Guan Xin Dan Shen formulation protects db/db mice against diabetic cardiomyopathy via activation of Nrf2 signaling

Abstract

Guan Xin Dan Shen formulation (GXDSF) is a widely used treatment for the management of coronary heart disease in China and is composed of three primary components: Dalbergiae odoriferae Lignum, Salviae miltiorrhizae Radix et Rhizoma and Panax notoginseng Radix et Rhizoma. However, the potential use of GXDSF for the management of diabetic cardiomyopathy (DCM) has not been previously assessed. The present study aimed to assess the effects of GXDSF on DCM, as well as the underlying mechanism. In the present study, db/db mice were used. Following treatment with GXDSF for 10 weeks, fasting blood glucose, insulin sensitivity, serum lipid levels and cardiac enzyme levels were detected. Cardiac pathological alterations and cardiac function were assessed by performing hematoxylin and eosin staining and echocardiograms, respectively. TUNEL assays were conducted to assess cardiomyocyte apoptosis. Additionally, reverse transcription‑quantitative PCR and western blotting were performed to evaluate the expression of apoptosis‑associated genes and proteins, respectively. In the model group, the db/db mice displayed obesity, hyperlipidemia and hyperglycemia, accompanied by noticeable myocardial hypertrophy and diastolic dysfunction. Following treatment with GXDSF for 10 weeks, serum triglyceride levels were lower and insulin sensitivity was enhanced in db/db mice compared with the model group, which indicated improvement in condition. Cardiac hypertrophy and dysfunction were also improved in db/db mice following treatment with GXDSF, resulting in significantly increased left ventricular ejection fraction and fractional shortening compared with the model group. Following treatment with metformin or GXDSF, model‑induced increases in levels of myocardial enzymes were decreased in the moderate and high dose groups. Moreover, the results indicated that, compared with the model group, GXDSF significantly inhibited cardiomyocyte apoptosis in diabetic heart tissues by increasing Bcl‑2 expression and decreasing the expression levels of Bax, cleaved caspase‑3 and cleaved caspase‑9. Mechanistically, GXDSF enhanced Akt phosphorylation, which upregulated antioxidant enzymes mediated by nuclear factor erythroid 2‑related factor 2 (Nrf2) signaling. Collectively, the results of the present study indicated that GXDSF attenuated cardiac dysfunction and inhibited cardiomyocyte apoptosis in diabetic mice via activation of Akt/Nrf2 signaling. Therefore, GXDSF may serve as a potential therapeutic agent for the management of DCM.

Keywords: Guan Xin Dan Shen formulation; diabetes mellitus; diabetic cardiomyopathy; heart; myocardial apoptosis.

Figure 1.

Effects of GXDSF on body weight and insulin sensitivity in db/db mice. (A) Schematic diagram indicating the timeline of the in vivo animal experiments. (B) Body weight and (C) fasting blood glucose levels in each group. Insulin sensitivity was evaluated by performing the intraperitoneal ITT and (D) quantified and (E) expressed as AUC. Data are represented as the mean ± SD (n=10). ^##P<0.01 vs. control; *P<0.05 and **P<0.01 vs. model. GXDSF, Guan Xin Dan Shen formulation; ITT, insulin tolerance test; AUC, area under the curve; FBG, fasting blood glucose.

Figure 2.

Effects of GXDSF on serum lipid levels in db/db mice. Serum (A) TG, (B) TC and (C) LDL levels in each group. Data are expressed as the mean ± SD (n =10). ^##P<0.01 vs. control; *P<0.05 and **P<0.01 vs. model. GXDSF, Guan Xin Dan Shen formulation; TG, triglyceride; TC, total cholesterol; LDL, low-density lipoprotein cholesterol.

Figure 3.

Effects of GXDSF on the cardiac function of db/db mice. (A) Echocardiograms were obtained via echocardiography. (B) LVIDd, (C) LVIDs, (D) % EF and (E) % FS. Data are presented as the mean ± SD (n=5). ^#P<0.05 vs. control; *P<0.05 and **P<0.01 vs. model. GXDSF, Guan Xin Dan Shen formulation; LVIDd, left ventricular end diastolic diameter; LVIDs, left ventricular end systolic diameter; % EF, percentage ejection fraction; % FS, percentage fractional shortening.

Figure 4.

Effect of GXDSF on pathological alterations to the heart in db/db mice. (A) Representative images of hearts isolated from db/db mice in each group. (B) Heart weight of db/db mice in each group. (C) Representative images of hematoxylin and eosin staining for identification of the pathological structure of the heart (n=5; scale bar, 50 µm). (D) Reverse transcription-quantitative PCR was performed to measure the impact of GXDSF (high-dose) on the mRNA expression levels of cardiac hypertrophy-related genes. Serum (E) CK-MB, (F) LDH and (G) AST levels in db/db mice. Data are presented as the mean ± SD (n=10). ^##P<0.01 vs. control; *P<0.05 and **P<0.01 vs. model. GXDSF, Guan Xin Dan Shen formulation; CK-MB, creatine kinase MB; LDH, lactate dehydrogenase; AST, aspartate transaminase; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; α-MHC, α-myosin heavy chain; β-MHC, β-myosin heavy chain.

Figure 5.

Effect of GXDSF on cardiac apoptosis in each group. Cardiac apoptosis was (A) determined by performing TUNEL staining (magnification, ×200; scale bar, 50 µm) and (B) quantified in each group (n=5). Reverse transcription-quantitative PCR was performed to measure the impact of GXDSF (high-dose) on the mRNA expression of apoptosis-related genes, including (C) Bcl-2, (D) Bax, (E) caspase-3 and (F) caspase-9 (n=10). Protein expression levels were (G) determined via western blotting and semi-quantified for (H) Bcl-2/Bax, (I) cleaved caspase-3 and cleaved caspase-9 (n=3). Data are presented as the mean ± SD. ^##P<0.01 and ^###P<0.001 vs. control; *P<0.05, **P<0.01 and ***P<0.001 vs. model. GXDSF, Guan Xin Dan Shen formulation.

Figure 6.

GXDSF-induced activation of the Akt-mediated Nrf2 signaling pathway. Western blotting was performed to determine the impact of high dose of GXDSF on the protein expression of (A) p-Akt, (B) cytoplasmic Nrf2, (C) nuclear Nrf2, (D) HO-1, (E) NQO-1 and (F) γ-GCS (n=3). (G) Schematic illustration indicating the mechanism underlying the protective effects of GSDXF against diabetic cardiomyopathy. Data are presented as the mean ± SD. ^#P<0.05 and ^##P<0.01 vs. control; *P<0.05 and **P<0.01 vs. model. GXDSF, Guan Xin Dan Shen formulation; p, phosphorylated; Nrf2, nuclear factor-erythroid 2-related factor 2; HO-1, heme oxygenase-1; γ-GCS, γ-glutamylcysteine synthetase heavy subunit; NQO-1, NAD(P)H quinone oxidoreductase-1; NS, not significant; cyto, cytoplasmic; nucl, nuclear; Keap1, kelch like ECH associated protein.