地塞米松缓释剂对藏红花红O肾病家兔的保护作用及其机制研究

发布时间：2018-08-07 21:41

【摘要】：肾病综合征(nephrotic syndrome,NS)的临床表现可以概括为“三高一低”,即高蛋白尿、高血脂、高度水肿、低血蛋白。原发性的NS以肾小球肾病最为常见,发生与双侧肾脏肾小球,病因各不相同。地塞米松(dexamethasone,Dex)为糖皮质类激素的代表药物,具有强大的抗炎、抗毒、抗过敏等作用,临床应用广泛,同时对下丘脑-垂体-肾上腺轴(the hypothalamic-pituitary-adrenal axis,HPA)抑制作用较明显。地塞米松可以抑制肾内的炎性反应,缓解一系列的症状,治疗方法为开始大剂量静脉给药后期口服维持治疗。长期使用易产生较严重的毒副作用,治病的同时也给治疗过程中的病人增加痛苦。所以,我们设想对于肾病的治疗,若使药物在肾脏局部浓度高,而在全身的血药浓度低,是否不仅可以增加疗效,又能减轻毒副作用。故本实验使用的药物剂型为地塞米松缓释剂(sustained-release of dexamethasone,SR Dex),药物到达局部后,缓慢释放,维持时间长。使用的动物模型为耳缘静脉注射藏红花红O(safranine O)诱导的家兔肾小球肾病,藏红花红O能选择性的引起近端和远端肾小管上皮细胞广泛坏死,形成的肾病模型与临床表现较为一致。目的:探讨地塞米松缓释剂对藏红花红O肾病家兔的保护作用、对HPA轴和肾上腺功能的影响及糖皮质激素类药物治疗肾病的可能机制。方法:家兔肾病模型的制备方法为连续两次间隔3小时耳缘静脉注射臧红花红O,每次15mg。地塞米松缓释剂分别采用手术肾囊内一次性注射给药(1.48,0.74,0.37 mg/kg),阳性对照组地塞米松灌胃给药(0.4mg/kg,qd×8w)。每天观察造模前及造模后家兔一般状况,每周检测体重、24h尿蛋白,每周检测血蛋白(总蛋白、白蛋白)、血脂(总胆固醇)、血肾功能(血肌酐、尿素氮)、血尿酸及血电解质(NA+、K+、Cl ),每两周检测肾上腺功能指标(血液指标:促肾上腺皮质激素、皮质酮、尿液指标:17-羟基皮质类固醇)。HE染色观察肾、肾上腺组织的病理学变化;PAS染色观察肾小球系膜(glomerular mesangum,GB)、基底膜(glomerulus basement membrane,GBM)的形态改变;SR染色观察肾间质胶原纤维的变化;免疫组织化学法检测肾组织nephrin、TRPC6蛋白的表达;实时荧光定量PCR(real-time PCR)检测肾组织nephrin、TRPC6 m RNA的表达量。结果:地塞米松缓释剂可显著降低肾病家兔升高的尿蛋白,改善肾功能(降低血尿素氮和血肌酐),降低血总胆固醇及尿酸水平,升高血总蛋白、白蛋白,改善肾组织病理学变化。另外,地塞米松缓释剂对肾病家兔血促肾上腺皮质激素和皮质酮含量无明显影响,可不同程度降低尿17-羟基皮质类固醇含量,对肾上腺病理组织学也无明显影响。结论:地塞米松缓释剂肾囊用药对家兔藏红花红O肾病具有较好的保护作用,其机制可能与改变TRPC6蛋白通路及nephrin蛋白的表达与分布,改善肾小球滤膜的完整性有关。另外,地塞米松缓释剂肾囊用药对HPA轴无明显抑制,对肾上腺形态及功能未见明显影响。
[Abstract]:The clinical manifestations of nephrotic syndrome can be summarized as high proteinuria, hyperlipidemia, high edema and low blood protein. Primary NS, glomerular nephropathy is the most common, and bilateral renal glomeruli, the etiology is different. Dexamethasone Dex is the representative drug of glucocorticoid. It has powerful anti-inflammatory, anti-toxic and anti-allergic effects. It is widely used in clinic and has obvious inhibitory effect on the hypothalamic-pituitary-adrenal axis-adrenal axis. Dexamethasone can inhibit the inflammatory reaction in the kidney and relieve a series of symptoms. Long-term use can lead to severe side effects and increase pain in the course of treatment. Therefore, we imagine that for the treatment of nephropathy, if the drug concentration in the kidney is high, and the blood drug concentration in the whole body is low, it can not only increase the curative effect, but also alleviate the side effects. Therefore, sustained-release of dexamethasone SR Dex), was released slowly and maintained for a long time. The animal model used was rabbit glomerular nephropathy induced by injecting saffron O (safranine O) into ear margin. Saffron O could selectively cause extensive necrosis of proximal and distal renal tubular epithelial cells. Objective: to investigate the protective effect of dexamethasone sustained-release agent on saffron O nephropathy in rabbits, the effects of dexamethasone on HPA axis and adrenal function and the possible mechanism of glucocorticoid in the treatment of nephropathy. Methods: the rabbit model of nephropathy was established by injecting Zang Hua Hong O15 mg every time. Dexamethasone sustained-release agents were injected intraperitoneally into the renal sac for one time (1.48g / kg, 0.37 mg/kg), while those in the positive control group were given dexamethasone (0.4mg / kg / kg QD 脳 8w). The general condition of rabbits before and after modeling was observed every day, the body weight and 24 hours urine protein were measured weekly, the blood protein (total protein, albumin), blood lipid (total cholesterol), blood renal function (serum creatinine) were detected weekly. Urea nitrogen, serum uric acid and serum electrolytes (na) were used to detect adrenal function index (blood index: adrenocorticotropic hormone, corticosterone, urine index: 17 hydroxy corticosteroids). He staining was used to observe the renal function. The pathological changes of adrenal tissue were observed by pas staining, the morphological changes of glomerular Mesangium (GB) and basement membrane (glomerulus basement) were observed by SR staining, and the expression of nephrinTRPC6 protein in renal tissue was detected by immunohistochemistry. The expression of nephrinnin TRPC6 m RNA in renal tissue was detected by real time fluorescence quantitative PCR (real-time PCR). Results: dexamethasone sustained-release agent could significantly reduce the elevated urinary protein, improve renal function (reduce blood urea nitrogen and serum creatinine), decrease serum total cholesterol and uric acid levels, increase serum total protein, albumin, and increase the level of serum total protein and albumin in rabbits with nephropathy. To improve renal histopathological changes. In addition, dexamethasone sustained release had no significant effect on the contents of corticotropin and corticosterone in the blood of nephropathy rabbits, but could decrease the urinary 17-hydroxycorticosteroid content in varying degrees, and had no obvious effect on the histopathology of the adrenal gland. Conclusion: Dexamethasone sustained release agent has a better protective effect on saffron O nephropathy in rabbits. The mechanism may be related to changing the expression and distribution of TRPC6 protein pathway and nephrin protein and improving the integrity of glomerular filter membrane. In addition, Dexamethasone sustained release agent did not inhibit the HPA axis, but had no obvious effect on the morphology and function of adrenal gland.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R965

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