LPS致家兔发热诱导的HSF1聚合对体温及脑内AVP含量的影响

发布时间：2018-05-01 12:09

本文选题：热休克因子1 + 精氨酸加压素　；参考：《中国医科大学》2007年硕士论文

【摘要】： 前言恒温动物和人在发热时体温很少超过一定限度，例如，人体体温很少超过41℃，称为热限现象，提示体内存在着调控体温的自限机构，但其机制尚不十分清楚。脂多糖(lipopolysaccharide，LPS)作为外源性致热原可致体温升高。精氨酸加压素(arginine vasopressin，AVP)是一种内源性解热物质，主要作用于腹侧中隔区(vent ral septal area，VSA)。有报道向VSA注射AVP能显著地抑制家兔的内毒素(endotoxin，ET)性发热反应。热休克因子(Heat shock transcription factor，HSF)是一组结构和功能具有广泛同源性，普遍存在于真核生物细胞中的核蛋白，包括HSF1、HSF2、HSF3、HSF4。其中HSF1对温度刺激敏感，被激活时由单体结合成三聚体，具有调节热休克蛋白(Heat shock protein，HSP)及多种生物活性物质转录的功能。本研究室以往的研究结果显示，HSF1能够抑制单核细胞内生性致热原的产生及中枢发热介质的产生。但是HSF1的激活是否能够调控中枢AVP的水平，进而，AVP发挥对体温的负调节作用，相关的研究还未见报道。本实验通过复制家兔LPS发热模型，观察发热诱导的HSF1聚合对发热过程及脑内AVP含量的影响，并通过中枢给予槲皮素(quercetin，QCT)抑制HSF1的活化，旨在进一步探讨发热时HSF1限控体温的相关中枢调节机制。材料与方法 1、动物分组、模型及标本制备 (1)动物分组健康雄性新西兰白兔70只，随机分为四组：正常对照组(N)、槲皮素组(Q)、脂多糖组(L)和槲皮素+脂多糖组(Q+L)。 (2)脑室插管在应用1％戊巴比妥钠(3 ml·kg~(-1))静脉麻醉下，将动物固定在SN-2型脑立体定位仪上。按Sawyer氏兔脑图谱，在P1L4-5H5(侧脑室)处埋藏一外径为1mm的塑料管，用牙科水泥固定，术后将管端中封闭。术后家兔单笼饲养，常规喂养1wk后进行实验。 (3)复制发热模型用无热原的生理盐水将精制的大肠杆菌内毒素-LPS稀释成0.5μg·ml~(-1)，按1μg·kg~(-1)体重，经家兔耳缘静脉注射。 (4)取材N组和Q组于测温后60 min处死家兔，其它两组分别在给予LPS后0、30、60、180、240和360 min时处死家兔，迅速取出家兔下丘脑及腹中隔区，取一部分放入生理盐水中，煮沸5 min。制备组织匀浆，离心后取上清置-20℃保存待进行AVP含量的测定；另取一部分迅速放入液氮中，20 min后置-70℃深度冰箱中保存，待进行HSF1和HSP70表达量的测定。 2、检测指标实时监测体温的变化，体温变化情况采用△T，TRI进行分析；应用Western印迹法检测不同实验条件下下丘脑HSF1和HSP70的表达；用放射免疫法检测下丘脑及腹中隔区AVP含量的变化。 3、数据分析所有实验数据均用均数±标准差((?)±s)表示，采用SPSS11.0软件统计分析，相关分析用相关性检验分析法。实验结果 1、家兔的体温变化家兔静脉注射LPS(1μg·kg~(-1))后引起双峰热。即60 min时体温上升形成第一个高峰，体温变化值为1.32±0.24℃。在180 min时，体温上升形成第二个高峰，体温变化值为1.84±0.35℃。各组体温变化最大值(△Tmax)由低至高顺序为：Q组＜N组＜L组＜Q+L组；其中，Q+L组与L组比较，在给予LPS后240～360min期间，△T及TRI_6均呈显著性增高(P＜0.05)，且可见发热时程延长。 2、下丘脑及VSA中AVP的含量变化各组VSA中的AVP含量均呈现随发热幅度增大而增加，二者之间呈明显的正相关(r=0.913，P＜0.01)。Q+L组与L组比较，VSA中的AVP含量显著增高(240～360 min)(P＜0.05)，但下丘脑中AVP的含量均无显著变化(P＞0.05)。 3、下丘脑及腹中隔区HSF1和HSP70的表达在LPS致热过程中，可见从给予LPS后60 min(△T：1.32±0.24℃)起HSF1三聚体的表达显著增高，达到体温最高值后随着HSF1三聚体的表达水平的进一步升高，，体温逐渐下降。在各组HSF1的总体含量无显著变化。应用QCT可显著地抑制HSF1的聚合，同时可见HSP70表达水平相应降低。 4、HSF1三聚体含量与体温及AVP含量变化的相关性分析 LPS致发热过程中，在达到体温高峰以后(180～360 min)，下丘脑中HSF1三聚体的含量与体温变化呈负相关(r=-0.897，P＜0.01)；与VSA中的AVP含量也呈负相关(r=-0.774，P＜0.01)。结论 1、VSA区AVP的含量随发热幅度增大而增加。 2、下丘脑HSF1三聚体的形成可一定程度地抑制LPS引起的发热反应，并可见VSA区AVP的含量减少。
[Abstract]:Preface
The temperature of the thermostatic animals and people is rarely more than a certain limit, for example, the body temperature is rarely more than 41 degrees C, which is called the heat limit phenomenon, suggesting that there is a self limiting mechanism for regulating body temperature, but its mechanism is not very clear. Lipopolysaccharide (LPS) as an exogenous pyrogen can increase the body temperature. Arginine vasopressin (arginin E vasopressin, AVP) is an endogenous antipyretic substance which is mainly used in the ventral septum (vent ral septal area, VSA). It is reported that the injection of AVP to VSA can significantly inhibit the endotoxin (endotoxin, ET) sex fever reaction in rabbits.
Heat shock factor (Heat shock transcription factor, HSF) is a group of structures and functions that are widely homologous and ubiquitous in eukaryotic cells, including HSF1, HSF2, HSF3, HSF4. in which HSF1 is sensitive to temperature stimuli and is activated by a monomer, which regulates heat shock proteins (Heat shock) and Previous studies in this study show that HSF1 can inhibit the production of endogenous pyrogen and the production of central fever medium in monocyte, but whether activation of HSF1 can regulate the level of central AVP, and then AVP plays a negative regulatory role on body temperature, and the related research has not been reported.
In this experiment, the effect of fever induced HSF1 polymerization on the fever process and the AVP content in the brain was observed by replicating the rabbit LPS fever model, and the activation of HSF1 was inhibited by the central administration of quercetin (quercetin, QCT). The purpose of this study was to further explore the central regulatory mechanism of HSF1 limited temperature controlled by HSF1 in fever.
Materials and methods
1, animal grouping, model and specimen preparation
(1) 70 healthy male New Zealand white rabbits were randomly divided into four groups: normal control group (N), quercetin group (Q), lipopolysaccharide group (L) and quercetin + lipopolysaccharide group (Q+L).
