Metabolomics Analysis Reveals Gut Microbiota-Associated Sakuranin Modulates Endometrial Stem Cell Differentiation and Inflammation to Alleviate Pain in Endometriosis

Wen Shi,1– 3,* Minyi Wang,1– 3,* Zhuang Jin,1– 3 Xiaochuan Chen,1– 3 Jinbo Li,1– 3 Huiling Lai,1– 3 Xiao Li,1– 3 Qiyu Zhong,1– 3 Ye Chen,1– 3 Shuqin Chen1– 3 1Department of Gynecology, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, People’s Republic of China; 2Key Laboratory of Human Microbiome and Chronic Diseases (Sun Yat-sen University), Ministry of Education, Guangzhou, Guangdong, People’s Republic of China; 3Biomedical Innovation Center, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, People’s Republic of China*These authors contributed equally to this workCorrespondence: Shuqin Chen, Department of Gynecology, Block B, 12th Floor, Building 1, The Sixth Affiliated Hospital of Sun Yat-sen University, No. 26, Yuancun Erheng Road, Tianhe District, Guangzhou, Guangdong, 510655, People’s Republic of China, Tel +86 13825039699, Email [email protected]: Endometriosis (EMS) is characterized by pain symptoms that seriously affect patients’ quality of life. Gut microbiome-related metabolites (GMRM) play an important role in the process of EMS. However, the role of GMRM in endometrial stem cells and EMS-related pain remains unclear.Methods: An untargeted metabolomics approach was employed to analyze the fecal samples of 10 healthy individuals (heal), 11 EMS patients without dysmenorrhea (pless), and 14 EMS patients with dysmenorrhea (pain). The impact of potential key metabolite sakuranin on EMS-related pain was further investigated in vitro and in vivo.Results: We identified 33 metabolites that were commonly changed in the painful group compared to the health and pless groups, and these metabolites were associated with differential microorganisms. Among them, sakuranin was downregulated in the painful group and exhibited a notably inverse correlation with the degree of pain. ROC curve revealed that sakuranin had a relatively high predictive value for EMS-related pain (AUC=0.8027). Functionally, sakuranin inhibited differentiation, migration, and inflammatory cytokine production, and decreased the expression of VEGF and ALCAM in SUSD2-positive primary endometrial cells. In EMS mice, sakuranin suppressed ectopic lesion growth, reduced inflammation, modulated angiogenesis and proliferation markers (VEGF, ALCAM, Ki-67), and regulated sympathetic and sensory nerve markers, resulting in alleviated pain behaviors.Conclusion: We delineated the metabolic landscape related to EMS-related pain and uncovered that sakuranin has the potential to inhibit the growth of EMS and alleviate EMS-related pain. This finding offers therapeutic strategies of sakuranin in alleviating the pain symptoms associated with EMS.Keywords: endometriosis, endometriosis-related pain, gut microbiome, metabolomics, sakuranin