E3 Ubiquitin Ligase FBXO32 Promotes LPS-Induced Cardiac Injury by Regulating ANXA1/PI3K/AKT Signaling

Abstract

Sepsis-induced cardiomyopathy (SIC) is a severe complication of sepsis. Therefore, understanding SIC pathogenesis and developing new therapeutic targets are of great significance. This study investigated the role of F-box-only protein 32 (FBXO32) in SIC pathogenesis. LPS-induced cardiac injury models were established in rats and H9c2 cells using lipopolysaccharide. The effects of FBXO32 on myocardial apoptosis and mitochondrial structure and function were determined using electron microscopy, reactive oxygen species detection, and JC-1 staining. The molecular mechanism was elucidated using western blotting and co-immunoprecipitation. The results showed elevated FBXO32 expression in both in vivo and in vitro LPS-induced cardiac injury models. Fbxo32 knockdown alleviated apoptosis and mitochondrial and cardiac dysfunction. Mechanistic analysis revealed that FBXO32 promoted ubiquitination and degradation of annexin A1 (ANXA1), inhibiting the phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) pathways. Rescue experiments demonstrated that Anxa1 knockdown reversed the effects of Fbxo32 knockdown. This study suggests that FBXO32 exacerbates LPS-induced cardiac injury progression by mediating ANXA1 ubiquitination and inhibiting the PI3K/AKT signaling pathway.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10753-025-02273-w.

Introduction

Sepsis-induced cardiomyopathy (SIC) is one of the most severe complications of sepsis and is a leading cause of mortality among affected patients. Current treatment strategies for SIC include infection control, fluid management, pharmacological treatments with vasoactive drugs and inotropes, and non-pharmacological interventions such as circulatory support [1]. Despite advances in SIC treatment, the high costs and substantial consumption of medical resources have seriously compromised patient safety and quality of life [2]. Therefore, developing new therapeutic targets to overcome the bottlenecks in SIC treatment is crucial.

Studies have confirmed that apoptosis plays a critical role in sepsis-induced myocardial damage, and inhibiting apoptosis can partially reverse the myocardial dysfunction caused by sepsis [3, 4]. Apoptosis can be regulated through the ubiquitin–proteasome system (UPS), which is vital in cardiovascular diseases such as heart failure and ischemia–reperfusion injury [5, 6]. As key components of the UPS, E3 ubiquitin ligases exert their biological functions by specifically recognizing target proteins and regulating the ubiquitination of substrate proteins.

F-box only protein 32 (FBXO32), an E3 ubiquitin ligase and member of the F-box protein family, is highly expressed in myocardial tissue and plays a significant role in cardiac-related diseases such as myocardial hypertrophy, ischemia, and heart failure by regulating the degradation of myocardial substrate proteins [7, 8]. Research [7] has demonstrated that FBXO32 expression is significantly elevated in myocardial tissue during acute myocardial infarction in mice. FBXO32 plays a critical role in mediating excessive inflammation, including neutrophil infiltration, inflammasome formation, and the production of pro-inflammatory cytokines through the activation of nuclear factor-κB (NF-κB), thereby promoting cardiac rupture post-myocardial infarction. Al-Yacoub et al. [9] have found that by binding to its target protein and activating transcription factor 2 (ATF2), FBXO32 can mediate its ubiquitination. Moreover, the upregulation of ATF2 due to Fbxo32 mutations is associated with the activation of the endoplasmic reticulum stress apoptosis pathway, further contributing to the progression of dilated cardiomyopathy.

Xie et al. [10] established a myocardial ischemia–reperfusion injury model using primary cultured neonatal rat cardiomyocytes. Using this model, they found that FBXO32 promotes the ubiquitination and degradation of the substrate protein mitogen-activated protein kinase phosphatase-1, activating the c-Jun N-terminal kinase (JNK) signaling pathway and exacerbating cardiomyocyte apoptosis. This finding revealed that FBXO32 is a critical mediator in controlling cardiomyocyte apoptosis after ischemia–reperfusion injury. However, the effect of FBXO32 on myocardial apoptosis in SIC remains unclear.

In this study, we investigated the role of FBXO32 in myocardial apoptosis in lipopolysaccharide (LPS)-induced cardiac injury, providing a theoretical basis for identifying effective preventive and therapeutic targets for SIC.

Results

Fbxo32 is Upregulated in the Myocardial Cells of LPS-Induced Cardiac injury Rats

To identify potential ubiquitination molecules associated with the pathogenesis of SIC, RNA-seq was conducted on H9c2 cells treated with LPS for 24 h, followed by a comparison with untreated H9c2 cells. According to our sequencing results, a total of 221 differentially expressed genes were screened, including 86 upregulated genes and 135 downregulated genes (Supplement 1 a). Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were performed on these differentially expressed genes, with the 30 most significant pathways for further analysis (Supplement 1 b and c). In the GO dataset, the biological processes mainly focus on response to lipolysis, inflammatory response, and hypoxia response, aligning closely with the content of our study. Regarding cellular composition, it is predominantly associated with the extracellular space, collagen containing extracellular matrix, and extracellular matrix. The molecular functions mainly involve identity protein binding, insulin-like growth factor II binding, and integrin binding. Additionally, KEGG analysis revealed that the significance of signaling pathways such as immune system, infectious disease (viral), signal transduction, and signaling molecules and interactions was high. Notably, the immune system and infectious disease (viral) pathways were highly consistent with the focus of our study.The data showed that FBXO32 was upregulated in the LPS group (Figs. 1a-c). To validate the RNA-seq results, qRnT-PCR analysis was conducted, revealing that the mRNA expression of FBXO32 was upregulated in LPS-treated H9c2 cells (Fig. 1d), consistent with the sequencing results. Additionally, the expression of FBXO32 was elevated in LPS-treated cells based on LPS intervention (Fig. 1e). Similarly, the mRNA and protein expression levels of FBXO32 were enhanced in the myocardial tissues of LPS-treated rats (Figs. 1f, g).

Fig. 1.

