| BackgroundWith the development of social economy, arteriosclerotic cardiovascular disease has become the first rank of fatal diseases in china. The pathology of acute coronary syndrome is rupture or erosion of coronary atherosclerosis plaque, and leading to complete or incomplete occlusive thrombosis, which causes myocardial ischemia. In the treatment of myocardial ischemia, revascularization is the key to restore blood flow. But after restoring blood supply, ischemic tissues produce excess free radicals which cause more severe injury to the ischemia tissues. That is so called ischemia-reperfusion injury.Atorvastatin is widely used in clinical treatment of arteriosclerotic cardiovascular disease. Atorvastatin has multiple effect except lipid-lowering, including improving endothelial function, reducing inflammatory factor, preventing lipid oxidation, et al. Clinical studies have found that atorvastatin can improve prognosis of arteriosclerotic cardiovascular disease. Pretreatment of atorvastatin can reduce myocardial infarction area and ischemia-reperfusion injury. ARMYDA series studies has found that pretreatment of atorvastatin before PCI could reduce the markers of myocardial injury, lower cardiovascular events. Clinical studies and animal experiment indicated that atorvastatin provide cardioprotection by mimicking mechanism of ischemic preconditioning (IPC) which may be relevant with mitochondria. Atorvastatin may be the ideal drug for pharmacological preconditioning.Uncoupling protein 3(UCP3) are located in the mitochondrial inner membrane and acts as anion carrier proteins. Activation of UCP3 can reduce ATP synthesis by uncoupling oxidative phosphorylation, lowering proton gradient, decreasing ROS generation, transporting fatty acid anions, reducing peroxide damage. UCP3 was thought to be the first line to prevent mitochondrial injury.UCP3 was increased in ischemia-reperfusion myocardium. UCP3 play a critical role in both cardioprotection against I/R injury and induction of ischemia preconditioning. Overexpression of UCP3 can provide cardioprotection against I/R injury by reducing cardiomyocyte injury, reducing myocardial infarction area, reducing the incidence of I/R induced arrhythmia. But the mechanism of cardioprotection by UCP3 was unknown. Studies about the mechanism of UCP3 showed that UCP3 can protect mitochondria through inhibiting mPTP opening via the interaction with adenine nucleotide translocator(ANT).mPTP locate on the inside and outside mitochondria membrane, which was consisted of voltage dependent anion channel(VDAC), adenine nucleotide translocase(ANT), cyclophilin D(Cy D). mPTP opened abnormally after ischemia reperfusion which would damage the structure of mitochondria, affecting the function of mitochondria. The mPTP inhibitor can protect the I/R myocardium.The mPTP opener lonidamine reduce the cardioprotection of IPC.Atorvastatin and UCP3 were confirmed to have cardioprotection in ischemia reperfusion injury which may be related to opening of mPTP, but the exact mechanism was unknown.whether UCP3 pathway involving in inhibiting the opening of mPTP was unknown. In theory, Atorvastatin significantly unregulate expression of peroxisome proliferator activated receptor(PPAR).Activation of PPAR significantly unregulate expression of UCP3 and inhibite the opening of mPTP. But there were no studies about the effect of pretreatment with single dose of atorvastatin on UCP3. So we hypothesize that UCP3 was one of the potential regulator of atorvastatin. Atorvastatin may reduce mitochondrial injury by inhibiting the opening of mPTP through unregulating the expression of UCP3. Atorvastatin and UCP3 have inhibitory effect of mPTP opening.But mPTP was thought to be the last regulator of mitochondria. All the protective effect from regulation of upstream regulator could affect the opening of mPTP. So the role of mPTP can not explain the physiological function of atorvastatin and UCP3. There were no related research about whether atorvastatin protect myocardium through regulating UCP3. So we aim to observe whether atorvastatin provide cardiopretection via regulating expression and function of UCP3, to observe the effect