Combination decoction of Astragalus mongholicus and Salvia miltiorrhiza mitigates pressure-overload cardiac dysfunction by inhibiting multiple ferroptosis pathways

Abstract

Background: Astragalus mongholicus (AM) and Salvia miltiorrhiza (SM) are commonly used in traditional Chinese medicine to treat heart failure (HF). Ferroptosis has been studied as a key factor in the occurrence of HF. It remains unclear whether the combined use of AM and SM can effectively improve HF and the underlying mechanisms.

Objective: This study aims to explore whether the combined use of AM and SM can improve HF by inhibiting ferroptosis. It also examines the roles and interactions of the pathways associated with GPX4, FSP1, and DHODH.

Methods: In vitro experiments used angiotensin II-induced (4 μM for 48 h) hypertrophic H9c2 cells, while in vivo studies employed a rat model of transverse aortic constriction-induced (to 1 mm for 8 weeks) HF. Interventions included decoctions of AM and SM (for animal experiments) and medicated serum (for cell experiments), along with specific pathway inhibitors such as erastin, FSP1 inhibitor and brequinar. Subsequently, various molecular biology methods were used to measure the protein levels of GPX4, FSP1, and DHODH, as well as each sample group's ferroptosis-related and HF-related indicators, to elucidate the underlying mechanisms.

Results: The combined use of AM and SM can effectively restore the levels of GPX4, FSP1, and DHODH that are reduced after HF, as well as improve indicators related to ferroptosis and HF. When GPX4, FSP1, or DHODH is inhibited, the ferroptosis-inhibiting effect and the ability of AM and SM to improve HF are both weakened. When two of the three proteins are inhibited, the protective effect of HDC is strongest when GPX4 is retained, followed by FSP1, and weakest when DHODH is retained.

Conclusion: This study confirms that the combined use of AM and SM inhibits ferroptosis and alleviates HF by increasing GPX4, FSP1, and DHODH levels. It shows that the protective effect is strongest through GPX4, followed by FSP1, and weakest through DHODH. These findings provide new insights into the therapeutic mechanisms of this combination of botanical drugs.

Keywords: Astragalus mongholicus; DHODH; FSP1; GPX4; Salvia miltiorrhiza; ferroptosis; heart failure.

FIGURE 1

HDC metabolite identification and molecular docking. (A) Chromatograms of HDCH, Control_S, HDCH_S, and HDCL_S under NEG and POS ion modes; (B) Venn diagram of metabolites in different samples. (C) Molecular Docking Results of Astragaloside IV and Salvianolic Acid B with DHODH, FSP1, and GPX4. HDCH, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; Control_S, Normal Rat Serum; HDCH_S, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; HDCL_S, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; NEG, Negative Ion Mode; POS, Positive Ion Mode.

FIGURE 2

HDC effectively improves AngII-induced cell dysfunction and HF due to TAC. (A) Relative cell viability of different groups of H9c2 cells compared to the control group, and NT-proBNP content per mg of total cellular protein; (B) Gross images of rat hearts from each group for direct comparison of heart size, and comparison of heart weight to tibia length ratio in each group; (C) Parasternal long-axis M-mode echocardiographic images of the left ventricle in rats from each group, and quantitative comparison of LVEF; (D) Comparison of serum NT-proBNP levels in rats from each group; (F) Microscopic images of Masson’s trichrome-stained myocardium in rats from each group (×50, ×100, ×200), where the blue-stained areas represent collagen deposition, indicating myocardial fibrosis; (E) Quantification of myocardial fibrosis as indicated by Masson’s trichrome staining, expressed as the percentage of fibrotic area. p^ns ≥ 0.05, p* < 0.05, p** < 0.01, p*** < 0.001, p**** < 0.0001. represents an individual sample data point. HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; AngII, Angiotensin II; HF, Heart failure; F, Ferrostatin-1; E, Erastin; L-S, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; H-S, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; LVEF, Left Ventricular Ejection Fraction; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; TAC, Transverse Aortic Constriction; L, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; H, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction.

