法舒地尔对内毒素引起的急性肾损伤大鼠影响及机制

发布时间：2018-08-16 08:49

【摘要】：目的：脓毒血症引起的急性肾损伤(acute kidney injury, AKI)是临床上导致肾功能段时间内出现进行性恶化的重要因素之一。内毒素(lipopolysaccharide; LPS)可以通过介导下游多种症介质、信号通路参与肾脏损伤的发生,越来越多的证据表明内质网应激可能是肾脏病发病的一个重要机制,法舒地尔(fasudil)从药物分类上讲是Rho激酶特异性抑制剂,它主要的药物作用机制就是在炎症反应过程中阻断Rho/Rho激酶途径,该药物不仅具有抗凋亡、扩血管、抗炎等一系列的药理作用外,还被证实通过抑制过度内质网应激(endoplasmic reticulum;ERS),在多种肾脏疾病病理生理过程中起到非常明显的器官和细胞保护作用,然而在LPS所致的AKI过程中的作用鲜有报道；本研究观察LPS参与AKI发病过程中病理生理变化,并探讨fasudil对LPS所致AKI大鼠的影响及其潜在作用机制。方法：1.实验大鼠每4-5只一笼,饲养在恒温(22±2)℃、恒湿(55±5)%,人工光照明暗各12小时的饲养室内,标准饲料和自来水自由饮食。2.经上述环境适应性喂养1周后54只大鼠采用随机数字法分为3组,各组小鼠均为18只,正常对照组(Control):不做任何处理；模型组(LPS)：采用经静脉内注射内毒素(6mg/kg)建立急性肾损伤模型；法舒地尔组(LPS+FAS),建模前每天给予静脉内注射法舒地尔(30mg/kg),连续给药3天。3.血清Scr、BUN及24h-Ualb检测：血清肌酐(Scr)、尿肌酐(Ucr)、血清尿素氮(BUN)、分别由全自动生化分析仪检测,肌酐清除率(Ccr)采用如下计算公式：Ccr=U×V/P, U表示尿肌酐含量(μmol/L),V表示尿量(ml/min/100), P表示血肌酐水平(μmol/L), Ccr单位为ml/min/kg。4.肾组织及血液中TNF-α、IL-6检测：肾组织与3倍体积的磷酸盐缓冲液冰浴匀浆,离心后取上清液,采用双抗体夹ELISA法检测肾组织及血液中TNF-α、IL-6水平,具体步骤严格按试剂盒(RD公司；96人份)使用说明书进行操作。通过绘制标准曲线得出样品中相应细胞因子含量,该法检测TNF-α、IL-6的灵敏度为15pg/mL。5.HE染色：取各组肾组织标本制成3μm厚石蜡切片,常规行苏木素-伊红染色,在普通光学显微镜下观察各组大鼠肾组织病理变化。6. TUENL检测：按Dead End TM Colorimetric TUNEL System试剂盒(罗氏公司)说明书进行操作,检测肾脏组织凋亡细胞数目,期间在荧光显微镜观察拍照。7. Western印迹：采用Western-blot方法检测肾脏组织GRP78, CHOP, caspase-12及ROCK-1蛋白表达,采用Image J软件进行灰度分析,以β-actin作为内参,结果用目的蛋白与β-actin的比值表示。结果：1.各组大鼠肾功能及炎性指标变化：与Control组比较,AKI组Scr、BUN、TNF-α及IL-6水平明显升高；LPS+FAS组Scr、Bun水平较对照组升高,但显著低于LPS组(P0.05)；对于CrCl水平,LPS组较Control组明显降低,而LPS+FAS组Ccrl水平较模型组显著升高(P0.0 5)；见表1。2.病理学改变：内毒素所致的AKI的病理变化在肾髓质及皮质均有累及。与对照组组比较,模型组髓质区远端小管及皮质近端小管出现较多的肾小管上皮细胞脱落、坏死, 炎性浸润及空泡样改变,部分管腔出现蛋白管型、细胞管型,甚至闭塞变形,在皮质区,部分肾小球基底细胞脱落,甚至部分小球有炎性浸润；LPS+FAS组较模型组上述病理改变明显减轻(见图1)。3. TUNEL法荧光标记的凋亡细胞水平：在荧光显微镜下,细胞核呈亮绿色者为阳性细胞,肾脏髓质及皮质均有细胞凋亡。Control组内仅见少许阳性细胞核；相对于Control组,模型组可见大量的亮绿色荧光凋亡细胞核；法舒地尔能明显抑制肾脏组织细胞凋亡(见图2)；4. GRP78、CHOP、Caspase-12及ROCK-1蛋白免疫印迹电泳表达：电泳结果显示(分别见图3、4、5、6)：与Control组相比,LPS组GRP78、CHOP、caspase-12及ROCK-1蛋白表达均明显下调(P0.05)；与模型组组比较,LPS+FAS组GRP78、CHOP、Caspase-12及ROCK-1蛋白表达上调(P0.05)。结论：Fasudil可以改善内毒素所致的AKI后肾功能和病理损伤,降低血液内及肾脏中炎症因子(TNF-α、IL-6),减轻炎性反应；并且fasudil通过直接抑制ROCK1活性,并下调GRP78、CHOP等蛋白,抵抗内质网过度应激所致的损伤,二者相辅相成共同作用影响肾脏功能。fasudil有望成为一种新的治疗AKI的辅助药物。
[Abstract]:Objective: Acute kidney injury (AKI) caused by sepsis is one of the important factors leading to progressive deterioration of renal function in clinic. LPS can participate in the occurrence of renal injury by mediating many downstream pathogenic mediators and signaling pathways. Plasma reticulum stress may be an important mechanism in the pathogenesis of kidney disease. Fasudil is a specific inhibitor of Rho kinase in terms of drug classification. Its main mechanism of action is blocking the Rho/Rho kinase pathway in the process of inflammation. The drug not only has a series of pharmacological effects, such as anti-apoptosis, vasodilation, anti-inflammation, but also has some other effects. It has been proved that endoplasmic reticulum stress (ERS) plays an important role in the pathophysiological process of various kidney diseases. However, the role of LPS in the pathogenesis of AKI is rarely reported. Effects of PS on AKI rats and its potential mechanisms. Methods: 1. The experimental rats were fed in a cage of 4-5 rats at constant temperature (22 The model group (LPS): acute renal injury model was established by intravenous injection of endotoxin (6mg/kg), and the fasudil group (LPS + FAS) was given fasudil (30mg/kg) intravenously every day for 3 days before modeling. 