of administration of lonidamine on cardiopretection of atorvastatin and expression of UCP3 after canceling the protective effects of mPTP.Part 1:Cardiopretection of pretreatment of a single high-dose of atorvastatin in myocardial ischemia/reperfusion model in ratsObjective:Myocardial ischemia/reperfusion model in rats were builded.We aimed to observe whether pretreatment with atorvastatin can provide cardioprotection and the effect of lonidamin on cardioprotection of atorvastatin.Methods:1.Protocol of Ischemia-reperfusion(I/R)model:SD rats were used for I/R model, rats were general anesthesia.Thoracotomy was performed on the left side.5-0 nylon suture was used to ligate the LCA, the heart was subjected to ischemia for 30 min and then followed by 120 min of reperfusion.2.Groups:SD rats were randomly distributed into four groups(n=5 in each group) In sham group, the suture was placed around the coronary artery, but without I/R. In I/R group, rats were subjected to ischemia for 30min and then followed by 120 min of reperfusion. In Ator group, atorvastatin was administrated with a single dose(80 mg/kg, i.g.) 12h before I/R, In Ator+LND group, atorvastatin was administrated with a single dose(80 mg/kg, i.g.) 12h before I/R, lonidamine was administrated with 50mg/kg 10min before reperfusion.3.Observed indicator:Weights of Rats and left ventricular were recorded.Heart rate and rhythm of heart were recorded for the whole process.Myocardial ischemia area and infarct area were measured using area calculation method by Evans blue and TTC double staining. The myocardial ultrastructures and the damage degree of mitochondria in different groups were observed by transmission electron microscope.4. Statistical method:All data was analysised using spssl9. Data were expressed as mean±Standard Deviation. One-way analysis of variance (ANOVA) was used for statistical comparisons. Homogeneity test of variance was tested using Levene method, LSD was used for statistical comparison between groups if homogeneity test of variance was equal. Tamhane method was used for statistical comparison between groups if homogeneity test of variance was’t equal. The comparison of rates between groups using chi-square test.P values less than 0.05 were considered statistically significant.Results:1.Baseline data:There was no statistically difference in body weight in different groups (sham:244.08±8.81g,I/R:243.92±10.41 g,Ator:246.69±8.23g,Ator+LND: 247.62±8.14g, P>0.05). There were no statistically difference in weight of left ventricular in different groups(sham:0.408±0.004g,I/R:20.405±0.008g,Ator: 0.408±0.006g,Ator+LND:0.409±0.006g, P>0.05). Basal heart rate before openning chest were no statistically difference in different experimental groups(sham: 354.14±20.76 beats/min,I/R:368.07±16.63 beats/min,Ator:349.95±21.43 beats/min,Ator+LND:346.43±27.48 beats/min, P>0.05)2.The incidence of arrhythmia in I/R protocol:Compared to the sham group, The incidence of arrhythmia after reperfusion in I/R group were increased significantly(Sham vs I/R:7.7% vs 46.2%, P=0.027), The incidence of arrhythmia in Ator group and Ator+LND group were increased,but there were statistically significant difference(Sham vs Ator:7.7% vs 23.1%, P=0.277; Sham vs Ator+LND: 7.7% vs 38.5%, P=0.063). The incidence of arrhythmia in Ator+LND group was higher than Ator group and less than I/R group, but there were no statistically significant difference using chi-square(P>0.05).3.Area at risk/left ventricle(AAR/LV) was used to express myocardial area at risk, Infarct area/area at risk(IA/AAR) was used to express myocardial infract area. Compared with I/R group, AAR/LV of Ator group and Ator+LND group were significantly reduced (I/R vs Ator:46.76±1.42% vs 40.78±1.40%,P<0.001;I/R vs Ator+LND:46.76±1.42% vs 43.86±1.07%, P=0.043), IA/AAR of Ator group and Ator+LND group were significantly reduced(I/R vs Ator:29.16±1.21% vs 21.47±1.65%,P<0.001;I/R vs Ator+LND:29.16±1.21% vs 26.04±1.27%, P=0.024), Compared to Ator group, AAR/LV of Ator+LND group were significantly increased(Ator vs Ator+LND:40.78±1.40% vs 43.86±1.07%, P=0.03). and IA/AARf Ator+LND group were significantly increased(Ator vs Ator+LND:21.47±1.65% vs 