Effects and mechanisms of Bmal1 on myocardial ischemia-reperfusion injury in diabetic rats
Deng Jie1, He Jiandong2, Han Chongfang2
1 School of Anesthesiology, Shanxi Medical University, Taiyuan 030000, China; 2 Department of Anesthesiology Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Taiyuan 030000, China
Abstract：Objective To explore the effect and related mechanism of Bmal1 on myocardial ischemia-reperfusion injury in diabetic rats. Methods Sixty-five male pathogen-free Sprague-Dawley rats were selected and randomly divided into non-diabetic sham operation group (N-S group, n=10), non-diabetic ischemia-reperfusion group (N-I/R group, n=10), non-diabetic + SR8278 ischemia-reperfusion group (NS-I/R group, n=10), and type 2 diabetes model group (35). The type 2 diabetes model was established by high-fat diet feeding and intraperitoneal streptozotocin injection. Thirty rats were successfully modeled and divided into Sham diabetic group (DM-S group), diabetic ischemia-reperfusion group (DM-I/R group), and diabetic +SR8278 ischemia-reperfusion group (DMS-I/R group) by random number table method with 10 rats in each group. Six groups of rats were randomly divided into subgroup A and B according to different observation items with 5 rats in each group. Myocardial ischemia-reperfusion models were prepared for each group, in which the N-S and DM-S groups only simulated the operation process without the ligation of the anterior descending branch. Rats in the NS-I/R and DMS-I/R groups were intraperitoneally injected with orphan nuclear receptor Rev-ERB α antagonist, SR8278 (50 mg/kg), once a day for 7 days before myocardial ischemia-reperfusion model preparation. The myocardial ischemia-reperfusion model was successfully maintained for 24 h, and the left ventricle was isolated from rats in subgroup A for pathological staining and ultrastructural observation by transmission electron microscopy. The pathological sections of the separated left ventricle were prepared for myocardial infarction volume measurement. In subgroup B, myocardial tissue from the apical area was collected and subjected to Western blot to determine the expression of myocardial Bmal1, Sirt3, Nlrp3, and Bnip3 proteins. Results (1) Measurement of myocardial infarction volume. The N-S and DM-S groups had no myocardial infarction, whereas the N-I/R, NS-I/R, DM-I/R, and DMS-I/R group had myocardial infarction with volumes of (0.48±0.08), (0.34±0.05), (0.65±0.06), and (0.46±0.06) cm3, respectively. Compared with the N-I/R group, the volume of myocardial infarction decreased in the NS-I/R group and increased in the DM-I/R group with statistically significant differences(all P values <0.05). Compared with the NS-I/R group, myocardial infarction volume increased in the DM-I/R and DMS-I/R groups, and the differences were statistically significant (all P values <0.05). Compared with the DM-I/R group, the myocardial infarction volume in the DMS-I/R group decreased, and the difference was statistically significant (P<0.05). (2) Pathological examination of myocardial tissue. The N-S group had a complete myocardial tissue structure, neatly and densely arranged myocardial cells, and uniformly stained cytoplasm. The DM-S group had a slightly disordered myocardial fiber arrangement, slightly swollen cytoplasm, and mild inflammatory cell infiltration. The N-I/R group had irregular cell morphology; disordered or broken myocardial fibers; swollen or ruptured cytoplasm; and misaligned, fragmented, or dissolved nuclei. The DM-I/R group had a deeper degree of myocardial pathological damage than the N-I/R group, broken myocardial fibers, disintegrated myocardial, dissolved nucleus, deep staining, and a large number of inflammatory cell infiltration. Compared with the N-I/R and DM-I/R groups, the NS-I/R and DMS-I/R groups had restored myocardial cell morphology and myocardial fiber arrangement and greatly reduced inflammatory cell infiltration. (3) Ultrastructure of myocardial tissue under electron microscope. The N-S group had normal morphology and number of mitochondria and had no autophagosomes. The DM-S group had autophagosomes. In the N-I/R group, the number of normal mitochondria in the cardiomyocytes was reduced, the mitochondria were remarkably swollen and contained many autophagosomes, and phagosomes and some autophagosomes encapsulated undegraded mitochondrial structure and residual cellular components. Compared with the N-I/R group, the NS-I/R group had a relatively complete mitochondrial structure, slightly swollen mitochondria, and less damage. The DM-I/R group had abnormal mitochondrial crista structure, mitochondrial crista separation, swelling and dissolution, and mitochondrial area/perimeter ratio increased, mitochondrial autophagy function was impaired, and autophagosomes increased. Compared with the DM-I/R group, the DMS-I/R group had a more regular arrangement of muscle fibers, decreased number of abnormal mitochondrial crest, and decreased number of autophagosomes. (4) Protein expression levels of Bmal1, Sirt3, Nlrp3, and Bnip3 in myocardial tissue. Compared with the N-S group, Bmal1 and Sirt3 protein expression showed a downward trend, whereas Nlrp3 and Bnip3 protein expression showed an upward trend in the N-I/R, NS-I/R, DM-S, DM-I/R, and DMS-I/R groups, and the differences were statistically significant (all P values <0.05). Compared with the DM-S group, the expression of Bmal1 and Sirt3 proteins was downregulated and the expression of Nlrp3 and Bnip3 proteins was upregulated in the DM-I/R group; the expression of Nlrp3 protein was downregulated and the expression of Bnip3 protein was upregulated in the DMS-I/R group; and the differences were statistically significant (all P values <0.05). Compared with the DM-I/R group, the Bmal1, Sirt3, and Bnip3 protein expression were upregulated and the Nlrp3 protein expression was downregulated in the DMS-I/R group, and the differences were statistically significant (all P values <0.05). Conclusion Bmal1 has a protective effect on the myocardium during myocardial ischemia-reperfusion injury in diabetic rats. Its protective mechanism may be related to the activation of the Sirt3 pathway to induce mitochondrial autophagy and inhibit inflammatory response.
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