Dapagliflozin attenuates LPS-induced myocardial injury by reducing ferroptosis

doi:10.21203/rs.3.rs-3958232/v1

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Dapagliflozin attenuates LPS-induced myocardial injury by reducing ferroptosis

https://doi.org/10.21203/rs.3.rs-3958232/v1

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published 14 May, 2024

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Lipopolysaccharide induces sepsis in vivo, with a significant proportion of septic patients progressing to septic cardiomyopathy. Previous studies have reported the involvement of ferroptosis in the pathogenesis of septic cardiomyopathy. SGLT2 inhibitors such as dapagliflozin have been demonstrated to have cardioprotective effects, with reports indicating a reduction in myocardial ischemia-reperfusion injury through the attenuation of ferroptosis. However, the role of ferroptosis-induced myocardial injury in the context of LPS-induced sepsis remains unclear. Therefore, our study aims to investigate the therapeutic effects of dapagliflozin on LPS-induced iron-overload cardiac injury. Our results indicate that dapagliflozin inhibits the translation of key proteins associated with ferroptosis, including GPX4, FTH1, and SLC7A11, while reducing the transcription of lipid peroxidation-related mRNAs PTGS2 and ACSL4, as well as iron metabolism genes TFRC and HMOX1. Additionally, both compounds alleviate potential mitochondrial membrane damage. Furthermore, dapagliflozin has been shown to mitigate LPS-induced cardiac injury burden. Based on these findings, we conclude that dapagliflozin can alleviate LPS-induced iron dysregulation-mediated cardiac dysfunction, expanding the clinical indications for SGLT2 inhibitors.

lipopolysaccharide

dapagliflozin

ferroptosis

cardiomyopathy

Sepsis is a condition characterized by organ dysfunction caused by a dysregulated host response to infection(Singer et al., 2016). It remains a leading cause of mortality in intensive care units(Ravikumar et al., 2021). Lipopolysaccharide (LPS), also known as endotoxin, triggers sepsis by interacting with specific receptors on host effector cells(Solov'eva et al., 2013). A significant proportion of sepsis patients develop sepsis-induced cardiac dysfunction, which represents the most severe and lethal complication of sepsis, with mortality rates ranging from 70–90%(Li et al., 2022; Zhong et al., 2019). The pathogenesis of septic-induced cardiomyopathy(SIC) primarily involves cellular apoptosis, mitochondrial damage, autophagy, excessive inflammatory response, oxidative stress, pyroptosis, and ferroptosis(Lin et al., 2020; Lv and Wang, 2016; Rudiger et al., 2013; Sun et al., 2023; Yang and Zhang, 2021; Zanotti-Cavazzoni and Hollenberg, 2009).

Ferroptosis is a non-apoptotic, iron-dependent, and peroxide-driven form of cell death(Stockwell et al., 2017; Yagoda et al., 2007; Yang and Stockwell, 2008). In the past decade, ferroptosis has been increasingly recognized as a critical process mediating the onset and progression of numerous cardiovascular diseases, including atherosclerosis, drug-induced heart failure, myocardial ischemia-reperfusion injury(Del Re et al., 2019; Dixon et al., 2012; Fang et al., 2023). During the process of lipopolysaccharide-induced myocardial injury, oxidative stress and reactive oxygen species(ROS) increase, leading to an augmented release of free iron, subsequently causing mitochondrial damage and exacerbating lipid peroxidation. These multiple factors interact and exacerbate each other. Therefore, selectively reducing ferroptosis represents an effective approach to mitigate myocardial injury. Most studies have been conducted in animal models, targeting molecular and metabolic pathways involved in cellular defense against oxidative stress to prevent cardiomyopathy(Bajic et al., 2019; Chen and Zweier, 2014). Currently, these findings have not yet been translated into clinical applications. Thus, exploring drugs within the realm of clinically approved medications that can inhibit ferroptosis to alleviate LPS-induced myocardial injury represents a promising shortcut.

