血红蛋白氧载体抑制铁死亡减轻大鼠蛛网膜下腔出血后早期脑损伤

Hemoglobin-based oxygen carriers alleviate early brain injury after subarachnoid hemorrhage in rats by inhibiting ferroptosis

目的探讨血红蛋白氧载体(HBOC)对蛛网膜下腔出血(SAH)后早期脑损伤的保护作用及机制。方法采用血管内穿孔法制备大鼠SAH模型。将150只成年雄性SD大鼠采用随机数字表法分为假手术组(sham组)、SAH组、HBOC组、铁死亡促进剂组(HBOC+erastin组)和蛋白激酶B抑制剂组(HBOC+MK2206组), 每组30只。HBOC组术后立即经股静脉泵入HBOC, SAH组大鼠输注等体积乳酸林格钠液。术后24 h行SAH评分、神经功能评分及脑含水量的测定;采用尼氏染色检测神经元损伤;检测右侧颞叶皮质谷胱甘肽(GSH)、丙二醛(MDA)和组织铁含量;利用蛋白免疫印迹检测蛋白激酶B(Akt)通路蛋白及谷胱甘肽过氧化酶4(GPX4)的表达。组间比较采用t检验。结果 SAH组神经功能评分低于HBOC组[(12.50±2.48)分比(16.70±0.45)分, t=3.734, P<0.05)。尼氏染色结果显示正常神经元细胞计数SAH组明显少于sham组和HBOC组[(17.60±2.41)个/视野比(50.40±4.39)、(27.80±3.35)个/视野, t=14.640、5.532, P<0.05)。SAH组脑含水量高于sham和HBOC组[(79.65±0.13)%比(78.60±0.16)%、(79.05±0.27)%, t=11.420、4.486, P<0.05]、GSH明显低于sham和HBOC组(0.43±0.12比0.80±0.07、0.65±0.06, t=6.048、3.793, P<0.01)、MDA高于sham和HBOC组(4.69±0.85比1.34±0.34、2.14±0.88, t=8.146、4.652, P<0.01)、组织铁含量高于sham和HBOC组[(0.02±0.01) mg/g prot比(0.11±0.03)、(0.05±0.01) mg/g prot, t=7.194、4.496, P<0.05]、磷酸化Akt(pAkt)/Akt比值(0.75±0.05比2.19±0.04、2.035±0.04, t=5.855、3.545, P<0.05)和GPX4含量低于sham和HBOC组(0.31±0.01比1.81±0.05、1.28±0.04, t=73.290、55.810, P<0.05), 使用HBOC后改善了由SAH造成的指标变化。结论 HBOC可通过激活Akt信号通路抗氧化应激并抑制铁死亡减轻蛛网膜下腔出血后早期脑损伤。

Objective To investigate the protective effect and mechanism of hemoglobin-based oxygen carriers(HBOC)in early brain injury after subarachnoid hemorrhage(SAH).Methods Intravas-cular perforation was used to prepare rat SAH models.A total of 150 adult male SD rats(purchased from Changsha Tianqin Biotechnology Co.,Ltd.)were randomly divided into sham operation group(sham group),SAH group,HBOC group,ferroptosis promoter group(HBOC+erastin group)and protein kinase B inhibitor group(HBOC+MK2206 group)by a numerical random method.Rats in HBOC group were ad-ministered with HBOC via the femoral vein immediately after the operation,while rats in the SAH group were administered with an equal volume of lactated Ringer's solution.SAH score,neurological function score,and brain water content were measured after 24 h of surgery.Neuronal damage was detected by Nisslstaining.The corresponding kit was used to measure glutathione(GSH),malondialdehyde(MDA),and iron content in the right temporal lobe brain tissue.The expression of protein kinase B(Akt)pathway proteins and glutathione peroxidase 4(GPX4)were detected using Western blotting.The t-test was used for comparison between groups.Results The neurological function score of the SAH group was lower than that of the HB0C group(12.50±2.48 vs.16.70±0.45,t=3.734,P<0.05).Nissl staining showed that the normal neuronal cell count in the SAH group was significantly less than that in the sham group and HB0C group[(17.60±2.41)cells/field vs.(50.40±4.39),(27.80±3.35)cells/field,t=14.640.5.532,P<0.05].Compared with the sham and HBOC groups,the cerebral water content in the SAH group increased[(79.65±0.13)%vs.(79.05±0.27))%,(79.05±0.27)%,t=11.420,4.486,P<0.05],and CSH decreased significantly(0.43±0.12 vs.0.80±0.07.0.65±0.06,t=6.048,3.793.P<0.01),MDA increased(4.69±0.85 vs.1.34±0.34,2.14±0.88,t=8.146,4.652,P<0.01),iron content increased in tissues[(0.02±0.01)mg/g prot vs.(0.11±0.03),(0.05±0.01)mg/g prot,t=7.194,4.496,P<0.05],phosphorylated Akt(pAkt)/Akt ratio(0.75±0.05 vs.2.19±0.04,2.035±0.04,t=5.855,3.545,P<0.05)and GPX4 content decreased(0.31±0.01 vs.1.81±0.05,1.28±0.04,t=73.290,55.810,P<0.05).Conclusion HBOC alleviate early brain injury after SAH by activating the Akt signaling pathway,antioxidative stress,and inhibiting ferroptosis.