羅格列酮對(duì)糖尿病腎病大鼠腎小管上皮整合素連接激酶和α-平滑肌肌動(dòng)蛋白表達(dá)的影響

劉愛(ài)林，張皎月

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羅格列酮對(duì)糖尿病腎病大鼠腎小管上皮整合素連接激酶和α-平滑肌肌動(dòng)蛋白表達(dá)的影響

劉愛(ài)林1，張皎月2*

目的探討羅格列酮對(duì)糖尿病腎病大鼠腎小管上皮整合素連接激酶和α-平滑肌肌動(dòng)蛋白(α-SMA)表達(dá)的影響。方法30只健康雄性SD大鼠隨機(jī)分為正常對(duì)照組、陽(yáng)性對(duì)照組和羅格列酮處理組，每組10只，正常對(duì)照組給予普通營(yíng)養(yǎng)飼料喂養(yǎng)，陽(yáng)性對(duì)照組和羅格列酮處理組給予高脂飼料喂養(yǎng)，連續(xù)喂養(yǎng)12周后，陽(yáng)性對(duì)照組和羅格列酮處理組大鼠腹腔注射30 mg/kg鏈脲佐菌素，正常對(duì)照組給予等量枸櫞酸緩沖液。成功制備DKD模型后，羅格列酮處理組大鼠給予羅格列酮3 mg/kg灌胃，正常對(duì)照組和陽(yáng)性對(duì)照組給予等量生理鹽水灌胃。連續(xù)給藥28周后，對(duì)各組大鼠進(jìn)行稱重，檢測(cè)FBG、HbA1c和24 h尿蛋白水平，實(shí)時(shí)熒光定量PCR檢測(cè)右腎上極皮質(zhì)組織中ILK和α-SMA表達(dá)。結(jié)果羅格列酮處理組和陽(yáng)性對(duì)照組大鼠體重均低于正常處理組，而相對(duì)腎重均高于正常對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)，羅格列酮處理組大鼠體重均高于陽(yáng)性對(duì)照組，而相對(duì)腎重低于陽(yáng)性對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)；羅格列酮處理組大鼠FBG、HbA1c和24 h尿蛋白水平均高于正常對(duì)照組，但均低于陽(yáng)性對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)；羅格列酮處理組大鼠右腎上極皮質(zhì)組織中ILK mRNA和α-SMA mRNA相對(duì)表達(dá)量均高于正常對(duì)照組，但均低于陽(yáng)性對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論DKD大鼠腎組織中ILK和α-SMA呈高表達(dá)，羅格列酮可下調(diào)DKD大鼠腎組織中ILK和α-SMA表達(dá)，可能通過(guò)抑制腎小管上皮細(xì)胞轉(zhuǎn)分化發(fā)生而延緩腎小管間質(zhì)纖維化。

糖尿病腎??；羅格列酮；上皮整合素連接激酶；α-平滑肌肌動(dòng)蛋白

0　引言

糖尿病腎病(Diabetic kidney disease，DKD)是糖尿病所致最嚴(yán)重且危害最大的慢性并發(fā)癥，患者可出現(xiàn)腎小球硬化、漸進(jìn)性腎功能損害及蛋白尿，晚期發(fā)生腎功能衰竭，是造成患者死亡的重要原因[1]。研究表明，腎小管上皮細(xì)胞轉(zhuǎn)分化在DKD發(fā)生、進(jìn)展中發(fā)揮關(guān)鍵性作用[2]。腎小管上皮細(xì)胞轉(zhuǎn)分化過(guò)程涉及諸多信號(hào)傳導(dǎo)通路及細(xì)胞因子，整合素連接激酶(Integrin-linked kinase，ILK)作為具有多種功能的絲氨酸-蘇氨酸蛋白激酶，參與整合素介導(dǎo)的信號(hào)傳導(dǎo)，是腎小管上皮細(xì)胞轉(zhuǎn)分化過(guò)程中關(guān)鍵性效應(yīng)物[3]。α-平滑肌肌動(dòng)蛋白(α-smooth muscle actin，α-SMA)作為肌成纖維細(xì)胞的標(biāo)志物，亦是腎小管上皮細(xì)胞轉(zhuǎn)分化過(guò)程的標(biāo)志蛋白[4]。羅格列酮是臨床上常用的抗糖尿病藥物，但其對(duì)糖尿病腎病進(jìn)展過(guò)程是否有影響，鮮有報(bào)道。本研究通過(guò)分析羅格列酮對(duì)糖尿病腎病大鼠腎小管上皮ILK和α-SMA表達(dá)的影響，探討羅格列酮在糖尿病腎病進(jìn)展中的意義，以期為臨床實(shí)踐提供基礎(chǔ)資料。

