【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出現(xiàn)進行性惡化的重要因素之一。內毒素(lipopolysaccharide; LPS)可以通過介導下游多種癥介質、信號通路參與腎臟損傷的發(fā)生,越來越多的證據表明內質網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質網應激(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,標準飼料和自來水自由飲食。2.經上述環(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)：采用經靜脈內注射內毒素(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：內毒素所致的AKI的病理變化在腎髓質及皮質均有累及。與對照組組比較,模型組髓質區(qū)遠端小管及皮質近端小管出現(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,腎臟髓質及皮質均有細胞凋亡。Control組內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(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(P0.05)。結論：Fasudil可以改善內毒素所致的AKI后腎功能和病理損傷,降低血液內及腎臟中炎癥因子(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GRP78、CHOP等蛋白,抵抗內質網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

【相似文獻】

相關期刊論文 前10條

1 張琳;張驊;;法舒地爾臨床應用的研究進展[J];臨床肺科雜志;2011年04期

2 孫敏;孫曉艷;陳雪霽;呂軍;;法舒地爾治療非動脈炎性缺血性視神經病變[J];國際眼科雜志;2014年02期

3 張明;孫曉川;;法舒地爾的藥理作用及其臨床應用的研究進展[J];山西醫(yī)科大學學報;2007年04期

4 張愛華;全正翔;劉福中;;法舒地爾對急性缺血性腦卒中患者血液流變學的影響[J];中南藥學;2008年03期

5 趙克勤;夏友華;嚴忠文;周學海;;法舒地爾治療急性缺血性腦卒中的臨床觀察[J];中國醫(yī)院藥學雜志;2009年06期

6 崔學軍;楊少輝;宋洪波;邵建芳;;法舒地爾治療急性缺血性腦血管病60例[J];中國新藥與臨床雜志;2009年04期

7 張永珍;王軍;李海峰;;法舒地爾治療椎基底動脈供血不足性眩暈[J];中國實用神經疾病雜志;2009年17期

8 林福軍;陳玉蘭;;法舒地爾對急性動脈粥樣硬化性腦梗死的療效及對血清CRP的影響[J];中國醫(yī)院用藥評價與分析;2009年12期

9 陳鳳慧;肖麗萍;繳克華;;法舒地爾治療后循環(huán)供血不足的療效觀察[J];河北醫(yī)藥;2010年07期

10 武多嬌;張怡;高平進;朱鼎良;;法舒地爾抗動脈粥樣硬化作用及其機制[J];中國臨床藥學雜志;2010年02期

相關會議論文 前10條

1 葉紅偉;高琴;王曉梅;康品方;于影;李正紅;;乙醛脫氫酶2在法舒地爾心肌保護中的作用探討[A];中國生理學會第九屆全國青年生理學工作者學術會議論文摘要[C];2011年

2 趙青;;法舒地爾臨床應用的研究進展[A];2013年中國藥學大會暨第十三屆中國藥師周論文集[C];2013年

3 李琴;黃先菊;郭蓮軍;;甲磺酸法舒地爾對局灶性腦缺血損傷的保護作用[A];中國藥理學會第九次全國會員代表大會暨全國藥理學術會議論文集[C];2007年

4 章楊龍;聶福根;;法舒地爾對壓力超負荷性大鼠左室重構的影響[A];江西省第四次中西醫(yī)結合心血管學術交流會論文集[C];2008年

5 姜純國;黃慧;劉佳;王艷勛;徐作軍;;法舒地爾對肺纖維化的保護作用[A];中華醫(yī)學會呼吸病學年會——2013第十四次全國呼吸病學學術會議論文匯編[C];2013年

6 劉袁媛;邸欣;孫毓慶;;馬來酸法舒地爾與鹽酸法舒地爾注射液在犬體內的藥動學比較研究[A];第八次全國藥物與化學異物代謝學術會議論文摘要[C];2006年

7 張子新;余陸嬌;張立敏;李莎;;法舒地爾對糖尿病合并動脈粥樣硬化大鼠的心臟保護作用[A];中華醫(yī)學會第十五次全國心血管病學大會論文匯編[C];2013年

8 蔡輝;趙凌杰;張靜;趙智明;沈思鈺;;法舒地爾升高壓力超負荷大鼠心肌血管緊張素轉化酶2的表達[A];第九次全國中西醫(yī)結合基礎理論研究學術研討會論文匯編[C];2013年

9 王曉明;魏愛宣;邵延坤;徐忠信;;法舒地爾對慢性腦缺血大鼠學習記憶功能的影響[A];第十一屆全國神經病學學術會議論文匯編[C];2008年

10 王曉明;魏愛宣;趙虹;徐忠信;;法舒地爾對慢性腦缺血大鼠認知功能功能及皮層海馬自由基代謝的影響[A];第十一屆全國神經病學學術會議論文匯編[C];2008年

相關重要報紙文章 前1條

1 遼寧諾康生物心血管事業(yè)部總經理 孫一夫 林琳 整理;像川威一樣思考[N];醫(yī)藥經濟報;2013年

相關博士學位論文 前9條

1 高靜;Rho激酶抑制劑法舒地爾通過調控心肌細胞鈣清除及肌動蛋白重構改善糖尿病心功能不全[D];浙江大學;2016年

2 瞿星光;法舒地爾對內毒素引起的急性腎損傷大鼠影響及機制[D];武漢大學;2015年

3 姜純國;法舒地爾對肺纖維化的保護作用和吡非尼酮治療肺纖維化的副作用[D];北京協(xié)和醫(yī)學院;2013年

4 劉愛軍;法舒地爾對PDGF誘導的人肺動脈平滑肌細胞增殖的抑制作用及機制研究[D];北京協(xié)和醫(yī)學院;2011年

5 王娜;RhoA/ROCK信號通路在心肌肥厚和心力衰竭中的作用及法舒地爾的干預效應[D];河北醫(yī)科大學;2011年

6 馬志紅;Rho激酶信號通路對大鼠動脈粥樣硬化形成的影響及法舒地爾的心血管保護作用[D];河北醫(yī)科大學;2011年

7 劉煥龍;ROCK抑制劑法舒地爾對脂多糖致大鼠肺微血管內皮細胞損傷的保護作用及機制研究[D];河北醫(yī)科大學;2014年

8 鄧林;Rho激酶抑制劑法舒地爾抑制人腦膠質瘤的體內外實驗研究[D];山東大學;2010年

9 李琴;甲磺酸法舒地爾Rho激酶抑制劑對缺血性腦損傷的保護作用及機制研究[D];華中科技大學;2009年

相關碩士學位論文 前10條

1 蔡佳盈;法舒地爾對血管緊張素Ⅱ誘導足細胞骨架重構的干預作用[D];福建醫(yī)科大學;2015年

2 王盼;法舒地爾對脂多糖誘導的人臍靜脈內皮細胞損傷的影響[D];蚌埠醫(yī)學院;2015年

3 賈少勛;法舒地爾抗白血病機制相關研究[D];延安大學;2015年

4 趙振;法舒地爾對膿毒癥所致急性肺損傷及肺組織中TNF-α和HMGB1表達的影響[D];桂林醫(yī)學院;2015年

5 李世云;法舒地爾聯(lián)合羅格列酮對脂多糖誘導人冠脈內皮細胞MCP-1和VCAM-1表達的影響[D];廣西醫(yī)科大學;2016年

6 馬宗侖;Rho激酶抑制劑法舒地爾對微血管心絞痛患者的療效觀察[D];青島大學;2016年

7 江金W，

本文編號：2185491