Combination Therapy of Alpha-Lipoic Acid, Gliclazide and Ramipril Protects Against Development of Diabetic Cardiomyopathy via Inhibition of TGF-β/Smad Pathway

Abstract

Background: Diabetic cardiomyopathy (DCM) is a major long-term complication of diabetes mellitus, accounting for over 20% of annual mortality rate of diabetic patients globally. Although several existing anti-diabetic drugs have improved glycemic status in diabetic patients, prevalence of DCM is still high. This study investigates cardiac effect of alpha-lipoic acid (ALA) supplementation of anti-diabetic therapy in experimental DCM. Methods: Following 12 h of overnight fasting, 44 male Sprague Dawley rats were randomly assigned to two groups of healthy control (n = 7) and diabetic (n = 37) groups, and fasting blood glucose was measured. Type 2 diabetes mellitus (T2DM) was induced in diabetic group by intraperitoneal (i.p.) administration of nicotinamide (110 mg/kg) and streptozotocin (55 mg/kg). After confirmation of T2DM on day 3, diabetic rats received monotherapies with ALA (60 mg/kg; n = 7), gliclazide (15 mg/kg; n = 7), ramipril (10 mg/kg; n = 7) or combination of the three drugs (n = 7) for 6 weeks while untreated diabetic rats received distilled water and were used as diabetic control (n = 9). Rats were then sacrificed, and blood, pancreas and heart tissues were harvested for analyses using standard methods. Results: T2DM induction caused pancreatic islet destruction, hyperglycemia, weight loss, high relative heart weight, and development of DCM, which was characterized by myocardial degeneration and vacuolation, cardiac fibrosis, elevated cardiac damage markers (plasma and cardiac creatine kinase-myocardial band, brain natriuretic peptide and cardiac troponin I). Triple combination therapy of ALA, gliclazide and ramipril preserved islet structure, maintained body weight and blood glucose level, and prevented DCM development compared to diabetic control (p < 0.001). In addition, the combination therapy markedly reduced plasma levels of inflammatory markers (IL-1β, IL-6 and TNF-α), plasma and cardiac tissue malondialdehyde, triglycerides and total cholesterol while significantly increasing cardiac glutathione and superoxide dismutase activity and high-density lipoprotein-cholesterol compared to diabetic control (p < 0.001). Mechanistically, induction of T2DM upregulated cardiac expression of TGF-β1, phosphorylated Smad2 and Smad3 proteins, which were downregulated following triple combination therapy (p < 0.001). Conclusion: Triple combination therapy of ALA, gliclazide and ramipril prevented DCM development by inhibiting TGF-β1/Smad pathway. Our findings can be extrapolated to the human heart, which would provide effective additional pharmacological therapy against DCM in T2DM patients.

Keywords: alpha-lipoic acid (ALA); anti-diabetic therapy; diabetic cardiomyopathy (DCM); triple combination therapy; type 2 diabetes mellitus (T2DM).

FIGURE 1

Changes in body weight, HbA1c level and pancreas histology. (A) Percentage change in body weight, (B) HbA1c level and (C) representative photomicrographs of PAS-stained images of pancreatic tissues from all groups. Arrows point to damaged pancreatic islet. Magnification ×400. Healthy control (n = 7); DM Untreated = Untreated diabetes mellitus rats (n = 9); DM + GLC = Diabetic rats treated with gliclazide (n = 7); DM + ALA = Diabetic rats treated with alpha-lipoic acid (n = 7); DM + RAM = Diabetic rats treated with ramipril (n = 7); DM + ALA + GLC + RAM = Diabetic rats treated with alpha-lipoic acid, gliclazide and ramipril (n = 7). *p < 0.05 vs. diabetic control, **p < 0.01 vs. diabetic control, ***p < 0.001 vs. diabetic control.

FIGURE 2

Cardiac histology and damage markers (A) Representative photomicrograph of PAS-stained cardiac tissue from all groups. Arrows indicate cardiomyocyte vacuolation and arrow heads indicate myocardial degeneration. ×400 magnification in PAS staining. (B) Quantification of cardiomyocyte vacuolation, (C) relative heart weight, (D) plasma cardiac troponin I (cTnI), (E) plasma brain natriuretic peptide (BNP), (F) cardiac creatine kinase myoglobin band (CK-MB) and (G) plasma creatine kinase myoglobin band (CK-MB). Healthy control (n = 7); DM Untreated = Untreated diabetes mellitus rats (n = 9); DM + GLC = Diabetic rats treated with gliclazide (n = 7); DM + ALA = Diabetic rats treated with alpha-lipoic acid (n = 7); DM + RAM = Diabetic rats treated with ramipril (n = 7); DM + ALA + GLC + RAM = Diabetic rats treated with alpha-lipoic acid, gliclazide and ramipril (n = 7). *p < 0.05 vs. diabetic control, **p < 0.01 vs. diabetic control, ***p < 0.001 vs. diabetic control.

FIGURE 3

Lipid profile and inflammation. Levels of (A) plasma plasma triglycerides, (B) total cholesterol, (C) plasma high-density lipoprotein (HDL), (D) plasma interleukin-1beta (IL-1β), (E) plasma interleukin-6 (IL-6) and (F) plasma tumor necrosis factor-alpha (TNF-α). Healthy control (n = 7); DM Untreated = Untreated diabetes mellitus rats (n = 9); DM + GLC = Diabetic rats treated with gliclazide (n = 7); DM + ALA = Diabetic rats treated with alpha-lipoic acid (n = 7); DM + RAM = Diabetic rats treated with ramipril (n = 7); DM + ALA + GLC + RAM = Diabetic rats treated with alpha-lipoic acid, gliclazide and ramipril (n = 7). *p < 0.05 vs. diabetic control, **p < 0.01 vs. diabetic control, ***p < 0.001 vs. diabetic control.

FIGURE 4

Cardiac antioxidant status. Levels of (A) cardiac tissue malondialdehyde (MDA), (B) plasma malondialdehyde (MDA), (C) cardiac glutathione (GSH) content and (D) cardiac superoxide dismutase (SOD) activity. Healthy control (n = 7); DM Untreated = Untreated diabetes mellitus rats (n = 9); DM + GLC = Diabetic rats treated with gliclazide (n = 7); DM + ALA = Diabetic rats treated with alpha-lipoic acid (n = 7); DM + RAM = Diabetic rats treated with ramipril (n = 7); DM + ALA + GLC + RAM = Diabetic rats treated with alpha-lipoic acid, gliclazide and ramipril (n = 7). *p < 0.05 vs. diabetic control, **p < 0.01 vs. diabetic control, ***p < 0.001 vs. diabetic control.

FIGURE 5

Cardiac fibrosis showing (A) Western blot image, and quantification of (B) transforming growth factor-beta 1 (TGF-β1), (C) phosphorylated Smad2 and (D) phosphorylated Smad3. Healthy control (n = 7); DM Untreated = Untreated diabetes mellitus rats (n = 9); DM + GLC = Diabetic rats treated with gliclazide (n = 7); DM + ALA = Diabetic rats treated with alpha-lipoic acid (n = 7); DM + RAM = Diabetic rats treated with ramipril (n = 7); DM + ALA + GLC + RAM = Diabetic rats treated with alpha-lipoic acid, gliclazide and ramipril (n = 7). *p < 0.05 vs. diabetic control, **p < 0.01 vs. diabetic control, ***p < 0.001 vs. diabetic control.