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Sepsis, as a systemic inflammatory response syndrome (SIRS) subtype, is generally characterized by infection. Emerging evidence has highlighted dysregulated microRNAs (miRNAs) are involved in the progression of sepsis. The aim of the study was to investigate the effects of miR-335-5p on inflammatory responses in a septic mouse model. The hypothesis was subsequently asserted that the FASN gene and AMPK/ULK1 signaling pathway may participate in the regulation of miR-335-5p. A septic mouse model was established in order to validate the effect of miR-335-5p on the inflammatory response by means of suppressing the endogenous expression of FASN by siRNA against FASN in endothelial cells. A target prediction program and luciferase activity was employed to ascertain as to whether miR--335-5p targets FASN. The levels of inflammatory factors including IL-6 and IL-1β were determined by means of ELISA assay. RT-qPCR and western blot analysis were used to determine the AMPK/ULK1 signaling pathway-, apoptosis- and autophagy-related genes. Flow cytometry was employed in order to evaluate sepsis-induced cell apoptosis in response to miR-335-5p and FASN alternations. FASN was identified as a target gene of miR--335-5p. Gain- and loss-of-function studies revealed that miR-335-5p acted to enhance autophagy, reduce cell apoptosis, promote cell cycle entry in endothelial cells, and reduce inflammatory response through the modulation of pro- and anti-apoptotic factors in endothelial cells. The effect of miR-335-5p on endothelial cells was increased when FASN was suppressed by siRNA as well as when the AMPK/ULK1 signaling pathway was activated, suggesting that miR-335-5p influences sepsis by targeting and inhibiting FASN, and activating the AMPK/ULK1 signaling pathway. Our study provides evidence indicating that overexpressed miR-335-5p enhances autophagy by targeting FASN through activation of the AMPK/ULK1 signaling pathway working to alleviate the inflammatory response in septic mouse models, emphasizing the value of the functional upregulation of miR-335-5p as therapeutic strategy for sepsis. [ABSTRACT FROM AUTHOR]