L26/P-326 Targeting SPHK1 aggravates excessive stress and autophagy in endometriosis

Abstract Study question Is SPHK1 involved in the progression of endometriosis and its mechanisms? Summary answer SPHK1 knockdown induces excessive stress and autophagy via PERK degradation in ectopic endometrial stromal cells. What is known already Endometriosis (EMS), characterized by endometrium-like tissue outside the uterus, affects approximately 10% of women of reproductive age worldwide. Key clinical manifestations include chronic pelvic pain, dysmenorrhea, dyspareunia, infertility, and progressive ovarian failure, among others. Altered sphingolipid metabolism has been linked to EMS pathogenesis, especially involving key enzymes such as sphingosine kinase 1 (SPHK1) and sphingosine kinase 2 (SPHK2). These diacylglycerol kinase family members catalyze the conversion of ceramide to sphingosine-1-phosphate (S1P). Study design, size, duration Specimens and primary endometrial stromal cells (ESCs) from normal controls (n = 30) or endometriotic patients (n = 30) were analyzed. The subsequent experiments were performed using a controlled design with SPHK1-knockdown ectopic endometrial stromal cells (EcESCs) and negative control cells. An in vivo mouse model of endometriosis was also employed to evaluate the effects of Sphk1 knockdown on the progression of the disease. Participants/materials, setting, methods SPHK1 expression in normal and ectopic endometrium was analyzed by qRT-PCR, western blot, and immunofluorescence. Cell viability was assessed via CCK-8 and transwell assays in SPHK1-knockdown and control EcESCs. Results from Sphk1-knockdown mouse model of endometriosis and its controls encompassed both the number and size of peritoneal lesions. RNA-seq and transmission electron microscopy (TEM) were used for transcriptomic and ultrastructural analyses. Rescue was performed by PERK overexpression, and key proteins were detected by western blot. Main results and the role of chance SPHK1 was found to be upregulated in ectopic endometrium, and its knockdown inhibited the proliferation and migration of EcESCs. In a mouse model of endometriosis, SPHK1 knockdown also reduced the number and size of peritoneal lesions. RNA-seq analysis further demonstrated that SPHK1 knockdown altered the PERK mediated transcriptional profile in these cells. Ultrastructural examination via TEM showed endoplasmic reticulum expansion and increased autophagosomes/autolysosomes formation following SPHK1-knockdown. Consistent with these observations, dysregulation of endoplasmic reticulum stress and autophagy markers-including PERK, phospho-PERK, ATF6, phospho-IRE1, p62, and LC3II/I-suggested that SPHK1 promoted cell proliferation and migration by alleviating endoplasmic reticulum stress and suppressing autophagy. Notably, while treatment with the downstream product of SPHK1-S1P-failed to reverse the suppressive effect of its knockdown on cell viability, PERK overexpression restored cell viability. Further mechanistic studies indicated that PERK overexpression mitigated autophagy in SPHK1-knockdown EcESCs through the mTOR pathway and reduced oxidative stress via the NRF2-SOD1/CAT axis. Cycloheximide (CHX) chase assays confirmed that SPHK1 knockdown decreased PERK protein stability, an effect reversible upon treatment with either autophagy or proteasome inhibitors. Collectively, these results supported a model in which excessive autophagy and PERK downregulation formed a self-sustaining vicious cycle in SPHK1-deficient EcESCs. Limitations, reasons for caution A key limitation of this study is its reliance on cell and animal models, which may not fully replicate human endometriosis pathophysiology, necessitating further validation in human subjects. Wider implications of the findings Understanding the non-classical role of SPHK1 in EcESCs could lead to novel non-hormonal therapies for endometriosis, offering a potential strategy to reduce associated health risks. Trial registration number No