Sodium-glucose co-transporter 2 inhibitor(SGLT2i) potentially improve the prognosis of patients with heart failure (HF) independently of their hypoglycemic effects(Bajic et al., 2019). SGLT2i can inhibit myocardial inflammation, reduce oxidative stress, regulate autophagy, improve mitochondrial function, and decrease cardiomyocyte apoptosis(Lin et al., 2022; Nikolaou et al., 2021; Nikolaou et al., 2022; Yu et al., 2021). Based on these mechanisms, it can be speculated that SGLT2i may inhibit iron-mediated cell death. Therefore, SGLT2i holds promise as cardioprotective agents. However, the therapeutic effects of dapagliflozin in inhibiting iron-mediated cell death in LPS-induced SIC have not been extensively studied. The aim of our study is to investigate whether dapagliflozin can alleviate iron overload in LPS-induced cardiomyocytes and mitigate the symptoms of SIC.

Animal and animal model

The Ethics Review Committee of Anhui Medical University has approved all animal experiments in accordance with the guidelines for the protection and use of experimental animals issued by the National Institute of Health Research.

Male C57BL/6 mice (20-28g, 6–9 weeks old) were purchased from Gempharmatech and housed in a pathogen-free, temperature-controlled environment (20℃-25℃) with a specific facility providing a 12-hour light-dark cycle. Each cage accommodated a maximum of 6 mice and they were fed a standard mouse diet.

The mice were randomly divided into four groups: Control group (intraperitoneal injection of sterile saline), LPS group (intraperitoneal injection of 10mg/kg LPS), LPS + ferrostatin-1 group (10mg/kg LPS + 1mg/kg ferrostatin-1), LPS + dapagliflozin group (dapagliflozin 10 mg/kg/day + intraperitoneal injection of 10mg/kg LPS). LPS(MCE) and ferrostatin-1(MCE) were administered via intraperitoneal injection, with LPS administered first followed by ferrostatin-1. Dapagliflozin(MCE) was dissolved in sterile PBS and stored at -80℃. Dapagliflozin was administered via gastric lavage once daily for a consecutive period of five days. On the 5th day, after oral gavage, intraperitoneal injection of LPS (10mg/kg) was performed, and 18 hours later, the mice were anesthetized with 50 mg/kg pentobarbital sodium. Blood samples were collected from the eyeballs. After anesthetizing the mice, the heart was perfused with PBS to obtain cardiac tissue.

Echocardiography

The details of transthoracic echocardiography procedure have been extensively described in previous studies[Andrew Rhodes1*, #663]. In brief, after depilation of the thoracic and abdominal regions, mice were maintained under 1.5% isoflurane anesthesia. Transthoracic echocardiography was performed using the Vevo 2100 477

transthoracic echocardiography (Visualsoics, Canada), with the use of a MX400 (30MHz) transducer in B-mode. Subsequently, parameters such as heart rate (HR,bpm), left ventricle ejection fraction (LVEF,%), and left ventricle fractional shortening (LVFS,%) were measured and calculated using the Vevo imaging software. Data from 3–5 cardiac cycles were collected.

Quantitative real-time polymerase chain reaction(PCR)

Total RNA was extracted using the commercial RNA-easy isolation reagent (Vazyme Biotech Co. Ltd). First, the cardiac tissue was homogenized using enzyme-free steel beads, and then RNA extraction was performed according to the manufacturer's instructions. The concentration and purity of the samples were measured using the Unano-1000 ultramicro spectrophotometer (Hangzhou UMI instrument, China). Reverse transcription was performed using the HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme Biotech Co. Ltd), following the manufacturer's instructions. Quantitative PCR was conducted using the Applied Biosystems SimpliAmp PCR thermal cycler and the ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co. Ltd). The PCR primer sequence is as follows: ACSL4(F:5′-CCCCAGACACACCGATTCAT-3′, R: 5′-AGCGCCAACTCTTCCAGTAG-3′); PTGS2(F:5′-ATGCTACCATCTGGCTTCGG-3′, R: 5′-TGGAACAGTCGCTCGTCATC-3′); HMOX1(F:5′-GCCTGGTTCAAGATACTACCTCT-3′, R: 5′-CTGAGTGTGAGGACCCATCG); TFR1(F:5′-CGGCCTATATGCTTGGGTAGG-3′, R: 5′-GAAGCACCTCCTCAGTCCG-3′); GAPDH(F:5′-GACATGCCGCCTGGAGAAAC-3′, R: 5′-AGCCCAGGATGCCCTTTAGT-3′). The cycle threshold (Ct) values were determined using the comparative Ct method and normalized to the level of GAPDH.