1　資料與方法

1.1實(shí)驗(yàn)動(dòng)物及分組30只健康雄性SD大鼠購(gòu)自河南省實(shí)驗(yàn)動(dòng)物中心，4～5周齡，體重110～130 g，實(shí)驗(yàn)前血糖、尿糖及尿蛋白檢測(cè)顯示為陰性。利用隨機(jī)數(shù)字表隨機(jī)分為正常對(duì)照組(n=10)、陽(yáng)性對(duì)照組(n=10)和羅格列酮處理組(n=10)。

1.2方法

1.2.1喂養(yǎng)方法及DKD大鼠模型制備正常對(duì)照組大鼠給予普通營(yíng)養(yǎng)飼料喂養(yǎng)，熱卡為351 kcal/100 g。陽(yáng)性對(duì)照組和羅格列酮處理組大鼠則給予高脂飼料喂養(yǎng)，熱卡為436 kcal/100 g，成分比例：脂類22.3%、糖類44.4%、蛋白質(zhì)18.6%，其余為維生素及礦物質(zhì)。飼料購(gòu)自山東華康飼料公司。連續(xù)喂養(yǎng)12周后，陽(yáng)性對(duì)照組和羅格列酮處理組大鼠腹腔一次性注射30 mg/kg鏈脲佐菌素[5](購(gòu)自美國(guó)Sigma公司)，正常對(duì)照組則給予等量枸櫞酸緩沖液。3 d后對(duì)三組大鼠測(cè)量尾靜脈血糖，若陽(yáng)性對(duì)照組和羅格列酮處理組大鼠連續(xù)3次隨機(jī)尾靜脈血糖≥16.7 mmol/L則造模成功[5]，造模過(guò)程中陽(yáng)性對(duì)照組有1只大鼠出現(xiàn)死亡，其余大鼠均造模成功。

1.2.2給藥方法DKD大鼠模型制備成功后，羅格列酮處理組大鼠給予羅格列酮(批準(zhǔn)文號(hào)：國(guó)藥準(zhǔn)字H20030569，生產(chǎn)單位：成都恒瑞制藥有限公司)3 mg/kg灌胃，正常對(duì)照組和陽(yáng)性對(duì)照組則給予等量生理鹽水灌胃，每天1次，連續(xù)給藥28周。

1.2.3標(biāo)本采集及處理給藥28周后，對(duì)各組大鼠進(jìn)行稱重，將大鼠進(jìn)行單籠喂養(yǎng)，禁食不禁水，收集24 h尿液，利用雙縮脲法對(duì)24 h尿蛋白定量；尾靜脈取血1 mL，用于空腹血糖(FBG)和糖化血紅蛋白(HbA1c)測(cè)定；用40 mg/kg戊巴比妥鈉腹腔內(nèi)注射麻醉后，快速取出左腎，去掉背膜，濾紙吸干血跡后，用電子天平稱重，計(jì)算相對(duì)腎重=腎重重量(mg)/體重(g)；取右腎上極皮質(zhì)，保存于-70 ℃液氮中以備檢。

1.2.4腎臟組織病理學(xué)觀察用4%多聚甲醛對(duì)腎組織固定24 h后，利用乙醇進(jìn)行梯度脫水，用二甲苯透明、浸蠟、包埋。常規(guī)切片后，用HE染色觀察腎小管上皮細(xì)胞變化。

