RNA结合蛋白PCBP1通过铁死亡途径影响干眼的机制研究OA

Objective To investigate the mechanism by which RNA-binding protein polycytosine binding protein 1(PCBP1)mediates dry eye disease(DED)-induced corneal epithelial injury through the ferroptosis pathway.Methods A dry eye model of human corneal epithelial cells(HCECs)was established using hypertonic stress(94 mmol·L-1 NaCl).PCBP1 was knocked down via lentiviral short hairpin RNA(shRNA),with a negative control vector expressing a non-targe-ted sequence(shCtrl)as the control.Experimental groups included the shCtrl group,the shPCBP1 group,the NaCl+shCtrl group,and the NaCl+shPCBP1 group,all at a NaCl concentration of 94 mmol·L-1.The CCK-8 assay was used to detect cell viability;flow cytometry was employed to measure the apoptosis rate of cells;dihydroethidium fluorescent probe was used to assess reactive oxygen species(ROS)expression levels;Western blot was applied to evaluate the expression levels of ferroptosis-related proteins[glutathione peroxidase 4(GPX4),solute carrier family 7 member 11(SLC7A11),and ferritin heavy chain 1(FTH1)];and an iron assay kit was used to determine intracellular free iron content.Results Compared with the shCtrl group,the shPCBP1 group exhibited decreased PCBP1 expression level,apoptosis rate,ROS-positive rate,and intracellular free iron level,while the NaCl+shCtrl group showed increased PCBP1 expression level,apoptosis rate,ROS-positive rate,and intracellular free iron level,along with reduced cell viability.All differences were statistically signifi-cant(all P<0.001).Compared with the NaCl+shPCBP1 group,the shPCBP1 group demonstrated decreased PCBP1 expres-sion level,apoptosis rate,ROS-positive rate,and intracellular free iron level,whereas the NaCl+shCtrl group exhibited in-creased PCBP1 expression level,apoptosis rate,ROS-positive rate,and intracellular free iron level,along with reduced cell viability.All differences were statistically significant(all P<0.001).Compared with the shCtrl group,the shPCBP1 group showed increased relative expression levels of FTH1,GPX4,and SLC7A11 proteins,the NaCl+shPCBP1 group showed in-creased relative expression levels of FTH1 and SLC7A11 proteins,while the NaCl+shCtrl group exhibited decreased relative expression levels of FTH1,GPX4,and SLC7A11 proteins.All differences were statistically significant(all P<0.001).Con-clusion PCBP1 is a key regulator of ferroptosis and oxidative stress in DED.It inhibits the expression of GPX4,SLC7A11,and FTH1 proteins,promotes ferroptosis,and facilitates the development and progression of DED.