Protective Effect of Ginsenoside Rg5 on Hypoxia/Reoxygenation- Induced H9c2 Cardiomyocytes by Correcting Free Fatty Acids β- oxidation

doi:10.21203/rs.3.rs-2812492/v1

Clinically, the in-hospital mortality rate of cardiogenic shock (CS) is approximately 50%. In CS, metabolic pathways in the body undergo “embryonal transformation,” in which metabolic processes transform free fatty acids (FFAs) to glucose. Shenfu injection (SFI) treatment has exhibited a positive effect on cardiogenic shock, and ginsenoside Rg5 (G-Rg5) is the anticardiogenic shock component of SFI. In this study, we aimed to investigate the myocardial protective effect and mechanism of G-Rg5 in myocardial energy metabolism, especially from the perspective of FFA β-oxidation, one of the most crucial lipid metabolism pathways. Cardiomyocytes were exposed to hypoxia-reoxygenation (H/R) and treated with G-Rg5. MTT analysis was used to determine the viability of H9c2 cardiomyocytes under different interventions with G-Rg5. The levels of lactate dehydrogenase (LDH) and FFA were detected using ELISA, and adenosine triphosphate (ATP) was determined using high-performance liquid chromatography (HPLC). Furthermore, the expression of NR4A1 was determined by RT-qPCR, and the levels of AMPKα, p-AMPKα, and GPX4 were detected by western blotting to explore the underlying mechanism. We observed that G-Rg5-protected H9c2 cardiomyocytes exhibited better FFA β-oxidation regulation, thereby producing an increased abundance of ATP. G-Rg5 may correct FFA β-oxidation by regulating the levels of NR4A1, AMPKα, p-AMPKα, and GPX4. Therefore, G-Rg5 is a promising drug for CS treatment.

Ginsenoside Rg5

Hypoxia/Reoxygenation

Cardiogenic shock

H9c2 cardiomyocytes

Myocardial energy metabolism

Cardiogenic shock (CS), a cardiovascular disease, is primarily caused by heart pump failure and is accompanied by ischemia, hypoxia, and energy metabolic disorders [1]. However, the current treatment plan has not yet achieved the desired goals. CS, the most severe form of acute heart failure [2], is the leading cause of death due to acute myocardial infarction. A survey conducted by the Federal Statistical Office of Germany revealed that the incidence of CS increased from 33.1/100,000 population in 2005 to 51.7/100,000 population in 2017 [3]. The morbidity and mortality rates associated with CS remain high.

Abnormal myocardial energy metabolism is a critical mechanism in cardiovascular disease [4–6]. The heart, the center of energy supply, consumes more than seven times its weight of adenosine triphosphate (ATP) each day [7]. ATP is mainly produced by free fatty acid (FFA) β-oxidation. Zhao [8] reported that trimethyl-5-aminovaleric acid produced by the gut microbiota accelerates myocardial hypertrophy by reducing fatty acid oxidation. FFA produces much more ATP than glucose metabolism, but glucose metabolism consumes less oxygen than FFA [9]. The heart returns to a fetal gene program and modifies its substrate preference from FFA to glucose under ischemic and hypoxic conditions [10], thereby increasing the FFA levels in cells and leading to ATP deficiency. ATP deficiency reduces the activity of Ca²⁺-ATPase in the cell membrane and sarcoplasmic reticulum, which prevents efficient Ca²⁺ transport and calcium overload. An increase in the intracellular free calcium concentration can activate calcium-dependent phospholipases and increase the permeability of the mitochondrial inner membrane. Phospholipases hydrolyze membrane phospholipids, which damages the membrane structure and releases FFA, further increasing FFA content [11]. Collectively, an increase in FFA concentration in cardiomyocytes leads to cardiac lipotoxicity [12] and metabolic abnormalities [13].

One of the most effective ways to target CS is to regulate FFA β-oxidation to maintain cardiac function [14]. Many regulatory factors are involved in FFA β-oxidation. As a transcription factor, nuclear receptor subfamily 4 group A member 1 (NR4A1) is a vital shock target involved in regulating glucose and lipid metabolism. AMP-activated protein kinase (AMPK) is a receptor that maintains cellular energy metabolism, and the AMPKα isoform is a regulatory factor in FFA β-oxidation [15]. p-AMPK is the active form of AMPK, and changes in AMPK activity have been evaluated by measuring p-AMPK protein levels. Glutathione peroxidase 4 (GPX4) is a crucial regulator of glutathione (GSH) in the regulation of ferroptosis. It participates in the hydrolysis of lipid peroxide by scavenging free radicals to protect cells from lipid peroxide accumulation [16]. Prior studies [17–20] have also reported that traditional Chinese medicine has obvious advantages in the treatment of cardiovascular disease, which can improve the prognosis of patients, reduce the incidence of complications, and improve the safety of the medication. The representative compound preparation for the clinical treatment of CS in China is Shenfu injection (SFI). SFI can effectively treat CS by protecting the myocardium, improving hematology, adjusting myocardial energy metabolism, and anti-shock and anti-arrhythmia [21–27]. SFI is a traditional Chinese medicine injection prepared from the extraction of red ginseng and Radix Aconiti, and its components include ginsenosides and alkaloids. Ginsenoside Rg5 (G-Rg5, the chemical structure of which is shown in Fig. 1(a)) is a ginsenoside that has been proven to promote angiogenesis, improve endothelial function [28], have anticancer properties [29–32], lower glucose [33–36] and protect against myocardial injury [35]. However, few studies have investigated the beneficial effects of G-Rg5 on correcting myocardial energy metabolism, especially from the perspective of FFA β-oxidation, one of the most critical lipid metabolism pathways. Furthermore, H/R-induced cardiomyocytes have pathophysiological processes similar to those of CS. Therefore, we chose an H/R-induced cardiomyocyte model to evaluate the protective effects of G-Rg5 on FFA β-oxidation in cardiomyocytes from the perspective of energy metabolism. The findings of this study provide a theoretical basis for the development of new drugs for the treatment of CS.

