Shikonin Modulates the NOD2/CARD9 Pathway to Ameliorate Ulcerative Colitis Through Inhibiting M1 Macrophage Polarization

Given the critical involvement of intestinal macrophages in ulcerative colitis (UC) pathogenesis and Shikonin's (SHK) established anti-inflammatory properties, this study investigated whether SHK inhibits macrophage proinflammatory (M1) polarization in UC and elucidated its downstream mechanisms. Dextran sulfate sodium (DSS)-induced UC model in mice was treated with SHK by gavage, and the therapeutic effect of SHK was evaluated by observing the changes in body weight, colon length, and Disease Activity Index (DAI) in mice. The pathological changes of colon tissue were observed by HE staining, and tight junction (TJ) proteins and inflammatory cytokines in colon tissue were detected. In vitro experiments were conducted to observe the inhibitory effect of SHK intervention on M1 macrophage polarization using LPS/IFN-γ-induced RAW264.7 cell model. Immunofluorescence, RT-qPCR, and Western blot were used to detect changes in NOD2 and CARD9 levels. SHK treatment significantly ameliorated murine colitis, evidenced by reduced DAI, attenuated DSS-induced colon histopathology, and preserved TJ integrity. SHK downregulated colonic expression of macrophage activation markers (F4/80), M1 polarization markers (iNOS, MCP-1), and pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), while elevating anti-inflammatory mediators (IL-10, Arg1). In LPS/IFN-γ-stimulated RAW264.7 macrophages, SHK consistently suppressed M1 polarization markers/pro-inflammatory mediators (TNF-α, IL-6, MCP-1, iNOS) while enhancing M2 markers (IL-10, Arg1, CD206, Ym1). Mechanistically, SHK inhibited the NOD2/CARD9 pathway in both in vivo and in vitro models. Crucially, NOD2/CARD9 overexpression attenuated SHK's therapeutic effects in murine colitis and blocked its suppression of M1 polarization in vivo and in vitro. This study demonstrates that SHK alleviates UC by inhibiting NOD2/CARD9-mediated macrophage M1 polarization, revealing a novel therapeutic mechanism for UC management.

Inflammation; M1 macrophage polarization; NOD2/CARD9; Shikonin; Ulcerative colitis.