP, SOD, MDA and NO.Method: Totally 16 minipin of 30-40 Kg were randomly divided into anisodamine groups (A, n=8) and anisodamine plus simvastatin group (A+S, n=8). Pigs of A+S group were prtreated with oral simvastatin (1 mg/Kg) for 7 days, while pigs in S groups were given oral placebo.Seven days later, after coronary angiography were performed, a minicatheter was superselected into middle LAD,and the dopper wire was placed at the midle LAD, 3 cm far from the catheter in order to record the changes of blood velocity. The pressure of aorta was monitored all the time. PMBS was injected (5ml each time) for 4 times with an interval of 10 minutes. Anisodamine (5000u) was injected into the LAD 2 minutes before PBMS was injected. CAG was reperformed 5 minutes after the PMBS was injected to record the TIMI blood flow, TMPG and CTFC to evaluate the myocardial perfusion. Then the animal was killed and the smple of myocardium was take at the joint area of ischemic and normal myocardium in order to measure the tissue level of SOD,MDA and NO and to perform pathological examination.Blood sample was take before and after the expeiment to measure the level of CK-MB,InI and hs-CRP. The percent of necrostic myocardium was calculated by myocardium stain method.Results: The TIMI blood flow was better in S group and TFCs was lower in A group (P<0.05). At the early stage of PMBS injection, the heart rate was increased in both the groups than their baseline (P<0.05). There was no difference between the two groups. There after, the HR was further increased in A group, while the HR in A+S group remain unchanged.The Pa was increased in both the two groups after PMBS injection at the early stage (P<0.01). Then it begain to decrease in A group (P<0.05), while it remain its high level in A+S group (P=0.042). In the late stage, the Pa did not decrease in A+S group, however, it was further decreased in A group.In the early stage after PMBS injection, the bAPV was increased in both groups, which was more obvious in the S group.The hAPV was decreased in S group while it remain its high level in A+S group (P=0.000). After the third injection of PMBS, comparing with the baseline, the hAPV was sinificantly decreased in both groups (P<0.01), but it was still higher than that in S groups (P=0.000). After the forth injection, the hAPV was further decreased with no difference between the two groups.After the first and second injection of PMBS, the CFR in A+S group was not changed, while it was decreased continously in S group (P=0.006).The CFR was continously deceased after the third and the fourth injection (p<0.05), but it was higher in A+S group (P=0.025).The h-MR in S group was elevated in S group after the first and the second injection of PMBS (P=0.032), while it not changed in A+S group.After the third injection, the h-MR increased in both group (P=0.030) with the higher level in A+S group (P=0.010). After the fourth injection, the h-MR was further increased (P=0.024), with no difference between two groups.The level of serum cholesterol 7 days later was 4.44±0.47mmol/L and 3.93±0.53mmol/L in S group and A+S group (P=0.063). The CK-MB, TnI, hs-CRP and MDA were increased 60 minutes after the experiment, but they were higher in S group. Level of NO was also increased (P=0.000), with the higher level in A+S group (P=0.006). SOD was decreased (P=0.000) in both groups, but it was lower in A group (P=0.000).The infarcted myocardium in S grous was 18.5±3.1%, while it was 11.3±2.9% in A+S group (P<0.05).Conclusion: Simvastatin combined with anisodamine treatment can significantly improves myocardial blood perfusion and porotect the myocardium against ischemic injury during percutaneous coronary intervention. The possible mechanism involves improving of coronary hemodynamics, antiinflammation and antioxidation.Part III Protective effects of intensive statin pretreatment on renal function in patients with acute coronary syndrome undergoing percutaneous interventionObjectives: To evaluate the protective effects of higher dose statin on renal function and the incidence of CIN and to probe its possible mechanisms.Methods: Two hundreds and twenty eight patients with acute coronary syndrome undergoing delayed percutaneous coronary intervention were randomly divided into standard statin group (SSG n=115) and intensive statin group (ISG n=113). Patients in SSG group were given simvastatin 20 mg/day and patients in ISG were given simvastatin 80 mg/day for at least 7 days befor PCI, Serum creatinine was measured at admission, 24 hours and 48 hours after PCI. Creatinine clearance was calculated by Cochcroft-Gault formula. The changes of high sensitive C reaction protein (hs-CRP), intercellular cell adhesion molecule 1 (ICAM-1) and P-selectin level before and after the procedure were also measured.Results: Serum creatinine underwent significant increase after