Menispermum dauricum rhizome alkaloids ameliorate colitis by inhibiting mitochondrial DNA synthesis via the JAK2-STAT4-AK4 pathway

p-STAT4 directly binds to the Ak4 promoter to drive its transcription. • STAT4-AK4 signaling engages in the production of mitochondrial DNA (mtDNA). • TAMR inhibits Ak4 transcription and mtDNA synthesis in a STAT4-dependent manner. • TAMR ameliorates UC by suppressing the JAK2-STAT4 signaling pathway. • The JAK2-STAT4-AK4-mtDNA axis represents a novel therapeutic target for UC. Background Ulcerative colitis (UC), a chronic inflammatory bowel disease with increasing global incidence, has inadequate existing treatments, highlighting the urgent need for new therapeutic agents and strategies. The total alkaloids from Menispermi Rhizoma (TAMR) has been clinically used to treat inflammatory diseases in traditional Chinese medicine, but its specific therapeutic effects and mechanisms in UC remain poorly elucidated. Purpose This study aimed to systematically clarify the therapeutic efficacy of TAMR against UC and elucidate its potential molecular mechanisms. Methods A dextran sulfate sodium (DSS)-induced murine colitis model was used to evaluate the efficacy of TAMR, with RNA sequencing providing mechanistic insights. A variety of molecular biology experiments were conducted for subsequent pathway validation in RAW264.7 macrophage cells. STAT4-AK4 interactions were further explored via Stat4 knockdown and chromatin immunoprecipitation (ChIP) assays. Results TAMR treatment effectively ameliorated colonic pathological lesions and reestablished M1/M2 macrophage polarization in DSS-challenged mice. Integrated RNA-seq and Western blot profiling revealed that TAMR specifically suppressed JAK2-STAT4 signaling cascade and AK4 expression, whose inhibition curbed excessive mitochondrial DNA (mtDNA) synthesis in inflammatory macrophages. Notably, STAT4 overexpression entirely negated TAMR-driven AK4 and mtDNA suppression. ChIP experiments confirmed that p-STAT4 directly bound to the Ak4 promoter, and STAT4 silencing abrogated LPS-induced Ak4 transcription and mtDNA synthesis. Conclusions TAMR exerts its anti-colitis effects by inhibiting the JAK2-STAT4 signaling pathway, which in turn suppresses AK4-mediated mtDNA overproduction. This study provides a scientific basis for the clinical application of TAMR in UC treatment and identifies a novel JAK2-STAT4-AK4-mtDNA regulatory axis as a potential therapeutic target for UC. Download: Download high-res image (436KB) Download: Download full-size image