Materials and Reagents

G-Rg5, deslanoside, and ATP were purchased from Vakic Biotechnology Co., Ltd. (purity >98%, Sichuan, China). Dulbecco’s modified Eagle medium (DMEM), glucose-free DMEM, and dimethyl sulfoxide (DMSO) were obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). 0.25% Trypsin-EDTA, fetal bovine serum (FBS) and PBS phosphate buffer were bought from Genview (Florida, USA), Tianhang Biotechnology Co., Ltd. (Zhejiang, China), and Dingguo Changsheng Co., Ltd. (Nanchang, China), respectively. K₂HPO₄ and NaH₂PO₄ was obtained from Sinopharm Chemical reagent Co., Ltd. (Analytical level, Shanghai, China). LDH and Rat FFA ELISA kit were purchased from Nanjing Bioengineering Institute (Nanjing, China) and BIM (California, USA), respectively. CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) was acquired from Promega Biological Co., Ltd. (Beijing, China). RIPA lysate, bicinchoninic acid (BCA) kit, twelve alkyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Kit, polyvinylidene fluoride (PVDF) membranes (0.45 μm), skim milk, ECL kit, β-actin, RNA extraction, Servicebio®RT First Strand cDNA Synthesis Kit, and 2nd 2esis KitqPCR Master Mix (HighROX) were supplied by Servicebio Co., Ltd. (Wu Han, China). AMPKα and p-AMPKα were purchased from Cell Signaling Technology (Massachusetts, USA). GPX4 was purchased from Abcam (Cambridge, UK).

Culture of H9c2 Cardiomyocytes

Rat H9c2 cardiomyocytes were obtained from the Cell Resource Center of the Chinese Academy of Sciences. H9c2 cardiomyocytes were cultured in DMEM supplemented with 15% fetal bovine serum (FBS; v/v) at 37°C in a humidified atmosphere (Smartor118, Hua Yi Ning Chuang Co., Ltd, Ningbo, China). The culture medium was replaced regularly.

Establishment of an H/R Model

The H/R model was established according to our previous method [36]. An orthogonal experiment was conducted to optimize the experimental conditions. Hypoxia time (6, 8, and 10 h), glucose content (0, 2, and 4 mg/mL), and FBS content (0, 10, and 20%) were used as experimental control factors. Cell activity and lactate dehydrogenase (LDH) content were used as indices. According to the comprehensive score, lower cell vitality and higher LDH content were desirable. As shown in Table 1, the model had the lowest cell viability and the highest LDH release under 6 h hypoxia, 0 mg/mL glucose content, and 0% FBS. Therefore, cardiomyocytes in the H/R group were cultured in glucose-free DMEM with 0% FBS (v/v) at 37°C in a humidified atmosphere, followed by 6 h of hypoxia (94% N₂, 5% CO₂, and 1% O₂) and 4 h of reoxygenation (95% O₂ and 5% CO₂).

Table 1: Result of orthogonal experiment（M ± SD, n = 6).

Test number	Orthogonal experiment factors
Test number	Hypoxia time A (h)	Glucose content B (mg/mL)	FBS content C (%)	Experimental group	Cell viability (%)	LDH viability (U/L)
1	A1	B1	C1	A1B1C1 (6 h, 0 mg/mL, 0%)	52.7±9.92^##	90.00±19.19^##
2	A1	B2	C2	A1B2C2 (6 h, 2 mg/mL, 10%)	91.02±7.40	10.35±9.55^##
3	A1	B3	C3	A1B3C3 (6 h, 4 mg/mL, 15%)	98.68±21.92	-97.54±26.87^##
4	A2	B1	C2	A2B1C2 (8 h, 0 mg/mL, 10%)	93.18±4.80	10.62±18.00^##
5	A2	B2	C3	A2B2C3 (8 h, 2 mg/mL, 15%)	68.83±3.21^##	78.14±4.60^##
6	A2	B3	C1	A2B3C1 (8 h, 4 mg/mL, 0%)	85.63±14.96^#	-72.98±13.92^##
7	A3	B1	C3	A3B1C3 (10 h, 0 mg/mL, 15%)	86.8±4.64^#	-72.07±16.57^##
8	A3	B2	C1	A3B2C1 (10 h, 2 mg/mL, 0%)	85.3±4.76^#	-24.65±8.81^##
9	A3	B3	C2	A3B3C2 (10 h, 4 mg/mL, 10%)	71.03±1.26^##	54.80±9.79^##

^#P<0.05 vs. control group; ^##P<0.01 vs. control group.

Determination of Cell Viability

Experimental Protocols

To determine the effect of G-Rg5 on H9c2 cardiomyocytes, we measured the viability of H/R-induced cardiomyocytes under different G-Rg5 conditions. Cardiomyocytes in the logarithmic phase were randomly divided into different groups. The control group was cultured in DMEM supplemented with 15% FBS (v/v). The H/R group was cultured under the experimental conditions used for the H/R model. The G-Rg5 treatment group (H/R+G-Rg5) was treated with 1, 3.33, 10, 33.3, or 100 μmol/L G-Rg5 for 12, 24, or 48

MTT Cell Viability Assay

Cell viability was determined using an MTS assay. After reoxygenation, 20 μL MTS was added to each well, mixed, and incubated at 37°C for 4 h. The absorbance of each well was measured at a wavelength of 490 nm using a microplate reader. The cell viability was expressed as a percentage of the absorbance of the control group.

Determination of LDH Release

Experimental Protocols

We determined the optimal intervention conditions for G-Rg5 using time- and effect-dose experiments. The control group was cultured in DMEM supplemented with 15% FBS (v/v). The H/R group was cultured under the experimental conditions used for the H/R model. The H/R+G-Rg5 and H/R+desianoside groups were pretreated under optimal administration conditions. After reaching the intervention time, the H/R, H/R+G-Rg5, and H/R+desianoside groups were subjected to hypoxia and reoxygenation for 6 h and 4 h, respectively.

LDH Level Determination

After reoxygenation, 20 μL of the supernatant was collected from each well, and the operation was carried out according to the instructions of the LDH kit. The absorbance of each well was determined at 450 nm using a microplate reader. LDH content (U/L)=(test well-control well)/(standard well-blank well)×standard concentration×1000.

ATP Measurements using High-Performance Liquid Chromatography (HPLC)

Liquid Chromatography

An Agilent 1260 system was used for liquid chromatography (Agilent Technologies, California, USA). The samples were separated on an Agilent ZORBAX SB-AQ liquid chromatographic column (4.6 mm×150 mm, 5 μm). The mobile phase was a 50 mmol/L phosphate buffer solution with a pH of 6.3. Separations were performed at a temperature of 30°C, a flow rate of 1.0 mL/min, injection volume of 20 μL, and detection wavelength of 258 nm.

Preparation of a Standard Solution

The ATP standard was dissolved in ultrapure water to prepare the reserve solution. The standard solution was diluted with ultrapure water to concentrations of 20, 15, 10, 5.0, 2.5, 1.0, and 0.5 μg/mL.