PCI, the peak value occurred at 24 hours, and then began to decrease. At 48 hours after PCI, the creatinine level significantly decreased (P<0.001 compared with the level at 24 hours) to baseline level (P=0.94 compared with the level at baseline) in ISG, whereas in SSG the creatinine level failed to decrease significantly (P=0.11) at 48 hours. Serum creatinine at admission was not significantly different between the two groups. But at 24 and 48 hours after PCI, it was lower in ISG than SSG (P<0.05 at 24 hours and P<0.001 at 48 hours). The creatinine clearance significantly decreased after PCI, the lowest value occurred at 24 hours, and then it began to increase. In SSG, the creatinine clearance increased significantly (P=0.03 compared with the level at 24 hours) at 48 hours, but still significantly lower than baseline level (P<0.001 compared with the level at baseline). In ISG, the creatinine clearance increased significantly (P<0.001 compared with level at 24 hours) at 48 hours and recover to the level at baseline (P=0.87 compared with level of baseline). Creatinine clearance improved much more in ISG at 24 and 48 hours than that in SSG (P<0.001 at 24 hours and at 48 hours). After PCI, the serum level of hs-CRP, P-selectin and ICAM-1in ISG were significantly lower than SSG (all P<0.001). Although procedure caused significant increase in hs-CRP, P-selectin and ICAM-1 (P<0.001), the increase in ISG was smaller than SSG (P<0.001).Conclusion: Pretreatment with intensive statin dosage before PCI can further decrease the occurrence of postprocedural contrast induced nephropathy compared with standand statin therapy. This benefit is associated with the lowering of hs-CRP, P-selectin and ICAM levels.Part IV: Establishment of contrast induced nephropathy model in ratsObjectives: with the widespread application of intravascular contrast, contrast induced nephropathy (CIN) has became to the third rank cause of intra-hospital-attained acute kidney injury. Study of the mechanism and its prevention has become the hot research point in the field of interventional cardiology. So far, the accepted animal model of CIN has not been established.The purpose of this stuy was to establish an rat model of CIN and to evaluate its efficacy.Methods: Totally 24 SD rats of 250-270 grams were randomly allocated into experimental group (group A, n=12) and control group (group B, n=12). Rats in the two groups were further divided into group of 12 hours and group of 24 hours. All the rats were normally raised for 7 days in order to accommodate to the new environment.After dehydration of three days, rats in group A were given intravenous Meglumine Diatrizoate and Diatrizoate Sodium (MDDS), while rats in group B were given intraveous normal saline (NS). Then, all rats get normal water-drinking to the end of this study. Animals were killed at 12 hours (group of 12 hours) and 24 hours (group of 24 hours).Renal ultrasonic examination was performed before this study, after dehydration and 3, 6, 12 and 24 hours after contrast media injection to observe the morphologic changes and the changes of renal dimensions (length,width and thickness), diameters of renal artery, peak systolic velocity (PSV), end diastolic velocity (EDV), ratio of PSV/EDV (S/D) and velocity-time index of left kidney. The heart rate was also recorded at the corresponding time points.Blood samples were taken before the study, after dehydration and 12 hours (Group A) , 24 hours (Group B) to meseaure the level of serum creatinine. Animals were killed at 12 hours (group A) and 24 hours (group B) and the tissue of kidney were incised for microscope and electron microscope study.Results: The dimensions of the two groups before and after dehydration were not different. It gradually enlarged at 3, 6, 12 and 24 hours after CM injection. These changes were the most obvious at 6 and 12 hours, which did not recover at 24 hours.The renal diameters of the two groups were not different. The PSV, EDV, S/D and VTI were lowerest at 6 hours and then recover to normal level at 24 hours. RI was increased after CN injection, the lowest occurred at 6 hours, and recover to normal level at 24 hours. The heart rate was obviously increased after hehydration, but it remained unchanged during the following time period. Serum creatinine was signicicantly elevated after dehydration, the highest level occurred at 12 hours and then begain to recover at 24 hours.Microscope examination to renal sample at 12 hours found patch disapperence of tubular structure, widely congestion at medullar area. High degeneration of the tubular endothelium, infiltration of fibromyocyte and mononuclear cells were also found. At 24 hours, the obove changes were still present, but become much mild. No pathological glomerular changes were found under microscope. Electron microscope examination found desquamation, sparseness of microvillous