26.04±1.27%,P=0.009).4.The myocardial ultrastructures observed by transmission electron microscope as followed:In sham groups, the structure of mitochondrial was normal. In I/R group,the myocardial ultrastructures were damaged obviously.The structure of mitochondrial was oedema. Cavitations in mitochondrial were found obviously.Many were ruptured. The damage in the Ator group was significantly reduced. Mitochondria were slightly swollen. Cavitations and rupture in mitochondrial could be found unfrequently. The damage in the Ator+LND group was between I/R group and Ator group. Mitochondria were moderately swollen. Cavitations and rupture in mitochondrial could be found commonly.Conclusion:1.Myocardial ischemia/reperfusion model in vivo was builded successfully. Pretreatment with atovastatin(80 mg/kg) orally before I/R protocol could reduce I/R injury. Lonidamine partially abolished the protective effect of atorvastatin.2.Pretreatment with atorvastatin can reduce the incidence of arrhythmia induced by I/R.3.Pretreatment with atorvastatin can alleviate the damage of untralstruture of mitochondria induced by I/R.Part 2:The effect of Pretreatment with atorvastatin on uncoupling protein3 in myocardial ischemia/reperfusion model in ratsObjective:We aimed to explore the effect of pretreatment with atorvastatin on myocardium uncoupling protein3, to observe the effect of atorvastatin on protection of mitochondria and the infruence of lonidamin to UCP3 and mitochondria function.Methods:SD rats were randomly distributed into four groups(n=5 in each group):Sham group, I/R group, and Ator group. Myocardial ischemia/reperfusion model in vivo was builded.After 2H reperfusion, blood samples were collected, and the heart was then excised.UCP3 gene transcription level was determined by real-time RT-PCR. UCP3 protein was determined using Western blot. The ATP content of ischemia myocardium was also determined.Serum LDH,MDA,SOD,FFA were measured. At last we campared all the indicator in different groups.All data was analysised using spss19.0 and Graphpad Prism. Data were expressed as mean±Standard Deviation. One-way analysis of variance (ANOVA) was used for statistical comparisons. Homogeneity test of variance was tested using Levene method, LSD was used for statistical comparison between groups if homogeneity test of variance was equal. Tamhane method was used for statistical comparison between groups if homogeneity test of variance was’t equal. P values less than 0.05 were considered statistically significant.Results:1. UCP3 gene transcription level and UCP3 protein in different groupsCompared to the Sham group, UCP3 gene transcription level in I/R group,Ator group and Ator+LND group were increased significantly(Sham vs I/R:1.02+0.17 vs 1.42±0.21, P=0.003; Sham vs Ator:1.02±0.17 vs 1.94±0.31, P<0.001; Sham vs Ator+LND:1.02±0.17 vs 1.70±0.28, P<0.001); UCP3 protein in I/R group,Ator group and Ator+LND group were increased significantly(Sham vs I/R:0.286±0.086 vs 0.420±0.092,P=0.005; Sham vs Ator:0.286±0.086 vs 0.584±0.099, P<0.001; Sham vs Ator+LND:0.286±0.08 vs 0.549±0.068, P<0.001). Compared to I/R group, UCP3 gene transcription level in Ator group and Ator+LND group were increased significantly(I/R vs Ator:1.42±0.21 vs 1.94±0.31,P<0.001; I/R vs Ator+LND:1.42±0.21 vs 1.70±0.28, P=0.029), UCP3 protein in Ator group and Ator+LND group were increased significantly(I/R vs Ator:0.420±0.092 vs 0.584± 0.099, P=0.001; I/R vs Ator+LND:0.420±0.092 vs 0.549±0.068, P=0.006). UCP3 gene transcription level and UCP3 protein in Ator+LND group was slightly less than Ator group,but there was no statistically significant difference.2.Serum LDH activity in different groupsCompared to the Sham group, LDH activity in I/R group, Ator group and Ator+LND group were increased significantly(Sham vs I/R:392.59±55.56 vs 3864.15±162.92U/L,P<0.001; Sham vs Ator:392.59±55.56 vs 3056.17±136.22 U/L, P<0.001; Sham vs Ator+LND:392.59±55.56 vs 3578.01±239.41 U/L, P <0.001). Compared to I/R group, LDH activity in Ator group and Ator+LND group were reduced significantly(I/R vs Ator:3864.15±162.92 vs 3056.17±136.22 U/L, P<0.001; I/R vs Ator+LND:3864.15±162.92 vs 3578.01±239.41 