Ginsenoside Rd alleviates lipopolysaccharide-induced myocardial injury via modulating the MAPK and NF-κB pathways in cardiomyocytes and macrophages

doi:10.21203/rs.3.rs-4933370/v1

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Ginsenoside Rd alleviates lipopolysaccharide-induced myocardial injury via modulating the MAPK and NF-κB pathways in cardiomyocytes and macrophages

https://doi.org/10.21203/rs.3.rs-4933370/v1

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Cardiac dysfunction is a common complication of sepsis that manifests as uncontrolled inflammatory responses and myocardial injury. Ginsenoside Rd possesses various biological activities, with neuroprotective effects being most commonly reported. This study aimed to investigate the protective effects of ginsenoside Rd on lipopolysaccharide (LPS)-induced myocardial injury and its underlying mechanisms. Here, the cell counting kit-8 (CCK-8) assay was used to detect the cytotoxicity of ginsenoside Rd on mouse macrophages (RAW264.7) and rat cardiomyocytes (H9C2). Furthermore, the expression of the inflammatory cytokines interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α in LPS-stimulated RAW264.7 cells was detected through enzyme-linked immunosorbent assay (ELISA). LPS was also used to induce damage in H9C2 cells, and CCK-8 assay and Hoechst staining were used to assess cell viability and apoptosis. Cardiac cell injury, oxidative stress, and inflammation were determined by measuring lactate dehydrogenase (LDH), Ca²⁺, malondialdehyde (MDA), reactive oxygen species (ROS), and nitric oxide (NO) levels. Moreover, western blotting was used to detect the expression of normal and phosphorylated forms of the mitogen-activated protein kinase (MAPK)signaling components extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 and the nuclear factor kappa-B (NF-κB) signaling components p65 and phospho-p65 as well as the nuclear translocation of p65 in RAW264.7 and H9C2 cells. Interestingly, the results illustrated that ginsenoside Rd significantly reduced the release of TNF-α, IL-6, and IL-1β in a concentration-dependent manner. Ginsenoside Rd improved the survival rate of myocardial cells, which might be attributable to reduced apoptosis. Ginsenoside Rd decreased the levels of LDH, Ca²⁺, ROS, MDA, and NO in myocardial cells. Mechanistically, ginsenoside Rd inhibited the phosphorylation of ERK, JNK, and p38 and the phosphorylation and nuclear translocation of p65 in RAW264.7 cells. Similarly, ginsenoside Rd inhibited the phosphorylation of JNK, p38, and p65 in H9C2 cells. In conclusion, ginsenoside Rd can reduce the inflammatory response in macrophages, increase their survival rate, reduce apoptosis, and suppress oxidative stress and inflammation in cardiomyocytes by inhibiting the MAPK and NF-κB signaling pathways. Overall, the findings of this study indicate that ginsenoside Rd has the potential to be used for the treatment of septic myocardial injury.

Ginsenoside Rd

macrophages

cardiomyocytes

myocardial injury

Inflammation

Sepsis is a systemic inflammatory response syndrome characterized by excessive production of inflammatory cytokines and multiple organ damage¹. Approximately 5.3 million people die of sepsis annually, accounting for 10% of all deaths². Acute myocardial injury is a common cardiovascular complication in patients with sepsis, occurring in approximately 40%–50% of patients. Meanwhile, 20% of patients with sepsis experience severe heart failure, which is one of the important factors leading to death³. The treatment of sepsis primarily focuses on reducing the severe inflammatory state and cardiac dysfunction. During sepsis, macrophages, which act both as effector cells and antigen-presenting cells, are activated, and they release large amounts of inflammatory cytokines, thereby promoting the inflammatory cascade reaction and immune dysfunction⁴. This in turn leads to energy metabolism disorders, hypoxia, calcium homeostasis imbalance, and apoptosis in myocardial cells, which are important mechanisms of septic myocardial dysfunction⁵. Thus, macrophages and cardiomyocytes play crucial regulatory roles in septic myocardial injury.

