Elucidating the Novel Mechanism of Ligustrazine in Preventing Postoperative Peritoneal Adhesion Formation

Abstract

Postoperative peritoneal adhesion (PPA) is a major clinical complication after open surgery or laparoscopic procedure. Ligustrazine is the active ingredient extracted from the natural herb Ligusticum chuanxiong Hort, which has promising antiadhesion properties. This study is aimed at revealing the underlying mechanisms of ligustrazine in preventing PPA at molecular and cellular levels. Both rat primary peritoneal mesothelial cells (PMCs) and human PMCs were used for analysis in vitro. Several molecular biological techniques were applied to uncover the potential mechanisms of ligustrazine in preventing PPA. And molecular docking and site-directed mutagenesis assay were used to predict the binding sites of ligustrazine with PPARγ. The bioinformatics analysis was further applied to identify the key pathway in the pathogenesis of PPA. Besides, PPA rodent models were prepared and developed to evaluate the novel ligustrazine nanoparticles in vivo. Ligustrazine could significantly suppress hypoxia-induced PMC functions, such as restricting the production of profibrotic cytokines, inhibiting the expression of migration and adhesion-associated molecules, repressing the expression of cytoskeleton proteins, restricting hypoxia-induced PMCs to obtain myofibroblast-like phenotypes, and reversing ECM remodeling and EMT phenotype transitions by activating PPARγ. The antagonist GW9662 of PPARγ could restore the inhibitory effects of ligustrazine on hypoxia-induced PMC functions. The inhibitor KC7F2 of HIF-1α could repress hypoxia-induced PMC functions, and ligustrazine could downregulate the expression of HIF-1α, which could be reversed by GW9662. And the expression of HIF-1α inhibited by ligustrazine was dramatically reversed after transfection with si-SMRT. The results showed that the benefit of ligustrazine on PMC functions is contributed to the activation of PPARγ on the transrepression of HIF-1α in an SMRT-dependent manner. Molecular docking and site-directed mutagenesis tests uncovered that ligustrazine bound directly to PPARγ, and Val 339/Ile 341 residue was critical for the binding of PPARγ to ligustrazine. Besides, we discovered a novel nanoparticle agent with sustained release behavior, drug delivery efficiency, and good tissue penetration in PPA rodent models. Our study unravels a novel mechanism of ligustrazine in preventing PPA. The findings indicated that ligustrazine is a potential strategy for PPA formation and ligustrazine nanoparticles are promising agents for preclinical application.