电针刺对内毒素致急性肺损伤大鼠肺组织内质网应激影响

发布时间：2018-03-02 05:25

本文关键词： 电针刺内毒素急性肺损伤内质网应激氧化应激　出处：《天津医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：急性肺损伤(ALI)是临床上常见的疾病,多数患者由于感染、外伤、休克等引起,发病后临床表现为中性粒细胞浸润,严重者可发展为急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS),甚至造成多器官功能障碍综合征(multiple organ dysfunctions syndrome,MODS)等严重并发症。在临床上有较高的发病率和死亡率,目前对此尚无理想的治疗手段[1-3]。内质网是真核生物重要的细胞器,是细胞蛋白合成、加工、折叠、运送及钙离子储存的主要场所[4]。内质网具有维持细胞内环境稳态平衡的重要作用[5]。而内质网应激(endoplasmic reticulum stress,ERS)作为细胞对外界不良刺激的自身保护反应,可引起凋亡相关基因如葡萄糖调节蛋白78(GRP78)、CCAAT/增强子结合蛋白同源蛋白(CHOP)、caspase-12等表达上调,从而启动细胞凋亡,内质网应激(ERS)可能是肺部相关疾病关键的病理生理机制之一[6-9]。我们前期研究已经证实了电针刺激对内毒素所致的急性肺损伤(ALI)具有防治作用,但其对内质网应激(ERS)的具体作用尚不明确[10-13]。目的本课题拟从内质网应激(ERS)角度探讨内毒素急性肺损伤(ALI)时电针刺激在微环境层面的内源性分子保护作用机制,为电针刺激在脏器保护的机制研究开辟新的思路,也为电针刺激防治内毒素性急性肺损伤(ALI)提供新的理论依据。方法40只健康清洁级雄性SD大鼠,8周龄,随机数字表法分为对照组(C组)、内毒素急性肺损伤模型组(LA组)、电针刺激穴位+内毒素急性肺损伤模型组(ELA组)、非穴位电针刺激+内毒素急性肺损伤模型组(NLA组),每组10只。LA组、NLA组和ELA组大鼠将内毒素脂多糖(lipopolysaccharide,LPS)5mg/kg溶于0.5m L生理盐水经尾静脉注射,建立大鼠ALI模型;C组给予等容量生理盐水。ELA组于内毒素急性肺损伤(ALI)模型制备前1~4d及模型制备过程中电针刺激双侧足三里和内关穴(疏密波,频率2/15Hz,刺激电流1~2m A,波宽0.2~0.6ms,刺激强度以大鼠肢体出现轻微颤动为宜),1次/d,30min/次,刺激时间为上午9:30-10:30;NLA组以相同的参数电针刺激足三里和内关穴旁开0.5cm非经非穴位处。静脉注射脂多糖(LPS)或生理盐水后6h时,处死大鼠留取肺组织处理后保存。进行肺组织病理学观察及损伤评分;肺组织湿重/干重比值(W/D比值)测定;TUNEL法检测肺泡上皮细胞凋亡指数(AI);Western blot法检测肺组织葡萄糖调节蛋白78(GRP78)、CHOP及caspase-12蛋白表达水平。结果与C组比较,LA组、NLA组及ELA组肺损伤评分、W/D比值及肺泡上皮细胞凋亡指数(AI)升高,肺组织GRP78、CHOP和caspase-12蛋白表达水平上调(P0.05);与LA组比较,ELA组肺损伤评分、湿重/干重比值(W/D比值)和肺泡上皮细胞凋亡指数(AI)降低,肺组织GRP78、CHOP及caspase-12蛋白水平下降(P0.05),NLA组以上指标比较差异无统计学意义(P0.05)。结论电针刺激双侧足三里和内关穴可减轻大鼠内毒素致急性肺损伤(ALI),其机制与抑制大鼠肺组织内质网应激(ERS)有关。
[Abstract]:Acute lung injury (Ali) is a common clinical disease. Most of the patients suffer from infection, trauma, shock and so on. In severe cases, acute respiratory distress syndrome may develop into ARDS, and even cause multiple organ dysfunctions syndrome, such as mods. The endoplasmic reticulum (ER) is an important organelle of eukaryotes, which is the synthesis, processing and folding of cellular proteins. Major sites for transport and storage of calcium ions [4] .Endoplasmic reticulum (ER) plays an important role in maintaining homeostasis of intracellular environment [5]. Endoplasmic reticulum stress (ER) is the self-protective response of cells to adverse external stimuli. The expression of apoptosis-related genes such as Glucose-regulated protein 78, GRP78, CCAATA / enhancer binding protein homologue protein, CHOPP7, caspase-12, was up-regulated, thus inducing apoptosis. Endoplasmic reticulum stress (ERS) may be one of the key pathophysiological mechanisms of lung related diseases [6-9]. Our previous studies have confirmed that electroacupuncture has a preventive and therapeutic effect on endotoxin-induced acute lung injury (ALI). But the specific effect of ERS on endoplasmic reticulum stress (ERS) is unclear [10-13]. Objective to explore the mechanism of endogenous molecular protection of electroacupuncture stimulation in microenvironment from the perspective of endoplasmic reticulum stress (ERS). This study provided a new theoretical basis for the study of the mechanism of electroacupuncture stimulation in organ protection and for the prevention and treatment of endotoxin induced acute lung injury (ALII). Methods 40 healthy and clean male Sprague-Dawley rats were 8 weeks old. The method was randomly divided into three groups: control group (C group), endotoxin acute lung injury model group (LA group), electroacupuncture stimulation point endotoxin acute lung injury model group (ELA group) and non-acupoint electroacupuncture stimulation endotoxin acute lung injury model group (NLA group). 10 rats in each group were injected intravenously with lipopolysaccharide of lipopolysaccharide of lipopolysaccharide (LPS5 mg / kg) dissolved in 0.5 mL of normal saline in each group (n = 10) and ELA group (n = 10). Rats in ALI model group C were given isovolumic physiological saline. ELA group was treated with endotoxin-induced acute lung injury (Ali) 1 day before the establishment of the model and electroacupuncture stimulated bilateral Zusanli and Neiguan points (dense wave) during the preparation of the model. At the frequency of 2 / 15 Hz, the stimulation current is 12mA, the wave width is 0.2ms, and the stimulation intensity is as follows: slight tremor in the limbs of the rat is suitable for 30 mins per rout. The stimulation time was 9: 30-10: 30: 30: 30: NLA group with the same parameters to stimulate Zusanli and Neiguan acupoints with 0.5 cm open at non-acupoints. 6 hours after intravenous injection of lipopolysaccharide (LPS) or normal saline, The rats were sacrificed and preserved after the treatment of lung tissue. The histopathological observation and injury score of the lung were performed. Lung tissue wet weight / dry weight ratio (WR / D) was used to detect apoptosis index of alveolar epithelial cells by Tunel method and Western blot method to detect the expression of glucose-regulated protein 78GRP 78 and caspase-12 protein in lung tissue. Results compared with group C, the expression of glucose regulated protein 78 GRP 78 and caspase-12 protein were detected in LA group and ELA group. The ratio of W / D and the apoptosis index of alveolar epithelial cells were increased. Compared with LA group, lung injury score, wet / dry weight ratio (WR / D) and apoptosis index of alveolar epithelial cells (AI) were decreased. There was no significant difference in the above indexes between the two groups (P 0.05). Conclusion the stimulation of bilateral Zusanli and Neiguan points by electroacupuncture can reduce the acute lung injury induced by endotoxin in rats. Endoplasmic reticulum stress (ERS) is involved.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.8

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