重组人血清白蛋白-硫氧还蛋白融合蛋白对流感病毒所致急性肺损伤小鼠的保护作用

Protective effect of recombinant human serum albumin-thioredoxin fusion protein on mice with acute lung injury induced by influenza virus

目的:探讨重组人血清白蛋白(HSA)-硫氧还蛋白(Trx)融合蛋白(HSA-Trx)对流感病毒感染所致急性肺损伤(ALI)小鼠的保护作用。方法:利用毕赤酵母表达系统产生基因重组HAS-Trx融合蛋白,PR8(H1N1)流感病毒诱导ALI小鼠模型,设健康对照组、ALI组、ALI+Trx组和ALI+HSA-Trx组,每组10只。收集各组支气管肺泡灌洗液(BALF),计算细胞总数,测定肺泡中性粒细胞数量,考马斯亮蓝溶液法测定蛋白浓度,ELISA法测定BALF中干扰素γ(IFN-γ)含量。收集肺组织进行苏木精-伊红染色,免疫荧光法检测肺组织诱导型一氧化氮合酶(iNOS)、8-羟基脱氧鸟苷(8-OHdG)和3-硝基酪氨酸(NO2-Tyr)的表达。收集血浆,使用CR2000RC分析仪测量血浆中过氧化物浓度。结果:HSA-Trx治疗显著降低ALI小鼠BALF中总细胞、中性粒细胞数量和总蛋白的含量(P<0.05),降低肺组织中8-OHdG和NO2-Tyr水平和血浆中过氧化物浓度(P<0.05),但对肺组织iNOS和IFN-γ的表达无显著抑制效果(P>0.05)。结论:HSA-Trx能够抑制肺组织炎症反应和肺内一氧化氮的过量产生,这对流感病毒诱导的ALI小鼠具有一定的保护作用。

AIM:To investigate the protective effect of recombinant human serum albumin(HSA)-thioredoxin(Trx)fusion protein(HSA-Trx)on mice with acute lung injury(ALI)induced by influenza virus infection.METHODS:The recombinant HAS-Trx fusion protein was generated by Pichia pastoris expression system.ICR mice were used to establish the animal model of ALI induced by PR8(H1N1)influenza virus,and the experimental mice were divided into healthy control group,ALI group,ALI+Trx group and ALI+HSA-Trx group,with 10 mice in each group.The bronchoalveolar lavage fluid(BALF)in each group was collected,the total number of cells and the number of alveolar neutrophils were determined,the protein concentration was measured by Coomassie brilliant blue solution method,and the interferon-γ(IFN-γ)content in BALF was detected by ELISA.The lung tissues were collected for hematoxylin and eosin staining.The inducible nitric oxide synthase(iNOS),8-hydroxydeoxyguanosine(8-OHdG)and 3-nitrotyrosine(NO2-Tyr)in lung tissues were detected by immunofluorescence method.Peroxide concentration in plasma was evaluated using a CR2000RC analyzer.RESULTS:HSA-Trx treatment significantly reduced the total number of cells,neutrophils and total protein in BALF of ALI mice(P<0.05),and decreased the levels of 8-OHdG,NO2-Tyr in lung tissue and peroxide in plasma(P<0.05).However,it has no significant inhibitory effect on iNOS and IFN-γexpression(P>0.05).CONCLU?SION:HSA-Trx inhibits inflammatory response and excessive production of nitric oxide in the lung,thus protecting influenza virus-induced ALI mice.