肾脏中NLRP3炎性体在孕期LPS刺激致子代大鼠高血压中的作用研究

发布时间：2018-07-14 22:24

【摘要】：高血压是十分易见的慢性疾病,也是心脑血管病人死亡的主要原因。肾脏是具有内分泌功能的重要脏器,能够调节机体水、电解质的平衡,与高血压的发生有着非常密切的关系。在前期的实验中发现,孕期脂多糖(Lipopolysaccharide,LPS)刺激导致子代大鼠在三个月龄时发生高血压并伴有肾小球数量减少和肾功能下降,炎性因子表达增多,因此我们推测肾脏炎症在孕期炎症刺激致子代高血压中可能起着重要作用。NLRP3炎性体是NOD样受体(nucleotide-binding oligomerization domain-like receptors,NLRs)中最具代表性的炎性体之一,它能诱导机体细胞炎症或凋亡,产生大量的炎性因子,导致严重的炎症反应发生,而多巴胺(dopamine,DA)通过多巴胺D1受体可抑制NLRP3炎性体的激活。本研究通过复制孕期LPS致子代大鼠高血压动物模型,同时用多巴胺进行干预,研究肾脏NLRP3在孕期LPS刺激致子代大鼠高血压中的作用,为高血压及肾损伤的预防和治疗提供靶点。实验结果如下:1.孕期LPS刺激致子代大鼠高血压模型的建立及DA干预对孕期LPS致子代大鼠高血压的影响选取30只孕鼠随机分为三组:空白对照组、LPS组、LPS+DA组。选10只怀孕母鼠不做任何处理并饲喂普通饲料,将出生后子鼠做为空白对照组;10只母鼠在孕期第8、10、12天腹腔注射0.4 mg/kg的LPS,出生后的子鼠作为LPS组;10只母鼠在孕期第8、10、12天腹腔注射0.4 mg/kg LPS的同时注射1 mL/kg的多巴胺,出生后子鼠作为LPS+DA组。颈动脉穿刺测得三个月龄子代大鼠血压,实验结果显示,与对照组相比,LPS组子代雄鼠和雌鼠的平均动脉压都显著增高,表明孕期脂多糖刺激致子代高血压模型复制成功。DA+LPS组与LPS组相比显著降低。2.孕期LPS刺激及DA干预对子代大鼠肾功能及肾脏形态学的影响腹腔静脉采血,测定血清中尿素氮、肌酐、尿酸。结果表明,与对照组相比,雄性子鼠LPS组血清尿素氮含量显著降低,雄性子鼠和雌性子鼠LPS组尿酸含量显著增加,与LPS组相比,子代雌鼠LPS+DA组尿酸含量显著降低;血清中肌酐含量各组间相比无显著统计学意义。ELISA试验结果表明,与对照组相比,血清中IL-1β和IL-18的含量都显著增加,LPS+DA组与LPS组相比,IL-1β和IL-18的含量水平降低。HE染色结果显示,与对照组相比,LPS组子代大鼠部分血管球直径减小,肾小囊腔变大,囊腔中有散在的中度到重度肾小球系膜基质增生,且肾小球部分发生粘连,近曲小管和远曲小管中有散在的类似于蛋白样的物质存在,而LPS+DA组肾脏形态学特征接近于对照组。3.孕期LPS刺激及DA干预对子代大鼠肾脏NLRP3炎性体mRNA表达的影响荧光定量PCR结果表明,与对照组对比,LPS组子代大鼠中NLRP3的mRNA表达增高;与LPS组相比,LPS+DA组NLRP3的m RNA表达降低;在子代雌鼠肾脏ASC的mRNA表达中,LPS组相对于对照组有所增加,其它各组间相比没有统计学意义;子代大鼠中CASP1的mRNA表达各组间相比没有明显统计学意义。4.孕期LPS刺激及DA干预对子代大鼠肾脏NLRP3炎性体蛋白表达的影响免疫组织化学染色结果表明,NLRP3主要表达在近端小管和远端小管内皮细胞。与对照组相比,LPS组肾小球有少量NLRP3表达;ASC在子代大鼠中主要在肾小管和血管球中表达,子代雄性大鼠肾脏中的CASP1主要表达在血管球中,而雌鼠在血管球和肾小管中都有表达。同时光密度值(IOD)结果表明,与对照组和LPS+DA组相比,LPS组子代雌性大鼠肾脏中NLRP3、ASC和CASP1蛋白表达量显著增加;与对照组和LPS+DA组相比,子代雄性LPS组大鼠,仅NLRP3表达增高;Western blot的实验结果与免疫组织化学染色所测的光密度结果保持一致。综上所述,本研究通过复制孕期脂多糖致子代大鼠高血压动物模型,证实肾脏中NLRP3在孕期LPS刺激致子代大鼠高血压中具有重要作用,多巴胺干预对孕期LPS刺激致子代大鼠高血压和肾损伤有保护作用,可能与多巴胺抑制NLRP3的激活有关。
[Abstract]:Hypertension is a very easy to see chronic disease, also the main cause of death in cardiovascular and cerebrovascular patients. The kidney is an important organ with endocrine function, which can regulate the balance of the body's water and electrolytes. It has a very close relationship with the occurrence of hypertension. In the earlier experiment, the Lipopolysaccharide (LPS) stimulates the pregnancy. At the age of three months, induced hypertension in the offspring was accompanied by a decrease in the number of glomeruli and the decline of renal function, and the expression of inflammatory factors increased. Therefore, we speculate that renal inflammation may play an important role in NOD like receptor (nucleotide-binding oligomerization domain-like REC) during pregnancy induced inflammatory stimulation of the offspring. Eptors, NLRs), one of the most representative inflammatory bodies, can induce inflammation or apoptosis of the body cells, produce a large number of inflammatory factors and cause serious inflammatory reactions, and dopamine (dopamine, DA) can inhibit the activation of NLRP3 inflammatory body through the dopamine D1 receptor. At the same time, dopamine was used to study the role of kidney NLRP3 in hypertensive rats induced by LPS during pregnancy and to provide targets for prevention and treatment of hypertension and renal injury. The results of the experiment were as follows: 1. the establishment of hypertension model in offspring rats induced by LPS stimulation during pregnancy and the effect of DA intervention on the hypertension of offspring rats induced by LPS during pregnancy were selected 30. Only three pregnant rats were randomly divided into three groups: blank control group, group LPS and group LPS+DA. 10 pregnant female rats were selected as blank control group without any treatment and feeding, and 10 female rats were intraperitoneally injected with LPS of 0.4 mg/kg on day 8,10,12, after birth, as group LPS; 10 female mice were intraperitoneally injected 0 on day 8,10,12. .4 mg/kg LPS was injected with 1 mL/kg dopamine at the same time, and the postnatal rat was used as LPS+DA group. The blood pressure of the three month old rat was measured by the carotid artery puncture. The results