FIGURE 3

HDC effectively inhibits ferroptosis in HF. (A) Levels of antioxidants GSH and CoQ10H2 in H9c2 cells of each group; (B) Extent of lipid peroxidation (MDA content) in H9c2 cells of each group; (C) Levels of Fe²⁺ in H9c2 cells of each group; (D) Flow cytometry plots showing red and green fluorescence intensities after JC-1 staining of H9c2 cells from each group, and quantitative analysis of the proportion of green fluorescence signal; (E) GSH levels in myocardial tissue homogenates and serum CoQ10H2 levels in SD rats from each group; (F) MDA content in myocardial tissue homogenates of SD rats from each group; (G) Levels of Fe²⁺ in myocardial tissues of SD rats from each group; (H) TEM images of myocardial tissue from each group, with indicating one of the mitochondria in the image. p^ns ≥ 0.05, p* < 0.05, p** < 0.01, p*** < 0.001, p**** < 0.0001. represents an individual sample data point. HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; HF, Heart failure; AngII, Angiotensin II; F, Ferrostatin-1; E, Erastin; L-S, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; H-S, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; TAC, Transverse Aortic Constriction; L, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; H, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; GSH, Reduced Glutathione; CoQ10H2, Reduced Coenzyme Q10; MDA, Malondialdehyde; PE-A, Phycoerythrin Area; FITC-A, Fluorescein Isothiocyanate Area.

FIGURE 4

HDC ameliorates H9c2 cell dysfunction by inhibiting ferroptosis through the elevation of GPX4 levels. (A) Western blotting analysis of GPX4 levels in H9c2 cells from various groups to evaluate the effect of AngII and various doses of HDC on GPX4 content. (B–F) All panels represent the conditions of H9c2 cells under HDC intervention, assessing the effects of GPX4 inhibition and non-inhibition in varying FSP1 and DHODH states. (B–E) Measurements in H9c2 cells include GSH levels, MDA content, Fe²⁺ levels, cell viability, and NT-proBNP levels. (F) Flow cytometry images of H9c2 cells post JC-1 staining and quantitative analysis of the proportion of green fluorescence signal in flow cytometry. p* < 0.05, p** < 0.01, p**** < 0.0001. represents an individual sample data point. HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; GPX4, Glutathione Peroxidase 4; GAPDH, Glyceraldehyde 3-Phosphate Dehydrogenase; AngII, Angiotensin II; L-S, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; H-S, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; GSH, Reduced Glutathione; FSP1, Ferroptosis Suppressor Protein 1; DHODH, Dihydroorotate Dehydrogenase; E, Erastin; B, Brequinar; i, Inhibitor of FSP1; MDA, Malondialdehyde; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; PE-A, Phycoerythrin Area; FITC-A, Fluorescein Isothiocyanate Area.

FIGURE 5

HDC improves HF by inhibiting ferroptosis via an increase in GPX4 levels. (A) Western blotting analysis of GPX4 levels in the myocardium from SD rats of various groups to evaluate the impact of HDC on GPX4 content. (B–L) All panels depict the effects of GPX4 inhibition and non-inhibition under HDC treatment in rats with various FSP1 and DHODH states. (B–E, J) In HF rats, measurements include myocardial GSH levels, mitochondrial morphology (TEM images, indicates one of the mitochondria), MDA levels, Fe²⁺ content, and serum NT-proBNP levels. (F, G) The heart mass to tibia length ratio and gross cardiac images in HF rats. (H, I) M-mode echocardiography images of the left ventricle adjacent to the sternum and quantitative analysis of LVEF in HF rats. (L) Masson’s trichrome staining of myocardial tissue in HF rats (×50, ×100, ×200 magnifications), with blue staining indicating collagen deposition, suggestive of myocardial fibrosis. (K) Quantitative analysis of fibrotic area based on Masson’s trichrome staining. p*** < 0.001, p**** < 0.0001. represents an individual sample data point. HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; HF, Heart failure; GPX4, Glutathione Peroxidase 4; GAPDH, Glyceraldehyde 3-Phosphate Dehydrogenase; TAC, Transverse Aortic Constriction; L, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; H, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; FSP1, Ferroptosis Suppressor Protein 1; DHODH, Dihydroorotate Dehydrogenase; GSH, Reduced Glutathione; E, Erastin; B, Brequinar; i, Inhibitor of FSP1; TEM, Transmission Electron Microscopy; MDA, Malondialdehyde; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; HM, Heart Mass; TL, Tibia Length; LVEF, Left Ventricular Ejection Fraction.