1.2.5實(shí)時(shí)熒光定量PCR檢測(cè)右腎上極皮質(zhì)組織中ILK和α-SMA表達(dá)取右腎上極皮質(zhì)研磨后，用細(xì)胞裂解液進(jìn)行裂解，用Trizol總RNA提取試劑盒(購(gòu)自美國(guó)Invitrogen公司)、紫外分光光度計(jì)(購(gòu)自美國(guó)GE公司)對(duì)RNA純度進(jìn)行檢測(cè)，取A260/A280≥1.80標(biāo)本完成后續(xù)實(shí)驗(yàn)。利用逆轉(zhuǎn)錄試劑盒(購(gòu)自天根生化科技)進(jìn)行逆轉(zhuǎn)錄獲得模板cDNA，以cDNA為模板用PCR試劑盒(購(gòu)自天根生化科技)完成PCR。ILK、α-SMA及內(nèi)參引物均由上海生工生物公司合成。ILK引物：上游 5′-GTGCCGGGAGGGCAACGCGGTG-3′，下游 5′-CGGGGCCTTGTGCGAGTGG-3′；α-SMA引物：上游 5′-GATGTACCCTGGGATCGCTGAC-3′，下游 5′-AAGCATTTGCGGTGGACGAT-3′；β-actin引物：上游 5′-CATTGCCGACAGGATGCAG-3′，下游 5′-CTCGTCATACTCCTGCTTGCTG-3′。PCR反應(yīng)條件：94 ℃ 60 s，92 ℃ 45 s，56 ℃ 30 s，74 ℃ 30 s，連續(xù)進(jìn)行38個(gè)循環(huán)。用2-△△Ct法獲得右腎上極皮質(zhì)中ILK mRNA和α-SMA mRNA相對(duì)表達(dá)量。

2　結(jié)果

2.1三組大鼠體重和相對(duì)腎重羅格列酮處理組和陽(yáng)性對(duì)照組大鼠體重均低于正常處理組，而相對(duì)腎重均高于正常對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，羅格列酮處理組大鼠體重高于陽(yáng)性對(duì)照組，而相對(duì)腎重低于陽(yáng)性對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)，見(jiàn)表1。

表1　三組大鼠體重和相對(duì)腎重

注：與正常對(duì)照組比較，*P<0.05；與陽(yáng)性對(duì)照組比較，#P<0.05

2.2三組大鼠FBG、HbA1c和24 h尿蛋白比較羅格列酮處理組大鼠FBG、HbA1c和24 h尿蛋白水平均高于正常對(duì)照組，但均低于陽(yáng)性對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，見(jiàn)表2。

2.3腎小管病理形態(tài)學(xué)觀察腎臟組織病理學(xué)檢查顯示，與正常對(duì)照組比較，陽(yáng)性對(duì)照組大鼠腎小管上皮細(xì)胞出現(xiàn)排列紊亂、脫落、壞死，空泡樣、顆粒樣變性，部分腎小管出現(xiàn)萎縮；羅格列酮處理組腎小管上皮細(xì)胞病變較陽(yáng)性對(duì)照組減輕，見(jiàn)圖1。

2.4三組大鼠右腎上極皮質(zhì)組織中ILK和α-SMA表達(dá)羅格列酮處理組大鼠右腎上極皮質(zhì)組織中ILK mRNA和α-SMA mRNA相對(duì)表達(dá)量均高于正常對(duì)照組，但均低于陽(yáng)性對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)，見(jiàn)表3。

表2　三組大鼠FBG、HbA1c和24 h尿蛋白比較

注：與正常對(duì)照組比較，*P<0.05；與陽(yáng)性對(duì)照組比較，#P<0.05

圖1　腎臟組織病理學(xué)觀察(HE，×400。A.正常對(duì)照組，B.陽(yáng)性對(duì)照組，C.羅格列酮處理組)

組別例數(shù)ILKmRNAα-SMAmRNA羅格列酮處理組101.36±0.15*#1.31±0.12*#陽(yáng)性對(duì)照組91.79±0.18*1.68±0.14*正常對(duì)照組101.07±0.221.03±0.09F值26.54566.631P值<0.001<0.001