Sample Pretreatment

The experimental protocols were consistent with the “Experimental Protocols” subsection. We referred to published methods with minor revisions for sample treatment [37] as follows:

The cell suspension was diluted to a concentration of 3×10⁵cells/mL.
250 μL of precooled 0.4 mol/L HClO₄ solution was added into 250 μL of cell suspension.
The above mixture was vortexed for 20 s, chilled on ice for 10 min, and re-vortexed for 20 s, then centrifuged at 4°C with 12,000 rpm for 10 min using the high speed refrigerated centrifuge (Thermo Scientific SL 8R, Thermo Fisher Scientific, Massachusetts, USA).
The supernatant was adjust pH to neutral with 1mol/L NaOH, and centrifuged at 4°C with 12,000 rpm for 10 min.
A 0.22 μm microporous membrane was used for filtration. The above sample treatment was processed at 4°C.
Store the supernatant at -80°C for subsequent analysis.

Determination of FFA Content

The experimental protocols were consistent with that described in the “Determination of LDH Release: Experimental Protocols” section.

The cell suspension was centrifuged at 1,200 rpm for 5 min by the high speed refrigerated centrifuge and the supernatant was discarded.
Cells resuspended to 1×10⁶ cells/mL with PBS.
Samples were then subjected to water bath sonication (The cell suspension was ultrasonically performed at 300W power. Each ultrasound was performed for 3-4 s, with a pause of 7-8 s, and the process was repeated for 10-15 times).
Centrifugation at 2,000 rpm for 5 min, and the supernatant was taken.
ELISA was performed according to the instructions.
The absorbance at 450 nm was read with the microplate analyzer.

The FFA content in each group was calculated according to the standard curve.

RT-qPCR Analysis

RT-qPCR was performed to detect the expression of NR4A1. The total RNA of cells was extracted according to the manufacturer’s instructions. RNA was reverse-transcribed into cDNA using the Servicebio®RT First Strand cDNA Synthesis Kit. PCR amplification was performed using SYBR Green qPCR Master Mix (HighROX). The β-actin transcript level (ACTB) was used to normalize NR4A1 expression levels, and the 2⁻^ΔΔCt method was used to calculate the expression of NR4A1.

Western Blotting

Approximately 250 μL of RIPA lysate was added to 10⁶ cells, after which the sample was incubated on ice for 30 min and centrifuged at 12,000 ×g at 4°C for 15 min, and the supernatant was collected. The protein concentration was detected using a BCA kit. Equal amounts of protein samples were separated using 10% SDS-PAGE and then transferred to PVDF membranes. Membranes were blocked with 5% skim milk for 30 min at 20-25°C and incubated with primary antibodies against β-actin (1:3000), AMPKα (1:1000), p-AMPKα (1:1000), and GPX4 (1:1000) at 4°C overnight. The membranes were then incubated with an HRP-labeled secondary antibody (1:3000) for 30 min at 20-25°C. Enhanced ECL was used to visualize protein expression. The relative expression of the target protein was reported as the ratio of the target protein to actin.

Statistical Analysis

Results are shown as mean ± standard deviation (M ± SD). Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software, California, USA). Differences for multi-groups were analyzed by one-way analysis of variance (ANOVE). T-test were used to evaluate the statistical differences between the groups. There was statistical significance when P<0.05.

Estimation of Optimal G-Rg5 Administration Conditions

The molecular formula of G-Rg5 is C₄₂H₇₀O₁₂, and its chemical structure is shown in Figure 1(a). The survival rate of cells is directly proportional to the measured cell viability [38]. We first explored the time and concentration dependence of G-Rg5 by performing an MTT assay and observed that the protective effect of G-Rg5 on H/R-induced H9c2 cardiomyocytes did not depend on the dose and time. As shown in Figure 1(b), the H/R intervention significantly decreased the viability of H9c2 cardiomyocytes (P<0.01); 12 h-1 μmol/L of G-Rg5 reversed this effect, whereas other intervention conditions had no effect, and even decreased cell viability. Hence, 12 h-1 μmol/L was selected as the optimal intervention condition for G-Rg5.

Deslanoside is a cardiac glycoside used as a positive drug in this experiment. The effects of different concentrations of deslanoside on the activity of cardiomyocytes after 12 h were studied. As shown in Figure 2, compared to that in the control group, the cell activity of cardiomyocytes in the H/R group was significantly decreased (P<0.01). Deslanoside at 12 h-1 μmol/L, 12 h-3.33 μmol/L, and 12 h-33.3 μmol/L increased the cell activity of H/R-induced cardiomyocytes (P<0.01). Cell viability was the highest at a deslanoside time/dose of 12 h-1 μmol/L; therefore, it was selected as the administration condition for deslanoside.

G-Rg5 Reduced LDH Release from H/R-Induced H9c2 Cardiomyocytes

To further identify the protective effect of G-Rg5 on H/R-induced H9c2 cardiomyocytes, we measured LDH release, where LDH is an indicator of the degree of cellular damage. We selected the optimal administration conditions for G-Rg5 and deslanoside. As shown in Figure 3, LDH release from H9c2 cardiomyocytes significantly increased after H/R-induction (P<0.01); however, G-Rg5 reduced LDH release from H/R-induced H9c2 cardiomyocytes (P<0.01).

Effect of G-Rg5 on Energy Metabolism of H9c2 Cardiomyocytes

Abnormal energy metabolism is one of the main pathogenic factors of CS. To investigate the role of G-Rg5 in H/R-induced myocardial energy metabolism disorder, we measured ATP and FFA levels, two important indices of energy metabolism. Similarly, the intervention conditions for G-Rg5 and deslanoside were 12 h-1 μmol/L. ATP content was determined by HPLC, and FFA content was measured using a rat FFA ELISA kit. The HPLC chromatograms are shown in Figures 4(a) and 4(b). ATP levels were analyzed qualitatively and quantitatively according to the retention time and standard curve of the reference substance. ATP concentrations exhibited a good linear relationship in the range of 0.5-20 μg/mL（r2=0.9999).

H/R-Induced Disorders of NR4A1, AMPKα, p-AMPKα, and GPX4 Expression Levels were Suppressed by G-Rg5 Treatment

As mentioned in the introduction, FFA β-oxidation is a vital lipid metabolism pathway. The ATP produced by this pathway can provide energy to the myocardium. Our experiment revealed that G-Rg5 could correct the levels of ATP and FFA (Figures 4(c) and 4(d)). To explore the regulatory effect of G-Rg5 on FFA β-oxidation, we measured the levels of regulatory factors related to lipid metabolism, including NR4A1, AMPKα, p-AMPKα, and GPX4. As shown in Figures 5(a) and 5(b), compared to that in the control group, the expression of AMPKα, p-AMPKα, and GPX4 in H/R-induced H9c2 cardiomyocytes decreased, whereas the level of NR4A1 increased. Furthermore, G-Rg5 suppressed these effects. Based on these results, we speculate that G-Rg5 might correct FFA β-oxidation by regulating the levels of NR4A1, AMPKα, p-AMPKα, and GPX4.

