Role of CTGF-LRP1 in Impaired Healing of Cesarean Section Incisions

Uterine niche (niche) is a long-term complication following a cesarean section, which may increase the risk during subsequent pregnancies, and its pathogenesis remains incompletely understood. In this study, we utilized single-cell RNA sequencing to analyze 43,384 individual cells obtained from adjacent myometrium tissue of niche (Adjacent), well-healed cesarean scar tissue (Control), and niche tissue (Niche). Our analysis revealed that fibroblasts (FB) could be classified into three subgroups. FB3 in the Niche exhibited a reduced capacity for extracellular matrix synthesis. Cell communication analysis has identified that the secretion of CTGF by Endo3 and the expression of LRP1 by FB3 are involved in the receptor-ligand interaction of uterine fibroblasts during wound healing. Our research findings were supported by tissue staining and in vitro experiments, indicating that silencing LRP1 may prevent CTGF from promoting the synthesis of extracellular matrix by primary uterine fibroblasts. Furthermore, animal models have indicated that CTGF can promote the healing of uterine scars.These results provide insights into the cellular environment and mechanistic background underlying the formation of the uterine niche and poor wound healing following a cesarean section, laying the groundwork for future investigations into therapeutic approaches for the uterine niche. Overall design: LRP1 siRNA (5'-GTTTCGAGGTGGTGATTCA-3') and a negative control siRNA (5'- TTCTCCGAACGTGTCACGT-3') were sourced from Tsingke Biological Technology in China. The siRNA reagent was prepared in a stock solution at a concentration of 20 uM. JetPRIME (Polyplus) was utilized as an efficient transfection reagent. The transfection complex, comprising 5.5 ul of siRNA stock solution, 200 ul of jetPRIME Buffer, and 4 ul of jetPRIME reagent, was combined, gently vortexed, and incubated for 10 minutes at room temperature. This transfection complex was then added to a 6-well plate containing 1.5×105 cells and gently mixed to achieve a final siRNA concentration of 50 nM in each well. Following transfection with LRP1 siRNA or negative-control siRNA for 48 hours, the cells were subsequently incubated with CTGF (HY-P70106, MCE) for 48 hours. RNA and protein were then extracted for subsequent analysis.