Expression of Fbxo32 in myocardial cells of rats subjected to LPS-Induced Cardiac injury. a, b Heatmap of differentially expressed genes (red indicates upregulated genes, blue indicates downregulated genes) and volcano plot for three pairs of LPS and Control group samples; c List of differentially expressed genes based on RNA-seq, highlighting Fbxo32 mRNA information; d Fbxo32 mRNA and protein expressions in myocardial cells detected by qRT-PCR; e Fbxo32 mRNA and protein expressions in myocardial cells detected by western blotting; f qRT-PCR and g Fbxo32 mRNA and protein expressions in rat heart tissue detected by western blotting. Data are expressed as the mean ± standard deviation, n = 3. **P < 0.01, ***P < 0.001

Knockdown of Fbxo32 Alleviates LPS-Induced Mitochondrial Dysfunction and Apoptosis in H9c2 Cells

To explore whether Fbxo32 knockdown affects LPS-induced apoptosis in H9c2 cells, lentivirus-mediated Short hairpin RNA (shRNA) was used to knockdown Fbxo32 in vitro, and successful transfection was confirmed by qRT-PCR and western blotting (Figs. 2a, b). In the cell viability experiment, we verified that the cell viability of the control group was basically consistent with that of the negative control group in the transfection group (Supplement 2 I). The Cell Counting Kit-8 (CCK-8) assay showed that cell viability in the LPS group was significantly lower than that in the control group. However, there was no significant difference in cell viability between the LPS and LPS + LV-sh-NC groups. Conversely, cell viability was significantly higher in the LPS + LV-sh-FBXO32 group than in the LPS + LV-sh-NC group (Fig. 2c). Further quantification of the effect of Fbxo32 knockdown on LPS-induced apoptosis in cardiomyocytes using Annexin V-FITC/PI flow cytometry revealed a significant increase in apoptosis in the LPS group compared with that in the control group. In the cell apoptosis experiment, the apoptosis rate of the control group was consistent with that of each negative control group (Supplement 2 J and K).There was no significant difference in apoptosis between the LPS and LPS + LV-sh-NC groups. In contrast, apoptosis was significantly decreased in the LPS + LV-sh-FBXO32 group compared with that in the LPS + LV-sh-NC group (Fig. 2d). Use western blotting technology to detect the effect of different doses of LPS (5, 10, 15 ug/ml) on the relative expression of FBXO32. It was found that with the increase of LPS dose, the relative expression of FBXO32 also increased (Supplement 2 N and S).Additionally, western blotting revealed that cleaved-Caspase-3 expression increased, and the Bax/Bcl-2 ratio increased in the LPS group compared with those in the control group. No significant difference was identified between the LPS and LPS + LV-sh-NC groups. Similarly, in the western blotting experiment, the ratio of Bax/Bcl-2 and the ratio of C-Caspase-3 were basically consistent between the control group and each negative control group (Supplement 2 L, M, O and P).However, the expression of cleaved-Caspase-3 and the Bax/Bcl-2 ratio decreased in the LPS + LV-sh-FBXO32 group compared with those in the LPS + LV-sh-NC group (Fig. 2e).

Fig. 2.

Effects of Fbxo32 on LPS-induced mitochondrial dysfunction and apoptosis in H9c2 cells. a Fbxo32 knockdown efficiency in stable H9c2 cell lines detected by qRT-PCR; b Fbxo32 knockdown efficiency in stable H9c2 cell lines detected by western blotting; c Effect ofFbxo32 knockdown on the viability of LPS-induced H9c2 cells detected by CCK8 assay; d Flow cytometry analysis of apoptosis in LPS-induced H9c2 cells after Fbxo32 knockdown; e Levels of apoptosis-related proteins in H9c2 cells after Fbxo32 knockdown detected by western blotting; f Assessment of ROS levels in LPS-induced H9c2 cells after Fbxo32 knockdown by DCFH-DA staining. g Assessment of mitochondrial membrane potential changes in LPS-induced H9c2 cells after Fbxo32 knockdown by JC-1 staining. Data are expressed as the mean ± standard deviation, n = 3. Ns indicates no statistical significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

To assess the effect of FBXO32 on mitochondrial function, the 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to measure reactive oxygen species (ROS) production in H9c2 cells in vitro. Flow cytometry results showed a significant enhancement in ROS fluorescence intensity in the LPS group compared with that in the control group. In contrast, ROS intensity was significantly lower in the LPS + LV-sh-FBXO32 group than in the LPS + LV-sh-NC group, indicating that Fbxo32 knockdown mitigated intracellular ROS production (Fig. 2f).

The effect of FBXO32 on the mitochondrial membrane potential in LPS-treated H9c2 cells was subsequently assessed using JC-1 staining. Flow cytometry results suggested that for cells in the LPS group, JC-1 failed to accumulate in the mitochondrial matrix, manifesting as a decrease in the red/green fluorescence ratio compared with that in the control group, indicating a significant reduction in the mitochondrial membrane potential. Moreover, the red/green fluorescence ratio increased substantially in the LPS + LV-sh-FBXO32 group compared with that in the LPS + LV-sh-NC group, suggesting that Fbxo32 knockdown improved the mitochondrial membrane potential. This indicates protection against LPS-induced mitochondrial membrane potential loss in H9c2 cells (Fig. 2g).

Knockdown of Fbxo32 Alleviates LPS-Induced Mitochondrial Dysfunction and Apoptosis in Rat Myocardial Cells

To determine whether Fbxo32 knockdown affects LPS-induced apoptosis in rat myocardial cells, in vivo experiments were conducted using a myocardial in situ injection of adeno-associated virus serotype 9 (AAV9) carrying Fbxo32-shRNA to knock down Fbxo32 expression. Measurements at four weeks revealed that Fbxo32 mRNA and protein expression levels were significantly reduced (Figs. 3a, b). TUNEL assays were also employed to detect the impact of Fbxo32 knockdown on myocardial cell apoptosis. The results suggested that the apoptosis rate of myocardial cells in the LPS group was significantly higher than that in the control group. The results showed that the control group had similar results to the negative control group (Supplement 2B and C).Conversely, the LPS + AAV9-sh-FBXO32 group exhibited a considerably lower rate of apoptosis than the LPS + AAV9-sh-NC group (Fig. 3c). In addition, western blotting was performed to measure the expression of apoptosis-related proteins in the myocardial tissues of the different groups of rats. The results indicated that cleaved-Caspase-3 levels and the Bax/Bcl-2 ratio were higher in the LPS group than in the control group. Moreover, these values were lower in the LPS + AAV9-sh-FBXO32 group than in the LPS + AAV9-sh-NC group (Fig. 3d). These consistent results from in vitro and in vivo experiments indicate that Fbxo32 knockdown reduces myocardial cell apoptosis.

