盐酸戊乙奎醚对新生大鼠内毒素血症时肠组织HIF-1α表达的影响

发布时间：2018-01-22 16:25

本文关键词： 盐酸戊乙奎醚内毒素肠损伤缺氧诱导因子-1α　出处：《郑州大学》2014年硕士论文　论文类型：学位论文

【摘要】：新生儿肠损伤是儿科常见的疾病之一，其发生的主要原因有早产、窒息引起的缺血缺氧以及感染等。其中严重感染常可导致危重患儿胃肠功能衰竭。肠道在全身炎症反应综合征（SIRS）和多器官功能障碍综合征(MODS)的发生发展过程中扮演着极其重要的角色。肠道感染或缺血缺氧会导致肠黏膜屏障功能受损，引起细菌及内毒素的移位，引发败血症和脓毒性休克，从而进一步加重肠组织的损伤，甚者可伴随远隔器官受累。脂多糖（LPS）作为典型的内毒素,可诱发内毒素血症，导致肠黏膜损害。国内外诸多研究发现缺氧诱导因子-1α（HIF-1α）在内毒素性肠损伤的病理改变过程中具有重要作用。有研究指出肠道缺血缺氧可以诱导肠黏膜HIF-1α持续高表达，这一结果至少在部分程度上是由于肠道细菌的移位及其产物（如LPS）的增加所造成的。HIF-1α能够通过激活其下游的相关基因转录并促进炎症因子的大量释放和引起细胞凋亡等，从而加重内毒素血症或缺血/再灌注时的组织损伤。盐酸戊乙奎醚（PHC）是我国自主研制的抗胆碱药。研究表明，盐酸戊乙奎醚因能改善微循环、降低毛细血管壁通透性和减少溶酶体释放而具有多器官保护作用。目前盐酸戊乙奎醚对内毒素性肺损伤的保护作用得到了广泛地证实和认可，我们通过大量的临床观察发现盐酸戊乙奎醚在解除肠痉挛、减轻肠道水肿方面同样具有良好的效果。基于以上背景，本课题旨在研究内毒素致新生大鼠肠损伤时盐酸戊乙奎醚预先给药对肠组织HIF-1α表达的影响，并对其肠保护作用机制进行初步探讨。目的探讨盐酸戊乙奎醚预先给药对内毒素致新生大鼠肠损伤时肠组织HIF-1α和其他相关细胞因子表达的影响及其发挥肠保护作用的可能机制，为临床防治新生儿肠损伤提供一条新的思路。材料与方法 1.研究对象 69只SPF级健康新生7日龄SD大鼠，由郑州大学河南省实验动物中心提供，雌雄不限，体重18±2g。 2.实验方法 2.1实验动物分组本实验分为两部分。第一部分：将30只幼鼠随机分为3组，每组10只。分别为生理盐水对照组（C组）、内毒素致肠损伤模型组（L组）和盐酸戊乙奎醚干预组（P组）。本部分实验主要进行肠组织湿/干重比的测定、肠组织HE染色后光镜下形态学改变的观察以及相关细胞因子的检测。第二部分：同样将另39只健康新生7日龄SD大鼠随机分为C组、L组和P组，每组13只。本部分实验用于观察造模后各组幼鼠行为活动的改变并计算每组24h生存率。 2.2动物模型的制备内毒素致肠损伤模型组腹腔注射内毒素（细菌脂多糖LPS，E.Coli055：B5，Sigma公司，美国）5mg/kg，配制浓度为0.5mg/ml；盐酸戊乙奎醚干预组在注射内毒素前30min，先腹腔注射盐酸戊乙奎醚（成都力思特制药股份有限公司，批号：120603）2mg/kg，配制浓度为0.05mg/ml；生理盐水对照组腹腔注射生理盐水10ml/kg。第一部分实验于腹腔注射内毒素或生理盐水后6h麻醉并解剖幼鼠。两部分实验均在造模完成后将幼鼠放回鼠笼与母鼠共同喂养。 2.3肠组织的标本采集在密闭玻璃容器中吸入七氟烷快速麻醉幼鼠后，将其固定于操作台，解剖，用1ml注射器迅速进行心脏采血，静置2小时，4℃下4000r/min离心15分钟，取上清液，-20℃冰箱中保存待检。距回盲部3cm向上取回肠组织7cm，4℃生理盐水冲洗肠管3遍，滤纸吸干肠组织表面水分后，将7cm回肠组织分为4段：上段1cm用于光镜下形态学观察，中上段2cm用于测量肠组织湿重和干重，中下段2cm用于制备肠组织匀浆测定相关细胞因子的表达，下段2cm用于RT-PCR检测肠组织HIF-1α mRNA的表达。 2.4检测指标 2.4.1计算肠组织湿/干重比（W/D）电子称重计称量中上段2cm回肠组织并记录湿重，随后放入70℃恒温烘干箱烘干48h至重量不再发生变化，测量干重，计算湿/干重比。 2.4.2肠组织形态学观察将上段1cm回肠组织放入4%多聚甲醛中保存待检。石蜡包埋后制作5μm病理切片，，行HE染色。400倍光镜下观察回肠组织黏膜上皮细胞形态学的改变。 2.4.3ELISA法测定肠组织中TNF-α、IL-6、HIF-1α、Gln、二胺氧化酶（DAO）及血清DAO的含量中下段2cm回肠组织称重后制备组织匀浆，4℃下3000r/min离心15分钟，取上清液，置于-20℃冰箱中保存待检。TNF-α、IL-6、HIF-1α、Gln和DAO的检测严格按照ELISA试剂盒（RD公司，美国）说明书进行。 2.4.4RT-PCR法测定肠组织HIF-1α mRNA的表达将下段2cm回肠组织经DEPC水冲净后滤纸吸干，经液氮迅速冷却后放入冻存管中，置于-80℃冰箱保存待检。经肠组织总RNA的提取、逆转录、DNA扩增和琼脂糖凝胶电泳等步骤检测幼鼠肠组织HIF-1α mRNA的表达情况。 3．统计学处理采用SPSS17.0统计软件包进行统计学分析，计量资料以均数±标准差(x±s)表示，多组间比较采用单因素方差分析，两两比较采用最小显著差异法（LSD-t），以α=0.05为检验水准，即P0.05时，组间差异有统计学意义。结果 1.肉眼观察，腹腔注射LPS后，1小时内L组与P组幼鼠开始出现浑身抖动，呼吸急促。2~3小时左右出现周身发凉，精神萎靡，活动度下降，并且进食减少。6小时左右出现明显的倦怠乏力，几乎不活动也不进食，甚者口唇青紫。P组幼鼠症状相对较轻，C组幼鼠活动、进食等情况正常。C组、L组及P组的24h生存率分别为100%、69.2%和92.3%，幼鼠死亡主要发生在注射LPS后12~24小时期间。 2.与C组比较，L组和P组肠组织湿/干重比均显著升高（P0.05）；与L组比较，P组的这一比值显著降低（P0.05）。差异均有统计学意义。 3.HE染色光镜结果显示：C组回肠绒毛结构完整，上皮细胞排列整齐，形态正常；L组可见绒毛细胞水肿，上皮细胞排列紊乱；P组绒毛水肿较L组明显减轻，上皮细胞排列较整齐。 4.与C组比较，L组和P组肠组织TNF-α、IL-6、HIF-1α的含量均升高（P0.05），而Gln的含量降低（P0.05）；与L组比较，P组肠组织Gln的含量较高（P0.05），而余各指标的含量均较低（P0.05）。差异均有统计学意义。 5.与C组比较，L组和P组肠组织DAO含量均显著降低，而血清DAO含量升高（P0.05）；与L组比较，P组肠组织DAO含量较高，而血清DAO含量较低（P0.05）。差异均有统计学意义。 6.与C组比较，L组和P组肠组织HIF-1α mRNA的表达量均升高（P0.05）；与L组相比，P组肠组织HIF-1α mRNA的表达量较低（P0.05）。差异均有统计学意义。结论盐酸戊乙奎醚预先给药能够减轻内毒素血症时的肠道水肿，减轻肠组织炎症反应，降低肠黏膜屏障的通透性。其肠保护作用机制可能与其下调HIF-1α的表达有关。