Ginsenoside Rd, a protopanaxadiol-type and dammarane-type triterpenoid structure, primarily exists in Panax notoginseng and Panax ginseng of the Araliaceae family. Ginsenoside Rd has poor water solubility and is easily oxidized and degraded under specific conditions, making it difficult to extract directly from medicinal plants. Therefore, ginsenoside Rd is commonly obtained through specific forms of biotransformation using glycosidase enzyme or through metabolism by microbial intestinal flora⁶. Ginsenoside Rd exhibits a wide range of pharmacological effects; however, its neuroprotective effects, mainly in diseases such as Alzheimer’s, ischemic stroke, Parkinson’s, mood disorders, and epilepsy, are the most studied⁷. Ginsenoside Rd exerts neuroprotective effects mainly by inhibiting the release of pro-inflammatory factors, reducing the production of inflammatory mediators⁸, resisting oxidative stress⁹ and apoptosis¹⁰, regulating Ca²⁺ overload¹¹, and maintaining mitochondrial function¹².

In cardiovascular studies, ginsenoside Rd was found to alleviate myocardial ischemia and reperfusion injury by inhibiting inflammation and apoptosis and improving mitochondrial biogenesis through the PI3K/Akt and WNT5A/Ca²⁺ signaling pathways^13,14. Ginsenoside Rd protects cerebral endothelial cells from oxygen–glucose deprivation/reoxygenation by inhibiting SLC5A1-mediated sodium influx¹⁵. In addition, ginsenoside Rd activates the Nrf2/HO-1 signaling pathway, thereby reducing the activity of creatine kinase (CK) and (LDH) in serum and improving heart contraction and relaxation¹⁶. In conclusion, ginsenoside Rd exhibits a significant cardioprotective effect. However, there have been no reports on the application of ginsenoside Rd in treating septic myocardial injury, and its molecular mechanism remains unclear. In the present study, a myocardial inflammatory injury model was established by treating RAW264.7 murine macrophages and H9C2 rat cardiomyocytes with lipopolysaccharide (LPS), and the protective effect of ginsenoside Rd on excessive inflammation in macrophages and myocardial cell injury and its mechanism were investigated. Our study demonstrated that ginsenoside Rd can reduce the overexpression of inflammatory factors and myocardial injury by inhibiting the MAPK and NF-κB signaling pathways, revealing its potential application in the treatment of sepsis-induced cardiac injury.

Chemicals and reagents

Ginsenoside Rd, with 99.07% purity based on high performance liquid chromatography (HPLC) analysis, was purchased from SUNLIGHT TECHNOLOGIES CO., LTD. (China). LPS from Escherichia coli O55:B5 was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture and treatment

Murine macrophages (RAW264.7) and rat cardiomyocytes (H9C2) were obtained from the China Center for Type Culture Collection (Wuhan, China) and Gineo Biotechnology Co., Ltd (China), respectively. Both cell lines were cultured in Dulbecco’s Modified Eagle’s medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco). The cells were maintained in a 5% CO₂ incubator at 37℃ until they reached approximately 80% confluence. Both cell lines were passaged twice a week.

Cell viability assay

The cell counting kit-8 (CCK-8) assay (Beyotime, Jiangsu, China) was performed to evaluate cell viability. First, the cytotoxicity of ginsenoside Rd on RAW264.7 and H9C2 cells was examined. RAW264.7 and H9C2 cells were seeded into 96-well plates at a density of 2 × 10⁴ cells/well and incubated overnight. RAW264.7 and H9C2 cells were treated with various concentrations of ginsenoside Rd for 24 h. Then, nontoxic concentrations of ginsenoside Rd were used to detect H9C2 cell survival following LPS treatment. H9C2 cells were seeded at a density of 2 × 10⁴ cells/well into a 96-well plate, treated with various nontoxic concentrations of ginsenoside Rd, and stimulated with 10 µg/mL LPS for 24 h. After incubation, the culture medium was supplemented with 10% CCK-8 reagent and incubated for 3 h in the dark. The absorbance at 450 nm was measured using a continuous wavelength microplate reader (Thermo Fisher Scientific).

Apoptosis assay

H9C2 cell apoptosis was determined using Hoechst staining. The cells were plated in confocal dishes at a density of 2 × 10⁵ cells/dish, treated with various concentrations of ginsenoside Rd, and concomitantly stimulated with 10 µg/mL LPS for 24 h. After incubation, the cell supernatant was removed and fixed with paraformaldehyde (Biosharp, Anhui, China). The cells were stained with Hoechst 33258 according to the instructions of the staining kit (Beyotime). The apoptotic cells were observed using a fluorescence microscope (Zeiss, Oberkochen, Germany).