Fig. 3.

Effects of Fbxo32 on LPS-induced mitochondrial dysfunction and apoptosis in the rat myocardium. a Fbxo32 knockdown efficiency in rat heart tissue detected by qRT-PCR (n = 3); b Fbxo32 knockdown efficiency in rat heart tissue detected by western blotting (n = 3); c Representative TUNEL staining images of myocardial tissue (scale bar = 50 μm) and quantitative comparison of TUNEL-positive nuclei (n = 6); d Levels of apoptosis-related proteins in rat heart tissue after Fbxo32 knockdown detected by western blotting (n = 3); e Myocardial cell ultrastructure in rats under TEM. Scale bar = 2 μm (low magnification) and 500 nm (high magnification). Data are expressed as the mean ± standard deviation. Ns indicates no statistical significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

To investigate the effect of FBXO32 on mitochondrial function in the rat myocardium, TEM was performed to observe mitochondrial morphological changes. In the control group, the mitochondria appeared to be oval-shaped and neatly arranged and had clear and straight cristae. Conversely, in the LPS and LPS + AAV9-sh-NC groups, many mitochondria were swollen, exhibiting reduced or broken cristae with a sparse matrix, and some even appeared vacuolated with a decreased electron density. In the LPS + AAV9-sh-FBXO32 group, the mitochondria appeared oval-shaped and neatly arranged, exhibiting clear and straight cristae, uniform electron density, and fewer swollen mitochondria, with a mildly dissolved and sparse matrix (Fig. 3e). These observations suggested that Fbxo32 knockdown significantly improved mitochondrial morphology.

Knockdown of Fbxo32 Mitigates LPS-Induced Cardiac Injury and Dysfunction

To evaluate whether Fbxo32 knockdown affects LPS-induced cardiac dysfunction, right common carotid artery catheterization was performed to measure hemodynamic parameters. The LPS group showed lower left ventricular ± dp/dt max and left ventricular systolic pressure (LVSP) and higher left ventricular end-diastolic pressure (LVEDP) than that of the control group. In contrast, the LPS + AAV9-sh-FBXO32 group exhibited higher ± dp/dt max and LVSP and lower LVEDP than the LPS + AAV9-sh-NC group (Fig. 4a), indicating improved cardiac function due to Fbxo32 knockdown. Further assessment of myocardial injury markers in serum demonstrated that the levels of inflammatory markers IL-6 and TNF-α, myocardial injury markers CK-MB and cTnT, and the cardiac function marker NT-proBNP were substantially higher in the LPS group than in the control group. In contrast, the LPS + AAV9-sh-FBXO32 group exhibited significantly lower levels of these markers than the LPS + sh-NC group (Fig. 4b). In addition, there was no significant difference in these blood test indicators between the control group and the negative control group (Supplement 2D-H).

Fig. 4.

Effects of Fbxo32 on LPS-induced cardiac injury and dysfunction. a Hemodynamic effects of Fbxo32 knockdown in LPS-treated rats (n = 6). b Effect of Fbxo32 knockdown on expression levels of serum biological markers (IL-6, TNF-α, CK-MB, cTnT and NT-proBNP) in LPS-treated rats (n = 6). c Representative HE staining images of myocardial tissue in rats. Scale bar = 50 μm (low magnification) and 20 μm (high magnification). Data are expressed as the mean ± standard deviation. Ns indicates no statistical significance, **P < 0.01, ***P < 0.001, ****P < 0.0001

Histological analysis using HE staining confirmed the protective effects of Fbxo32 knockdown on myocardial injury. In the LPS and LPS + AAV9-sh-NC groups, rat myocardial fibers exhibited blurred boundaries, disorganized arrangement, vacuolar degeneration, cytoplasmic swelling, and signs of nuclear dissolution. In contrast, in the LPS + AAV9-sh-FBXO32 group, the myocardial fibers appeared smoother with a more orderly arrangement, reduced vacuolar degeneration, and significantly reduced nuclear dissolution, with nuclei appearing more solidified (Fig. 4c). The results of LPS induced myocardial injury in normal rat models were consistent with those in AAV9 model rats (Supplement 2A). These data demonstrate that Fbxo32 knockdown reverses LPS-induced cardiac injury and dysfunction in vivo.

Knockdown of Fbxo32 Promotes Activation of the PI3K/AKT Signaling Pathway

To understand the impact of altered Fbxo32 expression on the PI3K/AKT signaling pathway, western blotting was conducted to detect changes in the phosphorylation levels of PI3K and AKT following Fbxo32 knockdown. The ratio of p-AKT/AKT and p-PI3K/PI3K did not show statistical significance in the comparison between the control group and each negative control group (Supplement 2 M, Q and R).In vitro, results showed lower p-PI3K and p-AKT protein levels in LPS-treated H9c2 cells than in the control group. Conversely, the LPS + LV-sh-FBXO32 group exhibited higher p-PI3K and p-AKT protein levels than the LPS + LV-sh-NC group (Fig. 5a).Fbxo32 knockdown reversed the decrease in p-PI3K and p-AKT levels in the myocardial tissues of LPS-treated rats in vivo (Fig. 5b). Collectively, these results indicated that FBXO32 knockdown promoted the activation of the PI3K/AKT signaling pathway.

Fig. 5.

