氨氯地平通过调节Smad的表达防止肾脏纤维化

发布时间：2018-04-25 19:14

本文选题：氨氯地平 + 庆大霉素　；参考：《武汉大学》2014年博士论文

【摘要】：第一部分氨氯地平通过调节Smad2,7的表达防止庆大霉素所致大鼠肾小管间质纤维化目的：在庆大霉素所致大鼠肾小管间质纤维化损伤整体模型上,观察氨氯地平通过调节Smad2,7的表达对庆大霉素所致大鼠肾小管损伤的影响。方法：取SD大鼠28只,分正常对照组,庆大霉素模型组,氨氯地平2.5mg/kg+庆大霉素组,氨氯地平5mg/kg+庆大霉素组,每组7只。氨氯地平2.5mg/kg/d和5mg/kg/d给药组每日灌胃(i.g.)给药一次,给药体积为10mL/kg/d,连续给药8天。给药第2天,模型组及氨氯地平两个给药组腹腔注射(i.p.)庆大霉素100mg/kg/d(体积4mL/kg/d),连续7天,对照组给予相应体积的生理盐水。给药第8天置代谢笼中收集24h尿,检测尿蛋白含量及N-乙酰-β-D-氨基葡萄糖苷酶(NAG)、丫-谷氨酸转换酶(GGT)、碱性磷酸酶(ALP)活性。大鼠称重后处死,收集血液标本检测血清尿素氮(BUN)和肌酐(SCr)含量。大鼠肾脏用10%福尔马林固定,石蜡包埋切片后,HE染色,光镜观察肾组织形态学变化；原位缺口末端标记法(d-UTP nick and labeling,TUNEL)检测肾小管细胞凋亡情况；免疫组化SP法检测肾小管Smad2,Smad7表达水平；其余肾组织制备肾皮质匀浆,测定其丙二醛(MDA)、一氧化氮(NO)含量及超氧化物歧化酶(SOD)、一氧化氮合酶(NOS)活性。结果：庆大霉素模型组大鼠的24h尿蛋白、血清BUN、SCr的含量明显升高,与正常对照组比较具有显著性差异(P0.05,P0.01)；肾脏病理组织学检查,庆大霉素模型组近曲小管上皮细胞浊肿,管腔内可见明显红细胞管型和蛋白管型,间质呈炎症反应,远曲小管腔内可见充血和红细胞管型,说明肾小管间质纤维化造模成功。氨氯地平呈剂量依赖性降低庆大霉素模型大鼠的24h尿蛋白含量,血清BUN、SCr含量及尿NAG、GGT、ALP活性的升高趋势,并且可明显降低肾组织MDA、 NO含量及NOS活力,提高SOD活性。氨氯地平给药组对庆大霉素引起的肾小管病理性损伤有明显的改善及恢复作用,仅见少量的蛋白管型和红细胞管型。同时,氨氯地平也明显抑制庆大霉素引起的肾小管细胞凋亡；氨氯地平给药组显著降低肾小管Smad2的表达而增加Smad7的表达。结论：氨氯地平通过调节Smad2,7的表达能防止庆大霉素所致大鼠肾小管间质纤维化的进程。第二部分氨氯地平通过调节Smad6,7的表达防止阿霉素所致大鼠肾小球系膜细胞的损伤目的：在阿霉素所致大鼠肾小球系膜细胞纤维化的模型上,观察氨氯地平通过调节Smad6,7的表达对阿霉素所致大鼠肾小球系膜细胞损伤的影响。方法：体外培养肾小球细膜细胞(MCs),并按照实验设计对细胞进行分组。分光光度法测定大鼠肾小球系膜细胞上清液LDH活性；Real-time PCR分析系膜细胞Smad6,7基因mRNA表达；Western blot分析系膜细胞Smad6,7蛋白表达；细胞免疫化学SP法检测系膜细胞Smad7的表达水平。结果：氨氯地平呈浓度依赖性降低阿霉素模型组细胞培养上清液中LDH的活性(P0.01)：对阿霉素+TGF-β1组所致LDH活性的增加也能呈浓度依赖性降低,与阿霉素组比较具有显著差异(P0.01)。Real-time PCR和Western blot分析显示,氨氯地平(10-7,10-6mol/L)显著上调阿霉素+TGF-β1组系膜细胞Smad6,7基因和蛋白的表达(P0.01)；细胞免疫化学分析表明,氨氯地平能显著上调阿霉素+TGF-β1组所致系膜细胞Smad7的表达。结论：氨氯地平通过调节Smad6,7的表达防止阿霉素所致大鼠肾小球系膜细胞的损伤。
[Abstract]:Part 1 amlodipine prevents gentamicin induced tubulointerstitial fibrosis in rats by regulating the expression of Smad2,7.
Objective: To observe the effect of amlodipine on renal tubule injury induced by gentamicin in rats induced by gentamicin induced renal tubulointerstitial fibrosis in rats. The effect of amlodipine on the expression of Smad2,7 was observed.
Methods: 28 SD rats were divided into normal control group, gentamicin model group, amlodipine 2.5mg/kg+ gentamicin group, amlodipine 5mg/kg+ gentamicin group, 7 rats in each group. Amlodipine 2.5mg/kg/d and 5mg/kg/d administration group were administered daily (i.g.), the dosage was 10mL/kg/d, and the drug was given for 8 days. The drug was given for second days, model group and amamlol chloride. The two administration groups were injected intraperitoneally (i.p.) gentamicin 100mg/kg/d (volume 4mL/kg/d) for 7 days, and the control group was given the corresponding volume of normal saline. 24h urine was collected in the metabolic cage for eighth days, and the content of urine protein and N- acetyl beta -D- aminoglucosidase (NAG), GGT, and alkaline phosphatase (ALP) activity in rats were detected. After weighing, the blood samples were collected to detect the content of serum urea nitrogen (BUN) and creatinine (SCr). Rats' kidneys were fixed with 10% formalin and paraffin embedded sections, HE staining, and light microscopy were used to observe the morphological changes of renal tissue; in situ nick end labeling (d-UTP Nick and labeling, TUNEL) was used to detect the apoptosis of renal tubule cells; immunohistochemical SP method was used. The expression of Smad2 and Smad7 in renal tubules was detected, and renal cortex homogenate was prepared in the rest of the renal tissue, and the content of malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD) and nitric oxide synthase (NOS) activity were measured.