注：與正常對(duì)照組比較，*P<0.05；與陽(yáng)性對(duì)照組比較，#P<0.05

3　討論

羅格列酮是2型糖尿病患者常用的噻唑烷二酮類降糖藥物，可增強(qiáng)胰島素敏感性，亦可特異性激活過(guò)氧化物增殖激活受體γ，在減少炎性因子形成和釋放中發(fā)揮重要作用[6]。有研究表明，羅格列酮可抑制TNF-α表達(dá)而減輕膿毒癥急性腎損傷[7]。亦有研究表明，羅格列酮在腎臟、肝臟等多個(gè)器官中具有抑制血管新生、抗纖維化作用[8]。目前，羅格列酮在控制2型糖尿病患者血糖中的作用已得到臨床認(rèn)可，本研究嘗試對(duì)羅格列酮在DKD發(fā)病、進(jìn)展中的功效及可能機(jī)制進(jìn)行探討。

糖尿病作為一種消耗性疾病，造模組大鼠出現(xiàn)“三多一少”癥狀。研究結(jié)果顯示，羅格列酮處理組和陽(yáng)性對(duì)照組大鼠體重均低于正常處理組，而相對(duì)腎重、FBG、HbA1c和24 h尿蛋白水平較高。腎臟組織病理學(xué)檢查顯示，陽(yáng)性對(duì)照組大鼠腎小管上皮細(xì)胞出現(xiàn)排列紊亂、脫落、壞死，空泡樣、顆粒樣變性，部分腎小管出現(xiàn)萎縮，提示利用注射鏈脲佐菌素的方法可成功構(gòu)建DKD大鼠模型，模型大鼠出現(xiàn)DKD相關(guān)癥狀表現(xiàn)。此外，羅格列酮處理組大鼠體重均高于陽(yáng)性對(duì)照組，而相對(duì)腎重、FBG、HbA1c和24 h尿蛋白水平較低，說(shuō)明羅格列酮處理DKD模型大鼠可有效減輕糖尿病相關(guān)癥狀及腎臟組織損傷，進(jìn)而減少蛋白尿，對(duì)延緩DKD進(jìn)展發(fā)揮積極作用，腎小管病理形態(tài)學(xué)觀察結(jié)果亦顯示，羅格列酮處理組腎小管上皮細(xì)胞病變較陽(yáng)性對(duì)照組減輕。

研究表明，DKD病情進(jìn)展與腎小管上皮細(xì)胞損傷及腎小管間質(zhì)纖維化密切相關(guān)[1]。研究發(fā)現(xiàn)，腎小管上皮細(xì)胞可通過(guò)轉(zhuǎn)分化轉(zhuǎn)化為具有間質(zhì)肌成纖維細(xì)胞的新表型，在加速間質(zhì)纖維化過(guò)程中發(fā)揮關(guān)鍵性作用[4]。α-SMA是肌成纖維細(xì)胞的蛋白標(biāo)志物，而鑒于病理情況下腎臟組織中肌成纖維細(xì)胞大多來(lái)源于腎小管上皮細(xì)胞轉(zhuǎn)分化，因此，α-SMA亦可作為腎小管上皮細(xì)胞發(fā)生轉(zhuǎn)分化的標(biāo)志物[9]。研究表明，轉(zhuǎn)化生長(zhǎng)因子在腎小管上皮細(xì)胞發(fā)生轉(zhuǎn)分化過(guò)程中發(fā)揮重要的正向調(diào)控作用[10]。而ILK作為重要的信號(hào)轉(zhuǎn)導(dǎo)蛋白激酶，是轉(zhuǎn)化生長(zhǎng)因子信號(hào)通路下游重要因子，可能參與了腎小管上皮細(xì)胞轉(zhuǎn)分化調(diào)控過(guò)程，本研究顯示，羅格列酮處理組和陽(yáng)性對(duì)照組大鼠右腎上極皮質(zhì)組織中ILK mRNA和α-SMA mRNA相對(duì)表達(dá)量均高于正常對(duì)照組(P<0.05)，說(shuō)明ILK和α-SMA在DKD模型大鼠腎組織中呈高表達(dá)，提示腎小管上皮細(xì)胞可能通過(guò)轉(zhuǎn)分化而加速DKD大鼠腎臟纖維化過(guò)程，本研究顯示，羅格列酮處理組大鼠右腎上極皮質(zhì)組織中ILK mRNA和α-SMA mRNA相對(duì)表達(dá)量均低于陽(yáng)性對(duì)照組(P<0.05)，說(shuō)明羅格列酮可通過(guò)抑制腎小管上皮細(xì)胞發(fā)生轉(zhuǎn)分化，而延緩DKD大鼠腎組織纖維化過(guò)程，從而保護(hù)DKD大鼠腎臟組織。