Effects of Fbxo32 on the PI3K/AKT signaling pathway. a, b Levels of PI3K/AKT signaling pathway proteins in myocardial cells and rat heart tissue detected by western blotting. Data are expressed as the mean ± standard deviation, n = 3. Ns indicates no statistical significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

FBXO32 Promotes Ubiquitination and Degradation of ANXA1

To further explore how FBXO32 influences apoptosis in LPS-induced cardiac injury, as well as co-immunoprecipitation and mass spectrometry (CoIP-MS) were employed to detect interactions between FBXO32 and other proteins in LPS-treated H9c2 cells for 24 h(Additional file 1), identifying ANXA1 as one of the proteins involved (Figs. 6a, b). Subsequent validation confirmed the reduced ANXA1 protein expression in both cell and animal models exposed to LPS (Figs. 6c, d). Given FBXO32’s role as an F-box protein, which is part of the ubiquitin E3 ligase complex, we hypothesized that FBXO32 regulates ANXA1 through ubiquitination. To verify this hypothesis, we examined how variations in Fbxo32 mRNA expression affected Anxa1 mRNA levels under LPS-induced conditions. The qRT-PCR results indicated that Fbxo32 knockdown did not significantly affect Anxa1 mRNA levels compared with those in the control group (Fig. 6e). However, further examination using western blotting revealed a notable increase in ANXA1 protein levels following Fbxo32 knockdown compared with those in the control group (Fig. 6f), suggesting that FBXO32 might regulate ANXA1 protein expression through post-translational modifications.

Fig. 6.

Mediating effects of FBXO32 on the ubiquitination and degradation of ANXA1. a COIP silver staining; b List of identified proteins from mass spectrometry analysis, highlighting ANXA1; c, d Expression of ANXA1 protein in myocardial cells and rat heart tissue detected by western blotting; e Anxa1 mRNA levels in H9c2 cells under LPS-induced conditions after Fbxo32 knockdown detected by qRT-PCR; f ANXA1 protein expression in H9c2 cells under LPS-induced conditions after FBXO32 knockdown detected by western blotting; g, h Degradation of ANXA1 protein via the proteasome pathway under LPS-induced conditions. H9c2 cells were treated with DMSO, MG132 (20 μM), or NH4Cl (25 mM) for 6 h before harvest, and ANXA1 protein levels were detected by western blotting; i ANXA1 expression variations in H9c2 cells with different FBXO32 expression levels detected by western blotting before and after MG132 (20 μM) treatment; j Interaction between FBXO32 and ANXA1 proteins in H9c2 cells under LPS-induced conditions; k Analysis of ANXA1 protein expression and quantification in H9c2 cells under LPS-induced conditions after Fbxo32 knockdown. H9c2 cells with varying levels of FBXO32 expression were treated with CHX (40 μM), and proteins were collected at designated time points for western blotting to detect FBXO32 and ANXA1 expression and analyze changes in ANXA1 protein levels; l Effects of Fbxo32 knockdown on ANXA1 ubiquitination levels under LPS-induced conditions. Data are expressed as the mean ± standard deviation, n = 3. Ns indicates no statistical significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Subsequently, FBXO32’s negative regulation of ANXA1 was investigated by examining the degradation pathways of ANXA1. Under LPS treatment, the proteasome inhibitor, MG132, effectively blocked ANXA1 protein degradation, whereas the lysosomal inhibitor, NH4Cl, exerted no such effect. This finding indicates that LPS triggers ANXA1 degradation predominantly through the proteasome pathway rather than the lysosome pathway (Figs. 6g, h). Moreover, Fbxo32 knockdown resulted in a more pronounced increase in ANXA1 protein expression after MG132 treatment than no Fbxo32 knockdown, confirming that FBXO32 modulates ANXA1 protein expression through proteasome-dependent regulation (Fig. 6i). Subsequent Co-IP experiments verified the direct binding between FBXO32 and ANXA1 proteins (Fig. 6j). Next, a gradient of CHX was administered to inhibit protein synthesis and evaluate the effect of FBXO32 on ANXA1 protein degradation. Fbxo32 knockdown significantly prolonged the half-life of ANXA1, suggesting that Fbxo32 knockdown attenuated its role in mediating ANXA1 degradation (Fig. 6k). Finally, Co-IP analysis was used to examine the impact of FBXO32 on ANXA1 ubiquitination, revealing a decrease following Fbxo32 knockdown (Fig. 6l).

FBXO32 Regulates LPS-Induced Apoptosis and Mitochondrial Dysfunction Through ANXA1

To investigate whether ANXA1 contributes to the pro-apoptotic effects of FBXO32 in LPS-induced myocardial injury, rescue experiments were conducted by knocking down Anxa1 in H9c2 cells with stable Fbxo32 shRNA using siRNA. qRT-PCR and western blotting results confirmed a significant reduction in ANXA1 expression with Anxa1 siRNA (Figs. 7a, b).

Fig. 7.

Regulatory effects of FBXO32 via ANXA1 on LPS-induced apoptosis and mitochondrial dysfunction. a Verification of Anxa1 siRNA interference efficiency by qRT-PCR; b Verification of Anxa1 siRNA interference efficiency by western blotting; c Comparison of cell viability among different groups; d Comparison of apoptosis rates among different groups; e Apoptosis-related protein expression levels among different groups; f Intracellular ROS changes among different groups; g Comparison of the mitochondrial membrane potential across different groups; h PI3K/AKT signaling pathway protein expression levels among different groups. Data are expressed as the mean ± standard deviation, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

The CCK-8 assay revealed higher cell viability in the LPS + sh-FBXO32 + si-NC group than in the LPS + sh-NC + si-NC group. Moreover, cell viability was lower in the LPS + sh-FBXO32 + si-ANXA1 group than in the LPS + sh-FBXO32 + si-NC group (Fig. 7c). In addition, annexin V-FITC/PI flow cytometry, western blotting, and DCFH-DA staining revealed lower apoptosis rates, expression of apoptosis-related proteins, and intracellular ROS in the LPS + sh-FBXO32 + si-NC group than in the LPS + sh-NC + si-NC group. Conversely, these levels were significantly higher in the LPS + sh-FBXO32 + si-ANXA1 group than in the LPS + sh-FBXO32 + si-NC group (Figs. 7d-f).

