Objectives:(1).To construct the replication-deficient recombinant adenovirus vector with Nrf2 gene. (2).To research the feasibility of transfection of human Nrf2 gene via coronary artery firstly.Then to observe the protective effect against myocardial ischemia reperfusion injury.(3).To explore its protective mechanism against ischemia reperfusion injuryMethods:(1).Construction of the replication-deficient recombinant adenovirus,Recombinant plasmids PDC-EGFP (containing enhanced green fluorescence protein EGFP gene),PDC-Nrf2 (containing human Nrf2 gene).Recombinant adenovirus Adv-EGFP,Adv-Nrf2 were produced by homologous recombinant in 293 cells,amplified in 293 cells on a large scale, and purified by Adeno-XTM Virus Purification Kit (BD Biosciences, Clontech). The concentration of recombinant adenovirus Adv-EGFP was 1.0E+12(PFU/mL); that of Adv-Nrf2 was 1.0E+12 (PFU/mL). According to preliminary results,the concentration of Adv-EGFP and Adv-Nrf2 were 1.0E+9(PFU/mL), dosage was 0.1ml.(2).Forty SD rats were randomly divided into four groups (n=10 in each group). Left thoracotomy was performed, then the apex of the heart and the base of aortic and pulmonary artery were exposured. The base of aortic and pulmonary artery were occluded by applying a nontraumatic vascular clamp to achieve complete cessation of outflow for ten seconds, at the same time,0.1 ml of Adv-Nrf2 or empty viral vector were injected into left ventricle cavity using a 27-gauge needle. Ten seconds later, the clamp was removed.0.1ml empty viral vector was injected into the rat heart in Groupl,â…¡,â…¢. Adv-Nrf2 was injected into the rat heart in Groupâ…£.Breasts were closed after operation.Then rats recovered and stabilized for three days.(3).Three days later, a thoracotomy was performed to expose the left auricle and arterial cone. Then the proximal of great cardiac vein and the left anterior descending coronary artery (LAD) were ligated for 30 min, following reperfusion for 120 min. Groupâ… were not ligated the LAD,but them were subjected the same surgical procedures as experimental group. Groupâ…¢were subjected to ischemia and reperfusion for 3 cycles before 30 min ischemia and 120 min reperfusion, which was induced LAD ischemia for 5 min followed by 5 min reperfusion.(4).Heart electricity activity and the mean arterial pressure of all animals were monitored by the BL-420F system.Area at risk (AAR) and infarct region were determined by Evan's blue dye perfusion and triphenyl tetrazolium chloride (TTC) staining.Changes of the cardiac inflammation were measured under light microscope.Changes of the myocardial ultrastructure were observed under electron microscope. Myocardial organization was used to measure MDA content by substrate TBA(Thibabituric Acid) and SOD activity by substrate XO(Xanthine Oxidase) and TNF-a content by Enzyme-linked immunosorbent assay. Determination of Bcl-2, Bax expression were performed with Western Blotting analysis. Myocytes apoptosis were measured by TUNEL method.Result:(1). There are no difference in each group about HR and MAP in the foundation state.Compared with Groupâ… , HR decreased significantly at the timepoint of T3, T5, T6(P<0.05).MAP decreased obviously at the timepoint of T1, T3, T4, T5, T6(P<0.05).Arrhythmia increased remarkably in the remaining group(P<0.05).However all of these were improved in Groupâ…¢,â…£. A reduction in the myocardial infarct size were observed in Groupâ…¢(26±1.5%) and Groupâ…£(26.6±1.0%), compared with Groupâ…¡(37.6±1.9%).(2).LDH, CK, CK-MB and cTnI increased remarkably in other groups(P<0.05) after ischemia/reperfusion compared with GroupI.It also had statistics significance between Groupâ…¡and Groupâ…¢,â…£(P<0.05). CK and CK-MB increased clearly in Groupâ…£compared with Groupâ…¢.(3).Groupâ…¡rendered typical pathological changes of myocardial structure under light microscope, such as cells edema, arrange disorder, cardiac fibrosis broken, a large number of red blood cells and neutrophil infiltration, etc; however, Compared with Groupâ…¡, Groupâ…¢and Groupâ…£reduced significantly about pathological damage. Groupâ…¡injuried most seriously under electron microscope, such as myocardial edema, cardiac fibrosis arrange disorder, part of the fibrosis fracture and dissolution, unclear light and dark, mitochondrial swelling, part of the ridge fracture and dissolved, and a wide range of vacuolar degeneration, etc; however, Compared with Groupâ…¡, Groupâ…¢and Groupâ…£reduced significantly with subcellular structures damage.(4).Injury caused by oxidation and inflammation:Compared with Groupl, after ischemia/reperfusion, MDA and TNF-a increased remarkably in the remaining group(P<0.05), it also has statistics significance between Groupâ…¡and Groupâ…¢,â…£(P<0.05); However, SOD decreased significantly in the remaining group(P<0.05), it also had statistics significance between Groupâ…¡and Groupâ…¢,â…£(P<0.05).(5).Expression of myocardial protein and apoptosis:Compared with Groupl, after ischemia/reperfusion, Bcl-2/Bax ratio decreased remarkably in the remaining group(P<0.05), it also has statistics significance between Groupâ…¡and Groupâ…¢,â…£(P<0.05); Compared with Groupâ… , after ischemia/reperfusion, cardiomyocyte apoptosis increased obviously in the remaining group(P<0.05).It also had statistics significance between Groupâ…¡and Groupâ…¢,â…£(P<0.05).Conclusion:(1). The Adv-EGFP and Adv-Nrf2 were constructed successfully and the foundation for further research was laid.(2)Transfection of Nrf2 gene via coronary arteria was feasible and demonstrated the protectiv effect against myocardial ischemia reperfusion injury.(3).Nrf2-Keapl-ARE signal pathway could be activated by overexpresstion of Nrf2 gene in rats cardiomyocytes, then against oxidative stress, apoptosis and inflammatory were enhanced to against myocardial ischemia reperfusion injury.(4).Compared Group Nrf2 with Group ischemic preconditioning, most indicators have no statistics differences. Overexpression of Nrf2 and ischemic preconditioning had approximative protective effect.
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