Sinomenine inhibits pyroptosis in atopic dermatitis by regulating TRIM32/IRF1/TRAF6 axis in HaCaT cells

Atopic dermatitis (AD) is a chronic, inflammatory skin disorder characterized by immune dysregulation and skin barrier dysfunction. Current therapies often have adverse effects or limited long-term compliance, highlighting the need for novel approaches. Pyroptosis, a highly inflammatory form of programmed cell death, is increasingly recognized for its role in driving inflammation in various diseases, including skin conditions. Understanding and targeting pyroptosis pathways, such as the NLRP3 inflammasome, presents a promising therapeutic avenue for AD. Sinomenine, a natural alkaloid with known anti-inflammatory properties, has not been thoroughly investigated for its specific effects on pyroptosis in AD.

Researchers established an AD-like in vitro model using HaCaT keratinocytes stimulated with IFN-γ (10 ng/mL) and TNF-α (10 ng/mL) to induce inflammation and pyroptosis. Cells were pretreated with Sinomenine, though the specific dose was not detailed in the abstract. The study evaluated Sinomenine's ability to attenuate inflammation, pyroptosis, and cell damage using a comprehensive panel of assays, including ELISA for cytokine levels, MTT for cell viability, LDH for cytotoxicity, flow cytometry for cell death markers, and Western blot for protein expression. Mechanistic investigations involved ChIP-qPCR, luciferase assays, co-immunoprecipitation, and immunofluorescence to explore protein interactions and transcriptional regulation.

IFN-γ/TNF-α stimulation robustly triggered pyroptosis in HaCaT cells, evidenced by significantly elevated levels of inflammatory cytokines such as IL-18, IL-6, IL-8, and IL-1β. This was accompanied by increased NLRP3 inflammasome expression, along with the activation markers cleaved Caspase-1 and GSDMD-N. Sinomenine pretreatment significantly reversed these detrimental effects, leading to improved cell viability and a marked reduction in inflammatory cytokine production and pyroptosis markers. Mechanistically, Sinomenine downregulated TRAF6 expression, a known activator of the NLRP3 inflammasome, by inhibiting its transcriptional regulator IRF1. IRF1 was confirmed to directly bind the TRAF6 promoter, promoting its transcription. Furthermore, a critical finding was that: > Sinomenine enhanced TRIM32 expression, which promoted the ubiquitination and proteasomal degradation of IRF1, thus interrupting the IRF1/TRAF6/NLRP3 axis. This multi-pronged action of Sinomenine effectively protected HaCaT cells from the induced pyroptosis.