Flow cytometry

Nitric oxide (NO), Ca²⁺, and reactive oxygen (ROS) levels in H9C2 cells were detected via flow cytometry. H9C2 cells were seeded into a six-well plate at a density of 4 × 10⁵ cells/well, treated with various concentrations of ginsenoside Rd, and concomitantly stimulated with 1 µg/mL LPS for 24 h. After incubation, the cell supernatant was discarded and cells were washed with PBS (Gibco). Then, the cells were digested with EDTA-free trypsin (Chaselection, China). Cells were obtained by removing the cell suspension after centrifugation. Fluorescent probes against NO, ROS, and Ca²⁺ (all from Beyotime) were added, and the procedures were performed according to the instructions of the kit. The stained cells were assessed via flow cytometry (BD, Franklin Lakes, NJ, USA). FlowJo software (FlowJo, Ashland, OR, USA) was used to process the result.

Enzyme linked immunosorbent assay (ELISA)

The levels of the inflammatory cytokines interleukin (IL)-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) in RAW264.7 cells were determined using ELISA. RAW264.7 cells were seeded at a density of 4 × 10⁵ cells/well into a six-well plate, treated with various concentrations of ginsenoside Rd, and concomitantly stimulated with 200 ng/mL LPS. After incubation for 24 h, the culture supernatant was collected. The concentrations of interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α were measured using ELISA kits (Dakewe, Shenzhen, China) according to the manufacturer’s instructions.

Lactate dehydrogenase (LDH), Malondialdehyde (MDA) assays

H9C2 cells were seeded into a 24-well plate at a density of 2 × 10⁵ cells/well, treated with various concentrations of ginsenoside Rd, and concomitantly stimulated with 1 µg/mL LPS for 24 h. After incubation, the culture supernatant was collected and the cells were collected after digestion. The cell supernatant was used to detect LDH (NJJCBIO, Nanjing, China) levels, whereas the collected cells were used to detect MDA (NJJCBIO, Nanjing, China) levels according to the manufacturer’ s protocols.

Western blot analysis

MAPK and NF-κB signaling pathway-related proteins, including their phosphorylated forms, in RAW264.7 and H9C2 cells were detected via western blotting. RAW264.7 and H9C2 cells were seeded into six-well plates at a density of 4 × 10⁵ cells/well, pretreated with various concentrations of ginsenoside Rd for 12 h, and stimulated with LPS (1 µg/mL in H9C2 cells and 200 ng/mL in RAW264.7 cells) for 3 h. The cellular proteins were extracted using RAPI lysate (containing a protease and phosphatase inhibitor mixture, Solarbio, Beijing, China). The protein concentration was determined via BCA (Biosharp). Total proteins were separated via SDS-PAGE (EpiZyme, Cambridge, MA, USA) by loading 30 µg of total proteins into each well. After electrophoresis, the proteins were transferred to a PVDF membrane (MilliporeSigma, Burlington, MA, USA). The membrane was blocked with 5% skimmed milk (Neofroxx, Einhausen, Germany) for 1 h and incubated with the primary antibodies anti-p65 (#8242, 1:1000), anti-p-p65 (#3033, 1:1000), anti-p38 (#9212, 1:1000), anti-p-p38 (#4511, 1:1000), anti-ERK (#4695, 1:1000), anti-p-ERK (#5726, 1:1000), anti-JNK (#9252, 1:1000), and anti-p-JNK (#4668, 1:1000) overnight at 4°C. All primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The membrane was rinsed with TBST, followed by incubation with an HRP-conjugated secondary antibody (ab6717, Abcam, Cambridge, UK) for 1 h. Blots were visualized using the Western Blot Detection System (LIUYI, Beijing, China), and each band’s density was analyzed using ImageJ (US National Institutes of Health, Bethesda, MD, USA).

Immunofluorescence staining

The localization of p65 in RAW264.7 cells was observed through immunofluorescence staining. The cells were plated in confocal dishes at a density of 2 × 10⁵ cells/dish. The cells were pretreated with various concentrations of ginsenoside Rd for 12 h and stimulated with LPS for 1 h. After fixation with paraformaldehyde and blocking with goat serum, the cells were incubated with anti-p65 (1:500) overnight at 4°C, rinsed with PBS (Gibco), and incubated with the FITC-conjugated secondary antibody for 1 h at 37°C. Finally, the cells were incubated with 4′,6-diamidine-2′-phenylindole dihydrochloride (Solarbio) for 2 min. The stained cells were observed under a laser confocal microscope (Zeiss).

