陳凱，張承英,，李建民，張建榮

(1. 安徽醫(yī)科大學(xué)武警總醫(yī)院臨床學(xué)院，合肥　230032；2. 中國(guó)人民武裝警察部隊(duì)總醫(yī)院，北京　100039；

3. 北京市中西醫(yī)結(jié)合醫(yī)院，北京　100039)

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纈沙坦對(duì)糖尿病大鼠腎臟中內(nèi)質(zhì)網(wǎng)應(yīng)激及炎癥反應(yīng)的抑制作用

陳凱1，張承英1,2，李建民3，張建榮2

(1. 安徽醫(yī)科大學(xué)武警總醫(yī)院臨床學(xué)院，合肥230032；2. 中國(guó)人民武裝警察部隊(duì)總醫(yī)院，北京100039；

3. 北京市中西醫(yī)結(jié)合醫(yī)院，北京100039)

【摘要】目的探討內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulum stress, ERS)及相關(guān)炎癥反應(yīng)在糖尿病大鼠腎臟損害中的作用及血管緊張素II受體拮抗劑纈沙坦對(duì)其的影響。 方法采用腹腔注射鏈脲佐菌素方法建立糖尿病腎病大鼠模型。將大鼠隨機(jī)分為對(duì)照組(Con組)、糖尿病組(DM組)、纈沙坦組(DM+V組)。纈沙坦組每日灌胃給予纈沙坦(10 mg/kg)共6周。應(yīng)用免疫組化法及Western blot方法檢測(cè)ERS相關(guān)蛋白P-IRE1α、P-JNK及中性粒細(xì)胞趨化因子MCP-1的表達(dá)及定位，實(shí)時(shí)熒光定量PCR(FQ-PCR)檢測(cè)IRE1α、JNK及MCP-1mRNA的表達(dá)變化，同時(shí)觀察各組大鼠尿蛋白、BUN、Scr等指標(biāo)的變化。 結(jié)果 與Con組相比，DM組大鼠腎臟病理炎細(xì)胞浸潤(rùn)加重，P-IRE1α、IRE1α、P-JNK、MCP-1蛋白表達(dá)上調(diào)，IRE1α mRNA、MCP-1mRNA表達(dá)水平上調(diào)；與DM組相比，DM+V組腎臟病理炎癥細(xì)胞浸潤(rùn)減輕，P-IRE1α、IRE1α、P-JNK、MCP-1蛋白表達(dá)下調(diào)，IRE1α mRNA、MCP-1 mRNA表達(dá)水平下調(diào)。3組間JNK mRNA及蛋白表達(dá)無(wú)明顯差異。 結(jié)論 糖尿病大鼠腎臟中存在內(nèi)質(zhì)網(wǎng)應(yīng)激和炎癥反應(yīng)的激活，纈沙坦可能部分通過(guò)抑制內(nèi)質(zhì)網(wǎng)應(yīng)激中的IRE1/JNK/MCP-1通路，減少炎癥反應(yīng)，從而發(fā)揮腎臟保護(hù)作用。

【關(guān)鍵詞】?jī)?nèi)質(zhì)網(wǎng)應(yīng)激；炎癥反應(yīng)；糖尿病腎??；纈沙坦

糖尿病腎病(diabetic nephropathy，DN)是糖尿病的微血管并發(fā)癥，也是導(dǎo)致終末期腎病(end-stage renal disease, ESRD)最常見(jiàn)的基礎(chǔ)疾病之一。其發(fā)病機(jī)制復(fù)雜，近年來(lái)，代謝性炎癥與糖尿病腎病的關(guān)系越來(lái)越受到重視。目前，以纈沙坦為代表的血管緊張素II受體拮抗劑(angiotension Ⅱ receptor blocker，ARB)在DN尤其是早期DN治療中的作用不斷被證實(shí)，研究表明，纈沙坦具有降低CRP、IL- 6 等炎癥因子的作用[1]，然而其作用機(jī)制尚未完全明了。本文從內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulum stress，ERS)的角度，研究觀察纈沙坦對(duì)早期糖尿病大鼠腎臟內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白IRE1α、JNK及其下游炎癥因子中性粒細(xì)胞趨化因子MCP-1表達(dá)的影響，探討其抑制炎癥反應(yīng)的相關(guān)機(jī)制。

1材料與方法

1.1材料

1.1.1實(shí)驗(yàn)試劑

鏈脲佐菌素(streptozotocin，STZ)購(gòu)于Sigma 公司。纈沙坦由北京諾華制藥有限公司提供。兔抗鼠IRE1α抗體，兔抗鼠P-IRE1α，兔抗鼠JNK，兔抗鼠P-JNK抗體均購(gòu)于美國(guó)Santa Cruz公司，兔抗鼠MCP-1抗體購(gòu)于北京博奧森公司，小鼠抗GAPDH單抗，羊抗兔二抗及羊抗小鼠二抗購(gòu)于北京中杉金橋公司。PCR試劑盒為北京天根生化科技有限公司產(chǎn)品。