Western blot

The cardiac tissue was extracted for total protein according to the instructions of the total protein extraction kit, using RIPA lysis buffer (KeyGEN Biotech). The entire process was performed on ice. The protein concentration was determined using the Pierce BCA protein assay kit (Thermo Scientific). Subsequently, the proteins were separated by 10%-15% SDS-PAGE electrophoresis. After transfer onto PVDF membranes, the membranes were blocked with 5% milk for 2 hours. The membranes were then incubated overnight at 4°C with GPX4(CST, 52455S, 1:1000), FTH1(CST, 4393S, 1:1000) and SCL7A11(BOSTER, M00248-1, 1:1000). GAPDH (Proteintech, 60004-1-Ig, 1:2000) was used as control. Immunoreactive bands were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 hours, followed by exposure using a gel imaging system (Bio-Rad, USA). Visualization of the bands was performed using the ECL detection kit(Abbkine), and quantitative analysis was conducted using ImageJ software version 1.8.0. The relative abundance of the target protein was normalized to GAPDH.

Immunofluorescence

For immunofluorescence staining, the mouse heart was fixed in 4% paraformaldehyde and dehydrated in a 30% sucrose solution. Subsequently, the hearts were embedded in OCT compound (SAKURA). Longitudinal tissue sections were obtained using a cryostat (Leica CM3050S), and the sections were stored at -20°C for long-term preservation. The tissue sections were blocked with goat serum blocking solution (Biosharp) for 1 hour, followed by overnight incubation of GPX4(CST, 52455S, 1:200), FTH1(CST, 4393S, 1:200) and SCL7A11(BOSTER, M00248-1, 1:200) at 4°C and subsequent 2-hour incubation of the secondary antibody at room temperature. Nuclear staining was performed using DAPI (Invitrogen). Images were captured using a confocal microscope (ZEISS, LSM 900 with Airyscan 2, Germany), and analysis was performed using Image-Pro Plus version 6.

Detection of malondialdehyde(MDA), superoxide dismutase(SOD) and iron levels in serum and cardiac tissue

The levels of MDA(Biosharp) and SOD(Biosharp) in serum and tissue were determined using colorimetric assays, following the instructions provided with the assay kits. Absorbance was measured at 532nm or 450nm using a spectrophotometer (Thermo Scientific Varioskan LUX, USA). To measure the levels of ferrous ions (Elabscience) in the tissue and serum. The experimental procedures were carried out according to the instructions, and absorbance was measured at 593nm.

Elisa assay for determination of Creatine kinase-MB(CK-MB) and Cardiac troponin T(cTnT)

Quantification of cTNT(FineTest) and CK-MB(FineTest) levels in cardiac tissue and serum using assay kits. The experiment was conducted following the instructions provided, and the OD values were measured at 450 nm.

Measurement of tissure section ROS

The fluorescent probe BBoxiProbe DHE was utilized to detect ROS (Bestbio). Perform ROS staining according to the provided instructions. Fluorescence intensity was examined under a confocal microscope (ZEISS, LSM 900, Airyscan 2, Germany) with an excitation wavelength of 535 nm and an emission wavelength of 610 nm. Finally, Image-Pro Plus version 6 was used to process the data, representing the difference in ROS levels by comparing the fluorescence intensity of the experimental group to that of the control group.