Cardiovascular disease is a global public health problem. The clinical treatment of CS mainly includes inotropic drugs, vasoactive drugs, PCI therapy, thrombolytic therapy, impella, tandem heart, extracorporeal membrane oxygenation, and intra-aortic balloon counterpulsation [39]; however, these treatments may cause complications. Thus, other treatments with improved safety must be identified. Prior studies have demonstrated that Chinese herbal medicines have great advantages in the treatment of diabetes [40–42], inflammation [43], nervous system diseases [19, 44–45], cancer [46–48], COVID-19 [46], and cardiovascular diseases [50]. Therefore, we turned our attention to Chinese herbal medicines and their prescriptions. In China, clinical trials [24, 50–54] have demonstrated that SFI has a good therapeutic effect and high safety. The main active components of SFI are ginsenosides and alkaloids. G-Rg5, a minor ginsenoside transformed from a major ginsenoside through deglycosylation, has high stability, bioavailability, and absorption [55]. G-Rg5 reportedly has therapeutic effects on cardiovascular diseases. It can increase the resistance of cardiomyocytes to ischemic injury and protect the integrity of mitochondrial morphology and function [35].

H/R-induced myocardial injury is a complex pathophysiological process often accompanied by cardiomyocyte necrosis, apoptosis, and autophagy. Important mechanisms include myocardial cell injury and abnormal energy metabolism [56]. First, we explored the protective effect of G-Rg5 on cardiomyocytes. Myocardial ischemia and hypoxia release a large number of oxygen free radicals and inflammatory factors, produce apoptosis-related proteins, and damage cell membranes and mitochondrial membranes, thus damaging cardiomyocytes [57]. This is consistent with the experimental results. Myocardial cell viability decreased under H/R, whereas 12 h-33.3 µmol/L of G-Rg5 intervention significantly increased cell viability and reduced LDH levels. LDH is a living intracytoplasmic enzyme released from the cytoplasm into the external environment when cells are injured as an indicator of the degree of cell damage [58] and can indirectly reflect cell viability [59]. This suggests that G-Rg5 can reduce H/R-induced H9c2 cardiomyocyte injury.

Myocardial energy metabolism disorder is a crucial factor in CS. We focused on indices related to myocardial energy metabolism, including ATP content and FFA levels. As an index of energy metabolism, ATP is a direct energy supply substance that maintains normal heart contraction. The heart is a high-energy organ, and cardiomyocytes have low energy reserves; therefore, a constant supply of ATP is required. Cardiomyocytes produce ATP through the oxidation of the energy-supplying substrate FFA, glucose, lactic acid, and pyruvate, among which FFA accounts for 60%-90% of metabolic substrates [60]. Van et al. [56] reported that metabolic aspects of fetal cardiac reprogramming occur in heart failure pathology: the heart switches from fatty acid oxidation to glucose oxidation with higher oxygen efficiency [61]. The metabolic model of glucose from FFA β-oxidation to anaerobic glycolysis results in substance metabolism disorders, leading to mitochondrial membrane damage and energy metabolism disorders [62]. FFA β-oxidation decreases with an increase in heart failure [63]. Furthermore, approximately 134 mol of ATP is produced by the complete oxidation of 1 mol of 20-carbon fatty acids; in contrast, approximately 30 mol of ATP is produced by 1 mol of glucose [64]. When myocardial energy metabolism changes FFA β-oxidation to anaerobic glycolysis of glucose, ATP production is significantly reduced. Moreover, ATP deficiency cannot meet the needs of the myocardium and results in the aggravation of myocardial damage [65]. Correcting FFA oxidation can improve cardiac function [66]. In our study, when H9c2 cardiomyocytes were exposed to hypoxia and reoxygenation, a marked increase in FFA levels and a decrease in ATP levels were observed. Therefore, we speculate that after H/R intervention, the energy metabolic substrate in cardiomyocytes changes. This may be caused by FFA β-oxidative conversion to glycolysis, which results in the decrease of ATP content in H9c2 cardiomyocytes and an increase in FFA levels. Notably, G-Rg5 inhibits this change and may correct FFA β-oxidation, an important pathway in lipid metabolism.

To further investigate lipid metabolism, we determined the levels of NR4A1, AMPKα, p-AMPKα, and GPX4. NR4A1 is a critical member of the orphan nuclear NR4A subfamily that is vital in regulating glucose and lipid metabolism [67–69]. NR4A1 deletion can lead to a decrease in glycolytic flux and the upregulation of lipoprotein lipase mRNA [70]. Some studies have indicated that NR4A1 knockdown improves cardiac function [67, 71, 72]. AMPK is a crucial regulator of fatty acid metabolism that can promote FFA β-oxidation by activating and improving the transcriptional activity of PPAR-α and the phosphorylation of PGC1-α [73, 74]. p-AMPK can regulate lipid metabolism through multiple pathways, including increasing fatty acid intake by promoting CD36 and LPL transport [75]. Metabolic disorders in lipid oxidative metabolism lead to the accumulation of lipid peroxide and induce ferroptosis [76]. Notably, Lee et al. [77] reported that AMPK activation inhibits ferroptosis. GPX4 is a crucial ferroptosis regulator and can protect H9c2 cardiomyocytes from iron death caused by lipid peroxidation [78–79]. RT-qPCR and western blotting analyses revealed that compared to that in the control group, NR4A1 was overexpressed in the H/R group, whereas AMPKα, p-AMPKα, and GPX4 levels decreased. Additionally, G-Rg5 treatment downregulated NR4A1 and upregulated AMPKα, p-AMPKα, and GPX4. Based on the above results, we speculated that G-Rg5 might correct lipid metabolism in H/R-induced H9c2 cardiomyocytes by modulating factors associated with lipid metabolism.

In this study, G-Rg5 protected H9c2 cardiomyocytes against H/R-induced damage. Furthermore, G-Rg5 corrected FFA β-oxidative stress in H/R-induced H9c2 cardiomyocytes, implicating the modulation of NR4A1, AMPKα, p-AMPKα, and GPX4 molecules. This suggests that we can explore the effects of related drugs on cardiovascular disease from lipid metabolism and related signaling pathways.