1.1.2實(shí)驗(yàn)動(dòng)物

SPF級(jí)SD雄性大鼠，體重190～210 g，購(gòu)自北京維通利華實(shí)驗(yàn)動(dòng)物有限公司【SCXK(京)2011-0011】，飼養(yǎng)于北京中醫(yī)藥大學(xué)東直門(mén)醫(yī)院屏障環(huán)境動(dòng)物室【SYXK(京)2009-0028】。

1.2方法

1.2.1模型制備及分組

34只SD雄性大鼠，隨機(jī)分為對(duì)照組(Con組，n=10只)、模型組(DM組，n=12只)、纈沙坦治療組(DM+V組，n=12只)。大鼠適應(yīng)性喂養(yǎng)一周后，DM組、DM+V組大鼠腹腔注射STZ(STZ 溶于10 mmol/L 的檸檬酸鹽溶液，pH 4.5，40 mg/kg)制作糖尿病模型，Con組只注射相同體積的枸櫞酸鈉緩沖液，2 d后尾靜脈采血，測(cè)定血糖≥16.7 mmol/L，尿糖+++～尿糖++++者確定為糖尿病大鼠。對(duì)照組血糖4～6 mmol/L 左右。所有大鼠試驗(yàn)期間不用外源性胰島素，避免胰島素干擾實(shí)驗(yàn)過(guò)程。DM+V組每天用纈沙坦灌胃6周(10 mg/kg)，Con組及DM組只灌等量蒸餾水。實(shí)驗(yàn)期間動(dòng)物自由進(jìn)食，整個(gè)實(shí)驗(yàn)過(guò)程中DM、DM+V組各有1只大鼠死亡，各有1只大鼠血糖未達(dá)到模型標(biāo)準(zhǔn)，予以剔除。最終共30只完成實(shí)驗(yàn)，其中C組10 只，DM組10 只，DM+V組10只。在給藥6周處死大鼠，處死前用代謝籠收集24 h尿, 用于測(cè)定24 h尿蛋白定量；下腔靜脈取血3 mL，用于測(cè)定血肌酐、血尿素氮、血漿白蛋白、血糖水平；留取雙側(cè)腎臟，稱(chēng)重，部分腎組織以10%中性福爾馬林溶液固定，其余組織置于-72℃冰箱中凍存。

1.2.2腎臟病理學(xué)檢查

觀察腎組織石蠟包埋后制成2 μm切片，常規(guī)HE染色，光鏡下觀察腎組織形態(tài)學(xué)改變及炎細(xì)胞浸潤(rùn)情況。

1.2.3免疫組織化學(xué)檢測(cè)

采用SP 法。2 μm 腎組織切片，常規(guī)脫蠟至水，3% H2O2處理清除內(nèi)源性過(guò)氧化物酶，抗原熱修復(fù)。正常山羊血清封閉。一抗 P-IRE1α、IRE1α、P-JNK、JNK( 1∶100) 稀釋?zhuān)篂樯锼鼗窖蚩雇肐gG，PBS 替代一抗作為陰性對(duì)照，DAB 顯微鏡控制下顯色，蘇木素復(fù)染。

1.2.4Westen blot檢測(cè)蛋白表達(dá)

取腎皮質(zhì)組織100 mg 左右, 用眼科剪剪碎, 加入RIPA 裂解緩沖液, 在玻璃研磨器中研磨, 冰浴1 h, 4℃、14000 r/min離心25 min, BCA法測(cè)定上清液蛋白濃度。取總蛋白50 μg, 十二烷基硫酸鈉- 聚丙烯酰胺( SDS- PAGE)凝膠電泳后電轉(zhuǎn)移至NC膜; 5%脫脂奶粉封閉NC膜2 h,洗膜后分別加入兔抗大鼠P-IRE1α抗體(1∶400)，兔抗大鼠IRE1α抗體(1∶500)、兔抗大鼠P-JNK抗體(1∶200)、兔抗大鼠JNK抗體(1∶250)，小鼠抗大鼠GAPDH抗體(1∶3000),4℃過(guò)夜, 洗膜后加辣根過(guò)氧化物酶標(biāo)記的羊抗兔抗體(1∶8000)或羊抗鼠抗體(1∶5000), 37℃孵育2 h; 洗膜后加ECL試劑, 然后將NC膜放入X光片暗盒, 壓片, 顯影, 定影。用ImageJ分析系統(tǒng)軟件對(duì)Western條帶進(jìn)行定量分析, 確定雜交條帶的吸光度值。

1.2.5實(shí)時(shí)熒光定量PCR(FQ-PCR)檢測(cè)mRNA表達(dá)

