【摘要】：目的：膿毒血癥引起的急性腎損傷(acute kidney injury, AKI)是臨床上導致腎功能段時間內(nèi)出現(xiàn)進行性惡化的重要因素之一。內(nèi)毒素(lipopolysaccharide; LPS)可以通過介導下游多種癥介質(zhì)、信號通路參與腎臟損傷的發(fā)生,越來越多的證據(jù)表明內(nèi)質(zhì)網(wǎng)應激可能是腎臟病發(fā)病的一個重要機制,法舒地爾(fasudil)從藥物分類上講是Rho激酶特異性抑制劑,它主要的藥物作用機制就是在炎癥反應過程中阻斷Rho/Rho激酶途徑,該藥物不僅具有抗凋亡、擴血管、抗炎等一系列的藥理作用外,還被證實通過抑制過度內(nèi)質(zhì)網(wǎng)應激(endoplasmic reticulum;ERS),在多種腎臟疾病病理生理過程中起到非常明顯的器官和細胞保護作用,然而在LPS所致的AKI過程中的作用鮮有報道；本研究觀察LPS參與AKI發(fā)病過程中病理生理變化,并探討fasudil對LPS所致AKI大鼠的影響及其潛在作用機制。方法：1.實驗大鼠每4-5只一籠,飼養(yǎng)在恒溫(22±2)℃、恒濕(55±5)%,人工光照明暗各12小時的飼養(yǎng)室內(nèi),標準飼料和自來水自由飲食。2.經(jīng)上述環(huán)境適應性喂養(yǎng)1周后54只大鼠采用隨機數(shù)字法分為3組,各組小鼠均為18只,正常對照組(Control):不做任何處理；模型組(LPS)：采用經(jīng)靜脈內(nèi)注射內(nèi)毒素(6mg/kg)建立急性腎損傷模型；法舒地爾組(LPS+FAS),建模前每天給予靜脈內(nèi)注射法舒地爾(30mg/kg),連續(xù)給藥3天。3.血清Scr、BUN及24h-Ualb檢測：血清肌酐(Scr)、尿肌酐(Ucr)、血清尿素氮(BUN)、分別由全自動生化分析儀檢測,肌酐清除率(Ccr)采用如下計算公式：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單位為ml/min/kg。4.腎組織及血液中TNF-α、IL-6檢測：腎組織與3倍體積的磷酸鹽緩沖液冰浴勻漿,離心后取上清液,采用雙抗體夾ELISA法檢測腎組織及血液中TNF-α、IL-6水平,具體步驟嚴格按試劑盒(RD公司；96人份)使用說明書進行操作。通過繪制標準曲線得出樣品中相應細胞因子含量,該法檢測TNF-α、IL-6的靈敏度為15pg/mL。5.HE染色：取各組腎組織標本制成3μm厚石蠟切片,常規(guī)行蘇木素-伊紅染色,在普通光學顯微鏡下觀察各組大鼠腎組織病理變化。6. TUENL檢測：按Dead End TM Colorimetric TUNEL System試劑盒(羅氏公司)說明書進行操作,檢測腎臟組織凋亡細胞數(shù)目,期間在熒光顯微鏡觀察拍照。7. Western印跡：采用Western-blot方法檢測腎臟組織GRP78, CHOP, caspase-12及ROCK-1蛋白表達,采用Image J軟件進行灰度分析,以β-actin作為內(nèi)參,結果用目的蛋白與β-actin的比值表示。結果：1.各組大鼠腎功能及炎性指標變化：與Control組比較,AKI組Scr、BUN、TNF-α及IL-6水平明顯升高；LPS+FAS組Scr、Bun水平較對照組升高,但顯著低于LPS組(P0.05)；對于CrCl水平,LPS組較Control組明顯降低,而LPS+FAS組Ccrl水平較模型組顯著升高(P0.0 5)；見表1。2.病理學改變：內(nèi)毒素所致的AKI的病理變化在腎髓質(zhì)及皮質(zhì)均有累及。與對照組組比較,模型組髓質(zhì)區(qū)遠端小管及皮質(zhì)近端小管出現(xiàn)較多的腎小管上皮細胞脫落、壞死, 炎性浸潤及空泡樣改變,部分管腔出現(xiàn)蛋白管型、細胞管型,甚至閉塞變形,在皮質(zhì)區(qū),部分腎小球基底細胞脫落,甚至部分小球有炎性浸潤；LPS+FAS組較模型組上述病理改變明顯減輕(見圖1)。3. TUNEL法熒光標記的凋亡細胞水平：在熒光顯微鏡下,細胞核呈亮綠色者為陽性細胞,腎臟髓質(zhì)及皮質(zhì)均有細胞凋亡。Control組內(nèi)僅見少許陽性細胞核；相對于Control組,模型組可見大量的亮綠色熒光凋亡細胞核；法舒地爾能明顯抑制腎臟組織細胞凋亡(見圖2)；4. GRP78、CHOP、Caspase-12及ROCK-1蛋白免疫印跡電泳表達：電泳結果顯示(分別見圖3、4、5、6)：與Control組相比,LPS組GRP78、CHOP、caspase-12及ROCK-1蛋白表達均明顯下調(diào)(P0.05)；與模型組組比較,LPS+FAS組GRP78、CHOP、Caspase-12及ROCK-1蛋白表達上調(diào)(P0.05)。結論：Fasudil可以改善內(nèi)毒素所致的AKI后腎功能和病理損傷,降低血液內(nèi)及腎臟中炎癥因子(TNF-α、IL-6),減輕炎性反應；并且fasudil通過直接抑制ROCK1活性,并下調(diào)GRP78、CHOP等蛋白,抵抗內(nèi)質(zhì)網(wǎng)過度應激所致的損傷,二者相輔相成共同作用影響腎臟功能。fasudil有望成為一種新的治療AKI的輔助藥物。
[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.
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R692

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