Proenkephalin modulates apoptosis and neuronal differentiation in enteric neural crest cells with implications for the TLR4 signaling pathway

BACKGROUNDHirschsprung's disease (HSCR) is a common congenital disorder in pediatric surgery, defined by the absence of the enteric nervous system (ENS) due to aberrant development of enteric neural crest cells (ENCCs).Proenkephalin (PENK), a precursor of endogenous opioid peptides, has known neuroprotective potential, but its function in ENS development and HSCR remains unknown. AIMTo elucidate the role of PENK in ENCC apoptosis and neuronal differentiation and to explore whether the toll-like receptor 4 (TLR4) signaling pathway is involved in mediating these effects, providing new insights into ENS developmental biology and HSCR pathogenesis. METHODSDifferential expression analysis was performed using HSCR-related datasets (GSE96854 and GSE98502), and PENK expression was further validated in clinical tissue samples.PENK knockdown and overexpression models were established in primary mouse ENCCs.The effects of PENK on ENCC apoptosis, differentiation, and cell cycle progression were evaluated using qRT-PCR, Western blot, immunofluorescence, flow cytometry, and transmission electron microscopy.RNA sequencing was employed to identify downstream pathways, followed by target validation using a TLR4-specific inhibitor (TAK-242).RESULTS Bioinformatics analysis and clinical validation showed reduced PENK expression in aganglionic intestinal segments of HSCR patients.PENK knockdown increased ENCC apoptosis and impaired neuronal differentiation marker expression, whereas PENK overexpression reduced apoptosis and enhanced differentiation marker 4 / 35 expression without a notable effect on the cell cycle.RNA sequencing and subsequent experiments demonstrated that PENK modulates the TLR4 signaling pathway.Pharmacological inhibition of TLR4 attenuated the anti-apoptotic and prodifferentiation effects of PENK overexpression.CONCLUSION PENK is associated with the regulation of ENCC apoptosis and neuronal differentiation, with TLR4 signaling implicated as a critical downstream mediator.Reduced PENK expression may contribute to the pathogenesis of HSCR.The PENK-TLR4 axis represents a candidate pathway for future mechanistic investigation.