Abstract
Diabetic cardiomyopathy (DCM), a common complication of diabetes mellitus, may eventually leads to irreversible heart failure. Metformin is the cornerstone of diabetes therapy, especially for type 2 diabetes. Statins are widely used to reduce the risk of cardiovascular diseases. In this study, we aimed to investigate whether the combined administration of metformin and atorvastatin could achieve superior protective effects on DCM and to elucidate its molecular mechanism. Here, db/db mice (9–10 weeks old) were randomly divided into four groups, including sterile water group (DM), metformin group (MET, 200 mg/kg/day), atorvastatin group (AVS, 10 mg/kg/day), and combination therapy group (MET + AVS). Mice were treated with different drugs via gavage once per day for 3 months. After 3 months of treatment, the pathological changes (inflammation, fibrosis, hypertrophy, and oxidative stress makers) were detected by histopathological techniques, as well as Western blotting. The H9C2 cardiomyocytes were treated with palmitate (PAL) to mimic diabetic condition. The cells were divided into control group, PAL treatment group, MET + PAL treatment group, AVS + PAL treatment group, and MET + AVS + PAL treatment group. The effects of MET and AVS on the cell viability and inflammation of H9C2 cells subjected to PAL condition were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, immunofluorescence staining, and Western blotting. Both MET and AVS prevented diabetes-induced fibrosis, hypertrophy, and inflammation. The combination therapy showed superior effects in protecting myocardial tissue against diabetes-induced injury. Mechanistically, the combination therapy significantly inhibited oxidative stress and the expression levels of inflammation-related proteins, e.g., NLRP3, caspase-1, interleukin-1β (IL-1β), Toll-like receptor 4 (TLR4), and P-p65/p65, in both cardiac tissues and H9C2 cells. TUNEL assay showed that the combination therapy significantly attenuated the apoptosis of cardiomyocytes; decreased the expression level of pro-apoptotic-related proteins, such as cleaved caspase-3 and BAX; and enhanced the expression level of anti-apoptotic protein (Bcl-2). Furthermore, the combination therapy remarkably upregulated the expression levels of 5′-AMP-activated protein kinase (AMPK) and SIRT1. Our findings indicated that the anti-inflammation and anti-apoptosis effects of the combination therapy may be related to activation of AMPK/SIRT1 signaling pathway.
Highlights
Clinical trials showed that the prevalence of myocardial dysfunction in diabetic patients varies from 19 to 26%, and the outcomes associated with myocardial dysfunction are remarkably worse in patients with diabetes than in those without diabetes (Jia et al, 2018)
The results showed that palmitate significantly increased the apoptosis of H9C2 cells apoptosis; besides, combination of metformin and atorvastatin could inhibit palmitate induced-apoptosis of H9C2 cells; as a result, the combined use of metformin and atorvastatin significantly decreased the apoptotic rate of H9C2 compared with treatment with either one
The results showed that the protein expression level of Toll-like receptor 4 (TLR4) and the phosphorylation level of NF-κB p65 were upregulated in H9C2 cells exposed to palmitate, while combined therapy with metformin and atorvastatin treatment remarkably inhibited the increase of the above-mentioned expression levels; the effects of combination treatment are more significant than either application of metformin or atorvastatin (Figure 3B)
Summary
Clinical trials showed that the prevalence of myocardial dysfunction in diabetic patients varies from 19 to 26%, and the outcomes associated with myocardial dysfunction are remarkably worse in patients with diabetes than in those without diabetes (Jia et al, 2018). A commonly used drug to treat diabetes for over 50 years, is significant to alleviate DCM. The positive effects of metformin on DCM are mainly associated with activation of 5′-AMP-activated protein kinase (AMPK) (Min et al, 2018), thereby, improving cardiac energy metabolism, as well as protecting heart against diabetic conditions. Statins could prevent DCM by alleviating left ventricular dysfunction and inhibiting myocardial fibrosis through anti-apoptosis and anti-inflammation pathways (AbdelHamid and Firgany, 2015; Al-Rasheed et al, 2017). A single therapeutic strategy may fail, or it only partially prevents or treats DCM; for instance, intensive glucose control in a clinic failed to protect against heart under diabetic conditions (Group, 1998)
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