Statistical analysis

All data are expressed as mean ± standard deviation. SPSS 22.0 (IBM, Armonk, NY, USA) was used for statistical analysis, and the results were analyzed using one-way analysis of variance. A p value of < 0.05 was considered statistically significant. All experiments were repeated at least three times.

Effects of ginsenoside Rd on the survival of RAW264.7 macrophages and H9C2 cardiomyocytes

The chemical structure of ginsenoside Rd is presented in Fig. 1a. As presented in Fig. 1b and 1c, ginsenoside Rd reduced the survival of RAW264.7 and H9C2 cells at concentrations exceeding 50 µM (p < 0.01) but not at lower concentrations. Therefore, the concentrations of ginsenoside Rd in subsequent experiments did not exceed 50 µM.

Effects of ginsenoside Rd on the viability of LPS-treated H9C2 cells

LPS causes cardiomyocyte injury and reduces the survival of cardiomyocytes. We explored the protective effect of ginsenoside Rd on LPS-induced cardiomyocyte death. As presented in Fig. 2, LPS reduced the viability of H9C2 cells, and these effects were reversed by ginsenoside Rd (p < 0.01, Fig. 2a). In addition, the percentage of apoptotic cells in the LPS treatment group was markedly higher than that in the normal group (p < 0.01). Compared with the findings in the LPS treatment group, ginsenoside Rd inhibited the pro-apoptotic effect of LPS (p < 0.01, Fig. 2b and 2c). In conclusion, ginsenoside Rd could reduce the apoptosis induced by LPS and improve the survival of H9C2 cells.

Effects of ginsenoside Rd on proinflammatory cytokines in LPS-treated RAW264.7 cells

Macrophage-induced inflammatory cytokines play a crucial role in sepsis. Thus, we investigated the effects of ginsenoside Rd on TNF-α, IL-1β, and IL-6 secretion in LPS-treated RAW264.7 cells. As illustrated in Fig. 3, the secretion of IL-1β (Fig. 3a), IL-6 (Fig. 3b), and TNF-α (Fig. 3c) was significantly higher in LPS-induced RAW264.7 cells than in normal cells (all p < 0.01). Conversely, the increased secretion of TNF-α, IL-1β, and IL-6 induced by LPS was significantly decreased by ginsenoside Rd treatment in a concentration-dependent manner (p < 0.01). Overall, these results suggested that ginsenoside Rd can inhibit the excessive secretion of proinflammatory cytokines in RAW264.7 cells induced by LPS in a concentration-dependent manner.

Effects of ginsenoside Rd on cell injury in LPS-treated H9C2 cells

The protective effects of ginsenoside Rd on LPS-induced cardiomyocyte injury in terms of myocardial injury (LDH and Ca²⁺), oxidative stress (MDA and ROS), and inflammation (NO) were investigated. As presented in Fig. 4, LPS markedly increased the levels of LDH (Fig. 4a), MDA (Fig. 4b), NO (Fig. 4c and 4d), ROS (Fig. 4e and 4f), and Ca²⁺ (Fig. 4g and 4h) compared with those in the normal group (all p < 0.01). Compared with the levels in the LPS treatment group, ginsenoside Rd significantly reduced LDH, Ca²⁺, MDA, ROS, and NO levels (all p < 0.05). Therefore, these results suggested that ginsenoside Rd can alleviate cardiomyocyte injury in LPS-treated H9C2 cells through several mechanisms.

Effects of ginsenoside Rd on NF-κB and MAPK activation in LPS-treated RAW264.7 and H9C2 cells

MAPK and NF-κB are important signaling pathways of myocardial injury in sepsis. Activated MAPK and NF-κB signaling pathways promote the release of inflammatory factors by macrophages, leading to increased oxidative stress and inflammation in cardiomyocytes and resulting in myocardial injury. We examined the effects of ginsenoside Rd on the regulation of the MAPK and NF-κB signaling pathways in LPS-treated RAW264.7 and H9C2 cells. The expression of proteins involved in MAPK and NF-κB signaling was detected via western blotting, and the localization of NF-κB p65 was observed through immunofluorescence staining. As presented in Figs. 5 and 6, LPS treatment increased the expression of p-JNK, p-ERK, p-p38, and p-p65 in RAW264.7 cells (all p < 0.01, Fig. 5a, 5b, 5d, and 5e) and increased the expression of p-JNK, p-p38, and p-p65 in H9C2 cells (all p < 0.01, Fig. 6a, 6b, 6c, and 6d). In addition, NF-κB p65 was mostly translocated to the nucleus in the LPS treatment group compared with that in the normal group in RAW264.7 cells (Fig. 5c). These data indicated that LPS could activate the MAPK and NF-κB signaling pathways in both RAW264.7 and H9C2 cells. However, treatment with ginsenoside Rd decreased the expression of p-JNK, p-ERK, p-p38, and p-p65 (all p < 0.01); partially inhibited the nuclear translocation of NF-κB p65 in RAW264.7 cells; and decreased the expression of p-JNK, p-p38, and p-p65 (all p < 0.01) in H9C2 cells. Overall, ginsenoside Rd attenuated LPS-induced septic myocardial injury by inhibiting the MAPK and NF-κB signaling pathways.

