长链脂酰辅酶A合成酶4通过驱动铁死亡加剧中耳炎炎症损伤的研究OA

OBJECTIVE To investigate the expression changes of long-chain acyl-CoA synthetase 4(ACSL4),a key enzyme involved in lipid metabolism,in otitis media,and to verify whether ACSL4 aggravates inflammatory injury by promoting ferroptosis.METHODS Human middle ear epithelial cells(HMEECs)were divided into five groups:control group,model group(LPS group),control transfection group(LPS+sh-NC group),ACSL4 inhibition group(LPS+sh-ACSL4 group),and ferroptosis activation group(LPS+sh-ACSL4+Erastin group).A total of 50 healthy male Sprague-Dawley(SD)rats aged 6-8 weeks of the same batch were randomly assigned into four groups(10 rats per group):healthy control group(Ctrl group),acute otitis media(AOM)model group,ACSL4 inhibition group(sh-ACSL4 group),and ACSL4 inhibition combined with ferroptosis activation group(sh-ACSL4+Erastin group).The AOM rat model was established by intratympanic injection of 40 µl lipopolysaccharide(LPS)at a concentration of 1.0 mg/ml,with 10 healthy SD rats serving as the Ctrl group.Inflammatory scores of rats were evaluated.The mRNA and protein expression levels of ACSL4 in HMEECs and middle ear mucosal tissues of rats were detected by RT-qPCR and Western blot(WB),respectively.The proliferation and apoptosis levels of HMEECs were determined by CCK-8 assay and flow cytometry.Pathological changes of rat middle ear mucosal tissues were observed via hematoxylin-eosin(HE)staining.Commercial assay kits were used to detect the levels of inflammatory factors including interleukin-1β(IL-1β),IL-6,IL-8 and tumor necrosis factor-α(TNF-α),oxidative stress indicators including malondialdehyde(MDA),glutathione(GSH)and reactive oxygen species(ROS),as well as Fe levels in cells and rat tissues.WB was performed to detect the expression levels of ferroptosis-related proteins,including 4-hydroxynonenal(4-HNE),glutathione peroxidase 4(GPX4),and solute carrier family 7 member 11(SLC7A11)in cells and rat middle ear mucosal tissues.RESULTS Cell experiments showed that LPS stimulation significantly reduced cell viability,GSH content,and protein expression of SLC7A11 and GPX4;meanwhile,it markedly increased the cell apoptosis rate,levels of inflammatory factors(TNF-α,IL-6,IL-1β,IL-8),oxidative stress indicators(ROS,MDA),and ferroptosis markers(4-HNE,Fe2+)(all P<0.05).Inhibition of ACSL4(LPS+sh-ACSL4 group)reversed the above abnormal changes,whereas further activation of ferroptosis(LPS+sh-ACSL4+Erastin group)deteriorated these indicators again.In animal experiments,compared with the healthy control group,the AOM group exhibited significantly higher inflammatory scores,levels of inflammatory factors,oxidative stress indicators,ferroptosis markers and ACSL4 expression(P<0.05),while GSH level and protein expression of SLC7A11 and GPX4 were significantly decreased(P<0.05).ACSL4 inhibition alleviated inflammatory responses and ferroptosis in rats,while ferroptosis activation reversed the protective effects and aggravated the pathological lesions.CONCLUSION ACSL4 is upregulated in otitis media,which drives ferroptosis by promoting lipid peroxidation and ultimately exacerbates inflammatory injury.