脂多糖诱导脓毒症大鼠远期脑线粒体损伤的初步研究

A preliminary study of long-term mitochondrial dysfunction in rat brain caused by lipopolysaccharide-induced sepsis

目的初步探讨脓毒症大鼠远期脑线粒体结构和功能损伤。方法将Wistar大鼠随机分为对照组和脓毒症组,通过腹腔注射革兰阴性菌脂多糖（LPS,10 mg/kg）建立脓毒症大鼠模型并进行生存率实验。脓毒症组大鼠分别在腹腔注射革兰阴性菌LPS后12、24、48和72 h各处死8只,对照组大鼠（n=8）在给予等量生理盐水腹腔注射后处死,取脑组织提取线粒体。通过流式细胞仪检测线粒体膜电位（MMP）及线粒体肿胀度,使用试剂盒检测呼吸链复合物酶（Ⅰ~Ⅴ）活性。通过苏木精-伊红（HE）染色及电镜观察脑组织和线粒体形态学变化。结果脓毒症组较对照组生存率明显下降（P〈0.01）。脓毒症组MMP和呼吸链复合物酶（Ⅰ~Ⅴ）活性较对照组明显下降（P〈0.05）,48 h降至最低,72 h部分恢复;脓毒症组线粒体肿胀度较对照组明显升高（P〈0.05）,48 h达到峰值,72 h部分恢复;HE染色可见脑组织结构明显破坏,同时电镜结果观察到线粒体肿胀,少量线粒体出现＂空泡样变＂。结论在LPS诱导脓毒症大鼠模型中,脑线粒体结构和功能均受损,损伤高峰可能发生在48 h左右。

Objective To preliminarily investigate the long-term structural and functional injuries of mitochondria in rat brain caused by sepsis. Methods Wistar rats were randomly assigned into sepsis and control groups. A rat model of sepsis was prepared by an intraperitoneal injection of 10 mg/kg lipopolysaccharide（LPS） of gram-negative bacteria, and the survival assay was performed. Eight rats in the sepsis group were sacrificed at 12, 24, 48, or 72 hours after LPS injection, while rats in the control group were sacrificed after an intraperitoneal injection of an equal volume of normal saline. Mitochondria were extracted from rat brain tissue. Mitochondrial membrane potential（MMP） and mitochondrial swelling level were determined by flow cytometry, and the activities of electron transport chain complexes（I-V） were measured using enzyme assay kits. Hematoxylin-eosin（HE） staining and electron microscopy were used to observe morphological changes in brain tissue and mitochondria. Results The sepsis group had a significantly lower survival rate than the control group（P0.01）. The MMP and activities of electron transport chain complexes（I-V） in the sepsis group, which were significantly lower than those in the control group（P0.05）, were reduced to the lowest levels at 48 hours and partially recovered at 72 hours. The mitochondrial swelling level in the sepsis group, which was significantly higher than that in the control group（P0.05）, increased to the peak level at 48 hours and partially recovered at 72 hours. Hematoxylin and Eosin staining revealed substantial damages in the structure of brain tissue, and electron microscopy showed mitochondrial swelling, and vacuolization in a few mitochondria. Conclusions In the rat model of LPS-induced sepsis, both structural and functional injuries are found in cerebral mitochondria, and achieve the peak levels probably at around 48 hours.