按照Trizol 試劑盒說(shuō)明書(shū)提取各組大鼠腎組織總RNA，并參照逆轉(zhuǎn)錄擴(kuò)增試劑盒操作程序進(jìn)行cDNA 合成。采用2-△△CT法進(jìn)行數(shù)據(jù)的相對(duì)定量分析。擴(kuò)增條件為: 94℃預(yù)變性10 min，活化Tag 酶，94℃ 15 s， 60℃ 60 s，45 個(gè)循環(huán)結(jié)束，每個(gè)cDNA 樣品在96孔板均設(shè)置3個(gè)復(fù)孔，反應(yīng)完成后于16℃ 保存，采用GAPDH作為內(nèi)參照。引物序列為：IRE1α：5’-CTGTGGAGACCCTACGCTAT-3’(上游)與 5’-AGTGGGCGTCAGTTTGCTC-3’(下游)；JNK：5’-TGATGACGCCTTACGTGGTA-3’(上游)與 5’-GGCAAACCATTTCTCCCATA-3’(下游)；MCP-1：5’-ACCTGCTGCTACTCATTCA-3’(上游)與 5’-GCTGCTGGTGATTCTCTTG-3’(下游)；GAPDH：5’-TCAAGAAGGTGGTGAAGCAG-3’(上游)與 5’-AGGTGGAAGAATGGGAGTTG-3’(下游)。

1.2.6統(tǒng)計(jì)學(xué)分析

2結(jié)果

2.1各組大鼠24 h蛋白定量、血漿白蛋白、血糖、尿素氮和血肌酐的比較

6周末，與Con組相比，DM組大鼠24 h尿蛋白定量、血尿素氮顯著增高(P<0.01)、血肌酐增高(P<0.05)，血漿白蛋白降低(P<0.05)。與DM組相比，DM+V組大鼠24 h 尿蛋白定量、尿素氮降低(P<0.01，P<0.05)，血漿白蛋白增高(P<0.05)，血肌酐降低，但差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)(見(jiàn)表1)。

Tab.1The 24 h urinary protein excretion, plasma albumin, serum glucose, BUN and Scr in each group

組別ProAlBGluBUNScrGroupsmg/24hg/Lmmol/Lmmol/Lμmol/LCon組12.64±2.0329.21±1.709.16±1.667.01±0.5520.06±3.11DM組36.46±7.34**23.91±1.46*26.35±5.32**15.60±2.43**29.50±6.42*DM+V組25.23±3.75##28.05±1.79#24.68±4.47**10.33±1.73#25.92±4.25

注：與Con組相比，*P<0.05，**P<0.01；與DM組相比，#P<0.05，# #P<0.01

Note.*P<0.05**P<0.01 vs. control group;#P<0.05，# #P<0.01 vs. DM group.

2.2各組大鼠腎臟組織病理學(xué)改變

光鏡下Con組腎組織未見(jiàn)明顯病理改變，DM組大鼠腎小球明顯肥大，腎小球細(xì)胞外基質(zhì)略增生，毛細(xì)血管袢明顯擴(kuò)張，系膜基質(zhì)明顯增多、基底膜增厚且系膜間隙增寬，腎小管上皮空泡變性伴部分小灶狀萎縮，間質(zhì)輕度水腫。DM+V組與DM組相比病變明顯減輕。(見(jiàn)圖1)。

2.3免疫組織化學(xué)檢測(cè) P-IRE1α、P-JNK、MCP-1蛋白在腎臟的定位表達(dá)

P-IRE1α、P-JNK、MCP-1在Con組大鼠腎皮質(zhì)均有表達(dá),陽(yáng)性細(xì)胞為細(xì)胞核呈棕黃色，在100 倍視野中連續(xù)不重疊的計(jì)數(shù)100 個(gè)腎小管的細(xì)胞總數(shù)和陽(yáng)性細(xì)胞數(shù)及50 個(gè)腎小球的陽(yáng)性細(xì)胞數(shù)，以腎小管細(xì)胞的陽(yáng)性率和單個(gè)腎小球切面的陽(yáng)性細(xì)胞數(shù)作為比較指標(biāo)。P-IRE1α在Con組弱表達(dá)，主要位于遠(yuǎn)端小管和集合管上皮細(xì)胞胞質(zhì)及胞核內(nèi)；DM組表達(dá)明顯增多，腎小球及腎小管胞質(zhì)胞核均明顯表達(dá)。P-JNK在Con組弱表達(dá)，主要位于近端及遠(yuǎn)端腎小管上皮細(xì)胞胞質(zhì)內(nèi)；DM組表達(dá)明顯增多，主要位于腎小球系膜細(xì)胞及腎小管上皮細(xì)胞內(nèi)，以腎小管上皮細(xì)胞表達(dá)明顯，部分上皮細(xì)胞腎小球呈強(qiáng)陽(yáng)性。MCP-1在Con組表達(dá)極弱; DM組表達(dá)明顯增強(qiáng)，尤以遠(yuǎn)曲小管胞質(zhì)最為明顯，部分腎小球內(nèi)細(xì)胞胞質(zhì)也呈強(qiáng)陽(yáng)性表達(dá)。DM+V組P-IRE1α、P-JNK、MCP-1表達(dá)部位與DM組大致相同，程度介于Con組、DM組之間(見(jiàn)圖2)。