Results: the content of 24h urine protein, serum BUN and SCr in the gentamicin group was significantly higher than that in the normal control group (P0.05, P0.01); the renal histopathology examination, the cloudy swelling of the proximal tubular epithelial cells in the gentamicin model group, the obvious tube type and the protein tube type in the lumen, and the interstitial inflammation in the lumen. It should be seen that hyperemia and erythrocyte tube can be seen in the canalicular cavity of the far curvature, indicating the success of renal tubulointerstitial fibrosis. Amlodipine has a dose-dependent reduction of 24h urine protein content in gentamicin model rats, serum BUN, SCr content and NAG, GGT, ALP activity in urine, and can obviously reduce the MDA, NO content and NOS activity of renal tissue, and improve S. OD activity. Amlodipine administration group has obvious improvement and recovery effect on renal tubular pathological injury caused by gentamicin, only a small amount of protein tube type and erythrocyte tube type. Amlodipine also obviously inhibits the apoptosis of renal tubule cells caused by gentamicin, and amlodipine administration group significantly reduces the expression of Smad2 in renal tubule. Add the expression of Smad7.
Conclusion: Amlodipine can prevent gentamicin induced tubulointerstitial fibrosis in rats by regulating the expression of Smad2,7.
The second part of amlodipine prevents doxorubicin induced glomerular mesangial cell injury by regulating the expression of Smad6,7.
Objective: To observe the effect of amlodipine on the damage of glomerular mesangial cells induced by adriamycin in rat glomerular mesangial cell fibrosis induced by doxorubicin (adriamycin).
Methods: the cultured glomerular mesangial cells (MCs) were cultured in vitro, and the cells were grouped according to the experimental design. The activity of LDH in the supernatant of glomerular mesangial cells was measured by spectrophotometric method; the Smad6,7 gene mRNA expression of the mesangial cells of the rat mesangial cells was analyzed by Real-time PCR; the Smad6,7 protein expression in the mesangial cells of the Western blot analysis; and the detection of the cell immunochemistry SP method. The expression level of Smad7 in mesangial cells.
Results: Amlodipine was a concentration dependent decrease in the activity of LDH in the supernatant of cell culture of adriamycin model group (P0.01): the increase of LDH activity caused by adriamycin +TGF- beta 1 groups decreased in a concentration dependent manner, and there was significant difference (P0.01).Real-time PCR and Western blot analysis, amlodipine (10-7,10-6mo, 10-7,10-6mo). L/L) significantly up-regulated the expression of Smad6,7 gene and protein in the mesangial cells of adriamycin +TGF- beta 1 group (P0.01). Cell immuno chemical analysis showed that amlodipine could significantly increase the expression of Smad7 in the mesangial cells induced by adriamycin +TGF- beta 1 group.
Conclusion: Amlodipine can prevent the injury of mesangial cells induced by adriamycin by regulating the expression of Smad6,7.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R965

【参考文献】