U/L, P=0.001). LDH activity in Ator+LND group was significantly increased as compared with Ator group(Ator vs Ator+LND:3056.17±136.22 vs 3578.01±239.41 U/L, P<0.001).3. Serum SOD in different groupsCompared to the Sham group, Serum SOD in I/R group, Ator group and Ator+LND group were reduced significantly(Sham vs I/R:178.80±.03 vs 121.17 +7.09 U/ml,P<0.001; Sham vs Ator:178.80±7.03 vs 159.53±8.64 U/ml, P< 0.001; Sham vs Ator+LND:178.80±7.03 vs 143.27±6.15 U/ml, P<0.001). Compared to I/R group, Serum SOD in Ator group and Ator+LND group were increased significantly(I/RvsAtor:121.17±7.09 vs 159.53±8.64 U/ml, P<0.001; I/R vs Ator+LND:121.17±7.09 vs 143.27±6.15 U/ml, P<0.001). Serum SOD in Ator+LND group was significantly reduced as compared with Ator group(Ator vs Ator+LND:159.53±8.64 vs 143.27±6.15 U/ml,P<0.001).4. Serum MDA in different groupsCompared to the Sham group, Serum MDA in I/R group, Ator group and Ator+LND group were increased significantly(Sham vs I/R:2.56±0.58 vs 5.02± 0.72nmol/ml, P<0.001; Sham vs Ator:2.56±0.58 vs 3.49±0.40nmol/ml, P=0.002; Sham vs Ator+LND:2.56±0.58 vs 4.45±0.47 nmol/ml, P<0.001). Compared to I/R group, Serum MDA in Ator group and Ator+LND group were reduced significantly(I/R vs Ator:5.02±0.72 vs 3.49±0.40nmol/ml, P<0.001; I/R vs Ator+LND:5.02±0.72 vs 4.45±0.47nmol/ml, P=0.046). Serum MDA in Ator+LND group was significantly increased as compared with Ator group(Ator vs Ator+LND: 3.49±0.40 vs 4.45±0.47 nmol/ml, P=0.002).5.Myocardial content of ATP in different groupsCompared to the Sham group, Myocardial content of ATP in I/R group, Ator group and Ator+LND group were reduced significantly(Sham vs I/R:15.62±1.18 vs 11.28 ±1.86umol/mL, P<0.001; Sham vs Ator:15.62±1.18 vs 14.13±0.90 umol/mL, P=0.031; Sham vs Ator+LND:15.62±1.18 vs 12.78±1.10 umol/mL, P<0.001). Compared to I/R group, Myocardial content of ATP in Ator group and Ator+LND group were increased significantly(I/R vs Ator:11.28±1.86 vs 14.13±0.90 umol/mL, P<0.001; I/R vs Ator+LND:11.28±1.86 vs 12.78±1.10 umol/mL, P=0.029). Myocardial content of ATP in Ator+LND group was significantly reduced as compared with Ator group(Ator vs Ator+LND:14.13±0.90 vs 12.78±1.10 umol/mL, P=0.049).6. Serum FFA in different groupsCompared to the Sham group, Serum FFA in I/R group, Ator group and Ator+LND group were increased significantly(Sham vs I/R:2384.82±369.11 vs 3494.79±392.49 umol/L, P<0.001; Sham vs Ator:2384.82±369.11 vs 2752.97± 299.00 umol/L, P=0.027; Sham vs Ator+LND:2384.82±369.11 vs 3239.28±245.22 umol/L, P< 0.001). Compared to I/R group, Serum FFA in Ator group and Ator+LND group were reduced significantly(I/R vs Ator:3494.79±392.49 vs 2752.97±299.00umol/L, P<0.001; I/R vs Ator+LND:3494.79±392.49 vs 3239.28 ±245.22 umol/L,P=0.118). Serum FFA in Ator+LND group was slightly higher than Ator group. but there was no statistically significant difference(Ator vs Ator+LND:2752.97±299.00 vs 3239.28±245.22 umol/L, P=0.005).Conclusion:1.UCP3 gene transcription level and UCP3 protein was increased in ischemia-reperfusion myocardium. Pretreatment with atorvastatin further unregulated UCP3 gene transcription level and UCP3 protein. Lonidamine partially abolished the unregulation effect of atorvastatin.2.Pretreatment with atorvastatin can reduce myocardium injury in I/R.The protective mechanism may relate with relieving lipid peroxidation damage, enhancing myocardial ability of scavenging free radicals and improving mitochondrial ATP quantities.but lonidamine could weaken the effect of atorvastatin.3.The role of Pretreatment with atovastatin on regulatation of UCP3 was not relevant with. serum FFA.Summary:Ischemia-reperfusion increased myocardium injury. Pretreatment with atovastatin orally 12h before I/R protocol could provide cardioprotection. lonidamine partially abolished the protective effect of atovastatin. Atovastatin may provide cardioprotection via regulating the expression and function of UCP3. The trend of FFA didn’t match the trend of UCP3 gene transcription and protein level. Atovastatin may not regulate UCP3 through serum FFA.The mechanism of regulation of atorvastatin to UCP3 still need to be revealed. |
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