注: A為Con組,腎組織未見(jiàn)明顯病理改變；B為DM組，腎小球明顯肥大，腎小球系膜細(xì)胞輕度增生,腎小管上皮細(xì)胞腫脹，可見(jiàn)空泡變性；C為DM+V組,腎小球及腎小管病變相對(duì)于DM組明顯減輕。　　圖1　各組大鼠腎臟皮質(zhì)HE染色(×100) Note: A：Representative photomicrograph showing normal kidney tissues in a control mouse；B：Glomerular hypertrophy, mesangial cell mild hyperplasia, renal tubular epithelial cell swelling with visible vacuoles degeneration in a rat of the DM group；C：DM+V group. Compared with the DM group, lesions of glomeruli and renal tubules were alleviated significantly.　　Fig.1　Histology of the renal cortex (HE staining,×100)

注：A1-A3為Con組；B1-B3為DM組；C1-C3為DM+V組；A1-C1為P-IRE1α免疫組化染色；A2-C2為P-JNK免疫組化染色；A3-C3為MCP-1免疫組化染色；圖中紅色箭頭指示腎小球目標(biāo)蛋白陽(yáng)性表達(dá)，黑色箭頭指示腎小管目標(biāo)蛋白陽(yáng)性表達(dá)?！　D2　各組大鼠腎皮質(zhì)免疫組化P-IRE1α、P-JNK及MCP-1蛋白表達(dá)變化(×100)Note. A1-A3.Con group；B1-B3.DM group；C1-C3.DM + V group；A1-C1. Comparison of immunohistochemical staining of P-IRE1α in different groups；A2-C2. Comparison of immunohistochemical staining of P-JNK in different groups；A3-C3. Comparison of immunohistochemical staining for MCP-1 in different groups；Red arrow point to target protein expressed in glomerulus positively, black arrow point to target protein expressed in renal tubule positively. 　　Fig.2　Immunohistochemical staining of P-IRE1α,P-JNK and MCP-1 protein in the rat renal cortex at week 6(×100)

2.4Western blot檢測(cè)IRE1α、P-IRE1α、P-JNK、JNK、MCP-1蛋白表達(dá)情況

JNK表達(dá)在各組均無(wú)明顯差異。與Con組相比，DM組IRE1α、P-IRE1α、P-JNK、MCP-1表達(dá)增高(P<0.01)。與DM組相比，DM+V組IRE1α、P-IRE1α、P-JNK、MCP-1表達(dá)降低(P<0.01)(見(jiàn)圖3)。

注：與Con組相比，** P<0.01；與DM組相比，##P<0.01　　圖3　6周末各組大鼠腎臟皮質(zhì)中P-IRE1α、IRE1α、P-JNK、JNK、MCP-1的表達(dá) Note. ** P<0.01 vs. Con group；##P<0.01 vs. DM group.　　Fig.3　Expression of P-IRE1α, IRE1α, P-JNK, JNK and MCP-1 proteins in the rat renal cortex at week 6 assayed by Western blot analysis.

2.5FQ - PCR法檢測(cè)腎組織mRNA 的表達(dá)情況

6周末，Con組大鼠腎組織IRE1α、JNK、MCP-1mRNA 僅有微量表達(dá)。與Con組比較，DM組IRE1α、MCP-1 mRNA表達(dá)明顯增高，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)，JNK mRNA表達(dá)亦有增高，差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05); 與DM組相比，DM+V組IRE1α、MCP-1mRNA表達(dá)量明顯降低(P<0.01,P<0.05), JNK mRNA表達(dá)差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)(見(jiàn)圖4)。

注：與Con組相比，** P<0.01；與DM組相比，##P<0.01，#P<0.05。　　圖4　各組大鼠腎組織IRE1α、JNK和MCP-1mRNA的表達(dá)變化Note. ** P<0.01 vs. Con group；##P<0.01, #P<0.05 vs. DM group.　　Fig.4　FQ-PCR results of IRE1α, JNK and MCP-1 of the renal cortex in each group

3討論

糖尿病腎病的發(fā)病機(jī)制除蛋白激酶C(PKC)學(xué)說(shuō)、氧化應(yīng)激(ROS)學(xué)說(shuō)、細(xì)胞因子學(xué)說(shuō)及遺傳分子學(xué)說(shuō)外[2]，炎癥學(xué)說(shuō)目前倍受關(guān)注。微炎癥反應(yīng)和隨后的細(xì)胞外基質(zhì)擴(kuò)張是上述機(jī)制在糖尿病腎病中進(jìn)展的共同途徑，炎癥通路在糖尿病腎病的進(jìn)展中發(fā)揮著核心作用[3]。