[Abstract]:Neonatal intestinal injury is one of the most common diseases in children, the main reasons for its occurrence are premature, caused by ischemia and hypoxia and asphyxia. The infection of severe infection often leads to failure in critically ill children. Gastrointestinal gut in systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS) plays a very important the role in the pathogenesis of intestinal infection or ischemia. Hypoxia can lead to intestinal mucosal barrier dysfunction, caused by the translocation of bacteria and endotoxin, sepsis and septic shock, so as to further increase the intestinal tissue damage, even accompanied with remote organ involvement. Lipopolysaccharide (LPS) as a typical endotoxin induced endotoxemia that lead to intestinal mucosal damage. Many studies found that hypoxia inducible factor -1 alpha (HIF-1 alpha), intestinal injury pathological change process plays an important role on. That intestinal anoxia can induce intestinal mucosal HIF-1 a high expression, this result is at least partly due to the shift of intestinal bacteria and their products (such as LPS) caused by an increase in.HIF-1 alpha through transcriptional activation of its downstream genes and promote the inflammatory factor release and induce cell apoptosis. In order to increase the tissue damage during reperfusion endotoxemia or ischemia. Penehyclidine hydrochloride (PHC) is China's self-developed anticholinergic drugs. The study shows that penehyclidine hydrochloride can improve microcirculation, reduce capillary wall permeability and reduce the release of lysosomal function has multiple organ protection. Current protection effect of penehyclidine hydrochloride on endotoxin induced acute lung injury has been widely recognized and confirmed, we found that penehyclidine hydrochloride in relieving intestinal spasm through a number of clinical observation, reduce intestinal edema. Based on the above background, the aim of this study is to investigate the effect of penehyclidine hydrochloride on the expression of HIF-1 alpha in intestinal tissue after intestinal injury induced by lipopolysaccharide in neonatal rats, and to explore the mechanism of intestinal protection.
objective
Objective to investigate the effect of penehyclidine hydrochloride on the expression of HIF-1 and other related cytokines in intestinal tissue of neonatal rats with endotoxin induced intestinal injury, and to explore the possible mechanisms of intestinal protection, so as to provide a new idea for the prevention and treatment of neonatal intestinal injury.
Materials and methods