3. Serum Scr, BUN and 24h-Ualb were detected: serum muscle. Scr, Ucr and BUN were measured by automatic biochemical analyzer. The creatinine clearance rate (Ccr) was calculated by the following formula: Ccr = U *V/P, U = urinary creatinine content (micromol/L), V = urine volume (ml/min/100), P = serum creatinine level (micromol/L), Ccr unit (ml/min/kg.4). The levels of TNF-a and IL-6 in kidney tissues and blood were detected by double antibody clip ELISA after centrifugation. The specific procedures were strictly operated according to the instructions of the kit (RD Company; 96 persons). The sensitivity of detecting TNF-a and IL-6 was 15pg/mL.5. HE staining: 3 micron thick paraffin sections were made from kidney tissue specimens of each group. Hematoxylin-eosin staining was performed routinely. Pathological changes of kidney tissue were observed under ordinary optical microscope. 6. TUENL detection: According to the instructions of Dead End TM Colorimetric TUNEL System Kit (Roche Company) Western blot: Western blot was used to detect the expression of GRP78, CHOP, caspase-12 and ROCK-1 proteins in kidney tissues. Image J software was used to analyze the gray level of GRP78, CHOP, caspase-12 and ROCK-1 proteins. The results were expressed by the ratio of target protein to beta-actin. Results: 1. Changes of renal function and inflammatory indexes in rats of each group: Compared with Control group, the levels of Scr, BUN, TNF-a and IL-6 in AKI group were significantly higher; the levels of Scr and Bun in LPS+FAS group were significantly higher than those in LPS group, but significantly lower than those in LPS group (P 0.05); the levels of CrCl in LPS group were significantly lower than those in Control group, while the levels of Ccrl in LPS+FAS group were significantly higher than those in model group (P 0.0). 5) See Table 1.2. Pathological changes: Endotoxin-induced AKI pathological changes in the renal medulla and cortex were involved. Compared with the control group, the model group showed more distal tubules and proximal cortical tubules of renal tubule epithelial cells shedding, necrosis, inflammatory infiltration and vacuole-like changes, some lumen protein tubules, cell tubules. Type I, or even occlusive deformation, in the cortical area, some of the glomerular basal cells exfoliated, and even some of the glomerular inflammatory infiltration; LPS + FAS group than the model group, the pathological changes significantly reduced (see Figure 1). 3. TUNEL fluorescent labeling of apoptotic cells level: under the fluorescent microscope, the nucleus was bright green for positive cells, kidney medulla and cortex There were only a few positive nuclei in the control group; compared with the control group, a large number of bright green fluorescent apoptotic nuclei were observed in the model group; Fasudil could significantly inhibit apoptosis in renal tissue (see Figure 2); 4. GRP78, CHOP, Caspase-12 and ROCK-1 protein immunoblot electrophoresis expression: electrophoresis results (see figure 2, respectively) 3,4,5,6: Compared with Control group, the expressions of GRP78, CHOP, caspase-12 and ROCK-1 in LPS group were significantly down-regulated (P 0.05); compared with model group, the expressions of GRP78, CHOP, Caspase-12 and ROCK-1 in LPS + FAS group were up-regulated (P 0.05). Conclusion: Fasudil can improve the renal function and pathological damage after AKI induced by endotoxin, and reduce inflammation in blood and kidney. Syndrome factor (TNF-alpha, IL-6) can alleviate inflammatory reaction, and fasudil can inhibit ROCK1 activity directly, and down-regulate GRP78, CHOP and other proteins to resist the damage caused by endoplasmic reticulum (ER) overstress. The two complement each other and affect renal function.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R692

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