The definition for sepsis from the third international consensus states that “sepsis is a lethal organ function disorder induced by an aberrant reaction to infections.”¹ Myocardial tissue is the most common target during the progression of sepsis, and myocardial tissue damage is also the starting point for multiple organ dysfunction syndrome in sepsis¹⁷. Gram-negative bacterial endotoxin (LPS) is a key mediator of sepsis-associated multiple organ dysfunction or mortality¹⁸. Therefore, endotoxemia is often used to simulate the acute inflammatory response associated with early sepsis¹⁹. Several experimental studies have established animal and cell models of sepsis using LPS-induced excessive inflammation and organ impairment^20–22. Macrophages and cardiomyocytes are the main effector cells in septic myocardial injury. After LPS induction, both macrophages and cardiomyocytes can release substantial amounts of inflammatory mediators²³.

Myocardial infiltration of immune cells is a recognized feature of severe sepsis, and the main cell types involved are monocytes/macrophages²⁴. Cytokines, especially IL-1β, IL-6, and TNF-α, are major contributors to septic myocardial injury²⁵. When LPS binds to its receptor expressed on macrophages, a cascade of inflammatory responses is triggered²⁶. In this study, we found that ginsenoside Rd can significantly reduce the LPS-induced release of inflammatory factors in RAW264.7 cells, including TNF-α, IL-6, and IL-1β, in a concentration-dependent manner. These results suggest that ginsenoside Rd exerts a significant inhibitory effect on the release of inflammatory factors from macrophages in sepsis, thereby preventing myocardial and organ damage.

Numerous clinical and animal studies have confirmed the presence of myocardial cell suppression in sepsis^27,28. The myocardium not only experiences inflammatory infiltration by immune cells but also produces an inflammatory response when induced by LPS, leading to myocardial cell apoptosis, calcium homeostasis imbalance²⁹, energy metabolism disorders³⁰, increased oxidative stress levels, and excessive NO production³¹. All these factors contribute to decreased cardiac contractility, diastolic dysfunction, reduced ejection fraction, and a lower cardiac index, which ultimately lead to myocardial injury³². In this study, we found that ginsenoside Rd can improve the survival rate of cardiomyocytes, possibly by reducing myocardial cell apoptosis. Excessive LDH production is a hallmark indicator of myocardial injury, and increased intracellular Ca²⁺ levels also disrupt calcium homeostasis and lead to myocardial injury³³. The findings of the present study indicated that ginsenoside Rd can reduce LDH and Ca²⁺ levels in cardiomyocytes, thereby alleviating LPS-induced myocardial injury. ROS production reflects the overall oxidative stress level of cells, and MDA content can indirectly reflect the degree of oxidative stress in the body or tissue cells³⁴. We herein observed that ginsenoside Rd could reduce ROS and MDA levels in cardiomyocytes. In addition, NO influences several aspects of the inflammatory cascade, and excessive NO production can induce cardiomyocyte death³⁵. We also found that ginsenoside Rd could reduce NO levels in cardiomyocytes. These results suggest that ginsenoside Rd can reduce the extent of LPS-induced myocardial cell damage, as evidenced by increased cell survival rates, reduced cell apoptosis, lower myocardial injury marker levels, and suppressed oxidative stress and inflammatory responses.