Assessment of mitochondrial membrane potential

The JC-1 fluorescent probe(Beyotime) can be used to detect mitochondrial membrane potential. Staining with JC-1 was performed according to the provided instructions. Fluorescence expression was observed using a confocal microscope (ZEISS, LSM 900, Airyscan 2, Germany). Green fluorescence represents JC-1 monomers, with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Red fluorescence represents JC-1 aggregates, with an excitation wavelength of 525 nm and an emission wavelength of 590 nm. Data was processed using Image-Pro Plus version 6.

Statistical analysis

The data are presented as mean ± standard deviation.For all the images in this study, data were obtained by selecting 5 fields of view per slide and averaging them as one "n" value. One slide per mouse was used. As for Western blot experiments, three replicates with consistent trends were performed, and the average value was calculated. Each mouse represented one "n" value, consistent with other experiments. GraphPad Prism 9.0.0 software was used for data analysis. Unpaired t-tests were used for comparisons between two groups, while one-way ANOVA tests were used for comparisons among three groups. P < 0.05 was considered statistically significant.

LPS causes myocardial injury through oxidative stress damage

Over the past few decades, a substantial body of clinical and experimental data has consistently supported the concept of oxidative stress, characterized by an imbalance between the excessive production of ROS and the antioxidant defense system, resulting in tissue damage(Belch et al., 1991; Hill and Singal, 1996, 1997; Mallat et al., 1998). Elevated levels of reactive oxygen species can lead to cellular dysfunction, protein and lipid peroxidation, and irreversible cell injury and death, implicating their involvement in various pathological cardiovascular conditions(Tsutsui et al., 2011). CK-MB and cTnT serve as biomarkers of myocardial injury. Following LPS injection, both serum and cardiac tissue levels of CK-MB and cTnT were significantly elevated compared to the control group (Fig. 1A-B). MDA, a metabolic byproduct of lipid peroxidation, accumulates as a result of lipid peroxidation. Meanwhile, SOD, as an important antioxidant enzyme, is excessively consumed in lipid peroxidation. Following LPS injection, there was a notable increase in MDA levels and a significant decrease in SOD levels in both serum and tissues compared to the control group (Fig. 1C-D). These findings indicate that LPS primarily induces myocardial cell dysfunction through oxidative stress-mediated damage.

Ferroptosis can induce myocardial oxidative stress damage, mitochondrial damage

Lipid peroxidation is generally closely associated with ferroptosis(Li et al., 2020b). In order to verify that LPS-induced myocardial injury is mediated by ferroptosis, we examined the expression of three critical proteins in iron metabolism. GPX4 can counteract the impact of oxidative stress, while SCL7A11 indirectly sustains the function of GPX4, thereby inhibiting the progression of ferroptosis. Additionally, FTH1 maintains the homeostasis of Fe²⁺ within the body, collectively preventing the occurrence of ferroptosis(Mancias et al., 2014; Mi et al., 2023; Yang et al., 2016).The results showed that GPX4, FTH1, and SLC7A11 were significantly decreased in the LPS-treated group compared to the control group, and this decrease could be partially rescued by the ferroptosis inhibitor(ferrostatin-1)(Fig. 2A-D). In addition to assessing protein expression levels using Western blot, we concurrently employed immunofluorescence for validation (Fig.S1E-F), which yielded consistent results. Our results showed that ferrostatin-1 can attenuate LPS-induced myocardial injury, providing evidence that ferroptosis partially contributes to the cardiac damage caused by LPS (Fig.S1A-B).

Next, at the gene level, we examined the mRNA expression levels of iron metabolism-related genes TFRC and HMOX1. We also assessed the expression of myocardial tissue lipid metabolism-related genes PTGS2 and ACSL4. It was evident that the mRNA expression levels of TFRC, HMOX1, PTGS2 and ACSL4 significantly increased following LPS injection. Importantly, the administration of ferrostatin-1 effectively alleviated the elevation of these four genes induced by ferroptosis (Fig. 2E and S1D).

