Investigating the combined effect of alpha lipoic acid and ischemic-postconditioning on cardioprotection and expression of proteins involved in fibrosis and apoptosis in myocardial infarction of type II diabetic rats

Abstract

Ischemia-reperfusion (IR) injury is one of the main causes of mortality. Preventing this damage in the diabetic heart is associated with many challenges. Diabetes is a major limiting factor for cardioprotective interventions. In this study, the effect of combined treatment with alpha lipoic acid (ALA) and Post on cardiac protection and signaling pathway of fibrosis and apoptosis in myocardial infarction of type II diabetic rats was investigated. Methods: Male Wistar rats were divided into five diabetic groups (twelve rats in each group). Diabetes was induced in rats for 12 weeks with a high-fat diet and also by injecting a 35 mg dose of streptozotocin. Pretreatment with alpha-lipoic acid by gavage (100 mg/kg/day) was performed for five weeks before induction of ischemia-reperfusion. The hearts were isolated and immediately placed on a Langendorff cardiac perfusion system. In this device, IR injury was induced by occluding the LAD coronary artery for 35 minutes and reopening it for 60 minutes. The Post protocol was applied immediately at the beginning of reperfusion in the hearts by the method of 6 cycles of 10 seconds of ischemia and 10 seconds of reperfusion. The tests are done in two groups. A grouping consisting of six rats in each group was used to investigate the infarct size and in another group six rats were examined for other physiological, histological and molecular studies. Cardiac infarction was measured with Evans Blue dye and TTC. Masson's trichrome staining was used to investigate tissue fibrosis. TGF-β and Smad3 proteins were analyzed by western blot method. LDH and BNP cardiac enzyme levels were investigated using ELISA and spectrophotometry. Tissue apoptosis was investigated using TUNEL test and expression of proteins involved in apoptosis (cleaved caspase-3, Bax and Bcl2) by western blot method. In this study, hematoxylin-eosin staining was used to examine the morphological changes of cells. Results: The Post protocol alone had no significant effect on improving the severity of left ventricular fibrosis, reducing apoptosis, and reducing infarct size. While pretreatment with alpha lipoic acid had a positive effect. However, application of Post in ALA-treated rats more effectively reduced injury markers compared to the untreated IR group. In addition, the combined treatment caused a further decrease in the expression of Bax, cleaved caspase-3, Smad3 and TGF-β and a further increase in the expression of Bcl2, which was associated with a decrease in IS and LDH levels compared to the untreated IR group. Also, histopathological findings were better in combination treatment than monotherapy.