Parthenolide ameliorates inflammation in sepsis via covalently targeting Trim33 and inhibiting NF-κB pathway

Sepsis, a life-threatening condition, arises from an aberrant or uncontrolled immune response of the host to severe infection. Timely and precise control of this inflammatory response is of paramount importance in sepsis prevention and constitutes the core of sepsis prevention efforts. Parthenolide (PA), recognized as an NF-κB inhibitor, has previously demonstrated its capacity to mitigate the inflammatory response. This study aimed to investigate whether and how PA can inhibit inflammation, thereby alleviating sepsis, and to profile functional target proteins using chemoproteomics strategy.

The cecal ligation and puncture (CLP) procedure was performed to induce a mouse model of sepsis, and lipopolysaccharide (LPS) was employed to establish a model of cellular inflammation in mouse mononuclear macrophage cells (RAW264.7) in vitro. Enzyme-linked immunosorbent assay (ELISA) and hematoxylin and eosin (H&E) staining were utilized to evaluate the therapeutic effects of PA on septic mice. In the in vitro experiments, streamlined cysteine activity-based protein profiling (SLC-ABPP) and proteomics techniques were employed to identify potential protein targets and molecular pathways responsible for the anti-inflammatory properties of PA.

We demonstrated that PA could enhance the survival rate, reduce inflammatory cytokines release, ameliorate histopathological changes, and balance macrophage polarization in a sepsis model. Similarly, PA could effectively alleviate the inflammatory response in LPS-stimulated RAW264.7 cells. Chemoproteomics profiling revealed that the Trim33 protein was a potential covalent target of PA via cysteine engagement, and the expression changes of global proteins indicated by proteomics analysis suggested that PA mitigated inflammation by inhibiting the NF-κB pathway. Finally, through a series of molecular biology experiments, we confirmed that PA suppresses the NF-κB pathway by functionally binding to Trim33 and subsequently reducing the ubiquitin dependent degradation of Smad4, thereby exhibiting anti-inflammatory activity.

Our findings demonstrated that PA significantly mitigated inflammation in sepsis by targeting the Trim33 protein and consequently suppressing ubiquitination on Smad4 as well inhibiting the NF-κB pathway, thus providing new insights into the clinical use of PA in treatment of inflammation related diseases.