Subsequently, we observed a significant increase in the serum Fe²⁺ concentration following LPS injection. However, administration of ferrostatin-1 effectively reduced the serum Fe²⁺ concentration (Fig. 2F). Alterations in MDA and SOD levels were observed in the animal model following LPS injection. Administration of ferrostatin-1 showed a trend towards normalization of MDA and SOD levels(Fig. 2G and S1C). Our findings further demonstrate that ferrostatin-1 can also alleviate the generation of ROS induced by LPS(Fig. 2H and Fig.S1F). Mitochondrial membrane potential (MMP) aberrations serve as crucial indicators of mitochondrial damage and early warning signs of iron overload(Fang et al., 2019). JC-1, a fluorescent dye, is commonly employed to assess changes in MMP. In the LPS group, the monomeric form of JC-1 exhibited a pronounced increase in green fluorescence, indicating a low MMP, while the control group shows normal high mitochondrial membrane potential with aggregated red fluorescence. Notably, treatment with ferrostatin-1 attenuated the LPS-induced green fluorescence, restoring the red fluorescence associated with normal membrane potential (Fig. 2I-J). Taken together, all of these results indicated that ferroptosis is closely related to oxidative stress and mitochondrial damage. Mitigating both can reduce the harm caused by ferroptosis.

The SGLT2 inhibitor dapagliflozin can mitigate ferroptosis-associated damage

In order to ascertain whether dapagliflozin can alleviate myocardial damage induced by LPS, we conducted an assessment of hallmark proteins associated with ferroptosis. Our findings unequivocally demonstrate that dapagliflozin partially mitigates the reduced protein expression of GPX4, FTH1, and SLC7A11 induced by LPS(Fig. 3A-B and S2B). Subsequently, we evaluated the expression of ferroptosis-related genes and observed that dapagliflozin treatment effectively reduced the expression of HMOX1 and TFRC(Fig. 3C). Simultaneously, the upregulation of lipid peroxidation-related genes PTGS2 and ACSL4 induced by LPS was partially rescued by the administration of dapagliflozin(Fig. 3D). In addition, we assessed the concentration of ferrous ions in both serum and tissues to validate the impact of dapagliflozin on free iron ions. Remarkably, Dapagliflozin significantly reduced the elevation of free iron ions induced by LPS, thereby preventing further damage(Fig. 3E). The increased levels of MDA in serum and heart tissue induced by LPS can be mitigated by dapagliflozin, and the compromised antioxidant effect of SOD can be partially restored by dapagliflozin as well(Fig. 3F-G and S2A). Furthermore, the fluorescence images demonstrate a reduction in the expression intensity of ROS due to administration of dapagliflozin(Fig. 3H and I). The utilization of dapagliflozin facilitates the transition of mitochondrial JC-1 from its monomeric form to its aggregated state(Fig. 3J and K).

The aforementioned data demonstrate that dapagliflozin, as an SGLT2i, exerts effects beyond diabetes treatment by exhibiting antioxidant properties and alleviating mitochondrial dysfunction in the myocardium. Additionally, it attenuates the impact of ferroptosis on cardiac tissue caused by LPS.

Dapagliflozin can effectively mitigate cardiac functional impairment induced by LPS