JC-1 staining indicated a higher mitochondrial membrane potential in the LPS + sh-FBXO32 + si-NC group than in the LPS + sh-NC + si-NC group. Conversely, it was lower in the LPS + sh-FBXO32 + si-ANXA1 group than in the LPS + sh-FBXO32 + si-NC group (Fig. 7g). Furthermore, western blotting revealed higher levels of p-PI3K and p-AKT in the LPS + sh-FBXO32 + si-NC group than in the control group, whereas these levels were lower in the LPS + sh-FBXO32 + si-ANXA1 group than in the LPS + sh-FBXO32 + si-NC group (Fig. 7h).

Collectively, these findings suggest that Anxa1 knockdown effectively reversed the effects of Fbxo32 knockdown on cell viability, apoptosis, ROS levels, and mitochondrial function. Additionally, it significantly mitigated the effects of FBXO32 on the PI3K/AKT signaling pathway.

Discussion

In this study, FBXO32 expression was significantly elevated in both in vivo and in vitro rat models of LPS-induced cardiac injury. Fbxo32 knockdown mitigated apoptosis, mitochondrial dysfunction, and cardiac dysfunction. Further mechanistic analyses revealed that FBXO32 promotes the ubiquitination and degradation of ANXA1 by binding to it, thereby inhibiting the activation of the PI3K/AKT pathway.

Apoptosis is a tightly regulated form of cell death in which mitochondria play a critical role in regulation and execution. Mitochondria are abundant in cardiomyocytes and are essential for maintaining cardiac function and cell survival. Mitochondrial dysfunction is a key factor that can promote apoptosis [11, 12] and significantly impacts the onset, progression, and prognosis of myocardial dysfunction in septic patients [1]. Research [13–15] has shown that bacterial toxins, such as LPS, can induce excessive production of cytokines and ROS, causing mitochondrial DNA oxidation and subsequent mitochondrial damage. In their study on the cytotoxicity and molecular mechanisms of LPS in human alveolar epithelial A549 cells, Chuang et al. [16] found that LPS increased ROS levels and reduced the mitochondrial membrane potential, thereby driving the intrinsic apoptotic pathway in the mitochondria. Xie et al. [10] demonstrated that FBXO32 promotes apoptosis in myocardial ischemia–reperfusion injury. In this study, Fbxo32 expression was significantly elevated in the LPS-induced cardiac injury models. Fbxo32 knockdown increased the mitochondrial membrane potential, inhibited intracellular ROS production, improved abnormal mitochondrial morphology, and alleviated apoptosis.

LPS stimulation has been confirmed to be a major mechanism underlying cardiac dysfunction through the production of pro-inflammatory cytokines [17]. Excessively activated inflammatory responses can induce cardiomyocyte apoptosis [18, 19]. As an inflammatory trigger, TNF-α is involved in the production of IL-1β and the subsequent induction of secondary inflammatory factors such as IL-6, ultimately leading to an inflammatory cascade reaction [20]. In this study, LPS induction resulted in elevated levels of the inflammatory cytokines TNF-α and IL-6 in rat plasma. Furthermore, these levels decreased following the Fbxo32 knockdown. HE staining demonstrated that Fbxo32 knockdown alleviated LPS-induced inflammatory cell infiltration in the rat myocardium.

The PI3K/AKT signaling pathway is crucial for regulating cardiomyocyte apoptosis and inflammatory responses. Xing et al. [21] discovered that activation of the β3-adrenergic receptor-mediated PI3K/AKT signaling pathway alleviated apoptosis and cardiac dysfunction in LPS-induced cardiac injury rats. Yang et al. [22] demonstrated that aloesin lowered mortality in mice with CLP-induced sepsis by activating the PI3K signaling pathway. A study on the cardioprotective effects of dexmedetomidine during sepsis [23] indicated that dexmedetomidine prevented sepsis-induced cardiac dysfunction in rats by regulating cardiomyocyte autophagy via the PI3K/AKT pathway. In this study, we evaluated the effect of FBXO32 on the activation of the PI3K/AKT pathway. The results indicated that in both the in vivo and in vitro LPS-induced cardiac injury models, p-PI3K and p-AKT protein levels decreased, whereas Fbxo32 knockdown reversed this reduction, suggesting that FBXO32 may influence LPS-induced cardiomyocyte apoptosis and inflammatory responses via the PI3K/AKT pathway.

ANXA1 is an endogenous anti-inflammatory protein involved in various cellular processes, including apoptosis, cell proliferation, inflammation, and carcinogenesis [24]. Qin et al. [25] found that in LPS-induced sepsis models, annexin-A1 short peptide (ANXA1sp) alleviated myocardial damage and reduced apoptosis by upregulating silent information regulator 3 (SIRT3). Alternatively, Zhang et al. [26] induced sepsis-related cardiac dysfunction in rat and H9c2 cell models using cecal ligation and puncture (CLP) and 10 μg/mL LPS stimulation. They observed that the ANXA1 mimetic peptide Ac2-26 mitigated sepsis-induced cardiomyocyte apoptosis in vivo and in vitro, potentially by modulating the lipoxin A4 (LXA4)/PI3K/AKT signaling pathway. In this study, Co-IP was performed to screen FBXO32 interacting proteins, revealing that ANXA1 not only served as a binding molecule for FBXO32 but also negatively regulated its expression. Additionally, ubiquitination experiments confirmed that Fbxo32 knockdown reduced the ubiquitination level of ANXA1, thereby verifying the impact of FBXO32 on the ubiquitination of ANXA1.

Subsequent rescue experiments in Fbxo32-knockdown H9c2 cells, involving additional Anxa1 knockdown, significantly reversed the increased cell viability and mitochondrial membrane potential. Furthermore, it mitigated the reduction in apoptosis rate, apoptosis-related proteins, and intracellular ROS levels associated with Fbxo32 knockdown. Moreover, the activation of the PI3K/AKT signaling pathway was also weakened. Collectively, these findings suggest that ANXA1 is a target of FBXO32, which may promote LPS-induced myocardial apoptosis by enhancing the ubiquitination and degradation of ANXA1.