(2) intraventricular intubation in the use of 1% pentobarbital sodium (3 ml. Kg~ (-1)) intravenous anesthesia, the animals were fixed on the SN-2 stereotaxis. According to the Sawyer rabbit brain atlas, a plastic tube with a external diameter of 1mm was buried in the P1L4-5H5 (lateral ventricle), fixed with dental cement and closed in the end of the tube after operation. The rabbits were fed in a single cage after the operation, and the 1wk was fed routinely after the operation. Do the experiment.
(3) the replicating fever model diluted the refined Escherichia Coli Endotoxin -LPS into 0.5 mu g / ml~ (-1) with the physiological saline of pyrogen, and injected the rabbit ear vein with 1 mu g. Kg~ (-1) body weight.
(4) the rabbits were killed in group N and group Q at 60 min after temperature measurement. The other two groups were killed at 0,30,60180240 and 360 min after LPS, and the rabbit hypothalamus and ventral septum were removed quickly. Some of them were put into the normal saline, and the tissue homogenate was prepared by boiling 5 min., and then collected at -20 C after centrifugation to determine the AVP content. Some of them were quickly placed in liquid nitrogen and stored in 20 min deep refrigerators at -70 C depth. The expression levels of HSF1 and HSP70 were determined.
2, detection index
The changes of body temperature were monitored in real time. The changes of body temperature were analyzed with delta T and TRI, and the expression of HSF1 and HSP70 in hypothalamus under different experimental conditions was detected by Western blotting, and the changes in the content of AVP in the hypothalamus and ventral septum were detected by radioimmunoassay.
3, data analysis
All the experimental data were expressed by mean + standard deviation ((+) s), and SPSS11.0 software was used for statistical analysis. Correlation analysis was conducted by correlation test method.
experimental result
1, the change of body temperature in rabbits
After intravenous injection of LPS (1 mu g. Kg~ (-1)), the heat of Shuangfeng was caused by the increase of temperature at 60 min. The temperature change was 1.32 + 0.24 C. At 180 min, the temperature rise formed second peaks, the temperature change was 1.84 + 0.35 degrees. The maximum temperature change of each group (delta Tmax) was from low to high in order of Q group < N < L < Q+L. In group Q+L, T and TRI_6 were significantly increased during the period from 240 to 360min after giving LPS, compared with group L (P < 0.05), and the duration of fever was longer.
2, changes in the content of AVP in the hypothalamus and VSA
The content of AVP in each group of VSA increased with the increase of the amplitude of fever, and there was a significant positive correlation between the two (r=0.913, P < 0.01). Compared with the L group,.Q+L group increased significantly (240~360 min) in VSA (P < 0.05), but the AVP content in the hypothalamus was not significantly changed (P > 0.05).
3, expression of HSF1 and HSP70 in the hypothalamus and ventral septum
During the heat process of LPS, the expression of HSF1 trimer increased significantly from the 60 min (delta T:1.32 + 0.24 C) after LPS, and the body temperature decreased gradually with the increase of the expression level of HSF1 trimer after the highest temperature of the body temperature. The total content of HSF1 in each group was not significantly changed. The application of QCT could significantly inhibit the polymerization of HSF1, at the same time The expression level of HSP70 was reduced accordingly.
4, the correlation between the content of HSF1 trimer and the changes of body temperature and AVP content
In the process of fever induced by LPS, after reaching the peak of temperature (180~360 min), the content of HSF1 trimer in the hypothalamus was negatively correlated with the change of body temperature (r=-0.897, P < 0.01), and negatively correlated with the AVP content in VSA (r=-0.774, P < 0.01).
conclusion
1, the content of AVP in VSA zone increases with the increase of heating rate.
2, the formation of hypothalamus HSF1 trimer can inhibit the fever reaction induced by LPS to some extent, and the content of AVP in VSA region can be reduced.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

【参考文献】