腎臟中NLRP3炎性體在孕期LPS刺激致子代大鼠高血壓中的作用研究
發(fā)布時(shí)間：2018-07-14 22:24
【摘要】：高血壓是十分易見的慢性疾病,也是心腦血管病人死亡的主要原因。腎臟是具有內(nèi)分泌功能的重要臟器,能夠調(diào)節(jié)機(jī)體水、電解質(zhì)的平衡,與高血壓的發(fā)生有著非常密切的關(guān)系。在前期的實(shí)驗(yàn)中發(fā)現(xiàn),孕期脂多糖(Lipopolysaccharide,LPS)刺激導(dǎo)致子代大鼠在三個(gè)月齡時(shí)發(fā)生高血壓并伴有腎小球數(shù)量減少和腎功能下降,炎性因子表達(dá)增多,因此我們推測腎臟炎癥在孕期炎癥刺激致子代高血壓中可能起著重要作用。NLRP3炎性體是NOD樣受體(nucleotide-binding oligomerization domain-like receptors,NLRs)中最具代表性的炎性體之一,它能誘導(dǎo)機(jī)體細(xì)胞炎癥或凋亡,產(chǎn)生大量的炎性因子,導(dǎo)致嚴(yán)重的炎癥反應(yīng)發(fā)生,而多巴胺(dopamine,DA)通過多巴胺D1受體可抑制NLRP3炎性體的激活。本研究通過復(fù)制孕期LPS致子代大鼠高血壓動(dòng)物模型,同時(shí)用多巴胺進(jìn)行干預(yù),研究腎臟NLRP3在孕期LPS刺激致子代大鼠高血壓中的作用,為高血壓及腎損傷的預(yù)防和治療提供靶點(diǎn)。實(shí)驗(yàn)結(jié)果如下:1.孕期LPS刺激致子代大鼠高血壓模型的建立及DA干預(yù)對孕期LPS致子代大鼠高血壓的影響選取30只孕鼠隨機(jī)分為三組:空白對照組、LPS組、LPS+DA組。選10只懷孕母鼠不做任何處理并飼喂普通飼料,將出生后子鼠做為空白對照組;10只母鼠在孕期第8、10、12天腹腔注射0.4 mg/kg的LPS,出生后的子鼠作為LPS組;10只母鼠在孕期第8、10、12天腹腔注射0.4 mg/kg LPS的同時(shí)注射1 mL/kg的多巴胺,出生后子鼠作為LPS+DA組。頸動(dòng)脈穿刺測得三個(gè)月齡子代大鼠血壓,實(shí)驗(yàn)結(jié)果顯示,與對照組相比,LPS組子代雄鼠和雌鼠的平均動(dòng)脈壓都顯著增高,表明孕期脂多糖刺激致子代高血壓模型復(fù)制成功。DA+LPS組與LPS組相比顯著降低。2.孕期LPS刺激及DA干預(yù)對子代大鼠腎功能及腎臟形態(tài)學(xué)的影響腹腔靜脈采血,測定血清中尿素氮、肌酐、尿酸。結(jié)果表明,與對照組相比,雄性子鼠LPS組血清尿素氮含量顯著降低,雄性子鼠和雌性子鼠LPS組尿酸含量顯著增加,與LPS組相比,子代雌鼠LPS+DA組尿酸含量顯著降低;血清中肌酐含量各組間相比無顯著統(tǒng)計(jì)學(xué)意義。ELISA試驗(yàn)結(jié)果表明,與對照組相比,血清中IL-1β和IL-18的含量都顯著增加,LPS+DA組與LPS組相比,IL-1β和IL-18的含量水平降低。HE染色結(jié)果顯示,與對照組相比,LPS組子代大鼠部分血管球直徑減小,腎小囊腔變大,囊腔中有散在的中度到重度腎小球系膜基質(zhì)增生,且腎小球部分發(fā)生粘連,近曲小管和遠(yuǎn)曲小管中有散在的類似于蛋白樣的物質(zhì)存在,而LPS+DA組腎臟形態(tài)學(xué)特征接近于對照組。3.孕期LPS刺激及DA干預(yù)對子代大鼠腎臟NLRP3炎性體mRNA表達(dá)的影響熒光定量PCR結(jié)果表明,與對照組對比,LPS組子代大鼠中NLRP3的mRNA表達(dá)增高;與LPS組相比,LPS+DA組NLRP3的m RNA表達(dá)降低;在子代雌鼠腎臟ASC的mRNA表達(dá)中,LPS組相對于對照組有所增加,其它各組間相比沒有統(tǒng)計(jì)學(xué)意義;子代大鼠中CASP1的mRNA表達(dá)各組間相比沒有明顯統(tǒng)計(jì)學(xué)意義。4.孕期LPS刺激及DA干預(yù)對子代大鼠腎臟NLRP3炎性體蛋白表達(dá)的影響免疫組織化學(xué)染色結(jié)果表明,NLRP3主要表達(dá)在近端小管和遠(yuǎn)端小管內(nèi)皮細(xì)胞。與對照組相比,LPS組腎小球有少量NLRP3表達(dá);ASC在子代大鼠中主要在腎小管和血管球中表達(dá),子代雄性大鼠腎臟中的CASP1主要表達(dá)在血管球中,而雌鼠在血管球和腎小管中都有表達(dá)。同時(shí)光密度值(IOD)結(jié)果表明,與對照組和LPS+DA組相比,LPS組子代雌性大鼠腎臟中NLRP3、ASC和CASP1蛋白表達(dá)量顯著增加;與對照組和LPS+DA組相比,子代雄性LPS組大鼠,僅NLRP3表達(dá)增高;Western blot的實(shí)驗(yàn)結(jié)果與免疫組織化學(xué)染色所測的光密度結(jié)果保持一致。綜上所述,本研究通過復(fù)制孕期脂多糖致子代大鼠高血壓動(dòng)物模型,證實(shí)腎臟中NLRP3在孕期LPS刺激致子代大鼠高血壓中具有重要作用,多巴胺干預(yù)對孕期LPS刺激致子代大鼠高血壓和腎損傷有保護(hù)作用,可能與多巴胺抑制NLRP3的激活有關(guān)。
[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.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R544.1
本文編號(hào)：2123150
[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.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R544.1
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