In conclusion, FBXO32 is upregulated in rat LPS-induced cardiac injury models and facilitates myocardial apoptosis in this context. The molecular mechanism involves FBXO32 interacting with ANXA1, which increases ANXA1 ubiquitination and promotes its degradation, thereby inhibiting the PI3K/AKT signaling pathway. This suggests that FBXO32 is an important regulator of LPS-induced cardiac injury cell apoptosis, positioning it as a promising therapeutic target for SIC.

Limitations of the study

The aim of this study was to evaluate the causal relationship and specific mechanisms underlying abnormal FBXO32 expression and apoptosis in LPS-induced cardiac injury cells. However, this study had certain limitations. First, although LPS-induced SIC models have been widely used, they do not fully replicate the pathophysiological state of patients with SIC. The relevant research results cannot fully replicate the mechanisms in the human body. Therefore, further human studies are warranted to assess the potential of FBXO32 as a therapeutic target for SIC. Secondly, although this study used Co-IP to confirm the interaction between FBXO32 and ANXA1, the specific binding sites between these two proteins require further investigation.

Methods

Cell Culture and Grouping

In this study, H9c2 cells ( provided by the Chinese Academy of Sciences Shanghai Cell Bank) were used as experimental cells in vitro. A complete culture medium was prepared using DMEM medium (C11995500BT, Thermo Fisher Scientific, USA), 10% fetal bovine serum (Thermo Fisher Scientific, USA), and 1% bispecific antibody (P1400, Solarbio, Beijing, China), and culture cells in a constant temperature incubator at 37℃ and 5% CO₂. When the cells reached approximately 80% confluence, they were digested with 0.25% trypsin solution (C0207; Beyotime, Shanghai, China) and passaged at a 1:3 ratio. H9c2 cells were divided into four groups: the LPS group, stimulated with 10 μg/mL LPS (L2360, Sigma, Germany) for 24 h to induce the LPS-induced cardiac injury model [27]; the LPS + LV-sh-NC group and the LPS + LV-sh-FBXO32 group, which underwent respective cell transfections followed by treatment with 10 μg/mL LPS for 24 h; and the Control group, which received an equal volume of culture medium.

Ribonucleic Acid Sequencing (RNA-seq)

Cell samples from the Control and LPS groups (with three biological replicates per group) were collected, and total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA purity and quantity were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Libraries were constructed using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA). After quality control using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), the libraries were sequenced on an Illumina HiSeq XTen platform by Shanghai OE Biotech. Co., Ltd. (Shanghai, China), and raw RNA-Seq data were exported in fastq format. Quality control and adapter removal were performed using Trimmomatic software, and differential gene expression analysis was conducted with the DESeq (2012) R package [28], applying screening criteria of log2 fold change (log2FC) > 1 or < −1 and FDR < 0.05.

Cell Transfection

shRNA lentiviruses targeting Fbxo32 were designed and synthesized by GeneChem Co., Ltd. (Shanghai, China), and small interfering RNAs targeting Anxa1 were designed and synthesized by GenePharma (Shanghai, China). According to the manufacturer’s instructions, HitransG A infection enhancer was employed to transfect sh-NC (control siRNA) and sh-FBXO32 (5’-GCAAAGTCACAGCTCACATCC-3’). Stable cell lines were established by continuous screening with 2 μg/mL puromycin for three generations, after which they were used for subsequent experiments. Alternatively, in sh-NC and sh-FBXO32 cell lines, si-NC and si-ANXA1 (5’-GCAGGAAUAUGUUCAAGCUTT-3’) were transfected with GP-transfect-Mate reagent. Following transfection, cells were cultured for another 48 h before being used in subsequent experiments.

Cell Viability Assay

Cell viability was assessed using the CCK-8 (Biosharp, Hefei, China). Specifically, H9c2 cells were seeded in 96-well plates at a density of 5 × 10³ cells/well. After experimental treatments, 10 μL of CCK-8 solution was added to each well, and plates were incubated at 37 °C for 2 h. Absorbance was measured at 450 nm using a microplate reader, and cell viability (%) was calculated [29].

Annexin V-FITC/PI Double Staining

H9c2 cells were seeded into 6-well plates. After treatment, the cells (including those in the culture supernatant) from different experimental groups were collected, centrifuged, washed, and resuspended in flow cytometry tubes. According to the instructions of the Annexin V-FITC/PI Apoptosis Kit (MULTI SCIENCES, Hangzhou, China), 5 μL of Annexin V-FITC and 10 μL of PI were added to each tube. Subsequently, the samples were incubated in the dark at room temperature for 5 min and then analyzed using a flow cytometer (NovoCyte, ACEA Biosciences, USA).

Intracellular ROS Detection

Intracellular ROS levels were detected using a ROS detection kit (S0033S; Beyotime, Shanghai, China). H9c2 cells were seeded in 6-well plates and treated according to the experimental groups. After treatment, cells were collected and incubated with 1 mL of 10 μmol/L DCFH-DA dilution at 37 °C in the dark for 20 min. The cells were subsequently washed thrice and analyzed using a flow cytometer (NovoCyte, ACEA Biosciences, USA).

Mitochondrial Membrane Potential Detection

The mitochondrial membrane potential was measured using a JC-1 kit (C2006, Beyotime, Shanghai, China) according to the manufacturer’s instructions. H9c2 cells were seeded in 6-well plates and treated according to the experimental groups. After treatment, the cells were collected, resuspended in 500 μL JC-1 working solution, and incubated at 37 °C for 20 min. The cells were washed twice and analyzed using a flow cytometer (NovoCyte, ACEA Biosciences, USA). According to the instructions, the maximum excitation wavelength of JC-1 monomer was 514 nm, and the maximum emission wavelength was 529 nm; The maximum excitation wavelength of JC-1 polymer (J-aggregates) was 585 nm, and the maximum emission wavelength was 590 nm. Therefore, FITC and PE channels were selected to detect the corresponding fluorescence signals.

