Modified Linggui Zhugan Decoction protects against ventricular remodeling through ameliorating mitochondrial damage in post-myocardial infarction rats

Figures

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  FIGURE 1. The ingredients of Modified Linggui Zhugan Decoction (MLZD) were identified, and 7 active components were finally obtained.

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  FIGURE 2. Modified Linggui Zhugan Decoction (MLZD) exerted protective effects on cardiac morphological abnormalities and dysfunctions. (A) Representative images of echocardiographic measurements in three groups. (B) Quantitative analysis of cardiac structure and function via echocardiography parameters, n = 9 or 10 per group. Data are mean ± standard deviation (x¯ ± s); **P < 0.01, ***P < 0.001, *⁣***P < 0.0001.

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  FIGURE 3. Modified Linggui Zhugan Decoction (MLZD) ameliorated cardiac fibrosis and reversed ventricular remodeling. (A) The appearance of hearts in three groups. (B) Ratios of heart weight to total body weight. (C) Representative images of HE and Masson trichrome staining, scale bar = 200 μm. (D) Quantification of myocardial fibrosis by fibrosis area, n = 3 per group. Data are presented as mean ± standard deviation (x¯ ± s); ***P < 0.001, ****P < 0.0001.

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  FIGURE 4. Modified Linggui Zhugan Decoction (MLZD) ameliorated apoptosis. (A) TUNEL staining for heart tissue, scale bar = 50 μm. (B) Quantitative analysis for apoptosis, n = 6 or 8 per group. (C,D) Immunoblot analyses of Bax, Bcl-2, Cyt c, and cleaved-caspase-3 in the hearts of different groups. The widths of the images have been compressed at a ratio of 1:0.6. n = 4 or 6 per group. (E,F) Immunohistochemical analyses for detecting Bax and Bcl-2 expression, n = 3 per group, scale bar = 100 μm. Data are represented as mean ± standard deviation (x¯ ± s); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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  FIGURE 5. Modified Linggui Zhugan Decoction (MLZD) inhibits the expression of inflammatory proteins. (A,B) Western blot analyses of the expression of NLRP3, caspase-1, ASC, IL-1β, NFκB p65, and NFκB p50 in cardiac tissue. The widths of the images have been compressed at a ratio of 1:0.6. n = 3 or 4 per group. Data are represented as mean ± standard deviation (x¯ ± s); *P < 0.05, **P < 0.01.

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  FIGURE 6. Modified Linggui Zhugan Decoction (MLZD) repairs mitochondrial damage. (A) The representative transmission electron microscope (TEM) images of mitochondria in the hearts at a magnification of 8.0k and 3.0k, respectively (Scale bar = 0.5 or 1 μm). (B) The mitochondrial number changes and the average mitochondrial area (μm2) under TEM in heart tissue, n = 20 visual fields (5 visual fields per rat, 4 rats per group). (C,D) Western blot analyses of the expression of p-Drp1, Mfn2, PGC-1α, and PPAR-γ in cardiac tissue. The widths of the images have been compressed at a ratio of 1:0.6. n = 4 per group. (E,F) Relative expression of p-Drp1 and Mfn2 in heart detected through immunohistochemical analyses, n = 3 per group, scale bar = 100 μm. (G,H) Western blot analyses of SOD2 expression in the heart. The widths of the images have been compressed at a ratio of 1:0.6. n = 4 per group. (I) Representative images of TMRM staining captured via fluorescent microscopy, scale bar = 200 μm. (J) Quantitation of fluorescence intensity in different groups, n = 3 per group. Data are mean ± standard deviation (x¯ ± s); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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  FIGURE 7. Modified Linggui Zhugan Decoction (MLZD) promoted SIRT3 expression in cardiac tissue. (A,B) Relative expression of SIRT3 detected via immunoblot analyses. The widths of the images have been compressed at a ratio of 1:0.6. n = 4 per group. (C,D) Immunohistochemical analysis for detecting SIRT3, n = 3 per group, scale bar = 100 μm. Data are represented as mean ± standard deviation (x¯ ± s); *P < 0.05, **P < 0.01, ***P < 0.001.

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  FIGURE 8. 3-TYP inhibited SIRT3 expression and prevented the cardiomyocyte protective effect of MLZD. (A,B) Relative expression of SIRT3 in cardiomyocytes detected via western blot. The widths of the images have been compressed at a ratio of 1:0.8. (C,D) Immunohistochemical analysis for detecting SIRT3, scale bar = 100 μm. (E) The cell viability in seven groups evaluated through the CCK-8 test kit. Data are represented as mean ± standard deviation (x¯ ± s); **P < 0.01, ****P < 0.0001 vs. CON; #P < 0.05, ##P < 0.01, ####P < 0.0001 vs. OGD; $$$P < 0.001, $$$$P < 0.0001, vs. OGD+MLZD.

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  FIGURE 9. 3-TYP reversed the regulation of mitochondria by Modified Linggui Zhugan Decoction (MLZD). (A,B) Detection of Drp1, p-Drp1, Mfn2, PGC-1α, and COX IV in cardiomyocytes by western blotting. The widths of the images have been compressed at a ratio of 1:0.8. (C,D) Detection of mitochondrial electron transport chain complex I and IV in cardiomyocytes. n = 8 per group. (E) Determination of ATP content in cardiomyocytes. n = 8 per group. (F,G) TMRM staining for assessing mitochondrial membrane potential, n = 3 per group, scale bar = 200 μm. Data are represented as mean ± standard deviation (x¯ ± s); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. CON; #P < 0.5, ##P < 0.01, ####P < 0.0001 vs. OGD; $P < 0.05, $$P < 0.01, $$$$P < 0.0001 vs. OGD + MLZD.

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  FIGURE 10. The mechanisms of Modified Linggui Zhugan Decoction (MLZD) in ventricular remodeling of left anterior descending coronary artery ligation-induced myocardial infarction models. Modified Linggui Zhugan Decoction (MLZD) intervention could exert protective effects on mitochondria, including enhancing mitochondrial antioxidant capacity, regulating mitochondrial dynamics disorder, and inhibiting mitochondrial membrane potential collapse, which contributed to preventing mitochondrial-mediated cardiomyocyte apoptosis. The heart-protective effect of MLZD might associate with SIRT3 activation in the cardiac tissue. MI, myocardial infarction; MMP, mitochondrial membrane potential; SIRT3, sirtuin 3; SOD2, superoxide dismutase 2; ROS, reactive oxygen species; Mfn2, mitofusin2; Drp1, dynamin-related protein 1; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator-1α; Cyt c, cytochrome c; PPAR-γ, peroxisome proliferator-activated receptor – γ; Bcl-2, B cell lymphoma-2; Bax, Bcl-2-associated X; Apaf-1, apoptotic peptidase activating factor 1.