In conclusion, our study revealed that G-Rg5, an effective component of SFI against CS, can enhance cell vitality and improve myocardial energy metabolism at a specific dose and intervention time. We have experimentally demonstrated that G-Rg5 can correct FFA β-oxidation in cardiomyocytes. These results suggest that G-Rg5 is a promising drug for the treatment of CS, exerting its cardioprotective function by correcting lipid metabolism.

Ethics approval and consent to participate

Not applicable.

Consent for Publication

Not applicable.

Availability of Data and Materials

The data used to support the findings of this study are included within the article. Data are available from the corresponding author upon request.

Competing Interests

The authors declare no conflict of interest.

Funding

This research was supported by grants of Jiangxi University of Chinese Medicine Science and Technology Innovation Team Development Program (CXTD22007), Chinese Medicine Research Project of Jiangxi Provincial Health and Family Planning Commission (2021B599), Chinese Medicine Research Project of Jiangxi Provincial Health and Family Planning Commission (2021B599), and Science and technology Project of Health Commission (SKJP_220210795).

Authors’ Contributions

Conceptualization: LJ, WJF and GLX.; data curation: LJ and WJF; funding acquisition: LJ and HNL investigation: YT, QYZ, GBS, and PN; methodology: LJ, WJF, XJY, WTZ, and GLX; writing—original draft: LJ, and WJF; writing—review and editing: LJ, and GLX; All authors have read and agreed to the published version of the manuscript.

Acknowledgements

Not applicable.

