内质网应激反应对豚鼠脑缺血再灌注后听皮质区损伤的作用

目的　观察豚鼠脑缺血再灌注后需肌醇酶1(inositol requiring enzyme 1，IRE1）、X盒结合蛋白1(Xbox binding protein 1，XBP-1）在其听皮层的反应以及血 液中C反应蛋白（C reactive protein，CRP）和免疫球蛋白(immunoglobulin M，IgM）的变化，探讨内质网应激及血液免疫反应在脑缺血再灌注后听皮层神经元凋亡中的作用。方法　选取健康豚鼠20只，随机分为5组，每组各4只，分别为正常对照组、缺血15 min组、缺血再灌注6 h组、缺血再灌注24 h组及缺血再灌注7 d组。后四组在相同条件下采用夹闭双侧颈总动脉的方法来建立脑缺血再灌注损伤模型，对照组不予手术。缺血15 min组术后马上行听性脑干反应(ABR)测试，其余实验组待再灌注至对应的时间点后行ABR测试。心脏采血酶标记免疫吸附 (enzymelinked immunosorbent assay，ELISA）检测血液CRP和IgM的浓度。断头取脑用免疫组织化学的方法测定IRE1、XBP-1表达。结果　ABR测试对照组为（10.0±2.47）dB SPL，缺血15 min组为（15±1.56）dB SPL，缺血再灌注6 h组为（30±2.03）dB SPL，缺血再灌注24 h组为（30±1.67）dB SPL，缺血再灌注7 d组为（15±1.14）dB SPL。缺血再灌注6 h组与24 h组行t 检验，无显著统计学差异，其余各组行方差分析，均有统计学差异（F =170.631，P <0.01）。ELISA法检测CRP对照组为（1198.96±50.81）μmol/ml，缺血15 min组为（1270.70± 34.76）μmol/ml，缺血再灌注6 h组为（1467.80±73.67）μmol/ml，缺血再灌注24 h组为（1352.83±175.71）μmol/ml，缺血再灌注7 d组为（1252.56±41.32）μmol/ml。各实验组行方差分析均有统计学差异（F =6.564，P <0.01）。IgM对照组为（987.32±39.13）μmol/ml，缺血15 min组为（1064.90±39.78）μmol/ml，缺血再灌注6 h组为（1118.03±57.75）μmol/ml，缺血再灌注24 h组为（1122.43±55.40）μmol/ml，缺血再灌注7 d组为（1010.70±55.95）μmol/ml。缺血再灌注6 h与24 h组行t检验无显著差异，其余各组行方差分析均有统计学差异（F =7.413，P <0.01）。HE染色显示各实验组凋亡细胞数 目不等，对照组为2.6±1.13，缺血15 min组为14.1±2.71，缺血再灌注6 h组为24.9±2.20，缺血再灌注24 h组为19.3±1.46，缺血再灌注7 d组为9.4±1.17。各实验组行方差分析均有显著差异（F =109.666，P <0.01）。免疫组化结果显示各组IRE1、XBP-1均有显色。 IRE1阳性颗粒数对照组为9.4±1.56，缺血15 min组为25.6±1.58，缺血再灌注6 h组为60.3±1.57，缺血再灌注24 h组为42.2±2.13，缺血再灌注7 d组为18.3±1.59；各实验组行方差分析均有显著差异（F =709.750，P <0.01）；XBP-1阳性颗粒数对照组为10.6±1.24，缺血15 min组为24.2±1.52，缺血再灌注6 h组为61.4±1.7，缺血再灌注24 h组为41.1±2.38，缺血再灌注7 d组为18.9±1.59；各实验组行方差分析均有显著差异（F =682.737，P <0.01）；IRE1与细胞凋亡率成正相关（r =0.947，P =0.000）；XBP-1与细胞凋亡率成正相关 （r =0.933，P =0.000）；CRP与IRE1成正相关（r =0.747，P =0.000）；CRP与细胞凋亡率成正相关（r = 0.755，P =0.000）；IgM与IRE1成正相关（r=0.695，P =0.000）；IgM与细胞凋亡率成正相关（r =0.765，P =0.000）。结论　在脑缺血再灌注损伤中，听皮层组织IRE1α、XBP1蛋白高表达，提示内质网应激参与了脑缺血再灌注损伤神经元细胞凋亡的发生发展，IRE1α和XBP1介导的信号转导通路是内质网应激导致脑缺血再灌注损伤神经元细胞凋亡的机制之一，脑缺血导致的听觉受损又有可能引起血液免疫学改变，内质网应激反应激发并加强了免疫炎性反应。

Effect of endoplasmic reticulum stress on auditory cortex after brain ischemia reperfusion in guinea pigs

OBJECTIVE The method was used to evaluate the IRE1, XBP1 positive expression in the guinea pigs auditory cortex and the ELISA was used to detect the change of CRP and IgM in blood, so as to research the effect of endoplasmic reticulum stress and the immunology reaction in the auditory cortex neuron apoptosis after brain ischemia reperfusion. METHODS Twenty healthy male guinea pigs were selected and divided randomly into 5 groups with 4 guinea pigs in each group： normal control group, ischemia in 15 minutes group, reperfusion for 6 hours group, reperfusion for 24 hours group, reperfusion for 7 days group. Cerebral ischemia reperfusion injury model was produced by occlusion of bilateral common carotid arteries, while the control group without treatment. The guinea pigs in the second group received ABR test after operation, the other groups received ABR test until the corresponding reperfusion time. ELISA method was used for measuring CRP and IgM and immunohistochemistry was used for detecting IRE1 and XBP-1. RESULTS ABR showed as dB SPL, and the control group was 10.0 ± 2.47, ischemia minutes group was 15~1.56, reperfusion 6h group was 30±2.03, reperfusion 24h group was 30 ± 1.67, and reperfusion 7d group was 15± 1.14. The CRP values measured by ELISA were 1198.96±50.81, 1270.70±34.76, 1467.80±73.67, 1352.83± 175.71 and 1252.56±41.32μmol/ml in control group, ischemia group, reperfusion 6h group, reperfusion 24h group and reperfusion 7d group respectively. The IgM were 987.32±39.13, 1064.90±39.78, 1118.03±57.75, 1122.43±55.40 and 1010.70±55.95 ~tmol/ml in control group, ischemia group, reperfusion 6h group, reperfusion 24h group and reperfusion 7d group respectively. HE staining showed the amount of apoptotic cells were different between each group. The amount of apoptotic cells was 2.6±1.13, 14.1±2.71, 24.9±2.20, 19.3±1.46 and 9.4± 1.17 in control group, ischemia group, reperfusion 6h group, reperfusion 24h group and reperfusion 7d group respectively. Immunohistochemical method results showed