重组人促红细胞生成素对脑出血大鼠脑组织损伤的影响

Effect of recombinant human erythropoietin on brain damage in rats with intracerebral hemorrhage

目的探讨重组人促红细胞生成素(EPO)调控细胞因子信号传导抑制蛋白3(SOCS3)/信号传导与转录激活因子3(STAT3)对脑出血大鼠脑组织损伤的影响。方法将90只SD大鼠随机分为假手术(Sham)组、模型(Model)组、EPO组(5 U/g)、STAT3激活剂Colivelin组(Colivelin组,1 mg/kg SOCS3/STAT3通路激活剂Colivelin)、EPO+Colivelin组(5 U/g EPO+1 mg/kg Colivelin),每组18只,Sham组、Model组注射等量生理盐水,1次/d,持续2周。采用Longa 5级评分法评估神经功能损伤情况;伊文思蓝测定血脑屏障通透性;试剂盒检测血清白细胞介素(IL)6、IL-1β、TNF-α、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)、丙二醛水平;苏木精-伊红、TUNEL法检测脑组织病理学及细胞凋亡;Western blot检测SOCS3/STAT3通路及酪氨酸蛋白激酶2(JAK2)蛋白表达。结果与Sham组比较,Model组神经功能评分、SOD、GSH-PX及SOCS3/GAPDH蛋白水平显著降低(P<0.05),伊文思蓝含量、IL-1β、IL-6、TNF-α、丙二醛、细胞凋亡率、STAT3/GAPDH、JAK2/GAPDH显著升高(P<0.05)。EPO组神经功能评分显著高于Model组[(4.67±0.25)分vs(1.83±0.07)分,P<0.05],伊文思蓝含量显著低于Model组[(3.37±0.12)μg/g vs(12.23±2.02)μg/g,P<0.05];与Model组比较,EPO组SOD、GSH-PX、SOCS3/GAPDH蛋白水平显著升高(P<0.05),IL-1β、IL-6、TNF-α、丙二醛水平、细胞凋亡率及STAT3/GAPDH、JAK2/GAPDH比例显著降低(P<0.05)。与EPO组比较,EPO+Colivelin组神经功能评分、SOD、GSH-PX水平及SOCS3/GAPDH蛋白水平显著降低(P<0.05),伊文思蓝含量、IL-1β、IL-6、TNF-α、丙二醛、细胞凋亡率及STAT3/GAPDH、JAK2/GAPDH显著升高(P<0.05)。结论EPO可能通过调控SOCS3/STAT3通路降低氧化应激,减轻炎性反应和神经元凋亡,对脑出血大鼠起到一定保护作用。

Objective To investigate the influences of recombinant human erythropoietin(EPO)on brain damage in rats with intracerebral hemorrhage(ICH)by regulating suppressor of cytokine signaling 3(SOCS3)/signal transducer and activator of transcription 3(STAT3).Methods Ninety SD rats were randomly grouped into sham operation(Sham)group,Model group,EPO group(5 U/g),and Colivelin group(SOCS3/STAT3 pathway activator,1 mg/kg),and EPO+Colivelin group(5 U/g EPO+1 mg/kg colivelin),with 18 animals in each group.The rats from the Sham group and Model group were injected with same amount of normal saline,1 time/d,for 2 weeks.Neurological impairment was assessed by Longa 5-level scale.Blood-brain barrier permeability was measured with Evans blue dye.Corresponding reagent kits were used to detect the levels of serum IL-6,IL-1β,TNF-α,superoxide dismutase(SOD),glutathione peroxidase(GSH-PX),and malondialdehyde(MDA).Brain histopathology and cell apoptosis were detected by hematoxylin eosin(HE)staining and TUNEL assay.With GAPDH as an internal reference gene,the protein levels of SOCS3/STAT3 pathway and tyrosine protein kinase 2(JAK2)were detected by Western blotting.Results The model group had less brain cells in irregular arrangment,lower neurological function score,decreased contents of SOD and GSH-PX and protein levels of SOCS3/GAPDH(P<0.05),more Evans blue dye,increased contents of IL-1β,IL-6,TNF-αand MDA,higher apoptotic rate and elevated ratios of STAT3/GAPDH and JAK2/GAPDH(P<0.05)when compared with the Sham group.Significantly higher neurological function score(4.67±0.25 vs 1.83±0.07,P<0.05),lower content of Evans blue dye(3.37±0.12μg/g vs 12.23±2.02μg/g,P<0.05),increased levels of SOD and GSH-PX and ratio of SOCS3/GAPDH(P<0.05),raised levels of SOD,GSH-PX and ratio of SOCS3/GAPDH protein(P<0.05),and reduced contents of IL-1β,IL-6,TNF-α,and MDA,apoptotic rate and ratios of STAT3/GAPDH and JAK2/GAPDH(P<0.05)were observed in the EPO group than the Model group.Colivelin treatment reversed above indicators significantly than EPO alone(P<0.05).Conclusion EPO may reduce oxidative stress,inflammatory response and neuronal apoptosis by regulating SOCS3/STAT3 pathway,and play a certain protective effect on ICH rats.