The MAPK and NF-κB signaling pathways play crucial roles in septic myocardial injury³⁶. These pathways are important in regulating inflammation, cell survival, apoptosis, and stress responses. The MAPK family mainly includes three key signaling proteins: ERK, JNK, and p38. ERK is mainly related to cell proliferation and differentiation³⁷. JNK and p38, known as stress-related protein kinases, are mainly involved in cellular inflammation, oxidative stress responses, and apoptosis³⁸. The classical NF-κB pathway involves a heterodimer composed of p65 and p50 that activates the expression of genes related to inflammatory responses through nuclear translocation³⁹. Mechanistically, there is an intersection between the MAPK and NF-κB signaling pathways. It is currently believed that the p38 MAPK signaling pathway is a major upstream factor that activates NF-κB⁴⁰. Upon activation, p38 MAPK can phosphorylate IκB, leading to its degradation and the release of the p50/p65 NF-κB heterodimer, directly triggering NF-κB pathway activation and nuclear translocation and driving the transcription of NF-κB target genes in the nucleus. These alterations promote the expression of the inflammatory factors TNF-α, IL-6, and IL-1β, thereby activating inducible nitric oxide synthase to promote NO expression⁴¹. Previous studies have reported that ginsenoside Rd can inhibit the NF-κB signaling pathway⁴². In the present study, we observed that LPS activated the MAPK and NF-κB signaling pathways in both macrophages and cardiomyocytes. In macrophages, ginsenoside Rd inhibited the phosphorylation of ERK, JNK, and p38 and suppressed the phosphorylation and nuclear translocation of p65. Similarly, in cardiomyocytes, ginsenoside Rd inhibited the phosphorylation of JNK, p38, and p65. These results indicate that ginsenoside Rd can inhibit the MAPK and NF-κB signaling pathways in both cardiomyocytes and macrophages, thereby preventing LPS-induced septic myocardial injury.

In summary, our study confirmed that ginsenoside Rd can reduce the inflammatory response of macrophages by inhibiting the MAPK and NF-κB signaling pathways, improve cardiomyocyte survival, reduce cardiomyocyte apoptosis, and inhibit myocardial oxidative stress and inflammatory responses (Fig. 7). Furthermore, our study demonstrated that ginsenoside Rd has the potential to be used in the treatment of septic myocardial injury. Further animal experiments are warranted to further confirm this conclusion.

LPS lipopolysaccharides

IL-6 interleukin-6

IL-1β interleukin-1β

TNF-α tumor necrosis factor-α

CCK-8 cell counting Kit-8

ELISA enzyme-linked immunosorbent assay

LDH lactate dehydrogenase

MDA malondialdehyde

ROS reactive oxygen

NO nitric oxide

ERK extracellular signal-regulated kinase

JNK c-Jun N-terminal kinase

MAPK mitogen-activated protein kinase

NF-κB nuclear factor kappa-B

CK creatine kinase

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Author contributions

CZ and YG conceived and designed the experimental protocol. CZ, HY and ZZ completed all the experimental operation and detection of H9C2 cells. TL and LG completed all the experimental operation and detection of RAW264.7 cells. ZH completed the detection of flow cytometry. CZ and YG carried out data analysis and wrote a draft of the manuscript. DW and HY contributed to critical review of the manuscript. All the authors have seen and approved the manuscript.

Funding

This work was supported by the Yunnan Fundamental Research Projects (grant NO. 202401AT070155), Key Research and Development Project of Yunnan Province (grant NO. 202203AC100008), and Caiyun Postdoctoral Program of Yunnan Province.

Competing interests

The authors declare no conflicts of interest in the publication of this paper.

Ethical approval

This paper does not contain any studies with human participants or animals performed by any of the authors.

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No competing interests reported.

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Ginsenoside Rd alleviates lipopolysaccharide-induced myocardial injury via modulating the MAPK and NF-κB pathways in cardiomyocytes and macrophages

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Abstract

Figures

Introduction

Materials and methods

Chemicals and reagents

Cell culture and treatment

Cell viability assay

Apoptosis assay

Flow cytometry

Enzyme linked immunosorbent assay (ELISA)

Lactate dehydrogenase (LDH), Malondialdehyde (MDA) assays

Western blot analysis

Immunofluorescence staining

Statistical analysis

Results

Effects of ginsenoside Rd on the survival of RAW264.7 macrophages and H9C2 cardiomyocytes

Effects of ginsenoside Rd on the viability of LPS-treated H9C2 cells

Effects of ginsenoside Rd on proinflammatory cytokines in LPS-treated RAW264.7 cells

Effects of ginsenoside Rd on cell injury in LPS-treated H9C2 cells

Effects of ginsenoside Rd on NF-κB and MAPK activation in LPS-treated RAW264.7 and H9C2 cells

Discussion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1