湖南省在治理“新官不理舊事”方面，拿出了很多力度頗大的措施，起到的效果也很明顯，這從湖南省從嚴(yán)治黨、依法治理方面就可見(jiàn)一斑。比如，記者近日在湖南省高級(jí)人民法院采訪，就了解到了一起“以新官不理舊事為由拒付貨款，村民起訴獲得法院支持”的案例。被告某村委會(huì)三年時(shí)間里在原告田某（某鎮(zhèn)郵電局的一名臨聘人員）處訂閱報(bào)紙，三年共欠原告報(bào)刊費(fèi)1409元，并向原告田某出具了一張條據(jù)。后來(lái)，該村村委會(huì)換屆，原告多次找被告某村委會(huì)催要此款，被告某村委會(huì)卻以“新官不理舊事”為由拒付款。原告田某向湘陰縣人民法院起訴請(qǐng)求法院判令被告某村委會(huì)向原告田某支付貨款1409元，湘陰縣人民法院一審作出了支持原告訴訟請(qǐng)求的判決。

DM患者腎臟存在局部RAS系統(tǒng)的激活，ANGII生成增多，降解減慢，從而導(dǎo)致腎臟局部ANGII的濃度增加[4]。體外實(shí)驗(yàn)已發(fā)現(xiàn)，ANGII可誘導(dǎo)腎小管細(xì)胞的NF-κB的激活，進(jìn)而引起炎癥反應(yīng)[5]。Titan SM[6]聯(lián)合應(yīng)用ACEI與ARB治療糖尿病腎病患者，尿MCP-1(中性粒細(xì)胞趨化因子1)呈下降趨勢(shì)。腎臟 MCP-1 的增加是糖尿病腎病患者腎臟損傷的顯著特點(diǎn)，是導(dǎo)致單核巨噬細(xì)胞浸潤(rùn)腎組織的重要因素[7]。本實(shí)驗(yàn)研究中我們也發(fā)現(xiàn)，DM組大鼠模型6周時(shí)腎組織中的MCP-1表達(dá)量增高，同時(shí)炎性細(xì)胞浸潤(rùn)明顯。DM+V組，MCP-1蛋白表達(dá)水平下調(diào)，同時(shí)炎癥細(xì)胞浸潤(rùn)減輕，并伴有24 h尿蛋白定量降低，提示纈沙坦具有減降低炎癥細(xì)胞聚集，抑制炎癥反應(yīng)的作用。

然而對(duì)于糖尿病腎病中炎癥反應(yīng)發(fā)生的原因，目前尚未完全明確。越來(lái)越多的研究顯示，內(nèi)質(zhì)網(wǎng)應(yīng)激(ERS)與炎癥反應(yīng)關(guān)系密切。內(nèi)質(zhì)網(wǎng)是多種蛋白的主要合成場(chǎng)所，當(dāng)各種因素導(dǎo)致內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài)被打破，引起的內(nèi)質(zhì)網(wǎng)中出現(xiàn)錯(cuò)誤折疊及未折疊蛋白在腔內(nèi)聚集的狀態(tài)稱(chēng)為內(nèi)質(zhì)網(wǎng)應(yīng)激。適度的ERS可以恢復(fù)ER及內(nèi)環(huán)境穩(wěn)態(tài)，保持細(xì)胞活性，但當(dāng)外界刺激因素持續(xù)存在時(shí)，過(guò)強(qiáng)或過(guò)長(zhǎng)時(shí)間的ERS將最終導(dǎo)致細(xì)胞的炎癥反應(yīng)及凋亡[8]。腎組織系膜細(xì)胞內(nèi)含有豐富的內(nèi)質(zhì)網(wǎng)系統(tǒng)，為ERS的發(fā)生提供了結(jié)構(gòu)基礎(chǔ)，同時(shí)糖尿病腎病患者伴隨著高血糖、氧化應(yīng)激、ANGII等多種破壞內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài)的因素[9]，提示持續(xù)存在的ERS可能參與了DN中炎癥反應(yīng)的發(fā)生。本研究檢測(cè)了ERS相關(guān)蛋白IRE1及下游炎癥相關(guān)轉(zhuǎn)錄因子激酶JNK，探討ANGII受體抑制劑纈沙坦抑制炎癥反應(yīng)，保護(hù)腎臟的潛在機(jī)制。