1. research objects
69 SPF grade healthy newborn 7 day old SD rats were provided by the experimental animal center of Henan province of Zhengzhou University. The male and female were not limited, and the weight was 18 + 2g.
2. experimental method
2.1 group of experimental animals
This experiment is divided into two parts. The first part: 30 rats were randomly divided into 3 groups, 10 rats in each group respectively. Normal saline control group (group C), endotoxin induced intestinal injury model group (L group) and penehyclidine hydrochloride group (group P). The main part of the experiment of intestine tissue wet / dry weight ratio determination, detection of intestinal tissue HE staining was used to observe the morphological changes under light microscopy and related cytokines. The second part: the same to the other 39 healthy SD rats of 7 days old were randomly divided into C group, L group and P group, 13 rats in each group. This part of the experiment for observation in different group activities after the model change and calculate each 24h survival rate.
The preparation of 2.2 animal models
Endotoxin induced intestinal injury model by intraperitoneal injection of endotoxin (lipopolysaccharide LPS, E.Coli055:B5, Sigma, 5mg/kg, USA) with a concentration of 0.5mg/ml; penehyclidine hydrochloride in the intervention group before the first injection of endotoxin 30min, intraperitoneal injection of penehyclidine hydrochloride (Chengdu Lisite pharmaceutical Limited by Share Ltd, batch number: 120603) 2mg/kg concentration was prepared 0.05mg/ml; saline control group received intraperitoneal injection of saline 10ml/kg. the first part of the experiment on intraperitoneal injection of endotoxin or saline 6h after anesthesia and the anatomy of rats. The two part experiments were made in rats after the rats were returned to the cage and the mother feeding.
Sample collection of 2.3 intestinal tissues
In a closed glass container seven inhalation halothane anesthesia in rats after rapid, which is fixed on the operating table, anatomy, with 1ml syringe rapid heart blood, standing 2 hours, at 4 4000r/min centrifuge for 15 minutes, the supernatant was detected, kept in the refrigerator. From -20 DEG 3cm to ileocecus intestinal tissue on the back 7cm, 4 C saline intestine 3 times, intestinal tissue surface water filter paper blot, 7cm ileum is divided into 4 sections: the upper 1cm for morphological observation under light microscope, in the upper 2cm used to measure intestinal tissue wet weight and dry weight, the lower section of 2cm for the preparation of expression of intestinal tissue homogenate were determined by cell the lower section of the 2cm factor, used to detect the expression of intestinal RT-PCR HIF-1 alpha mRNA.
