The mechanisms and therapeutic potential of clopidogrel in mitigating diabetic cardiomyopathy in db/db mice

Abstract

Clopidogrel has been shown to play a protective role against diabetic nephropathy. However, whether clopidogrel exerts a protective effect against diabetic cardiomyopathy (DCM) is unknown. Three-month-old male db/db mice were administered clopidogrel daily at doses of 5, 10, and 20 mg/kg by gavage for 5 months. Here, we showed that clopidogrel effectively attenuated diabetes-induced cardiac hypertrophy and cardiac dysfunction by inhibiting cardiac fibrosis, inflammatory responses, and oxidative stress damage in db/db mice. Diabetes-induced cardiac fibrosis was inhibited by clopidogrel treatment via blockade of the TGF-β1/Smad3/P2RY12 pathway and inhibition of macrophage infiltration in db/db mice. The protective effects of clopidogrel against oxidative damage were mediated by the induction of the Nrf2 signaling pathway. Taken together, our findings provide strong evidence that clopidogrel is a promising effective agent for the treatment of DCM by alleviating diabetes-induced cardiac hypertrophy and dysfunction. P2RY12 might be an effective target for the treatment of DCM.

Keywords: Biological sciences; Diabetology; Endocrinology; Natural sciences; Pathophysiology; Pharmacology; Physiology.

Graphical abstract

Figure 1

Physical and biochemical characteristics of the mice (A) Body weight of the mice (n = 6 per group). (B) Fasting blood glucose levels of the mice (n = 6 per group). (C) Serum triglyceride and total cholesterol levels of the mice (n = 5 per group). (D) Blood clotting time of the mice (n = 6 per group). (E) Heart weight/tibial length ratio (HW/TL) showing the relative heart weight (n = 6 per group). The data are presented as the mean ± SEM. Multiple comparisons were conducted with one-way ANOVA, followed by Tukey’s pairwise test. ∗∗∗p < 0.001 vs. the CON group; ###p < 0.001 vs. the DM group; ns (no significant difference).

Figure 2

Clo decreases DM-induced cardiac dysfunction and cardiac hypertrophy (A) Representative echocardiography and cardiac parameters measured via echocardiography (n = 5 per group). (B) WGA staining of the cardiac tissue for assessing cardiomyocyte size in cross-sections of the heart (n = 5 per group). Scale bar: 40 μm. (C) The protein and mRNA expression levels of MYH7 were used to evaluate cardiac hypertrophy (n = 6 per group). (D) The protein and mRNA expression levels of ANP were used to evaluate cardiac function (n = 6 per group). (E) Serum LDH, CK, and CK-MB levels in the mice (n = 5 per group). The data are presented as the mean ± SEM. Multiple comparisons were conducted with one-way ANOVA, followed by Tukey’s pairwise test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. the CON group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. the DM group; ns (no significant difference).

Figure 3

Negative correlation between Clo dosage and DM-induced cardiac fibrosis (A) Representative images of Masson’s trichrome staining followed by quantification of the percent positive area (n = 6 per group). Scale bar: 40 μm. (B) Western blotting and RT-qPCR analysis of α-SMA, COL1A1, COL3A1, and Fibronectin, and quantitation of their expression (n = 6 per group). (C) TGF-β1 and CTGF were analyzed using western blotting and RT-qPCR (n = 6 per group). The data are presented as the mean ± SEM. Multiple comparisons were conducted with one-way ANOVA, followed by Tukey’s pairwise test. ∗∗∗p < 0.001 vs. the CON group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. the DM group; ns (no significant difference).

Figure 4

The TGF-β1/Smad pathway is involved in Clo-induced decreases in DM-induced cardiac fibrosis (A) The levels of p-Smad2, Smad2, p-Smad3, and Smad3 were analyzed via western blotting, and the ratios of p-Smad2 to Smad2 and p-Smad3 to Smad3 were quantified (n = 4 per group). (B) P2RY12 was analyzed using western blotting and RT-qPCR (n = 6 per group). (C) Western blotting of ERK, p-ERK, JNK, p-JNK, P38, and p-P38 and quantitative analysis of the ratios of p-ERK to ERK, p-JNK to JNK, and p-P38 to P38 (n = 4 per group). The data are presented as the mean ± SEM. Multiple comparisons were conducted with one-way ANOVA, followed by Tukey’s pairwise test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. the CON group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. the DM group; ns (no significant difference).

Figure 5

Reduced DM-induced cardiac macrophage infiltration and cardiac inflammatory responses are implicated in Clo effectiveness (A) Representative fluorescence images of F4/80 and quantification of F4/80 expression. The white arrows indicate F4/80-positive cells (n = 5 per group). Scale bar: 20 μm. (B) Western blotting and mRNA expression of TNF-α, IL-1β, and IL-6 (n = 6 per group). (C) Western blotting and mRNA levels of MCP-1, ICAM1, and VCAM1 (n = 5–6 per group). (D) Serum levels of TNF-α, IL-1β, IL-6, and MCP-1 in mice (n = 6 per group). The data are presented as the mean ± SEM. Multiple comparisons were conducted with one-way ANOVA, followed by Tukey’s pairwise test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. the CON group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. the DM group; ns (no significant difference).

Figure 6

Clo-induced Nrf2 pathway activation prevents DM-induced cardiac oxidative stress damage (A) Western blotting and quantitative analysis of nuclear Nrf2 (nNrf2), Keap1, CAT, HO-1, and NQO-1 (n = 5–6 per group). (B) RT-qPCR and quantitative analysis of Nrf2, CAT, HO-1, and NQO-1 expression (n = 6 per group). (C) The levels of SOD, CAT, GSH, MDA, and 3-NT in heart tissues (n = 5 per group). The data are presented as the mean ± SEM. Multiple comparisons were conducted with one-way ANOVA, followed by Tukey’s pairwise test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. the CON group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. the DM group; ns (no significant difference).

Figure 7

Mechanism for the protective effects of clopidogrel on DCM T2DM-induced inflammatory responses, ROS accumulation, and cardiac fibrosis are hallmark mediators of DCM development. Clopidogrel attenuates DM-induced ROS accumulation by activating Nrf2 and its downstream effects (HO-1, NQO-1, SOD, and CAT). Clopidogrel attenuated the DM-induced increase in macrophage infiltration and hyperactive platelets by inhibiting P2RY12. Along these lines, the DM-induced increase in the expression of TGF-β1, which is partly secreted by macrophages and platelets, was inhibited by clopidogrel treatment. Diabetes-induced cardiac fibrosis can be alleviated by clopidogrel treatment via repression of the TGF-β1/Smad3/P2RY12 pathway.