IRE-1(肌醇依賴(lài)酶1)是定位于內(nèi)質(zhì)網(wǎng)膜上介導(dǎo)內(nèi)質(zhì)網(wǎng)信號(hào)的重要蛋白質(zhì)分子,與炎癥反應(yīng)的發(fā)生密切相關(guān)。生理?xiàng)l件下與內(nèi)質(zhì)網(wǎng)腔內(nèi)的蛋白質(zhì)GRP78結(jié)合而處于失活狀態(tài)；內(nèi)質(zhì)網(wǎng)應(yīng)激發(fā)生后,使內(nèi)質(zhì)網(wǎng)腔內(nèi)大量未折疊和/或錯(cuò)誤折疊的蛋白質(zhì)生成增加,可競(jìng)爭(zhēng)結(jié)合GRP78,使其與IRE-1解離,促進(jìn)IRE-1磷酸化為P-IRE1后活化[10]。本研究發(fā)現(xiàn)，DM組大鼠腎臟IRE1α蛋白及核酸表達(dá)上調(diào)，同時(shí)P-IRE1α蛋白量增加，提示DM大鼠腎臟6周時(shí)可能已發(fā)生ERS，6周時(shí)已有明顯炎癥反應(yīng)，可能持續(xù)存在的ERS促進(jìn)了炎癥反應(yīng)的發(fā)生。Cao等[11]研究發(fā)現(xiàn)，在8周時(shí)DM大鼠腎臟中存在GRP78 表達(dá)的增加，ERS激活，給予氯沙坦治療后，內(nèi)質(zhì)網(wǎng)應(yīng)激及相關(guān)凋亡反應(yīng)減輕，但未探及對(duì)炎癥反應(yīng)的影響。本實(shí)驗(yàn)研究表明給予纈沙坦的DM+V組大鼠腎臟IRE1α蛋白及核酸表達(dá)下調(diào)，P-IRE1α蛋白量減少，炎癥反應(yīng)減輕，提示纈沙坦可能減輕了DM大鼠腎臟中ERS狀態(tài)，進(jìn)而減輕了相關(guān)的炎癥反應(yīng)。

JNK (c-Jun 氨基端激酶)是負(fù)責(zé)轉(zhuǎn)錄因子 c-Jun 氨基端磷酸化的激酶。經(jīng)許多生理及病理信號(hào)刺激后(如生長(zhǎng)因子、環(huán)境因素、細(xì)胞因子等應(yīng)激)，JNK被激活并轉(zhuǎn)運(yùn)到細(xì)胞核，使核內(nèi)轉(zhuǎn)錄因子 c-Jun 的氨基末端磷酸化，進(jìn)而激活c-Jun而增強(qiáng)其轉(zhuǎn)錄活性。進(jìn)而引起下游炎癥因子(包括MCP-1)的激活[12]。因此，JNK 通路是介導(dǎo)巨噬細(xì)胞相關(guān)的腎損傷的重要途徑。ERS情況下，活化的IRE1α(P-IRE1α)結(jié)合TRAF2，募集并磷酸化下游蛋白JNK，促進(jìn)轉(zhuǎn)錄因子 C-Jun的激活，從而編碼多種炎癥介質(zhì)的基因表達(dá)[13]。本實(shí)驗(yàn)研究發(fā)現(xiàn)DM組、DM+V組大鼠腎臟JNK蛋白及核酸表達(dá)均無(wú)明顯變化，但是DM組P-JNK蛋白量增加，纈沙坦治療后DM+V組P-JNK蛋白量減少。提示在DM大鼠腎臟中由于ERS導(dǎo)致的JNK磷酸化的激活占主要作用，JNK蛋白表達(dá)并不明顯。纈沙坦可能是抑制了ERS，減少了JNK蛋白的磷酸化，進(jìn)而抑制了炎癥反應(yīng)。

對(duì)于ANGII受體抑制劑纈沙坦如何抑制了ERS，其作用機(jī)制尚不明確，可能與以下因素有關(guān)：①ANGII具有強(qiáng)大的促進(jìn)細(xì)胞增殖和肥大，增加ECM合成的效應(yīng)[14]。使得蛋白質(zhì)的大量合成，增加了內(nèi)質(zhì)網(wǎng)的蛋白負(fù)荷，進(jìn)而引起ERS；②ANGII可誘導(dǎo)系膜細(xì)胞活性氧自由基(Ros)的產(chǎn)生[15]，及血管內(nèi)皮細(xì)胞細(xì)胞內(nèi)Ca2+異常升高[16]，影響內(nèi)質(zhì)網(wǎng)的穩(wěn)態(tài)，引起ERS； AT1受體抑制與其受體結(jié)合，減弱了ERS的激活條件。另外，ARB類(lèi)藥物有明顯的降低尿蛋白的作用，減少了腎小管上皮細(xì)胞對(duì)尿蛋白的重吸收，進(jìn)而減輕腎小管上皮細(xì)胞的ERS的激活，最終減輕了ERS相關(guān)的炎癥反應(yīng)。

總之，我們的研究發(fā)現(xiàn)，糖尿病大鼠腎臟中存在內(nèi)質(zhì)網(wǎng)應(yīng)激和炎癥反應(yīng)的激活，纈沙坦可能部分通過(guò)抑制內(nèi)質(zhì)網(wǎng)應(yīng)激中IRE1/JNK/MCP-1通路，減輕炎癥反應(yīng)，從而發(fā)揮腎臟保護(hù)作用。

參考文獻(xiàn)

[1]Ruilope LM, Malacco E, Khder Y, et al. Efficacy and tolerability of combination therapy with valsartan plus hydrochlorothiazide compared with amlodipine monotherapy in hypertensive patients with other cardiovascular risk factors: The VAST study [J]. Clin Ther, 2005, 27(5): 578- 587.

[2]趙大鵬, 隋艷波, 欒中秋, 等. 糖尿病腎病發(fā)病機(jī)制的研究進(jìn)展 [J]. 中國(guó)醫(yī)藥導(dǎo)報(bào), 2012, 9(36): 47-48.

