Sotagliflozin Ameliorates LPS‑Induced Cardiomyocyte Injury by Regulating the PI3K‑Akt and Extracellular...

This study aimed to define the protective effects of sotagliflozin against lipopolysaccharide (LPS)-induced injury in H9C2 cardiomyocytes, and map the core molecular pathways driving these effects to support new therapeutic strategies for SRMI.

H9C2 rat cardiomyocytes were divided into a control group, LPS-challenged group, and LPS-challenged groups treated with 10, 20, and 30 μM sotagliflozin. Label-free quantitative Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) proteomics, coupled with bioinformatic analyses, was performed to map the drugregulated molecular network. Cell Counting Kit-8 (CCK-8) assay, biochemical kits, quantitative Real-Time PCR (qRT-PCR), Western blotting, immunofluorescence staining, and JC-1 probe assay were used to assess cell viability, cytotoxicity, oxidative stress markers, expression of inflammatory cytokines and NLRP3 inflammasome-related molecules, and mitochondrial membrane potential, respectively.

Proteomic profiling quantified 10,270 stably expressed proteins across all samples. Principal Component Analysis (PCA) revealed that PC1 and PC2 cumulatively explained 50.33% of intergroup variance, with clear separation between control and LPS groups, a pattern partially reversed by sotagliflozin treatment. In total, 630 differentially expressed proteins (DEPs; 252 upregulated, 378 downregulated) were identified in the LPS vs control comparison, and 210 DEPs (116 upregulated, 94 downregulated) in the sotagliflozin vs LPS comparison, with 49 core overlapping DEPs that were dysregulated by LPS and normalized by sotagliflozin. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that LPS challenge predominantly disrupted pathways, including extracellular matrix organization and the PI3K-Akt signaling cascade, which were targeted and restored by sotagliflozin. Functional validation demonstrated that sotagliflozin mitigated LPS-induced cardiomyocyte injury in a dose-dependent manner: it significantly improved cell viability (20 and 30 μM groups, P<0.01 and P<0.001, respectively), reduced lactate dehydrogenase (LDH) release (P<0.05 and P<0.001), and restored the antioxidant defense markers glutathione (GSH) and superoxide dismutase (SOD) (P<0.01 and P<0.001). Sotagliflozin also markedly downregulated mRNA levels of pro-inflammatory cytokines IL6, TNF, and NLRP3 (P<0.05 and P<0.01), and dose-dependently inhibited the aberrant upregulation of NLRP3 inflammasome activation markers NLRP3, cleaved Caspase-1, and cleaved IL-1β (30 μM group, P < 0.05), a regulatory effect further validated by immunofluorescence staining (P < 0.01). Additionally, sotagliflozin significantly ameliorated LPS-induced mitochondrial membrane potential collapse in cardiomyocytes (30 μM group, P<0.01).

SRMI is a major driver of adverse clinical outcomes in patients with sepsis, yet no disease-modifying targeted therapies have been successfully translated into clinical practice to date. Here, we present the first unbiased proteomic profiling of the cardioprotective effects of sotagliflozin, a dual SGLT1/2 inhibitor, in lipopolysaccharide (LPS)-injured cardiomyocytes, addressing a critical unmet need in defining its repurposing potential for SRMI. Our data demonstrate that sotagliflozin exerts concentration-dependent protective effects against LPS-induced cardiomyocyte injury, characterized by mitigated oxidative stress, suppressed inflammatory responses (including inhibition of NLRP3 inflammasome activation at high concentrations), and preserved mitochondrial function. At the molecular level, sotagliflozin reverses LPS-induced proteomic dysregulation in cardiomyocytes, with the PI3K-Akt signaling cascade and ECM homeostasis identified as its two core regulatory axes. Dysregulation of the prosurvival, anti-inflammatory, and antioxidant PI3K-Akt pathway is closely associated with concurrent amelioration of multiple SRMI-related pathological phenotypes, from reduced cell death to preserved mitochondrial function. We further identify ECM organization as a previously unrecognized, exploratory regulatory target of sotagliflozin in SRMI, extending our understanding of its cardioprotective actions beyond its well-characterized metabolic effects. The 49 core DEPs whose LPS-induced dysregulation is reversed by sotagliflozin, identified herein, not only delineate the molecular basis of its protective effects but also represent promising novel candidate targets for SRMI gene therapy. The precise causal regulatory mechanisms underpinning these effects remain to be validated in future in vivo studies and functional genomic experiments.

Sotagliflozin exerts cardioprotective effects against LPS-induced myocardial injury in H9C2 cardiomyocytes. This protection is mediated by the regulation of 49 core DEPs and key signaling pathways, most notably the PI3K/Akt pathway, which in turn attenuates excessive inflammatory responses, restores cellular redox balance, and ameliorates mitochondrial dysfunction.