Castillo Costa Y, Delfino F, Mauro V, D'Imperio H, Barrero C, Charask A, Zoni R, Macín S, Perna E, Gagliardi J. Clinical characteristics and evolution of patients with cardiogenic shock in Argentina in the context of an acute myocardial infarction with ST segment elevation. Data from the nationwide ARGEN-IAM-ST Registry. Curr Probl Cardiol. 2023;48(2):101468.
Rubini Gimenez M, Zeymer U, Desch S, et al. Sex-Specific Management in Patients With Acute Myocardial Infarction and Cardiogenic Shock: A Substudy of the CULPRIT-SHOCK Trial. Circ Cardiovasc Interv. 2020;13(3):e008537.
Schrage B, Becher PM, Goßling A, Savarese G, Dabboura S, Yan I, Beer B, Söffker G, Seiffert M, Kluge S, Kirchhof P, Blankenberg S, Westermann D. Temporal trends in incidence, causes, use of mechanical circulatory support and mortality in cardiogenic shock. ESC Heart Fail. 2021; 8(2):1295-1303.
Ketema EB, Lopaschuk GD. Post-translational Acetylation Control of Cardiac Energy Metabolism. Front Cardiovasc Med. 2021;8:723996.
Yu Q, Zhao G, Liu J, Peng Y, Xu X, Zhao F, Shi Y, Jin C, Zhang J, Wei B. The role of histone deacetylases in cardiac energy metabolism in heart diseases. Metabolism. 2023;142:155532.
Huang JR, Zhang MH, Chen YJ, Sun YL, Gao ZM, Li ZJ, Zhang GP, Qin Y, Dai XY, Yu XY, Wu XQ. Urolithin A ameliorates obesity-induced metabolic cardiomyopathy in mice via mitophagy activation. Acta Pharmacol Sin. 2023;44(2):321-331.
Selvaraj S, Kelly DP, Margulies KB. Implications of Altered Ketone Metabolism and Therapeutic Ketosis in Heart Failure. Circulation. 2020;141(22):1800-1812.
Zhao M, Wei H, Li C, Zhan R, Liu C, Gao J, Yi Y, Cui X, Shan W, Ji L, Pan B, Cheng S, Song M, Sun H, Jiang H, Cai J, Garcia-Barrio MT, Chen YE, Meng X, Dong E, Wang DW, Zheng L. Gut microbiota production of trimethyl-5-aminovaleric acid reduces fatty acid oxidation and accelerates cardiac hypertrophy. Nat Commun. 2022;13(1):1757.
Su Z, Liu Y, Zhang H. Adaptive Cardiac Metabolism Under Chronic Hypoxia: Mechanism and Clinical Implications. Front Cell Dev Biol. 2021;9:625524.
Taegtmeyer H, Sen S, Vela D. Return to the fetal gene program: a suggested metabolic link to gene expression in the heart. Ann N Y Acad Sci. 2010;1188:191-8.
Capone F, Sotomayor-Flores C, Bode D, Wang R, Rodolico D, Strocchi S, Schiattarella GG. Cardiac metabolism in HFpEF: from fuel to signalling. Cardiovasc Res. 2023;118(18):3556-3575.
Capone F, Sotomayor-Flores C, Bode D, Wang R, Rodolico D, Strocchi S, Schiattarella GG. Cardiac metabolism in HFpEF: from fuel to signalling. Cardiovasc Res. 2023;118(18):3556-3575.
Henderson GC. Plasma Free Fatty Acid Concentration as a Modifiable Risk Factor for Metabolic Disease. Nutrients. 2021;13(8):2590.
Du H, Zhao Y, Li H, Wang DW, Chen C. Roles of MicroRNAs in Glucose and Lipid Metabolism in the Heart. Front Cardiovasc Med. 2021;8:716213.
He Y, Huang W, Zhang C, Chen L, Xu R, Li N, Wang F, Han L, Yang M, Zhang D. Energy metabolism disorders and potential therapeutic drugs in heart failure. Acta Pharm Sin B. 2021;11(5):1098-1116.
Forcina GC, Dixon SJ. GPX4 at the Crossroads of Lipid Homeostasis and Ferroptosis. Proteomics. 2019;19(18):e1800311.
Shao-Mei W, Li-Fang Y, Li-Hong W. Traditional Chinese medicine enhances myocardial metabolism during heart failure. Biomed Pharmacother. 2022;146:112538.
Jiang Y, Zhao Q, Li L, Huang S, Yi S, Hu Z. Effect of Traditional Chinese Medicine on the Cardiovascular Diseases. Front Pharmacol. 2022;13:806300.
Chen Y, Tang M, Yuan S, Fu S, Li Y, Li Y, Wang Q, Cao Y, Liu L, Zhang Q. Rhodiola rosea: A Therapeutic Candidate on Cardiovascular Diseases. Oxid Med Cell Longev. 2022;2022:1348795.
Liu M, Long X, Xu J, et al. Hypertensive heart disease and myocardial fibrosis: How traditional Chinese medicine can help addressing unmet therapeutical needs. Pharmacol Res. 2022;185:106515.
Liu X, Liu R, Dai Z, Wu H, Lin M, Tian F, Gao Z, Zhao X, Sun Y, Pu X. Effect of Shenfu injection on lipopolysaccharide (LPS)-induced septic shock in rabbits. J Ethnopharmacol. 2019;234:36-43.
Xu P, Zhang WQ, Xie J, Wen YS, Zhang GX, Lu SQ. Shenfu injection prevents sepsis-induced myocardial injury by inhibiting mitochondrial apoptosis. J Ethnopharmacol. 2020;261:113068.
Wu J, Li Z, Yuan W, Zhang Q, Liang Y, Zhang M, Qin H, Li C. Shenfu injection improves cerebral microcirculation and reduces brain injury in a porcine model of hemorrhagic shock. Clin Hemorheol Microcirc. 2021;78(2):175-185.
Wu Y, Li S, Li Z, Mo Z, Luo Z, Li D, Wang D, Zhu W, Ding B. Efficacy and safety of Shenfu injection for the treatment of post-acute myocardial infarction heart failure: A systematic review and meta-analysis. Front Pharmacol. 2022;13:1027131.
Wu H, Dai Z, Liu X, Lin M, Gao Z, Tian F, Zhao X, Sun Y, Pu X. Pharmacodynamic Evaluation of Shenfu Injection in Rats With Ischemic Heart Failure and Its Effect on Small Molecules Using Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging. Front Pharmacol. 2019;10:1424.
Zhou W, Chen Z, Fang Z, Xu D. Network analysis for elucidating the mechanisms of Shenfu injection in preventing and treating COVID-19 combined with heart failure. Comput Biol Med. 2022;148:105845.
Wu H, Dai Z, Liu X, Lin M, Gao Z, Tian F, Zhao X, Sun Y, Pu X. Pharmacodynamic Evaluation of Shenfu Injection in Rats With Ischemic Heart Failure and Its Effect on Small Molecules Using Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging. Front Pharmacol. 2019;10:1424.
Cho YL, Hur SM, Kim JY, Kim JH, Lee DK, Choe J, Won MH, Ha KS, Jeoung D, Han S, Ryoo S, Lee H, Min JK, Kwon YG, Kim DH, Kim YM. Specific activation of insulin-like growth factor-1 receptor by ginsenoside Rg5 promotes angiogenesis and vasorelaxation. J Biol Chem. 2015;290(1):467-77.
Gao XF, Zhang JJ, Gong XJ, Li KK, Zhang LX, Li W. Ginsenoside Rg5: A Review of Anticancer and Neuroprotection with Network Pharmacology Approach. Am J Chin Med. 2022;50(8):2033-2056.
Wang YS, Li H, Li Y, Zhu H, Jin YH. Identification of natural compounds targeting Annexin A2 with an anti-cancer effect. Protein Cell. 2018;9(6):568-579.
Dong Y, Fu R, Yang J, Ma P, Liang L, Mi Y, Fan D. Folic acid-modified ginsenoside Rg5-loaded bovine serum albumin nanoparticles for targeted cancer therapy in vitro and in vivo. Int J Nanomedicine. 2019;14:6971-6988.