[3]Wada J, Makino H. Inflammation and the pathogenesis of diabetic nephropathy [J]. Clin Sci (Lond). 2013, 124(3): 139-152

[4]Furukawa M, Gohda T, Tanimoto M, et al. Pathogenesis and novel treatment from the mouse model of type 2 diabetic nephropathy [J]. Sci World J, 2013, 20(13): 917-922.

[5]Fang F, Liu GC, Kim C, et al. Adiponectin attenuates angiotensin II-induced oxidative stress in renal tubular cells through AMPK and cAMP-Epac signal transduction pathways [J]. Am J Physiol Renal Physiol, 2013, 304(11): 1366-1374 .

[6]Titan SMM, Vieira J Jr, Dominguez WV, et al. ACEI and ARB combination therapy in patients with macroalbuminuric diabetic nephropathy and low socioeconomic level: a double-blind randomized clinical trial [J]. Clin Nephrol. 2011, 76(4): 273-283.

[7]Chow FY, Ma FY, Ozols E, et al. Monocyte chemoattractant protein-1-induced tissue inflammation is critical for the development of renal injury but not type 2 diabetes in obese db/db mice [J]. Diabetologia, 2007, 50: 471-480.

[8]Hasnain SZ, Lourie R. The interplay between endoplasmic reticulum stress and inflammation [J]. Immunol Cell Biol, 2012, 90:260-270

[9]Liu G, Sun Y, Li Z, et al. Apoptosis induced by endoplasmic reticulum stress involved in diabetic kidney disease [J]. Biochem Biophys Res Commun, 2008, 370(4): 651-656.

[10] Zhang K, Kaufman RJ. From endoplasmic reticulum stress to the inflammatory response [J]. Nature, 2008, 454(7203): 455-462.

[11]曹延萍, 郝詠梅, 劉青娟, 等. 內(nèi)質(zhì)網(wǎng)應(yīng)激及其特有凋亡途徑Caspase-12與糖尿病大鼠腎組織固有細(xì)胞凋亡之間的關(guān)系. 中國(guó)應(yīng)用生理學(xué)雜志, 2011, 27(2):236-240.

[12]Hu E, Mueller E, Oliviero S, et al. Targeted disruption of the c-fos gene demonstrates c-fos-dependent and -independent pathways for gene expression stimulated by growth factors or oncogenes [J]. EMBO J, 1994, 13(13): 3094-3103.

[13]Liu CM, Zheng GH, Ming QL, et al. Protective effect of quercetin on lead-induced oxidative stress and endoplasmic reticulum stress in rat liver via the IRE1 /JNK and PI3K/Akt pathway [J]. Free Radic Res, 2013, 47: 192-201.

[14]Gorin Y, Ricono JM, Wagner B, et al. Angiotensin II-induced ERKl/ERK2 activation and protein synthesis are redox-dependent in glomerular mesangial cells [J]. Biochem J, 2004, 381(Pt 11): 231-239．

[15]Iturralde M, Gamen S, Pardo J, et al. Saturated free fatty acid release and intracellular ceramide generation during apoptosis induction are closely related processes [J]．Biochim Biophys Acta, 2003, 16(34): 40-51．

[16]Wang WZ, Pang L, Palade P, et al. Angiotensin lI causes endothelial dependent increase in expression of Ca protein in cultured arteries [J]. Eur Pharmacol, 2008, 599: 117-120．

·國(guó)際前沿·

【編者按】本文為丹尼爾整理2014年博士期間工作，發(fā)表在《neurobiology of disease》上的研究論文。他結(jié)合2014年本刊第6期向讀者解讀的：神經(jīng)功能原理模型對(duì)研究者的重要指導(dǎo)作用。從自身實(shí)際研究的角度，談了自己選題、設(shè)計(jì)、實(shí)施……的體會(huì)。本文有助于讀者和研究人員今后的研究實(shí)踐。

Topicsofinterest- ‘TargetingRegulatorsofG-proteinsignalling(RGS)proteinsinmovementdisorders’

Following on from the last article on the descriptions of the basal ganglia model in movement disorders (3rd edition); this related communication refers to our recently published research (Ko et al., 2014) on the pathophysiological roles of RGS proteins in Parkinson’s disease (PD) and L-DOPA-induced dyskinesia (LID).

Although the cause of LID remains unknown, evidence has suggested that repeated, pulsatile stimulation of the dopamine receptors in the basal ganglia contributes to the development of LID. This process is commonly referred to as ‘priming’ and is the abnormal long-term stimulation of dopamine receptor subtypes, which belong to the well-known class of proteins called G-protein coupled receptors (GPCRs). Ultimately, priming causes abrupt functional changes in second messenger signalling mechanisms (Aubert et al., 2005)that lead to the development and expression of dyskinesia.

