Methylene blue alleviates septic shock via FASN-mediated inhibition of neutrophil pyroptosis

Septic shock is a life-threatening condition characterized by excessive immune response to infection, often leading to organ failure and high mortality. Methylene blue (MB), a drug with rediscovered medicinal value despite historical concerns about mild toxic side effects, has shown potential in septic shock treatment. However, its underlying molecular mechanisms remain unclear. This study aimed to screen and characterize key genes associated with MB-mediated therapeutic effects against septic shock using multi-omics analysis, and to further validate the therapeutic efficacy and molecular regulatory mechanisms of MB through in vivo animal experiments. First, potential targets of MB were screened using the PubChem and SuperPred databases. Septic shock-related genes were retrieved from OMIM and GeneCards databases. Core intersection targets were identified by overlapping drug targets and disease-related genes, followed by validation via LASSO regression and SVM algorithms to confirm key genes. Single-cell RNA sequencing data (GSE175453) was analyzed to explore the expression of key genes in immune cells. Molecular docking was performed to verify the binding affinity between MB and key gene-encoded proteins. Subsequently, a septic shock mouse model was established using cecal ligation and puncture (CLP). Mice were divided into Control, sham, CLP, and CLP + MB groups. Survival rates, pathological changes in lung and spleen tissues, and expression levels of key genes were evaluated. Immunofluorescence, flow cytometry, enzyme-linked immunosorbent assay (ELISA), and western blotting were used to assess neutrophil infiltration, pyroptosis-related inflammatory factor secretion, and activation of the NOD-like receptor family pyrin domain-containing 3 (NLRP3)/apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/cysteine-aspartic acid protease-1 (Caspase-1)/Gasdermin D (GSDMD) pathway. Additionally, fatty acid synthase (FASN) expression was interfered with via lentiviral injection, and pyroptosis inhibitor was administered to verify the regulatory role of FASN and pyroptosis in MB-mediated therapy. A total of 51 potential targets of MB and 1610 septic shock-related genes were identified. Thirteen core intersection targets were obtained, and five key genes (ADORA2B, CTSD, CXCR4, FASN, PKN1) were confirmed after machine learning screening. Single-cell sequencing analysis showed that these key genes were mainly expressed in neutrophils, with FASN expression significantly downregulated in septic shock. MB treatment significantly improved the survival rate of CLP mice, alleviated lung and spleen tissue damage, reversed the abnormal expression of key genes (upregulated ADORA2B/CTSD and downregulated CXCR4/FASN/PKN1 in CLP mice), and reduced neutrophil infiltration. Moreover, MB inhibited the secretion of interleukin-1β (IL-1β) and IL-18 by neutrophils and suppressed the activation of the NLRP3/ASC/Cleaved-caspase1/GSDMD pathway. FASN silencing abrogated the protective therapeutic effects of MB, whereas pharmacological inhibition of pyroptosis effectively reversed the tissue damage induced by FASN knockdown. MB exerts a therapeutic effect on septic shock by up-regulating FASN expression, thereby inhibiting neutrophil pyroptosis. This study clarifies a novel molecular mechanism underlying MB-mediated septic shock treatment and provides promising therapeutic targets for clinical intervention against septic shock. Not applicable.