内质网应激相关性细胞凋亡在HMGB1介导严重烧伤小鼠脾脏树突状细胞功能障碍中的作用

Effect of endoplasmic reticulum stress-related apoptosis on HMGB1-mediated splenic dendritic cell dysfunction in severely burned mice

目的:研究严重烧伤小鼠体内高水平高迁移率族蛋白B1(HMGB1)诱导脾脏树突状细胞(DC)凋亡与内质网应激(ERS)的关系。方法:①复制严重烧伤小鼠模型,检测脾脏与血清中HMGB1的表达。②烧伤模型小鼠和假烫组(37℃水浴)均随机分为3组,分别给予抗HMGB1中和抗体、未免疫兔血清IgG和生理盐水,观察烧伤各组及生理盐水处理假伤组小鼠7 d生存率,并测定各组脾脏DC细胞ERS相关蛋白GRP78和XBP-1的表达。③正常小鼠尾静脉注射10或20μg重组HMGB1,48 h后处死,测定脾脏GRP78和XBP-1的表达。④以1、10、100 ng/ml HMGB1体外刺激健康小鼠的脾脏DC,并进一步应用细胞凋亡抑制剂Salubrinal(Sal)于HMGB1刺激1 h前进行干预,流式细胞术检测DC凋亡;Western blot法检测DC内ERS相关因子GRP78和CHOP的表达水平。分析HMGB1诱导DC细胞凋亡与ERS的内在联系。结果:小鼠烧伤后HMGB1蛋白表达水平增高。抗HMGB1中和抗体对严重烧伤小鼠有明确保护作用,可提高严重烧伤小鼠7 d存活率,显著降低烧伤小鼠脾脏DC内ERS标志分子GRP78以及ERS介导细胞凋亡的关键分子CHOP的蛋白水平。HMGB1(10 ng/ml)刺激可直接诱导DC凋亡以及ERS相关细胞凋亡关键分子CHOP蛋白表达升高;Sal干预可缓解ERS并抑制HMGB1诱导的DC凋亡(P<0.05)。结论:HMGB1刺激诱导DC凋亡与ERS相关性细胞凋亡通路激活密切相关,是严重烧伤小鼠脾脏DC功能障碍的重要发病机制。

Objective:To study the relationship between endoplasmic reticulum stress(ERS)and splenic dendritic cells(DC)apoptosis induced by high level high mobility group protein B1(HMGB1)in severely burned mice.Methods:a)Severe burn mice model was established and the expression of HMGB1 in spleen and serum was detected.b)Both mice model and sham control(37℃water bath)were randomly divided into three groups.Anti-HMGB1 neutralizing antibody,non-immunized rabbit serum IgG and normal saline were given respectively to mice of the three groups.The 7-day survival rate of mice in each burn group and sham control group with normal saline injection was observed,and the expressions of ERS related proteins(GRP78 and XBP-1)in splenic DCs of each group were detected.c)Normal mice were injected with 10 or 20μg recombinant HMGB1 via tail vein,and sacrificed 48 hours later.The expressions of GRP78 and XBP-1 in splenic DCs were determined.d)DCs were isolated from spleens of normal mice and stimulated with 1,10 or 100 ng/ml HMGB1 in vitro.The apoptosis inhibitor salubrinal(Sal,20μmol/L)was used 1 hour before HMGB1 stimulation to relieve ERS response.The apoptosis of DC was detected by flow cytometry,and the expression levels of ERS markers(GRP78 and CHOP)were detected by Western blotting to analyze the internal relationship between ERS and the apoptosis of DCs induced by HMGB1.Results:The expression level of HMGB1 protein increased in burned mice.Anti-HMGB1 neutralizing antibody showed a definite protective effect on severely burned mice,improved the 7-day survival rate,and obviously reduced the ERS marker GRP78 in spleen DCs of burned mice and the key molecule CHOP leading to the ERS-mediated apoptosis.HMGB1(10 ng/ml)stimulation directly induced DC apoptosis(P<0.01),and increased the key molecule CHOP level;Sal intervention could alleviate ERS and inhibit DC apoptosis induced by HMGB1(P<0.05).Conclusions:DC apoptosis induced by HMGB1 stimulation is closely related to the activation of ERSrelated apoptotic pathway,so is the important pathogenesis of splenic DC dysfunction in severely burned mice.