In our published paper (Ko et al., 2014), we focused our attention on RGS proteins which are known to modulate GPCRs (Hepler, 1999). Specifically, we investigated the role of RGS protein subtype 4 and its pathophysiological role in the expression of PD and LID motor symptoms. Our experiments utilised the well-established 6-hydroxydopamine (6-OHDA)-lesioned rat model (Cenci et al., 1998) and progressed from a series of in vivo to in vitro explorations, in an attempt to fully characterise the functional changes of RGS4 in PD and LID.

The main findings from our research demonstrated that RGS4 proteins were involved in the expression of LID. This was seen following correlation analyses (r=0.93,P<0.06) of RGS4 mRNA with abnormal involuntary movements (AIMs) in L-DOPA-treated 6-OHDA-lesioned rats. Thereafter, we innovatively suppressed the expression of RGS4 mRNA using antisense oligonucleotides, which were chronically delivered into the brain through osmotic mini-pumps. We found that dampening the expression of RGS4 mRNA was able to reduce the induction of AIMs and the subsequent development of marked molecular changes associated with LID, such as dopamine receptor super-sensitisation.Our key findings indicated that such second messenger signalling proteins may provide for novel therapeutic targets for the treatment of movement disorders and/ or other neurological disorders.

References

Aubert I, Guigoni C, H?kansson K, Li Q, Dovero S, Barthe N, Bioulac BH, Gross CE, Fisone G, Bloch B, Bezard E.Increased D1 dopamine receptor signaling in levodopa-induced dyskinesia. Ann Neurol. 2005;57(1):17-26.

Cenci MA, Lee CS, Bj?rklund A.L-DOPA-induced dyskinesia in the rat is associated with striatal overexpression of prodynorphin- and glutamic acid decarboxylase mRNA. Eur J Neurosci. 1998;10(8):2694-2706.

Hepler JR.Emerging roles for RGS proteins in cell signalling.Trends Pharmacol Sci. 1999;20(9):376-382.

Ko WK, Martin-Negrier ML, Bezard E, Crossman AR, Ravenscroft P. RGS4 is involved in the generation of abnormal involuntary movements in the unilateral 6-OHDA-lesioned rat model of Parkinson's disease. Neurobiol Dis. 2014;70: 138-148.

欄目主持：李秦

研究報(bào)告

Inhibitory effect of valsartan on endoplasmic reticulum

stress and inflammation in the diabetic rat kidney

CHEN Kai1, ZHANG Cheng-ying1,2, LI Jian-min3, ZHANG Jian-rong2

(1. Anhui Medical University, Hefei 230032, China; 2. the Armed Police General Hospital, Beijing 100039;

3. Beijing Hospital of Chinese Traditional and Western Medicine, Beijing 100039)

【Abstract】ObjectiveTo study the role of endoplasmic reticulum stress and related inflammation in the kidneys of rats with diabetic nephropathy and the effect of valsartan on these lesions. Methods The diabetic rat model was induced by intraperitoneal injection of streptozotocin. Thirty-four healthy male SD rats were randomly divided into normal control group (n=10), diabetic group (n=12), and valsartan group (n=12). Valsartan (10 mg/kg) was administered daily by gavage from the next day of the diabetes induction for 6 weeks. The expression and distribution of ERS-related proteins P-IRE1α, P-JNK, and MCP-1 were examined by immunohistochemistry and Western blot. Real-time fluorescence quantitative PCR was used to detect the mRNA expressions of IRE1α, JNK and MCP-1. The 24-hour urine protein excretion, Scr, and BUN were checked.ResultsCompared with the control group, infiltration of inflammatory cells was aggravated in the kidneys of DM+V group, the expressions of P-IRE1α,IRE1α,P-JNK,MCP-1 were significantly increased, and the levels of IRE1mRNA and MCP-1mRNA increased compared with the DM group, infiltration of inflammation cells was alleviated in the kidney of DM+V group, the protein expressions of P-IRE1α,IRE1α,P-JNK,MCP-1 were significantly reduced, the levels of IRE1mRNA and MCP-1mRNA were reduced. While there was no significant difference in the expression of JNK mRNA and protions among the three groups. ConclusionsERS and related inflammation are activated in the kidney of diabetic rats. Inhibition of the IRE1/JNK/MCP-1 pathway of ERS and related inflammation might be responsible for the protective effects of valsartan on the kidneys of diabetic rats.

【Key words】Endoplasmic reticulum stress; Inflammation; Diabetic nephropathy; Valsartan

[收稿日期]2014-11-19

Doi:10.3969/j.issn.1005-4847.2015.02.006

【中圖分類(lèi)號(hào)】Q95-33

【文獻(xiàn)標(biāo)識(shí)碼】A

【文章編號(hào)】1005-4847(2015) 02-0132-06

[通訊作者]張承英，碩士生導(dǎo)師。Email： zhangchy1969@126.com

[作者簡(jiǎn)介]陳凱(1986-)，男，碩士研究生，研究方向：糖尿病腎病的發(fā)病機(jī)制及治療。Email： chenk.ly@163.com

[基金項(xiàng)目]國(guó)家自然基金面上資助項(xiàng)目(No.81273706)。