showed that the mean arterial pressure of the male and female rats in the LPS group was significantly higher than that of the control group, indicating that the hypertensive model of the lipid polysaccharide stimulation during pregnancy was successful in the reproduction of.DA+LPS. Compared with group LPS, the effect of LPS stimulation on.2. during pregnancy and the effect of DA intervention on renal function and morphology of the offspring rats, and the blood urea nitrogen, creatinine and uric acid in serum were measured. The results showed that the serum urea nitrogen content of the male rat LPS group was significantly lower than that of the control group, and the uric acid content in the male and female rat LPS group was significantly lower than that of the control group. Compared with the LPS group, the uric acid content in the LPS+DA group was significantly lower than that of the LPS group, and the serum creatinine content was not statistically significant compared with the control group. Compared with the control group, the content of IL-1 beta and IL-18 in the serum increased significantly, and the level of IL-1 beta and IL-18 decreased in the LPS+DA group compared with the LPS group, and the level of IL-1 beta and IL-18 decreased in.HE staining nodes. Compared with the control group, compared with the control group, the diameter of the partial vascular ball in the LPS group was reduced, the renal capsule cavity became larger, the medium to the severe glomerular mesangial matrix hyperplasia was scattered in the capsule, and the glomeruli part adhered. The proximal convoluted tubules and the distal tubules were scattered in the presence of egg white substance, and the renal morphological characteristics of the LPS+DA group. The effect of LPS stimulation on.3. during pregnancy and the effect of DA intervention on the expression of mRNA in the renal NLRP3 inflammatory body of the offspring rats was close to that of the control group. The results showed that the mRNA expression of NLRP3 in the LPS group was higher than that of the control group, and the expression of NLRP3 in the LPS+DA group was lower than that in the LPS group; In the control group, there was no significant difference between the other groups. The mRNA expression of CASP1 in the offspring rats had no significant statistical significance compared with the LPS stimulation of.4. during pregnancy and the effect of DA intervention on the expression of NLRP3 inflammatory body protein in the kidneys of the offspring of the rat. The immunohistochemical staining results showed that the main expression of NLRP3 was in the proximal tubule. Compared with the distal tubule endothelial cells, there was a small amount of NLRP3 expression in the glomeruli in the LPS group compared with the control group; ASC was mainly expressed in the renal tubules and the vessels in the offspring rats. The CASP1 in the kidneys of the offspring male rats was mainly expressed in the glomus, while the female rats were expressed in the vessels and the renal tubules. Meanwhile, the result of the light density value (IOD) showed that the female rats were in the same vein. Compared with group LPS+DA, the expression of NLRP3, ASC and CASP1 protein in the kidneys of the LPS group female rats increased significantly. Compared with the control group and the LPS+DA group, the expression of NLRP3 only increased in the offspring male LPS group, and the experimental results of Western blot were consistent with the results of the light density measured by immunohistochemical staining. The animal model of hypertensive rat induced by gestation was replicated, and it was proved that NLRP3 in the kidney played an important role in the hypertension induced by LPS stimulation during pregnancy. The intervention of dopamine could protect the hypertension and renal injury induced by LPS stimulation during pregnancy, which may be related to the activation of dopamine inhibited NLRP3.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R544.1

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