| 1.Background and objectiveMyocardial ischemia and reperfusion represents a popular clinically relevant problem associated with thrombolysis, angioplasty, coronary bypass surgery and heart transplantation. The damage manifested as myocardial necrosis, apoptosis, mitochondrial dysfunction, increased lipid peroxidation and lead to clinical findings of myocardial stunning, reperfusion arrhythmias, myocardial and endothelial cell death and microvascular function no-reflow phenomenon and other related disorders. A large number of reperfusion therapys, such as coronary thrombolysis, after extensive coronary angioplasty, coronary artery bypass surgery and heart transplantation and so on, myocardial I/R injury to become the main factors affecting the effect of reperfusion therapy and general clinical problems. Therfore, the study of I/R injury in myocardial protection has been an important clinical and basic research in the cardiovascular field. Although the mechanism induced myocardial injury has not been fully elucidated, the results over the years has proved that oxygen free radicals, calcium overload, myocardial energy metabolism, neutrophils, endothelial cells, cell adhesion molecules and apoptosis all involved in I/R injury. These pathological mechanisms have relationships with each other, constituting a complex signaling networks, resulting in myocardial cell damage.Studies have shown that antioxidant，including upregulation or activation of the expression of antioxidase, scavenging active oxygen radicals, and so on, can help significantly to reduce the ischemia-reperfusion injury. Nanoceria have potentially regenerated ability of clearing oxygen radicals and antioxidant enzyme activity. We confirmed for the first time that Nanoceria can perform the function of inhibitjing myocardial oxidative stress, inflammatory response and endoplasmic reticulum stress. Recently, several studys show that Nanoceria can upregulate the expression of various antioxidant enzymes mediated by antioxidant response element (ARE). In this study, we will clarify the molecular mechanism of nanoceriaway regulating antioxidant enzymes by the way of HE、TUNEL、RT-PCR、Western Blotting. Meanwhile, explore the protective effects of nanoceria in myocardial oxidative injury through cytology and animal experiments and make further investigation, simulating clinical cardiopulmonary bypass, if Nanoceria can improve the ability of myocardial antioxidant, mitochondrial membrane structure and the myocardial systolic function. That is of great implications to develop Nanocera as as a new antioxidant to clinically prevent and treat free radical damage and promote the application rerearch of Nanocera in biomedicine.2.Materials and Methods2.1A total of forty health male Sprague-Dawley (SD) rats,250-300weight, were included in this study. Forty rats were randomly divided into five groups, Sham group, I/R group, I/R+CeO2preconditioning groups with three diverse nano size (1-10、10-25、50nm). Before surgery, sham and I/R group24h after intravenous injection of PBS buffer (0.2ml/100g); each pretreatment group at24h after preoperative intravenous injection of CeO2nano-suspensions (0.2mg/100g).2.2The rats were anesthetized with10%chlorate solution (300mg/kg) by intraperitoneal injection, and then back recumbent fixed. Intubated and connected to a small animal ventilator-assisted breathing, open the chest to expose the heart. In line with6-0proline pulmonary cone spacing aortic root and left atrial appendage about3-4mm at threading, to be regular rhythm with a fluted tube latex cushion between the blood vessels and coronary artery ligation in descending artery ligature, ligature after the remote myocardium becomes cyanotic color white for the first ligation success. After45min ischemia, cut the ligature and removed a small tube of latex restore coronary blood flow. The model is successful when myocardial ischemia color restored, and then reperfusion120min.2.3Myocardial tissue was observed by the method of HE staining, and recorded the changes of myocardial morphological under an optical microscope. Myocardial cell apoptosis were calculated by TUNEL assay. Conducted real-time Using the method of PCR amplification to detect the quantitative analysis of Bcl-2, Bax and caspase-3mRNA expression.2.4Analysis the expression of Nrf2and antioxidant genes HO-1, NQO1from expression levels of transcription and protein by RT-PCR and Western Blotting methods. Observe the effect of pretreatment on the heart Nanoceria endogenous protective molecules and apoptosis-related proteins.2.5The SPSS17.0software was used in this study for statistical analysis. Data were presented as mean±standard deviation (S.D.). One-way ANOVA was conducted across all groups first followed by a Bonferroni post-hoc correction between all group comparisons. Significance was accepted at p﹤0.05.3.Results3.1Compared with Sham group, I/R group and I/R+CeO2each pretreatment group appear to have a clear regional myocardial ischemia and myocardial infarction. I/R+CeO2(10-25nm) present the lightest degree of myocardial cell injury, and the apoptosis was significantly lower than the I/R group (P <0.01). The expression levels of Bcl-2mRNA in myocardial cells in the I/R+CeO2groups were significantly increased, compared with I/R group (P <0.01). While the expression levels of Bax and Caspase-3mRNA were significantly decreased（P<0.01）.3.2RT-PCR shows that the Nanoceria promote the mRNA expression levels of Nrf2, HO-1and NQO1increased. Although the expression levels of Nrf2mRNA elevated in model group, but the difference in each group was not statistically significant (p>0.05), for a description of ischemia-reperfusion injury in myocardial tissue of Nrf2transcriptional level had little effect. In the experimental group, mRNA expression levels of Nrf2, HO-1, NQO1increased (p <0.01), in which the transcriptional level in10-25nm particle group was the most obvious, compared with the model group, Nanoceria pretreatment can reduced myocardial apoptosis with the high significant, which was consistent with the results of antioxidant.3.3Detect the protein expression level of Nrf2in the cytoplasm and the nucleus of myocardial tissue by the method of Western Blotting. The results show that Nanoceria pretreatment promote Nrf2into the nucleus and increase the amount of its protein expression.4. Conclusion4.1To some extent, Nanoceria nanoparticles can inhibited ischemia-reperfusion induced myocardial apoptosis, in which10-25nm in CeO2nanoparticles protect the myocardial tissue is most significant. The mechanism of anti-apoptotic may be related to increased the expression of anti-apoptotic gene of Bcl-2and down-regulated the expression of pro-apoptotic genes Bax, Caspase-3.4.2The pretreatment of Nanoceria can increase the expression level of Nrf2in transcription and translation level, and promote the distribution of Nrf2protein transfer occurs from the cytoplasm to the nucleus.4.3After the ischemia-reperfusion injury, Nanoceria pretreatment can increases and activates the protein expression of Nrf2, meanwhile increase expression levels of its antioxidant and detoxifying enzymes in the downstream. Tip Nrf2-ARE pathway may be involved in the post-reperfusion of ischemic endogenous oxidative stress mechanism. |
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