2.4 detection index
2.4.1 calculation of the wet / dry weight ratio of intestinal tissue (W/D)
The electronic weigher weighed the upper and middle 2cm ileum tissue and recorded the wet weight. Then it was put into the 70 degree temperature drying oven and dried to 48h until the weight was no longer changed. The dry weight was measured and the wet / dry weight ratio was calculated.
Observation of 2.4.2 intestinal histomorphology
The upper 1cm ileum tissue was placed in 4% paraformaldehyde and kept for inspection. A 5 m pathological section was made after paraffin embedding, and HE staining was used to observe the morphological changes of ileum tissue epithelial cells after.400 staining.
Determination of TNF- alpha, IL-6, HIF-1 alpha, Gln, two amine oxidase (DAO) and the content of serum DAO in intestinal tissue by 2.4.3ELISA
The middle and lower 2cm ileum tissues were weighed and then homogenized, then centrifuged for 15 minutes at 4 degrees, and the supernatant was taken in the refrigerator at -20 degrees. The detection of.TNF-, IL-6, HIF-1 alpha, Gln and DAO was performed strictly in accordance with the instructions of ELISA Kit (RD company, USA). The contents of 3000r/min, IL-6 and HIF-1 were determined.
Determination of the expression of HIF-1 alpha mRNA in intestinal tissue by 2.4.4RT-PCR
The lower section of ileal tissue in 2cm by DEPC water rinse, dry with filter paper, rapid cooling by liquid nitrogen in freezing tube, placed in the refrigerator to be detected. C -80 extraction, the total RNA of intestinal tissue for reverse transcription to detect HIF-1 expression of alpha mRNA in intestinal tissue DNA amplification and agarose gel electrophoresis and other steps.
3. statistical treatment
The data were analysed by SPSS17.0 statistical software, measurement data to mean + standard deviation (x + s) said that the comparison among multiple groups using ANOVA, 22 compared with the least significant difference method (LSD-t), to test the level of a =0.05, namely P0.05 group, there was significant difference between two groups.
Result
1. eye, 1 hours after intraperitoneal injection of LPS, L group and P group rats began to appear all over the body was shaking, chills, shortness of breath apathetic about.2~3 hours, decreased activity, and eating less lassitude was.6 hours, almost no activity is not eating, even purple lips.P rats were relatively mild symptoms, C of rats in group activities, eating normal.C group, L group and P group 24h survival rates were 100%, 69.2% and 92.3%, neonatal death occurred mainly in LPS after injection of 12~24 hour period.
2. compared with group C, the wet / dry weight ratio of L group and P group increased significantly (P0.05). Compared with L group, the ratio of P group decreased significantly (P0.05). The difference was statistically significant.
3.HE staining and light microscopy showed that the villi of the C group were intact, the epithelial cells arranged neatly, and the morphology was normal. L group showed villous cell edema and epithelial cell disorder. The villus edema in group P was significantly lower than that in L group, and epithelial cells arranged orderly.
4. compared with group C, the contents of TNF-, IL-6, HIF-1 and alpha in intestinal tissue of group L and P increased (P0.05), while Gln content decreased (P0.05). Compared with L group, the content of P0.05 in intestinal tissue of the P group was higher than that of the L group, while the contents of all other indexes were all low.
5. compared with group C, DAO content in intestinal tissue of L group and P group was significantly decreased, while serum DAO level increased (P0.05). Compared with L group, DAO content in intestinal tissue of P group was higher, while serum DAO level was lower (P0.05). The difference was statistically significant.
6. compared with group C, the expression level of HIF-1 alpha and mRNA in intestinal tissue of L group and P group increased (P0.05). Compared with L group, the expression of HIF-1 mRNA mRNA in P group was low (P0.05). The difference was statistically significant.
conclusion
Penehyclidine hydrochloride can reduce the intestinal edema, reduce the inflammatory reaction and reduce the permeability of intestinal barrier in endotoxemia. The mechanism of intestinal protection may be related to its downregulation of the expression of HIF-1.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R726.1

【共引文献】