多聚腺苷二磷酸核糖聚合酶-1（poly (ADP-ribose) polymerase-1, PARP-1）抑制剂对热暴露介导的大鼠小肠上皮紧密连接屏障损害的保护性作用
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摘要
目的:建立稳定的热暴露大鼠模型,观察多聚腺苷二磷酸核糖聚合酶-1(PARP-1)抑制剂(pj34)是否影响热暴露大鼠肠上皮紧密连接屏障通透性,并通过观察肠上皮细胞紧密连接蛋白表达的变化,初步探讨pj34对肠上皮屏障通透性影响的机制。方法:将麻醉SD大鼠放置在环境温度恒定(42℃)通风的恒温箱中,利用温度计每10min测量一次肛温,建立稳定的热暴露模型,热暴露程度将24h死亡率控制在10-20%左右。先期预实验观察热暴露结束后0h,2h,6h,8h,12h,24h大鼠肠道上皮细胞屏障通透性及紧密连接蛋白occludin表达变化,选取变化最显著的热暴露结束后2h为最佳干预效果观察点。干预试验时,将SD大鼠随机分为三组(n≥6):热暴露前1小时给予pj34的热暴露组(Intervention组,I组)与安慰剂(生理盐水)对照的热暴露组(Vehicle组,V组)及常温对照组(Normal组,N组),给予热暴露。观察热暴露结束2小时后,pj34对大鼠肠上皮细胞屏障通透性的影响(血浆中荧光剂FD4、内毒素及炎性细胞因子浓度变化),并通过光镜(HE染色)及透射电镜观察小肠组织的大体及微观形态学改变。应用蛋白印迹杂交(Westem blot)技术,检测热暴露结束2小时后,各组肠上皮细胞紧密连接蛋白occludin表达的变化情况。并应用免疫荧光、免疫组化检测occludin蛋白表达的形态学变化情况。结果:预实验中,将大鼠麻醉后放入42℃恒温箱,核心体温上升速度约0.5℃/5min,热暴露50min的大鼠24h死亡率在16%,50min时肛温可达42℃左右。在6个观察时间点中,热暴露结束后2h内毒素水平最高(P＜0.05),紧密连接蛋白Occludin表达最低,因此该时间点热暴露的损伤作用达到最强,并选为干预效果观察点。干预试验中,热暴露结束后2h,给予生理盐水的热暴露大鼠小肠上皮细胞屏障通透性(FD4、内毒素及细胞因子水平)显著高于正常对照组(p＜0.05),而pj34干预组的血浆荧光剂FD4浓度显著低于安慰剂热暴露组(P＜0.05),两组间内毒素及炎性因子水平也具有显著差异(P＜0.05)。形态学方面,光镜观察空肠HE染色,与正常大鼠比较,安慰剂组及干预组均出现肠上皮细胞从微绒毛顶端滑塌,从多个镜下视野观察(≥6个视野/片),此种表现不具有普遍性,但干预组上皮细胞脱落程度明显减轻。透射电镜下,正常大鼠肠上皮细胞,紧密连接结构完整,呈致密的带状结构。热暴露结束2h后,安慰剂组紧密连接断裂,细胞间隙增宽,致密度减轻。干预组紧密连接带状结构完整,致密度增强。免疫荧光显示正常组大鼠肠上皮细胞,荧光信号沿细胞膜分布。在观察时间点,干预组荧光强度与正常组相似,安慰剂组荧光强度明显减弱。免疫组化及Western结果与此一致(P＜0.05)。结论:PARP-1抑制剂pj34引起紧密连接蛋白occludin的表达增加,从而加强了肠上皮细胞间的紧密连接,这是pj34防止肠上皮紧密连接屏障热损伤的作用位点,并因此抑制了热暴露引起的肠上皮细胞屏障通透性的增高,发挥保护性作用。
Objective:A stable rat model of heat exposure was established to investigate whether PARP-1 inhibitor(pj34) might affect the intestinal epithelial TJ barrier permeability,and to investigate the mechanism by observe the changes of TJ protein(occludin) expression.Methods:Anesthetized rats were exposed to 42℃in a ventilated chamber to establish a stable model of heat exposure.The rectal temperature was measured by a thermometer every 10 min.The heat exposure extent was controlled which produced about 10-20%mortality(24h) In preliminary experiment,changes of intestinal epithelial barrier permeability and TJ protein occludin expression were investigated on 0h,2h,6h,8h,12h,24h after heat exposure.2h after heat exposure had the most significant changes and was selected as the best intervention effect observing time point.In intervention experiment,SD rats were randomly divided into three groups(n≥6):Intervention group(I) which was given pj34 lh before heat exposure;Vehicle control group(V) which was given normal saline;and Normal control group(N).The three groups were given heat exposure.At 2h after heat exposure,the effect of pj34 on intestinal epithelial barrier permeability was observed through changes of plasma FD4,endotoxin and cytokines concentration.The general and micro pathology were observed by light microscope(hematoxylin and eosin-staining,HE staining) and transmission electron microscope(TEM).Occludin expression were investigated by western blot,immunofluorescent and immunochemistry.Results: In preliminary experiment,the anesthetic rats exposed to 42℃,the rectal temperature upgrade velocity was 0.5℃/5min.The rectal temperature reached about 42℃at 50min,the 24h mortality was 16%.In six observing time points, plasma endotoxin concentration was highest and occludin expression was lowest at 2h after heat exposure.So at 2h after heat exposure,the damage of heat exposure was strongest,and it's the best intervention effect observing time point. In intervention experiment,at 2h after heat exposure,intestinal epithelial barrier permeability(FD4,endotoxin and cytokines concentration) of vehicle control group was significantly higher than normal control group(p＜0.05),but plasma FD4 concentration of pj34 intervention group was significantly decreased than V group(P＜0.05),the plasma endotoxin and cytokines concentration between these two groups also had significant deviation(P＜0.05)o In morphology, observing the ight micrographs of HE jejunal tissue,the sloughing of epithelium off the basement membrane at the villus tips of the heat exposure groups tissue(I, V groups) compared with the normal control tissue.In many visual field(VF) (＞6VF / slice),this phenomenon was not universal.But the extent of epithelium sloughing in I group was obviously relieved.Under TEM,TJ of normal enterocytes was integrated with the compact zonal structure.At 2h after heat exposure,TJ of V group was break with widen intercellular space,and the density of TJ was decreased.In enterocytes of I