重组人促红细胞生成素对新生大鼠高体积分数氧肺损伤细胞炎症的影响

目的探讨人重组促红细胞生成素(rhEPO)对新生大鼠高体积分数氧(高氧)肺损伤炎症的影响。方法将72只新生SD大鼠按随机数字表法分为4组:对照组、支气管肺发育不良(BPD)组、高氧+低剂量促红细胞生成素EPO(L)]组、高氧+高剂量促红细胞生成素EPO(H)]组。BPD组、高氧+EPO(L)组和高氧+EPO(H)组新生大鼠暴露于850 mL/L氧气中,高氧+EPO(L)组和高氧+EPO(H)组分别于高氧暴露后1、3、7 d给予800 IU/kg、2000 IU/kg rhEPO皮下注射,对照组和BPD组注射等量9 g/L盐水。实验开始后3、7、14 d记录各组体质量。HE染色,光镜下观察其肺组织形态结构的改变,检测肺组织放射状肺泡计数(RAC)。免疫荧光染色法检测核因子-κB(NF-κB)的表达,Western blot技术检测肺组织磷酸化NF-κB(pNF-κB)、抑制蛋白(IκB)及Caspase-3的表达,酶联免疫吸附试验(ELISA)检测支气管肺泡灌洗液中白细胞介素-1β(IL-1β)的表达。结果1.BPD组新生大鼠14 d时体质量明显低于对照组(18.97±3.21)g比(27.97±2.30)g],差异有统计学意义(P<0.05);14 d时,高氧+EPO(L)组和高氧+EPO(H)组新生大鼠体质量(24.16±2.15)g、(26.04±1.97)g]均显著高于BPD组,差异均有统计学意义(均P<0.05)。2.HE染色观察肺组织形态学结构显示,与对照组比较,BPD组3 d肺泡间隔有少量炎性细胞渗出,7 d更为明显,肺泡腔有渗出,14 d出现肺泡数量减少,肺大疱形成,间隔增厚,RAC明显减少(5.50±1.29比14.33±2.80),差异均有统计学意义(P<0.01);高氧+EPO(L)组和高氧+EPO(H)组新生大鼠肺组织病理改变减轻,炎性细胞浸润减少,RAC值在14 d均明显高于BPD组,差异均有统计学意义(均P<0.05)。3.免疫荧光结果显示,BPD组NF-κB p65阳性细胞数目明显增多,荧光强度增强,给予EPO处理后可减少NF-κB p65阳性细胞数目,荧光强度减弱。4.Western blot结果显示,与对照组比较,BPD组pNF-κB p65及Cleaved Caspase-3显著增高,差异均有统计学意义(均P<0.05);IκB明显降低,差异有统计学意义(P<0.05);与BPD组比较,高氧+EPO(L)组和高氧+EPO(H)组pNF-κB p65及Cleaved Caspase-3显著降低,差异均有统计学意义(均P<0.05),IκB均明显升高,差异均有统计学意义(均P<0.05)。5.ELISA结果显示,与对照组比较,BPD组IL-1β表达明显增加,差异有统计学意义(P<0.05);高氧+EPO(L)组和高氧+EPO(H)组IL-1β表达则显著低于BPD组,差异均有统计学意义(均P<0.05)。结论EPO能通过NF-κB途径抑制细胞炎性反应,减轻高氧诱导的肺组织凋亡,对新生大鼠高氧肺损伤起保护作用。

Effect of human recombinant erythropoietin on inflammation of hyperoxic lung injury in neonatal rats

Objective To explore the effects of human recombinant erythropoietin(rhEPO)on inflammation of hyperoxic lung injury in neonatal rats.Methods Seventy-two neonatal rats were randomly divided into 4 groups:control group,BPD group,hyperoxia+low-dose recombinant erythropoietinEPO(L)]group,and hyperoxia+high-dose recombinant erythropoietinEPO(H)]group.The neonatal rats in BPD group,hyperoxia+EPO(L)group and hyperoxia+EPO(H)group were exposed to 850 mL/L oxygen.Then the neonatal rats in hyperoxia+EPO(L)group and hyperoxia+EPO(H)group were given 800 IU/kg and 2000 IU/kg rhEPO by subcutaneous injection respectively at 1 d,3 d and 7 d,while the control group and BPD group were given the same amount of 9 g/L saline water.Initially,the body weight of each group was recorded at 3 d,7 d and 14 d.The morphological structure changes of lung tissues were observed by HE staining under light microscope,and the radial alveolar count(RAC)in lung tissues were detected.The expression of nuclear factor kappa B(NF-κB)was detected by immunofluorescence staining;Western blot was applied to determine the protein expression of phosphorylated NF-κB(pNF-κB),inhibitor protein(IκB)and Caspase-3 in lung tissues;and the expression of interleukin-1β(IL-1β)in bronchoalveolar lavage fluid was determined using enzyme linked immunosorbent assay(ELISA).Results(1)On the 14th day,the body weight of neonatal rats in the BPD group was lower than that in the control group(18.97±3.21)g vs.(27.97±2.30)g],and the difference was statistically significant(P<0.01);however,the body weights of neonatal rats in the hyperoxia+EPO(L)group and hyperoxia+EPO(H)group(24.16±2.15)g,(26.04±1.97)g]was much heavier than that in the BPD group,and the differences was statistically significant(all P<0.05).(2)The morphological structure of lung tissues which was observed by HE staining showed that in the BPD group,there were a few inflammatory cells infiltration in alveolar septum on the 3rd day,the inflammatory response was more evident on the 7th day,when exudation could be seen in the alveolar cavity;and on the 14th day,the number of pulmonary alveoli decreased,pulmonary bulla formed,and septa were thickened.Besides,it was also observed that compared with control group,RAC was significantly decreased in BPD group on the 14th day(5.50±1.29 vs.14.33±2.80),and the difference was statistically significant(P<0.01).Pathological changes were ameliorated and the infiltration of inflammatory reaction cells was reduced in the hyperoxia+EPO(L)group and hyperoxia+EPO(H)group.RAC was remarkably higher in the hyperoxia+EPO(L)group and hyperoxia+EPO(H)group than that in the BPD group on the 14th day,and the differences were statistically significant(all P<0.05).(3)Immunofluorescence results showed that:the number of NF-κB p65 positive cells increased significantly and fluorescence intensity increased in the BPD group,while EPO could greatly reduce the number of NF-κB p65 positive cells and lower the fluorescence intensity.(4)Western blot results indicated that compared with the control group,the expressions of pNF-κB p65 and Cleaved Caspase-3 was significantly increased,and the differences were statistically significant(all P<0.05);and IκB was significantly lower,and the difference was statistically significant(P<0.05).The expression of NF-κB p65 and Cleaved Caspase-3 was significantly lower,but IκB was significantly higher in the hyperoxia+EPO(L)group and the hyperoxia+EPO(H)group than those in the BPD group,and the differences were statistically significant(all P<0.05).(5)ELISA results revealed that the expression of IL-1βin the BPD group was significantly higher than that in the control group,and the difference was statistically significant(P<0.05);Compared with BPD group,the expression of IL-1βwas significantly lower in the hyperoxia+EPO(L)group and hyperoxia+EPO(H)group,and the differences were statistically significant(all P<0.05).Conclusions EPO can reduce hyperoxia-induced lung tissue apoptosis and protect newborn rats against hyperoxic lung injury by decreasing the inflammatory response of cells through the NF-κB pathway on BPD.