of tubular endothelium, membrane confusion, disapperence, swelling, fragmentation of the MIT, with obstrcted tubular lumen and basal membrane sweling. Swelling, licking up, edema of basal membrane and interstitium of the endothelium of glomerular capillary were also found. It was most obvious a 12 hours. Conclusion: Combined with dehydration, intravenous injection of contrast lead to obvious acute kidney injury, with the changes of kidney tissue pathology, hemodynamics and kidney functions much similar to the characteristics of contrast induced nephropathy in humanbings. Thus, we established an eligible animl model of contrast induced nephropathy which mimiced the time course of contrast induced nephropathy in humanbeings.Part V: Renal protective effects of intensive statin in combined with anisodamine against contrast induced nephropathy.Objectives: With the widespread use of intravascular contrast, acute kidney injury was more and more found in clinical practice. Study on its mechanism and prevention has aroused great interest in the field of interventional cardiology. Statins possess pleiotrophic effects against renal injury, which are independent of its cholesterol lowering effects. Also, anisodamine have benefit features again renal injury such as antishock, improving microcirculation and antioxidation. But study of anisodamine in the prevention of CIN after percutaneous coronary intervention has not been carried out. Also, data about their combination for the prevention of CIN after percutaneous coronary intervention was absent. This study, based on the CIN rat model we established, was performed to evaluate the protective effect of simvastatin, anisodamine and their combination in the prevention of CIN and to probe the possible mechanism.Methods: Totally, 48 of SD rats were randomly divided into control group (control, C, n=12), anisodamine group (anisodamine, A, n=12), simvastatin group (simvastatin, S, n=12) and anisodamine+simvastatin, A+S group, n=12). Then, rats in the four group were futher divided into 12hrs subgroup and 24hrs subgroup (n=6 in each subgroup). Rats in S group and A+S group were given oral simvastatin 0.1mg/100g, while rats in group C and group A+S were given oral placebo for 7 days. After dehydration for 3 days, intravenous Meglumine Diatrizoate and Diatrizoate Sodium (MDDS) was given (0.8mg/100g), then normal water drinking was recovered.At 15 minutes before contrast injection, anisodamine (100ug/100g) was abdominally injected in group A and group A+S, while normal saline in the other two groups. During the 4 hours after contrast injection, anisodamine abdominal injection was performed every 1 hours in group A and group A+S, while normal saline was injected abdominally at the corresponding time point.During the time between 4 and 12 hours after contrast injection, anisodamine (100ug/100g) and normal saline was repeated every 4 fours as mentioned above.Kidney untrasound examination was performed at baseline, after dehydration, 3 hours, 6 hours after contrast injection to observe the mophorlogical changes, the renal dimensions and hemodynamic parameters such as diameter of renal artery, peak systolic velocity (PSV), end-diastolic velocity (EDV), resistant index (RI), velocity time index (VTI) and heart rate (HR). Serum creatinine, high sensitive C reactive protein (hs-CRP) were measured at baseline, after dehydration, 12 (12hrs subgroup), 24 (24hrs subgroup) hours after contrast injection. Animals were killed at 12 (12hrs subgroup) and 24 (24hrs subgroup) hours respectively. Renal samples were incised to measure level of superoxide dimutase (SOD), MDA, NOS, NO and GSH-Px.Results: All the dimensions in group C increased 6 hours afer contrast injection, but is remain unchanged in group A+S. Partial of the dimensions in group A and S increased. The echo decreased at 6 hours compared with 3 hous in group C, while it was abscent in group A+S. The echo changes in group A and S was mild. No dilation of collective system concentrating system was found in all the groups.At 3 hours, the PSV, EDV and VTI in group A+S were higher than the other groups, and were not less than that after dehydration. However, these parameters were much lower in group C, and lower that that after dehydration of the same group.These parameters was not changed in group A and S. At 6 hours, the PSV, EDV and VTI remain the highest among the four groups and were higher than that at 3 hours. However, these parameters in group C were still lower than the other groups and not recovered to the level after dehydration,Those parameters in group S were higher than that in group A.S/D was the lowest in group A+S and the higest in group C, while no difference was found between group S and A. At 3 and 6 hours after contrast injection, although these differences of S/D keep existed, it tends to recorver. At 6 hours, it has