Chen C, Lv Q, Li Y, Jin YH. The Anti-Tumor Effect and Underlying Apoptotic Mechanism of Ginsenoside Rk1 and Rg5 in Human Liver Cancer Cells. Molecules. 2021;26(13):3926.
Wei Y, Yang H, Zhu C, Deng J, Fan D. Hypoglycemic Effect of Ginsenoside Rg5 Mediated Partly by Modulating Gut Microbiota Dysbiosis in Diabetic db/db Mice. J Agric Food Chem. 2020;68(18):5107-5117.
Zhu Y, Zhu C, Yang H, Deng J, Fan D. Protective effect of ginsenoside Rg5 against kidney injury via inhibition of NLRP3 inflammasome activation and the MAPK signaling pathway in high-fat diet/streptozotocin-induced diabetic mice. Pharmacol Res. 2020;155:104746.
Yang YL, Li J, Liu K, Zhang L, Liu Q, Liu B, Qi LW. Ginsenoside Rg5 increases cardiomyocyte resistance to ischemic injury through regulation of mitochondrial hexokinase-II and dynamin-related protein 1. Cell Death Dis. 2017;8(2):e2625.
Chen Y, Li Y, Zhou ZY, Liu HN, Xu GL, Jiang L. Establishment and Optimization of Hypoxia/Reoxygenation Model of H9c2 Cardiomyocytes Based on Mixed Gas Method. Traditional Chinese Drug Research and Clinical Pharmacology.2020; 31(9):1123-1127.
Liao RH. Effects of rosiglitazone on H9c2 cardiomyocytes in high glucose microenvironment. https://kns.cnki.net/kcms2/article/abstract?v=qUVwNPMGfwpMvqucSAhuVVUpI4IONW_pgtf9WTV-D4l15fY4Tlp1cfkYBtNQS57ue1w6rHWee8CtLd0N5I0DCmheZnHWgSx3kTp7vngmpOqyHHJXH-d5fyt_1oy2VJx-MjG3cICA6p2un6-HtXV_mOT5Wla0vcTf&uniplatform=NZKPT&src=copy. Accessed 16 Jun 2018.
Galluzzi L, Aaronson SA, Abrams J, Alnemri ES, Andrews DW, Baehrecke EH, Bazan NG, Blagosklonny MV, Blomgren K, Borner C, Bredesen DE, Brenner C, Castedo M, Cidlowski JA, Ciechanover A, Cohen GM, De Laurenzi V, De Maria R, Deshmukh M, Dynlacht BD, El-Deiry WS, Flavell RA, Fulda S, Garrido C, Golstein P, Gougeon ML, Green DR, Gronemeyer H, Hajnóczky G, Hardwick JM, Hengartner MO, Ichijo H, Jäättelä M, Kepp O, Kimchi A, Klionsky DJ, Knight RA, Kornbluth S, Kumar S, Levine B, Lipton SA, Lugli E, Madeo F, Malomi W, Marine JC, Martin SJ, Medema JP, Mehlen P, Melino G, Moll UM, Morselli E, Nagata S, Nicholson DW, Nicotera P, Nuñez G, Oren M, Penninger J, Pervaiz S, Peter ME, Piacentini M, Prehn JH, Puthalakath H, Rabinovich GA, Rizzuto R, Rodrigues CM, Rubinsztein DC, Rudel T, Scorrano L, Simon HU, Steller H, Tschopp J, Tsujimoto Y, Vandenabeele P, Vitale I, Vousden KH, Youle RJ, Yuan J, Zhivotovsky B, Kroemer G. Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death Differ. 2009;16(8):1093-107.
Zhou X, Wu X, Qin L, Lu S, Zhang H, Wei J, Chen L, Jiang L, Wu Y, Chen C, Huang R. Anti-Breast Cancer Effect of 2-Dodecyl-6-Methoxycyclohexa-2,5-Diene-1,4-Dione in vivo and in vitro Through MAPK Signaling Pathway. Drug Des Devel Ther. 2020 Jul 7;14:2667-2684. doi: 10.2147/DDDT.S237699. Erratum in: Drug Des Devel Ther. 2020;14:4575-4577.
Willcox ML, Elugbaju C, Al-Anbaki M, Lown M, Graz B. Effectiveness of Medicinal Plants for Glycaemic Control in Type 2 Diabetes: An Overview of Meta-Analyses of Clinical Trials. Front Pharmacol. 2021;12:777561.
Fu S, Zhou Y, Hu C, Xu Z, Hou J. Network pharmacology and molecular docking technology-based predictive study of the active ingredients and potential targets of rhubarb for the treatment of diabetic nephropathy. BMC Complement Med Ther. 2022;22(1):210.
Wang J, Ma Q, Li Y, et al. Research progress on Traditional Chinese Medicine syndromes of diabetes mellitus. Biomed Pharmacother. 2020;121:109565.
Choi JH, Jang M, Nah SY, Oh S, Cho IH. Multitarget effects of Korean Red Ginseng in animal model of Parkinson's disease: antiapoptosis, antioxidant, antiinflammation, and maintenance of blood-brain barrier integrity. J Ginseng Res. 2018;42(3):379-388.
Gong L, Yin J, Zhang Y, Huang R, Lou Y, Jiang H, Sun L, Jia J, Zeng X. Neuroprotective Mechanisms of Ginsenoside Rb1 in Central Nervous System Diseases. Front Pharmacol. 2022;13:914352.
Cai Z, Liu M, Zeng L, Zhao K, Wang C, Sun T, Li Z, Liu R. Role of traditional Chinese medicine in ameliorating mitochondrial dysfunction via non-coding RNA signaling: Implication in the treatment of neurodegenerative diseases. Front Pharmacol. 2023;14:1123188.
Shang L, Wang Y, Li J, Zhou F, Xiao K, Liu Y, Zhang M, Wang S, Yang S. Mechanism of Sijunzi Decoction in the treatment of colorectal cancer based on network pharmacology and experimental validation. J Ethnopharmacol. 2023;302:115876.
Almatroodi SA, Almatroudi A, Khan AA, Alhumaydhi FA, Alsahli MA, Rahmani AH. Potential Therapeutic Targets of Epigallocatechin Gallate (EGCG), the Most Abundant Catechin in Green Tea, and Its Role in the Therapy of Various Types of Cancer. Molecules. 2020;25(14):3146.
Qiang M, Cai P, Ao M, Li X, Chen Z, Yu L. Polysaccharides from Chinese materia medica: Perspective towards cancer management. Int J Biol Macromol. 2023;224:496-509.
Motallebi M, Bhia M, Rajani HF, et al. Naringenin: A potential flavonoid phytochemical for cancer therapy. Life Sci. 2022;305:120752.
Bai J, Wang X, Du S, Wang P, Wang Y, Quan L, Xie Y. Study on the protective effects of danshen-honghua herb pair (DHHP) on myocardial ischaemia/reperfusion injury (MIRI) and potential mechanisms based on apoptosis and mitochondria. Pharm Biol. 2021 Dec;59(1):335-346. doi: 10.1080/13880209.2021.1893346IF: 3.889 Q1 . PMID: 35086399; PMCID: PMC8797739.
Guo B, Yang T, Nan J, Huang Q, Wang C, Xu W. Efficacy and safety of Shenfu injection combined with sodium nitroprusside in the treatment of chronic heart failure in patients with coronary heart disease: A protocol of randomized controlled trial. Medicine (Baltimore). 2021;100(7):e24414.
Gao Y, Gao Y, Zhu R, Tan X. Shenfu injection combined with furosemide in the treatment of chronic heart failure in patients with coronary heart disease: A protocol of randomized controlled trial. Medicine (Baltimore). 2021;100(3):e24113.
Jin YY, Gao H, Zhang XY, Ai H, Zhu XL, Wang J. Shenfu Injection () inhibits inflammation in patients with acute myocardial infarction complicated by cardiac shock. Chin J Integr Med. 2017;23(3):170-175.
Li ZE. Clinical research on effects of shenfu injection in different dosage in preventing heart failure occurred in patients of acute myocardial infarction with elevated ST segment. Chinese journal of integrated traditional and Western medicine. 2006;6(26):555-557.
Chen C, Lv Q, Li Y, Jin YH. The Anti-Tumor Effect and Underlying Apoptotic Mechanism of Ginsenoside Rk1 and Rg5 in Human Liver Cancer Cells. Molecules. 2021;26(13):3926.