CoIP-MS

Cell lysates from the LPS group were subjected to co-immunoprecipitation (Co-IP). The Co-IP products underwent western blotting and silver staining analyses. Subsequently, liquid chromatography-tandem mass spectrometry (LC–MS/MS) was performed by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). To identify FBXO32 interacting proteins, the resulting mass spectrometry data were analyzed using PEAKS software, searching against the UniProt rat proteome database (UniProt-proteome_UP000002494_20220418.fasta).

Co-IP

Co-IP was performed using an immunoprecipitation kit (P2179S; Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, cells were lysed in a buffer containing the protease inhibitor cocktail provided with the kit. An appropriate amount of supernatant was retrieved as an input control, whereas the remaining supernatant was reserved for subsequent Co-IP. Next, an antibody working solution was prepared, consisting of FBXO32 antibody (GTX47819, Gene Tex), ANXA1 antibody (21,990–1-AP, Proteintech), and Normal Rabbit IgG, all diluted to a ratio of 1:200). This solution was incubated with Protein A + G magnetic beads at room temperature for 1 h. Protein samples were then incubated with antibody-bound beads in a rotary mixer at 4 °C overnight. The beads were subsequently washed with lysis buffer, and the supernatant was discarded. After adding sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (1X), the samples were heated at 95 °C for 5 min. After magnetic separation, supernatants were subjected to western blot analysis.

Ubiquitination Level Analysis

Cells were pretreated with 20 μM MG132 (S2619, Selleck) for 6 h before collection. Next, the ubiquitination enzyme inhibitor N-ethylmaleimide (S3692, Selleck) was added to the protein lysis buffer at a final concentration of 10 μM. The product from cell lysis was then immunoprecipitated with protein A + G beads bound to ANXA1 antibody. Protein ubiquitination levels were detected by western blotting using an anti-ubiquitin (Ub) antibody (sc-8017, Santa Cruz).

Experimental Animals and Grouping

SPF-grade male SD rats aged 7–8 weeks and weighing 180–210 g were provided by the Animal Experiment Center of Lanzhou University and housed in the SPF-grade animal laboratory of Lanzhou University. Rats had ad libitum access to food and water in a controlled environment (temperature: 20–26 °C, relative humidity: 40–70%, 12-h light/dark cycle) and were acclimated for one week before experiments. All animal experiments complied with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH publication No. 85–23, revised 1996) and were approved by the Animal Ethics Committee of the First Hospital of Lanzhou University (Approval No. LDYYLL2024-405). The rats were divided into four groups using a random number table: Control, LPS, LPS + AAV9-sh-NC, and LPS + AAV9-sh-FBXO32, with each group containing six rats. In the LPS group, cardiac injury was induced by an intraperitoneal injection of 10 mg/kg LPS (diluted to 1 mL with saline) [30]. Alternatively, the LPS + AAV9-sh-NC and LPS + AAV9-sh-FBXO32 groups received in situ myocardial injections of AAV9-NC or AAV9-shRNA-FBXO32 before modeling. The Control group received an equal volume of saline via intraperitoneal injection at the same time points.

Myocardial Injection of Adeno-Associated Virus

AAV9 targeting Fbxo32 coupled with a cardiac troponin T (cTNT) promoter was designed and synthesized by Genechem Co., Ltd. (Shanghai, China). Myocardial injection of AAV9-NC or AAV9-shRNA-FBXO32 (5’-GCAAAGTCACAGCTCACATCC-3´) was subsequently performed. Rats were anesthetized with an intraperitoneal injection of 40 mg/kg sodium pentobarbital. Once anesthetized, the rats were placed in a supine position on a surgical table, intubated, and connected to a ventilator (HX-100E, TECHMAN, Chengdu, China) for assisted ventilation. The chest was opened to expose the heart, and a total of 25 μL of AAV9 viral solution (titer: 1.92E + 13 vg/mL) was injected into five different sites using a microsyringe. The chest was then closed, and the rats were allowed to recover naturally before being transferred to their cages. Four weeks later, the knockdown efficiency of Fbxo32 was verified using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and western blotting. Upon successful knockdown, LPS was administered intraperitoneally to induce the model.

Hematoxylin and Eosin (HE) Staining

The myocardial tissues from each group were fixed in 4% paraformaldehyde for 48 h, dehydrated using a graded series of ethanol, embedded in paraffin, and sectioned. The sections were deparaffinized, rehydrated, stained with HE, dehydrated, and mounted. The morphology of the myocardial tissue was examined under a microscope.

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL) Staining

Paraffin-embedded myocardial tissue sections were subjected to TUNEL staining according to the instructions of the TUNEL Apoptosis Detection Kit (C1091, Beyotime, Shanghai, China). The sections were observed and photographed under an Olympus microscope. Five random fields were selected from each section to calculate the percentage of TUNEL-positive cells. The rate of myocardial apoptosis was calculated by dividing the number of TUNEL-positive myocardial cells by the total number of cells, multiplied by 100.

Transmission Electron Microscopy (TEM) Observation of Myocardial Tissue Ultrastructure

Myocardial tissues were pre-fixed in 3% glutaraldehyde solution, post-fixed in 1% osmium tetroxide, washed, dehydrated using a graded ethanol series, and embedded in epoxy resin. Ultrathin sections were prepared, mounted on copper grids, stained sequentially with uranyl acetate and lead citrate, and observed under a transmission electron microscope (JEM-1400FLASH, JEOL, Japan) to assess ultrastructural changes in myocardial cells.