recovered to baseline in group A+S while it keeps higher in group C. The RI in group A+S at 3 and 6 hours was lower than that in group C, while there was no difference between group S and A. The HR increased in all groups after dehydration with the higher level in group A and A+S. The HR in group S was not different between 3 and 6 hours, while it was higher at 6 hours than 3 hours in group S.Serum creatinine increased after dehydration, 12 and 24 hours after contrast injection with the highest level occurred at 12 hours. Although it recovered significantly at 24 hours, only that in group A+S and S recovered to baseline level and group C remain the highest. At 12 hours, the higest level occurred in group C and the lowest level at group A+S, group A ranked the second and group S ranked the third.Microscope examination to renal sample at 12 hours found that in group C, there were patch disapperence of tubular structure, widely congestion at medullar area. High degeneration of the tubular endothelium, infiltration of fibromyocyte and mononuclear cells were also found. In group A, local changes of above mentioned, scattered tubular endothelium and a few fibromyocyte and mononuclear cells were found. In group S, the changes were much similar to group A. In group A+S, there were only spotted changes of above mentioned, few fibromyocyte and mononuclear cells and mild congestion. At 24 hours, the obove changes were still present in group C and group A, but it become much mild in group S. It recovered completely to normal apperence in group A+S. No pathological glomerular changes were found under microscope.Electron microscope examination found desquamation, sparseness of microvillous of tubular endothelium, membrane confusion, disapperence, swelling, fragmentation of the MIT, with obstructed tubular lumen and basal membrane sweling. Swelling, licking up, edema of basal membrane and interstitium of the endothelium of glomerular capillary were also found. It was most obvious a 12 hours. Among the four groups, group C was the most obvious and group A+S was the mildest. Although the level of hs-CRP increased in all the groups after dehydration, it was lower in group A+S and S. At 12 hours, hs-CRP reached its highest level. Among the four groups, group A+S was the lowest and group C was the highest. At 24 hours, the hs-CRP was lower than that of 12 hours in all the groups, but still higer than that after dehydration, among which, the lowerest occurred in group A+S, the highest in group C. It was lower in group S than that in group A.At 12 hours, the level of SOD was decreased in all the four groups, among which, the lowest in group C,the highest in group A+S. Although there was no difference between group A and S, they were all higher than that in group C. AT 24 hours, SOD was further decreased in group C and A, while it increased in group S and A+S. It has recovered to baseline level in group A+S.At 12 hours, level of MDA increased significantly. The highest occurred in group C and the lowest in group A+S. The level of SOD ranked the second in group A and the third in group S. At 24 hours, MDA was further decreased in all the groups, among which, the MDA in group has recovered to baseline, however, it was still higher the other three groups.At 12 hours, level of GSH-Px was the lowest in group C, the higest in group A+S.Group S rank the second and group A the third. At 24 hours, GSH-Px began to increase in all the four groups, among which, group A+S has recovered to baseline level and the other three groups remained lower than baseline.At 12 hours, the NOS deceased in group C and A, while group A+S increased. Group S did not change. At 24 hours, NOS in group C further decreased, NOS in group S and A+S further increased. Group A did not change. The most increasing occurred in group A+S.At 12 hours, iNOS increased significantly, with the highest in group C. At 24 hours, group C remain the highest and group A+S the lowest. There was no different between group A and S. Compared with 12 hours, level of iNOS at 24 hours in group C,A and S were not different, while level of iNOS in group A+S decreased.In group C, level of NO was lower at 12 hours and elevated at 24 hours. In group A, NO level was slightly but significantly higher, but decreased at 24 hours. In group S, NO level was significantly higher among the four groups at 12 hours, and further increased at 24 hours. In group A+S, NO level was the highest among the four groups at 12 hours.Althought there was a slight decrease at 24 hours, it was still higher than that at 12 hours, and further increased at 24 hours.Conclusion: Anisodamine combined with simvastatin can effectively prevent the contrast induced acute kidney injury. Improvement of renal hemodynamics, antiinflammation and antioxidation are the underlying mechanism. |
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