Berberine protects cardiac cells against ferroptosis

Abstract

Objectives: Cardiovascular diseases are one of the primary causes of death. Cardiomyocyte loss is a significant feature of cardiac injury. Ferroptosis is iron-dependent cell death, which occurs due to excess iron and reactive oxygen species (ROS) accumulation causing lipid peroxidation, and subsequent cell death. Ferroptosis has been confirmed to mediate ischemia/reperfusion-induced cardiomyopathy and chemotherapy-induced cardiotoxicity. Berberine (BBR) has been proven to protect the heart from cardiomyopathies, including cardiac hypertrophy, heart failure, myocardial infarction, and arrhythmias. It protects cardiomyocytes from apoptosis and autophagy. However, the relation between BBR and ferroptosis is still unknown. This study aimed to confirm if BBR reduces cardiac cell loss via inhibiting ferroptosis.

Materials and methods: We used erastin and Ras-selective lethal small molecule 3 (RSL3) to establish a ferroptosis model in an H9c2 cardiomyoblast cell line and rat neonatal cardiomyocytes to prove that BBR has a protective effect on cardiac cells via inhibiting ferroptosis.

Results: In H9c2 cardiomyoblasts, the results showed that BBR reduced erastin and RSL3-induced cell viability loss. Moreover, BBR decreased ROS accumulation and lipid peroxidation in cells induced with ferroptosis. Furthermore, quantitative polymerase chain reaction results showed that Ptgs2 mRNA was reduced in BBR-treated cells. In rat neonatal cardiomyocytes, BBR reduced RSL3-induced loss of cell viability.

Conclusion: These results indicated that BBR inhibited ferroptosis via reducing ROS generation and reducing lipid peroxidation in erastin and RSL3-treated cardiac cells.

Keywords: Berberine; Cardiomyocyte; Ferroptosis; Ischemia-reperfusion injury; Lipid peroxidation.

Figure 1

Erastin and ras-selective lethal small molecule 3 both induced ferroptosis in H9c2 cardiomyoblast cells. (a) Cells were treated with different concentrations of erastin (0.5, 1, 2 μM) or indicated inhibitors (Z-VAD-FMK, 10 μM; Fer-1, 1 μM; Lip-1, 2 μM) for 16 h. (b) Cells were treated with different concentrations of ras-selective lethal small molecule 3 (0.05, 0.1, 0.2 μM) or indicated inhibitors (Z-VAD-FMK, 10 μM; Fer-1, 1 μM; Lip, 2 μM) for 16 h. The cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. (n = 8) *P < 0.05. All data represent mean ± standard error of the mean. Con: Control, Veh: Vehicle, Era: Erastin, RSL-3: Ras-selective lethal small molecule 3

Figure 2

Berberine inhibited the erastin- and ras-selective lethal small molecule 3-induced cardiac cell loss via inhibition of ferroptosis. (a) H9c2 cells were treated with either erastin (1 μM) only or co-treated with erastin (1 μM) and different concentrations of berberine (2, 5, 10, 20 μM) for 16 h. (b) Cells were treated with ras-selective lethal small molecule 3 (0.1 μM) only or ras-selective lethal small molecule 3 (0.1 μM) co-treatment different concentrations berberine (2, 5, 10, 20 μM) for 16 h. The cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. (n = 8) *P < 0.05. All data represent mean ± standard error of the mean. Con: Control, Era: Erastin, BBR: Berberine, RSL-3: Ras-selective lethal small molecule 3

Figure 3

Analysis of apoptosis and necrosis by flow cytometry in berberine-treated H9c2 cardiomyoblast cells. Cells were treated with dimethyl sulfoxide, berberine (10 μM), or H₂O₂ (20 μM) for 1 h. Detection of apoptotic cells and necrotic cells by Annexin V and PI staining. (n = 6) *P < 0.05. All data represent mean ± standard error of the mean. Con: Control, BBR: Berberine

Figure 4

Berberine inhibited erastin- or ras-selective lethal small molecule 3-induced ferroptosis in H9c2 cardiomyoblast cells. (a) Cells treated 0.1% dimethyl sulfoxide, erastin (1 μM), ras-selective lethal small molecule 3 (0.1 μM), or berberine (10 μM) for 10 h. To measure intracellular reactive oxygen species by DCFDA assay (n = 4) (b) To measure lipid peroxidation measured by thiobarbituric acid reactive substances assay (n = 3). (c) To detect Ptgs2 mRNA expression by quantitative polymerase chain reaction. (n = 4) *P < 0.05. All data represent mean ± standard error of the mean. Con: Control, Era: Erastin, RSL-3: Ras-selective lethal small molecule 3, BBR: Berberine

Figure 5

Berberine reduced ras-selective lethal small molecule 3-induced cell viability loss of rat neonatal cardiomyocytes. (a) Cells were treated with 0.1% dimethyl sulfoxide or ras-selective lethal small molecule 3 (0.5 μM) for 24 h, then measured ras-selective lethal small molecule 3-induced apoptotic cells and necrotic cells were by Annexin V and PI (n = 4). (b) Cells were treated with ras-selective lethal small molecule 3 (0.5 μM), or indicated inhibitors (Fer-1, 1 μM; Lip-1, 2 μM; berberine, 10 μM) for 24 h. To measure the cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. (n = 8) *P < 0.05. All data represent mean ± standard error of the mean. Con: Control