Hemodynamic Monitoring of the Heart

Thirty minutes before the observation time point, the SD rats were weighed and anesthetized via an intraperitoneal injection of 40 mg/kg sodium pentobarbital. Once anesthetized, the rats were placed in a supine position on a surgical table. They were then intubated and connected to a ventilator (HX-100E, TECHMAN, Chengdu, China) to maintain assisted ventilation throughout the procedure. A PE50 polyethylene tube was connected to an integrated signal acquisition and processing system (BL-420I, TECHMAN, Chengdu, China) using a PT-102N pressure transducer. Next, the right common carotid artery was isolated, and the catheter was retrogradely inserted into the left ventricle through the common carotid artery and fixed. Once the waveform stabilized, the following cardiac parameters were recorded: LVSP, LVEDP, maximal rate of increase in left ventricular pressure during systole (+ dp/dtmax), and maximal rate of decrease in left ventricular pressure during diastole (-dp/dtmax).

Enzyme-Linked Immunosorbent Assay (ELISA)

According to the instructions provided with the ELISA kits, the levels of inflammatory cytokines IL-6 (ERC003.48, NeoBioscience, Shenzhen, China) and TNF-α (ERC102a.48, NeoBioscience, Shenzhen, China), as well as myocardial injury markers CK-MB (MB-6930B, Jiangsu Meibiao Biotechnology Co., Ltd, Jiangsu, China) and cTnT (MB-7278B, Jiangsu Meibiao Biotechnology Co., Ltd, Jiangsu, China), and the cardiac function marker NT-proBNP (MB-1870B, Jiangsu Meibiao Biotechnology Co., Ltd, Jiangsu, China) were measured in rat plasma. The optical density (OD) values at 450 nm were measured using a microplate reader (Multiskan Spectrum 1500, Thermo Scientific), and the concentrations of each sample were calculated based on a standard curve.

qRT-PCR

Total RNA was extracted from myocardial tissue or H9c2 cells using TRIzol reagent (15,596,026, Invitrogen). Subsequently, cDNA was synthesized using a reverse transcription kit (R223; Vazyme, Nanjing, China) and amplified using the ChamQ Universal SYBR qRT-PCR Master Mix (Q711, Vazyme, Nanjing, China) on a QuantStudio 5 Real-Time PCR System (Applied Biosystems). With Gapdh as the internal control gene, the relative expressions of target genes were then calculated using the 2^−ΔΔCT method. The primer sequences for PCR in this study were designed and synthesized by Sangon Biotech (Shanghai, China) as follows: Fbxo32 forward: 5’-ACTCATACGGGAACTTCTCCAGACC-3’; Fbxo32 reverse: 5’-GCTGCTGTTGCCAGTGTAGAGTG-3’; Anxa1 forward: 5’-GGAAGCCCCTGGATGAAACCTTG-3’; Anxa1 reverse: 5’-CCTTCATGGCAGCACGGAGTTC-3’; Gapdh forward: 5’-AGTTCAACGGCACAGTCAAGGC-3’; and Gapdh reverse: 5’-CGACATACTCAGCACCAGCATCAC-3’.’

Western Blotting

Proteins were extracted from myocardial tissue or H9c2 cells using western blotting and IP cell lysis buffer (P0013, Beyotime, Shanghai, China) and quantified using a BCA protein assay kit (P0012S, Beyotime, Shanghai, China). Proteins (30 μg) were separated using 12% SDS-PAGE (P1200, Solarbio, Beijing, China) and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were subsequently blocked with QuickBlock Western blocking solution and shaken at room temperature for 10 min. The membranes were then incubated overnight at 4 °C with different primary antibodies, including FBXO32 (GTX47819, Gene Tex, 1:500), ANXA1 (21,990–1-AP, Proteintech, 1:5000), Bcl-2 (Ab32124, Abcam, 1:1000), Bax (Ab32503, Abcam, 1:1000), Cleaved Caspase-3 (AF7022, Affinity, 1:1000), Caspase-3 (Ab32351, Abcam, 1:1000), AKT (YT0177, Immunoway, 1:500), p-AKT (Ab81283, Abcam, 1:1000), PI3K (Ab151549, Abcam, 1:1000), p-PI3K (bs-6417R, BIOSS, 1:1000), Ub (sc-8017, Santa Cruz, 1:500), and GAPDH (YN5585, Immunoway, 1:5000). The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (RS0002; Immunoway) at room temperature for 2 h. Finally, a hypersensitive chemiluminescent substrate (BL520B; Biosharp, Hefei, China) was evenly added to the protein side of the PVDF membranes. Membranes were visualized using a chemiluminescence imaging system (MiniChemi 610, SINSAGE, Beijing, China). The grayscale values of the protein bands were measured using ImageJ 1.52a software. The ratio of the grayscale values of the target protein to that of the internal control protein, GAPDH, was calculated to represent the relative expression of the target protein.

Statistical Methods

GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA, USA) was employed for graphing and statistical analysis. The results are presented as the mean ± standard deviation. The t-test was used for comparisons between two independent samples. One-way analysis of variance was adopted for comparisons among multiple groups, and Tukey’s method was used for pairwise comparisons. A two-tailed P-value < 0.05 was considered statistically significant.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

Conceptualization: Chen De, Liu Jian, and Liang Xuan; Methodology: Chen De, Liu Jian, and Liang Xuan; Formal analysis and investigation: Chen De, Liu Jian, and Liang Xuan; Writing—original draft preparation: Chen De; Writing—review and editing: Liu Jian, Zhang Lei, Zuo Kun, and Guo Hong; Funding acquisition: Chen De, Liu Jian, Liang Xuan, Zhang Lei, and Du Song; Resources: Zhang Jingjing, Gao Lina, Yan Dong, and Du Song; Supervision: Liu Jian, Zhang Lei, Zuo Kun, and Guo Hong. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China(82460379); the Natural Science Foundation of Gansu Province(24JRRA616); Wu Jieping Medical Foundation (320.6750.2024–2-3); and the Lanzhou Science and Technology Plan Project (2022–5-74).

Data availability

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA018060) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa.The original contributions presented in the study are included in the article/supplementary material; further inquiries can be directed to the corresponding author.

Declarations

Ethics Approval

All animal experiments complied with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH publication No. 85–23, revised 1996) and were approved by the Animal Ethics Committee of the First Hospital of Lanzhou University (Approval No. LDYYLL2024-405).

Competing Interests

The authors declare no competing interests.

Clinical Trial Number

Not applicable.