FIGURE 6

HDC ameliorates H9c2 cell dysfunction by increasing FSP1 levels to inhibit ferroptosis. (A) Western blotting analysis of FSP1 levels in H9c2 cells from various groups to assess the effects of AngII and different doses of HDC on FSP1 content. (B–F) These panels represent the HDC-treated cells, examining the effects of FSP1 inhibition and non-inhibition under varying GPX4 and DHODH states. (B–E) Measurements in H9c2 cells include CoQ10H2 levels, MDA levels, Fe²⁺ content, cell viability, and NT-proBNP levels. (F) Flow cytometry images of H9c2 cells post JC-1 staining and quantitative analysis of the green fluorescence signal proportion in flow cytometry charts. p** < 0.01, p**** < 0.0001. represents an individual sample data point. HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; FSP1, Ferroptosis Suppressor Protein 1; GAPDH, Glyceraldehyde 3-Phosphate Dehydrogenase; AngII, Angiotensin II; L-S, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; H-S, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; CoQ10H2, Reduced Coenzyme Q10; GPX4, Glutathione Peroxidase 4; DHODH, Dihydroorotate Dehydrogenase; i, Inhibitor of FSP1; B, Brequinar; E, Erastin; MDA, Malondialdehyde; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; PE-A, Phycoerythrin Area; FITC-A, Fluorescein Isothiocyanate Area.

FIGURE 7

HDC improves HF by inhibiting ferroptosis through the elevation of FSP1 levels. (A) Western blotting analysis of FSP1 levels in the myocardium from SD rats of various groups to evaluate the impact of HDC on FSP1 content. (B–K) Analysis in HDC-treated rats under various GPX4 and DHODH states, assessing the effects of both FSP1 inhibition and non-inhibition. (B–E, J) Measurements in HF rats include myocardial CoQ10H2 levels, mitochondrial morphology (TEM images, indicates one of the mitochondria), MDA levels, Fe²⁺ content, and serum NT-proBNP levels. (F, G) Heart mass to tibia length ratio and gross cardiac images in HF rats. (H, I) Parasternal long-axis M-mode echocardiography images of the left ventricle and quantitative analysis of LVEF in rats. (L) Masson’s trichrome staining of myocardial tissue in HF rats (×50, ×100, ×200 magnifications), where blue staining indicates collagen deposition, indicative of myocardial fibrosis. (K) Quantitative analysis of the fibrotic area based on Masson’s trichrome staining. p* < 0.05, p** < 0.01, p*** < 0.001, p**** < 0.0001. represents an individual sample data point. HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; HF, Heart failure; FSP1, Ferroptosis Suppressor Protein 1; GAPDH, Glyceraldehyde 3-Phosphate Dehydrogenase; TAC, Transverse Aortic Constriction; L, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; H, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; CoQ10H2, Reduced Coenzyme Q10; GPX4, Glutathione Peroxidase 4; DHODH, Dihydroorotate Dehydrogenase; i, Inhibitor of FSP1; B, Brequinar; E, Erastin; TEM, Transmission Electron Microscopy; MDA, Malondialdehyde; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; HM, Heart Mass; TL, Tibia Length; LVEF, Left Ventricular Ejection Fraction.

FIGURE 8

HDC ameliorates H9c2 cell dysfunction by inhibiting ferroptosis through the elevation of DHODH levels. (A) "Western blotting analysis of DHODH levels in H9c2 cells from various groups, assessing the effects of AngII and different doses of HDC on DHODH content. (B–F) Experiments conducted on cells treated with HDC, evaluating the impact of inhibiting or not inhibiting DHODH under different GPX4 and iFSP1 states. (B–E) Measurements in H9c2 cells include CoQ10H2 levels, MDA levels, Fe²⁺ content, cell viability, and NT-proBNP levels. Flow cytometry images of H9c2 cells post JC-1 staining and quantitative analysis of the green fluorescence signal proportion in flow cytometry charts. p^ns ≥ 0.05, p* < 0.05, p** < 0.01, p*** < 0.001, p**** < 0.0001. represents an individual sample data point. HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; DHODH, Dihydroorotate Dehydrogenase; WB, Western blotting; GAPDH, Glyceraldehyde 3-Phosphate Dehydrogenase; AngII, Angiotensin II; L-S, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; H-S, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; CoQ10H2, Reduced Coenzyme Q10; GPX4, Glutathione Peroxidase 4; FSP1, Ferroptosis Suppressor Protein 1; B, Brequinar; i, Inhibitor of FSP1; E, Erastin; MDA, Malondialdehyde; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; PE-A, Phycoerythrin Area; FITC-A, Fluorescein Isothiocyanate Area.