group,structure of TJ was integrated with higher density than V group.In normal small intestinal tissue,the fluorescence signal distribute along the cell membrane.The fluorescence intensity of I group was similar to N group,while the signal was obviously decreased in V group.The results of immunochemistry and western blot were coordinate with it (P＜0.05).Conclusion:PARP-1 inhibitor,pj34,increased occludin expression, strengthen the intestinal epithelial TJ,which is the prevention target of pj34 to intestinal epithelial tight junction barrier heat damage,so pj34 decreased the heat exposure induced high intestinal epithelial barrier permeability,produced a marked protective effect.
Objective:A stable rat model of heat exposure was established to investigate whether PARP-1 inhibitor(pj34) might affect the intestinal epithelial TJ barrier permeability,and to investigate the mechanism by observe the changes of TJ protein(occludin) expression.Methods:Anesthetized rats were exposed to 42℃in a ventilated chamber to establish a stable model of heat exposure.The rectal temperature was measured by a thermometer every 10 min.The heat exposure extent was controlled which produced about 10-20%mortality(24h) In preliminary experiment,changes of intestinal epithelial barrier permeability and TJ protein occludin expression were investigated on 0h,2h,6h,8h,12h,24h after heat exposure.2h after heat exposure had the most significant changes and was selected as the best intervention effect observing time point.In intervention experiment,SD rats were randomly divided into three groups(n≥6):Intervention group(I) which was given pj34 lh before heat exposure;Vehicle control group(V) which was given normal saline;and Normal control group(N).The three groups were given heat exposure.At 2h after heat exposure,the effect of pj34 on intestinal epithelial barrier permeability was observed through changes of plasma FD4,endotoxin and cytokines concentration.The general and micro pathology were observed by light microscope(hematoxylin and eosin-staining,HE staining) and transmission electron microscope(TEM).Occludin expression were investigated by western blot,immunofluorescent and immunochemistry.Results: In preliminary experiment,the anesthetic rats exposed to 42℃,the rectal temperature upgrade velocity was 0.5℃/5min.The rectal temperature reached about 42℃at 50min,the 24h mortality was 16%.In six observing time points, plasma endotoxin concentration was highest and occludin expression was lowest at 2h after heat exposure.So at 2h after heat exposure,the damage of heat exposure was strongest,and it's the best intervention effect observing time point. In intervention experiment,at 2h after heat exposure,intestinal epithelial barrier permeability(FD4,endotoxin and cytokines concentration) of vehicle control group was significantly higher than normal control group(p＜0.05),but plasma FD4 concentration of pj34 intervention group was significantly decreased than V group(P＜0.05),the plasma endotoxin and cytokines concentration between these two groups also had significant deviation(P＜0.05)o In morphology, observing the ight micrographs of HE jejunal tissue,the sloughing of epithelium off the basement membrane at the villus tips of the heat exposure groups tissue(I, V groups) compared with the normal control tissue.In many visual field(VF) (＞6VF / slice),this phenomenon was not universal.But the extent of epithelium sloughing in I group was obviously relieved.Under TEM,TJ of normal enterocytes was integrated with the compact zonal structure.At 2h after heat exposure,TJ of V group was break with widen intercellular space,and the density of TJ was decreased.In enterocytes of I group,structure of TJ was integrated with higher density than V group.In normal small intestinal tissue,the fluorescence signal distribute along the cell membrane.The fluorescence intensity of I group was similar to N group,while the signal was obviously decreased in V group.The results of immunochemistry and western blot were coordinate with it (P＜0.05).Conclusion:PARP-1 inhibitor,pj34,increased occludin expression, strengthen the intestinal epithelial TJ,which is the prevention target of pj34 to intestinal epithelial tight junction barrier heat damage,so pj34 decreased the heat exposure induced high intestinal epithelial barrier permeability,produced a marked protective effect.
引文
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