綜上所述，DKD大鼠腎組織中ILK和α-SMA呈高表達(dá)，提示DKD大鼠腎小管上皮細(xì)胞發(fā)生轉(zhuǎn)分化；羅格列酮可下調(diào)DKD大鼠腎組織中ILK和α-SMA表達(dá)，可能通過(guò)抑制腎小管上皮細(xì)胞轉(zhuǎn)分化發(fā)生而延緩腎小管間質(zhì)纖維化，達(dá)到保護(hù)腎功能的作用。

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Influence of rosiglitazone on expressions of integrin-linked kinase and α-smooth muscle actin in renal tubular epithelium in rats with diabetic kidney disease

LIU Ai-lin1,ZHANG Jiao-yue2*

(1.Department of Endocrinology,Xiaogan Central Hospital,Xiaogan 432000,China;2.Xiehe Hospital Affiliated to Tongji Medical College,Huazhong Technology University,Wuhan 432000,China)

ObjectiveTo investigate the effects of rosiglitazone on expressions of integrin-linked kinase and α-smooth muscle actin in renal tubular epithelium in rats with diabetic kidney disease (DKD).MethodsThirty healthy male SD rats were randomly divided into normal control group,the positive control group and rosiglitazone treatment group,each group had 10 rats.Rats in normal control group were given general nutrition diet,rats in positive control group and rosiglitazone treatment group were given high fat diet.After feeding for 12 weeks,the rats in positive control group and rosiglitazone treatment group were intraperitoneally injected 30 mg/kg streptozotocin,and the rats in normal control group received the same amount of citrate buffer.After the models of DKD rats were made successfully,rats in rosiglitazone treatment group were given rosiglitazone 3 mg/kg gavage,and the rats in normal control group and positive control group were given normal saline gavage.After being administered continuously for 28 weeks,the weights of rats in each group were recorded.The levels of FBG,HbA1c and 24 h urine protein were detected.The expression of ILK and α-SMA in cortical tissues of right kidney was detected by using real-time quantitative PCR.ResultsThe body weights of rats in rosiglitazone treatment group and positive control group were lower than those of normal control group,while the relative kidney weights were higher than normal control group,the differences being statistically significant (P<0.05).The body weights of rats in rosiglitazone treatment group were higher than those of positive control group,while the relative kidney weights were lower than those of positive control group,the differences being statistically significant (P<0.05).The levels of FBG,HbA1c and 24 h urine proteins of rats in rosiglitazone treatment group were higher than those of normal control group,and lower than those of positive control group,the differences being statistically significant (P<0.05).The relative expression levels of ILK mRNA and α-SMA mRNA in kidney cortex tissues in rosiglitazone treatment group were higher than those of normal control group and lower than those of positive control group,the differences being statistically significant (P<0.05).ConclusionThe ILK and α-SMA are highly expressed in DKD kidney tissues.Rosiglitazone can down-regulate the expression of ILK and α-SMA in renal tissues of DKD rat,and might inhibit the renal tubular epithelial-mesenchymal transition occurrence to delay tubulointerstitial fibrosis.

Diabetic kidney disease;Rosiglitazone;Epithelial integrin-linked kinase;α-smooth muscle actin

2016-02-27

1.孝感市中心醫(yī)院內(nèi)分泌科，湖北 孝感432000；2.華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬協(xié)和醫(yī)院內(nèi)分泌科，武漢 432000

10.14053/j.cnki.ppcr.201609007