FIGURE 9

HDC mitigates HF by inhibiting ferroptosis through the elevation of DHODH levels. (A) Western blotting analysis of DHODH levels in the myocardium from SD rats of various groups to assess the impact of HDC on DHODH content. (B–L) Analysis in HDC-treated rats under different GPX4 and FSP1 states, evaluating the effects of DHODH inhibition and non-inhibition. (B–E, J) Measurements in HF rats include myocardial CoQ10H2 levels, mitochondrial morphology (TEM images, indicates one of the mitochondria), MDA levels, Fe²⁺ content, and serum NT-proBNP levels. (F, G) Heart mass to tibia length ratio and gross cardiac images in HF rats. (H, I) Parasternal long-axis M-mode echocardiography images of the left ventricle and quantitative analysis of LVEF in rats. (L) Masson’s trichrome staining of myocardial tissue in HF rats (×50, ×100, ×200 magnifications), where blue staining indicates collagen deposition, suggestive of myocardial fibrosis. (K) Quantitative analysis of the fibrotic area based on Masson’s trichrome staining. p^ns≥0.05, p*<0.05, p**<0.01, p***<0.001, p****<0.0001. represents an individual sample data point. HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; HF, Heart failure; DHODH, Dihydroorotate Dehydrogenase; GAPDH, Glyceraldehyde 3-Phosphate Dehydrogenase; TAC, Transverse Aortic Constriction; L, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; H, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; CoQ10H2, Reduced Coenzyme Q10; GPX4, Glutathione Peroxidase 4; FSP1, Ferroptosis Suppressor Protein 1; B, Brequinar; i, Inhibitor of FSP1; E, Erastin; TEM, Transmission Electron Microscopy; MDA, Malondialdehyde; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; HM, Heart Mass; TL, Tibia Length; LVEF, Left Ventricular Ejection Fraction.

FIGURE 10

Comparative analysis of the effectiveness of GPX4, FSP1, and DHODH in mediating HDC’s improvement of H9c2 cell dysfunction. In this figure, H9c2 cells are divided into three groups: the AngII + H-S + i + E group, primarily mediated by DHODH for HDC’s protective effect; the AngII + H-S + B + E group, primarily mediated by FSP1 for HDC’s protective effect; and the AngII + H-S + B + i group, primarily mediated by GPX4 for HDC’s protective effect. (A–F) Measurements in these three groups of H9c2 cells include GSH levels, CoQ10H2 levels, MDA levels, Fe²⁺ content, cell viability, and NT-proBNP levels. (G) Flow cytometry images post JC-1 staining of the H9c2 cells and the quantitative analysis of the proportion of green fluorescence signal. p^ns≥0.05, p*<0.05, p**<0.01, p****<0.0001. represents an individual sample data point. GPX4, Glutathione Peroxidase 4; FSP1, Ferroptosis Suppressor Protein 1; DHODH, Dihydroorotate Dehydrogenase; HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; AngII, Angiotensin II; H-S, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; i (iFSP1), Inhibitor of FSP1; E, Erastin; B, Brequinar; GSH, Reduced Glutathione; CoQ10H2, Reduced Coenzyme Q10; MDA, Malondialdehyde; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; PE-A, Phycoerythrin Area; FITC-A, Fluorescein Isothiocyanate Area.

FIGURE 11

Comparative analysis of the effectiveness of GPX4, FSP1, and DHODH in mediating HDC’s amelioration of HF in SD rats. In this study, SD rats with HF were divided into three groups: TAC + H + i + E group, primarily mediated by DHODH for HDC’s protective effect; TAC + H + B + E group, primarily mediated by FSP1 for HDC’s protective effect; and TAC + H + B + I group, primarily mediated by GPX4 for HDC’s protective effect. (A–D) Measurements in these groups include myocardial GSH levels, MDA levels, Fe²⁺ content, and serum CoQ10H2 levels in SD rats. (E) Parasternal long-axis echocardiography images of the left ventricle in these groups of SD rats. (H) Quantitative calculation of LVEF based on echocardiography images. (F) Gross cardiac images of the rats in these groups. (I) Heart mass to tibia length ratio in these groups of SD rats. (G) TEM images of myocardial tissue, indicates one of the mitochondria. (J) Serum NT-proBNP levels in these groups of SD rats. (L) Masson’s trichrome staining of myocardial tissue (×50, ×100, ×200 magnifications), where blue staining indicates collagen deposition, suggestive of myocardial fibrosis. (K) Quantitative analysis of the fibrotic area ratio based on Masson’s trichrome staining images. p^ns≥0.05, p*<0.05, p**<0.01, p****<0.0001. represents an individual sample data point. GPX4, Glutathione Peroxidase 4; FSP1, Ferroptosis Suppressor Protein 1; DHODH, Dihydroorotate Dehydrogenase; HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; HF, Heart failure; TAC, Transverse Aortic Constriction; H, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; i, Inhibitor of FSP1; E, Erastin; B, Brequinar; GSH, Reduced Glutathione; CoQ10H2, Reduced Coenzyme Q10; MDA, Malondialdehyde; LVEF, Left Ventricular Ejection Fraction; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; PE-A, Phycoerythrin Area; FITC-A, Fluorescein Isothiocyanate Area.