The previous findings have demonstrated dapagliflozin's efficacy in mitigating ferroptosis and oxidative stress-induced myocardial tissue damage caused by LPS. Here, we conducted specific assessments of cardiac phenotypes. Echocardiography revealed a significant increase in LVEDD (%) and a notable decrease in LVEF (%) and LVFS (%) in the LPS group compared to the control group, with no significant impact on heart rate. Dapagliflozin administration partially reversed these effects, with minimal impact on heart rate (Fig. 4A-B). Additionally, we indirectly assessed heart failure severity by evaluating the wet-to-dry weight ratio of the lungs, which reflected reduced fluid retention and pulmonary edema due to decreased cardiac pumping function caused by LPS. Dapagliflozin reduced pulmonary edema (Fig. 4C). Other heart failure criteria, such as the ratio of heart weight to tibia length and the ratio of heart weight to body weight, showed varying degrees of increase due to LPS injection, which were alleviated by dapagliflozin (Fig. 4E). Furthermore, dapagliflozin significantly reduced CK-MB and cTnT levels compared to the LPS group (Fig. 4F). These multifaceted results collectively indicate dapagliflozin's protective effect on cardiac function.

LPS is a prototypical pathogen-associated molecular pattern (PAMP) and a potent mediator that contributes to the development of sepsis and septic shock. Therefore, deciphering the cascade of responses triggered by LPS has been the subject of intensive investigation(Downs et al., 2020). Early septic shock, induced by a cytokine storm and cardiac dysfunction, is a significant contributor to mortality in septic patients(Hotchkiss et al., 2013). The cardiac dysfunction caused by sepsis is known as SIC. The complex pathogenesis of SIC was involved in mitochondrial dysfunction and oxidative stress(Liu et al., 2017). Mitochondria are vital organelles involved in cellular energy metabolism. When mitochondrial function is impaired, cellular homeostasis is disrupted(Liesa et al., 2009; Yapa et al., 2021). The imbalance of intracellular redox homeostasis leads to increased iron ions and ROS(Li et al., 2020a). In our study, we observed that LPS administration led to an increase in the levels of the lipid peroxidation product MDA in the body, as well as excessive consumption of SOD. Additionally, LPS induced a decrease in mitochondrial membrane potential and an elevation in ROS expression. These phenomena were accompanied by a significant increase in markers of myocardial injury, indicating the involvement of oxidative stress in LPS-induced myocardial damage.

SIC is defined as a reversible impairment of both left and right ventricular systolic and diastolic function caused by myocardial depressants released by pathogens and hosts, as well as systemic ischemia following peripheral vasodilation(Antonucci et al., 2014). Considering the potential involvement of ferroptosis in LPS-induced myocardial injury, our research results have indeed confirmed this hypothesis. The first synthetic ferroptosis inhibitor reported in the literature is ferrostatin-1, which has been widely employed in studies over the past decade(Dixon et al., 2012). GPX4, as a structural protein and antioxidant enzyme, effectively inhibits lipid oxidation(Liu et al., 2023). In recent years, it has been recognized as a key regulatory factor in iron homeostasis(Yant et al., 2003). SLC7A11, a cystine transporter, is involved in intracellular cysteine-homocysteine exchange during iron metabolism(Chen et al., 2021). We investigated the cardioprotective effects of ferrostatin-1 against LPS-induced cardiac dysfunction. We found that ferrostatin-1 not only reduced the expression of cardiac injury markers but also decreased oxidative stress and lipid peroxidation in cardiac tissue. In terms of regulating iron metabolism, ferrostatin-1 reduced iron uptake by decreasing TFRC expression, increased iron sequestration by upregulating FTH1, and directly reduced levels of ferrous ions as indicated by their detection. ferrostatin-1 also minimized the abnormal mitochondrial membrane potential caused by ferroptosis. Based on these findings, we speculate that inhibiting ferroptosis could be a therapeutic strategy for treating LPS-induced cardiac dysfunction.

