Panax notoginseng flower protects against diabetic cardiomyopathy by regulating the ACSL4/ALOX15 pathway

Figures

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  FIGURE 1. Total ion chromatography of UPLC–QTOF–MS/MS analysis of SQH in negative mode.

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  FIGURE 2. SQH protected H9c2 cells against PA-induced oxidative stress. (A) Cell viability under different PA concentrations. (B) Cell viability under 0.2 mM PA with SQH treatment (bar = 100 μm). (C) Fluorescence staining of ROS (green) in H9c2 cells treated with SQH (100 and 200 μg/mL) or PBS (bar = 100 μm). The relative fluorescence intensity was analyzed using the ratio of green fluorescence intensity to the number of total cells in three randomly selected areas. (D) MDA level in H9c2 cells treated with SQH (100 and 200 μg/mL) or PBS. (E) SOD enzyme activity in H9c2 cells treated with SQH (100 and 200 μg/mL) or PBS. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. PA (+) group.

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  FIGURE 3. Protective effect of SQH was potentially mediated by the inhibition of ferroptosis. (A) PCA between the three groups (CON, PA, and SQH). (B) Venn diagram of differentially expressed genes. (C) KEGG enrichment of the top 20 signaling pathway. (D) Summary heatmap of quantitative RNA-seq analysis of genes related with ferroptosis in H9c2 cells treated with 100 μg/mL SQH or PBS. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. PA (+) group.

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  FIGURE 4. SQH treatment inhibited ferroptosis by alleviating lipid peroxidation. (A) GSH level in H9c2 cells treated with SQH (100 and 200 μg/mL) or PBS. (B) Fluorescence staining of BODIPY (green) in H9c2 cells treated with SQH (100 and 200 μg/mL) or PBS (bar = 100 μm). The relative fluorescence intensity was analyzed using the ratio of green fluorescence intensity to the number of total cells in three randomly selected areas. (C) Protein expressions for ALOX15 in SQH-treated H9c2 cells. (D) 12-HETE level of H9c2 cells under 0.2 mM PA. (E) Protein expressions for ALOX15 in SQH-treated H9c2 cells. (F) Protein expressions for GPX4 in SQH-treated H9c2 cells. (G) Quantitative RT-PCR analysis of genes Alox15, Acsl4, and Pgc1a. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. PA (+) group.

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  FIGURE 5. SQH protected mitochondrial function through PGC-1α. (A) Fluorescence staining of MitoPeDPP in H9c2 cells treated with SQH (100 and 200 μg/mL) or PBS (bar = 100 μm). The relative fluorescence intensity was analyzed using the ratio of green fluorescence intensity to the number of total cells in three randomly selected areas. (B) ATP levels in H9c2 cells treated with SQH (100 and 200 μg/mL) or PBS. (C) Western blots of Cyto c in H9c2 cells. (D) Fluorescence staining of TMRE (red) treated with SQH (100 and 200 μg/mL) or PBS. The relative fluorescence intensity was analyzed using the ratio of red fluorescence intensity to the number of total cells in three randomly selected areas. (E) Protein expressions for PGC-1α in SQH-treated H9c2 cells. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. PA (+) group.

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  FIGURE 6. SQH intervention improves glucose homeostasis and serum lipids in DbCM mice. (A) Schematic diagram depicting the experimental strategy for DbCM models. (B) Body weight gain of the mice in 12 weeks (left), and body weights of the mice at 12 weeks of age (right). (C) Weight of adipose tissue around epididymis. (D) Serum insulin level at 12 weeks. (E) Fasting blood glucose level at 12 weeks. (F) Glucose tolerance and associated AOC at 12 weeks. (G) Insulin tolerance and associated AOC at 12 weeks. (H) Serum TG levels. (I) Serum TC levels. (J) Serum HDL-C levels. (K) Serum LDL-C levels in different groups. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. DbCM group.

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  FIGURE 7. SQH intervention attenuates cardiac dysfunction in mice with T2D. (A) Representative images of Tissue Doppler and Pulse-wave Doppler in C57BL/6J mice with DbCM. (B) Tissue Doppler e’/a’ ratio, mitral E/A ratio, and E/e’ ratio assessed by ultrasound echocardiography. (C) Representative M-mode images and LV mass. (D) Left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular anterior wall diastole (LVAWd), and left ventricular posterior wall diastole (LVPWd) at end-systole were assessed via echocardiography. (E) Representative images of PSR-stained and WGA-stained sections. (F) Quantification of the cardiomyocyte fibrotic area and cross-sectional area based on PSR and WGA staining. (G) Serum levels of myocardial cell death marker LDH in DbCM and SQH-treated mice. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. the DbCM group.

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  FIGURE 8. SQH reduced serum 12-HETE levels and protected against experimental DbCM by the ACSL4/ALOX15/PGC-1α signaling pathway. (A) Protein expressions for ALOX15, ACSL4, GPX4, and PGC-1α in DbCM mice heart tissues. (B) Serum 12-HETE levels in DbCM mice with or without SQH treatment.

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  FIGURE 9. Schematic illustration showing the mechanisms underlying the prevention of PA-induced injury in cardiomyocytes by ACSL4/ALOX15/PGC-1α signaling. Panax notoginseng flower extract can ameliorate elevated ROS levels, lipid peroxidation, and mitochondrial dysfunction by regulating ACSL4/ALOX15/PGC-1α, thereby improving cardiac function and glucose homeostasis.