van der Pol A, Hoes MF, de Boer RA, van der Meer P. Cardiac foetal reprogramming: a tool to exploit novel treatment targets for the failing heart. J Intern Med. 2020;288(5):491-506.
Chen X, Li X, Zhang W, He J, Xu B, Lei B, Wang Z, Cates C, Rousselle T, Li J. Activation of AMPK inhibits inflammatory response during hypoxia and reoxygenation through modulating JNK-mediated NF-κB pathway. Metabolism. 2018;83:256-270.
Xu S, Wu B, Zhong B, et al. Naringenin alleviates myocardial ischemia/reperfusion injury by regulating the nuclear factor-erythroid factor 2-related factor 2 (Nrf2) /System xc-/ glutathione peroxidase 4 (GPX4) axis to inhibit ferroptosis. Bioengineered. 2021;12(2):10924-10934.
Deng H, Yao X, Cui N, Huang S, Ge Y, Liu R, Yang X. The protective effect of zinc, selenium, and chromium on myocardial fibrosis in the offspring of rats with gestational diabetes mellitus. Food Funct. 2023;14(3):1584-1594.
He Y, Huang W, Zhang C, Chen L, Xu R, Li N, Wang F, Han L, Yang M, Zhang D. Energy metabolism disorders and potential therapeutic drugs in heart failure. Acta Pharm Sin B. 2021;11(5):1098-1116.
Nabben M, Luiken JJFP, Glatz JFC. Metabolic remodelling in heart failure revisited. Nat Rev Cardiol. 2018;15(12):780.
Yang J, Zhang Y, Pan Y, Sun C, Liu Z, Liu N, Fu Y, Li X, Li Y, Kong J. The Protective Effect of 1,25(OH)2D3 on Myocardial Function is Mediated via Sirtuin 3-Regulated Fatty Acid Metabolism. Front Cell Dev Biol. 2021;9:627135.
Lopaschuk GD, Karwi QG, Tian R, Wende AR, Abel ED. Cardiac Energy Metabolism in Heart Failure. Circ Res. 2021;128(10):1487-1513.
He Y, Huang W, Zhang C, Chen L, Xu R, Li N, Wang F, Han L, Yang M, Zhang D. Energy metabolism disorders and potential therapeutic drugs in heart failure. Acta Pharm Sin B. 2021;11(5):1098-1116.
Greenwell AA, Gopal K, Ussher JR. Myocardial Energy Metabolism in Non-ischemic Cardiomyopathy. Front Physiol. 2020;11:570421.
Santos-Gallego CG, Requena-Ibanez JA, San Antonio R, et al. Empagliflozin Ameliorates Adverse Left Ventricular Remodeling in Nondiabetic Heart Failure by Enhancing Myocardial Energetics. J Am Coll Cardiol. 2019;73(15):1931-1944.
Deng S, Chen B, Huo J, Liu X. Therapeutic potential of NR4A1 in cancer: Focus on metabolism. Front Oncol. 2022;12:972984.
Crean D, Murphy EP. Targeting NR4A Nuclear Receptors to Control Stromal Cell Inflammation, Metabolism, Angiogenesis, and Tumorigenesis. Front Cell Dev Biol. 2021;9:589770.
Koenis DS, Medzikovic L, van Loenen PB, van Weeghel M, Huveneers S, Vos M, Evers-van Gogh IJ, Van den Bossche J, Speijer D, Kim Y, Wessels L, Zelcer N, Zwart W, Kalkhoven E, de Vries CJ. Nuclear Receptor Nur77 Limits the Macrophage Inflammatory Response through Transcriptional Reprogramming of Mitochondrial Metabolism. Cell Rep. 2018;24(8):2127-2140.
Chao LC, Wroblewski K, Zhang Z, Pei L, Vergnes L, Ilkayeva OR, Ding SY, Reue K, Watt MJ, Newgard CB, Pilch PF, Hevener AL, Tontonoz P. Insulin resistance and altered systemic glucose metabolism in mice lacking Nur77. Diabetes. 2009;58(12):2788-96.
Medzikovic L, Heese H, van Loenen PB, van Roomen CPAA, Hooijkaas IB, Christoffels VM, Creemers EE, de Vries CJM, de Waard V. Nuclear Receptor Nur77 Controls Cardiac Fibrosis through Distinct Actions on Fibroblasts and Cardiomyocytes. Int J Mol Sci. 2021;22(4):1600.
Medzikovic L, Schumacher CA, Verkerk AO, van Deel ED, Wolswinkel R, van der Made I, Bleeker N, Cakici D, van den Hoogenhof MM, Meggouh F, Creemers EE, Remme CA, Baartscheer A, de Winter RJ, de Vries CJ, Arkenbout EK, de Waard V. Orphan nuclear receptor Nur77 affects cardiomyocyte calcium homeostasis and adverse cardiac remodelling. Sci Rep. 2015;5:15404.
Lu K, Zhao L, Zhang Y, Yang F, Zhang H, Wang J, Li B, Ji G, Yu J, Ma H. Bupivacaine reduces GlyT1 expression by potentiating the p-AMPKα/BDNF signalling pathway in spinal astrocytes of rats. Sci Rep. 2022;12(1):1378.
Charlot A, Morel L, Bringolf A, Georg I, Charles AL, Goupilleau F, Geny B, Zoll J. Octanoic Acid-Enrichment Diet Improves Endurance Capacity and Reprograms Mitochondrial Biogenesis in Skeletal Muscle of Mice. Nutrients. 2022;14(13):2721.
Bian Y, Li X, Li X, Ju J, Liang H, Hu X, Dong L, Wang N, Li J, Zhang Y, Yang B. Daming capsule, a hypolipidaemic drug, lowers blood lipids by activating the AMPK signalling pathway. Biomed Pharmacother. 2019;117:109176.
Fang X, Ardehali H, Min J, Wang F. The molecular and metabolic landscape of iron and ferroptosis in cardiovascular disease. Nat Rev Cardiol. 2023;20(1):7-23.
Lee H, Zandkarimi F, Zhang Y, Meena JK, Kim J, Zhuang L, Tyagi S, Ma L, Westbrook TF, Steinberg GR, Nakada D, Stockwell BR, Gan B. Energy-stress-mediated AMPK activation inhibits ferroptosis. Nat Cell Biol. 2020;22(2):225-234.
Xiao Z, Kong B, Fang J, Qin T, Dai C, Shuai W, Huang H. Ferrostatin-1 alleviates lipopolysaccharide-induced cardiac dysfunction. Bioengineered. 2021;12(2):9367-9376. Zhang Z, Tang J, Song J, Xie M, Liu Y, Dong Z, Liu X, Li X, Zhang M, Chen Y, Shi H, Zhong J. Elabela alleviates ferroptosis, myocardial remodeling, fibrosis and heart dysfunction in hypertensive mice by modulating the IL-6/STAT3/GPX4 signaling. Free Radic Biol Med. 2022;181:130-142.

No competing interests reported.

supplementarydocument.docx

Protective Effect of Ginsenoside Rg5 on Hypoxia/Reoxygenation- Induced H9c2 Cardiomyocytes by Correcting Free Fatty Acids β- oxidation

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Materials and Reagents

Culture of H9c2 Cardiomyocytes

Establishment of an H/R Model

Determination of Cell Viability

Experimental Protocols

Determination of LDH Release

Experimental Protocols

LDH Level Determination

ATP Measurements using High-Performance Liquid Chromatography (HPLC)

Liquid Chromatography

Preparation of a Standard Solution

Sample Pretreatment

Determination of FFA Content

RT-qPCR Analysis

Western Blotting

Results

Estimation of Optimal G-Rg5 Administration Conditions

H/R-Induced Disorders of NR4A1, AMPKα, p-AMPKα, and GPX4 Expression Levels were Suppressed by G-Rg5 Treatment

Discussion

Conclusions

Declarations

Ethics approval and consent to participate

Consent for Publication

Availability of Data and Materials

Competing Interests

Funding

Authors’ Contributions

Acknowledgements

References

Additional Declarations

Supplementary Files

Status:

Version 1