SGLT2i was initially developed as anti-diabetic drugs based on their hypoglycemic effects. Subsequently, long-term cardiovascular efficacy and safety have been demonstrated in patients with type 2 diabetes(Reifsnider et al., 2021). However, even in the early stages of their development, the pharmacological properties of these drugs have shown benefits in non-diabetic individuals(van der Aart-van der Beek et al., 2022). Currently, four SGLT2i, namely dapagliflozin, empagliflozin, canagliflozin, and ertugliflozin, have been approved for clinical use in the European Union and the United States. In 2015, the first cardiovascular outcome trial, the EMPA-REG outcome trial, published its findings on empagliflozin, revealing a composite endpoint of major adverse cardiovascular events and hospitalization for heart failure associated with the use of empagliflozin. Subsequent cardiovascular outcome trials with other SGLT2i reported similar reductions in the relative risk of hospitalization for heart failure, such as dapagliflozin, canagliflozin, and others(Downs et al., 2020; Fernandez‑Balsells et al., 2017; Wiviott et al., 2019). In the context of LPS-induced cardiac dysfunction, we investigated the effects of pre-treatment with dapagliflozin on key proteins such as GPX4 involved in ferroptosis, as well as genes related to iron and lipid metabolism. We observed that dapagliflozin can inhibit LPS-induced ferroptosis in cardiac myocytes at both the protein and gene expression levels. Meanwhile, dapagliflozin could maintian intracellular ROS production and mitochondrial membrane potential under LPS. In the end, heart function was improved after dapagliflozin administration in animal model. These results collectively suggest that dapagliflozin can improve cardiac function by inhibiting ferroptosis in cardiac myocytes.

In conclusion, our study confirms the implication of ferroptosis in LPS-induced myocardial injury, which was partially attenuated by dapagliflozin. These results collectively underscore the therapeutic potential of dapagliflozin in addressing sepsis-induced cardiomyopathy.

Acknowledgements We thank everyone listed of authors.

Authorship contribution Jun Xie, Haiting Chen and Jinxuan Zhao conceived and designed the study. Ke Hu, Pin Jiang and Ya Hou performed the experiments. Ke Hu and Pin Jiang drafted the manuscript. Bing Song, Qianyu Gu, Meng Guo, Ningxin Peng and Jiayu Chen contributed to data collection and analysis. All authors have read and approved the manuscript, and all data were generated internally.

Funding This study was supported by the National Natural Science Foundation of China (92068116 and 82370305) and the Jiangsu Distinguished Young Scholars Fund (Approval No: BK20211501); the Funds for Distinguished Young Scientists in Nanjing (JQX22001); China Postdoctoral Science Foundation (2023M731631); Nanjing Postdoctoral Science Foundation for Jinxuan Zhao and Jiangsu “Mass Innovation and Entrepreneurship” Talent Program to Jinxuan Zhao; Anhui Medical University Research Enhancement Program Funding Project(2023xkjT029); Anhui Provincial Natural Science Research Project for Higher Education Institutions(KJ2021ZD0027) and Collaborative Project between the First Affiliated Hospital of Anhui Medical University and Hefei Zhongke Ion Medical Technology Equipment Co., Ltd.(CIM-HT-2018-327).

Data availability Data will be made available on request.

Ethics approval and contant participate All procedures with animals were approved by the Institution Ethics Committee of Anhui Medical University (Anhui, China) (No. LLSC20230846) and performed in accordance with the guidelines from Directive 2010/63/EU of the European Parliament.

Consent for publication Not applicable.

Competing interest The authors declare no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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No competing interests reported.

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Dapagliflozin attenuates LPS-induced myocardial injury by reducing ferroptosis

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Abstract

Figures

Introduction

Methods and materials

Animal and animal model

Echocardiography

Quantitative real-time polymerase chain reaction(PCR)

Western blot

Immunofluorescence

Detection of malondialdehyde(MDA), superoxide dismutase(SOD) and iron levels in serum and cardiac tissue

Elisa assay for determination of Creatine kinase-MB(CK-MB) and Cardiac troponin T(cTnT)

Measurement of tissure section ROS

Assessment of mitochondrial membrane potential

Statistical analysis

Results

LPS causes myocardial injury through oxidative stress damage

Ferroptosis can induce myocardial oxidative stress damage, mitochondrial damage

The SGLT2 inhibitor dapagliflozin can mitigate ferroptosis-associated damage

Dapagliflozin can effectively mitigate cardiac functional impairment induced by LPS

Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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