陳凱,張承英,,李建民,張建榮
(1. 安徽醫(yī)科大學武警總醫(yī)院臨床學院,合肥 230032;2. 中國人民武裝警察部隊總醫(yī)院,北京 100039;
3. 北京市中西醫(yī)結(jié)合醫(yī)院,北京 100039)
?
纈沙坦對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)科大學武警總醫(yī)院臨床學院,合肥230032;2. 中國人民武裝警察部隊總醫(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。將大鼠隨機分為對照組(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ERS相關(guān)蛋白P-IRE1α、P-JNK及中性粒細胞趨化因子MCP-1的表達及定位,實時熒光定量PCR(FQ-PCR)檢測IRE1α、JNK及MCP-1mRNA的表達變化,同時觀察各組大鼠尿蛋白、BUN、Scr等指標的變化。 結(jié)果 與Con組相比,DM組大鼠腎臟病理炎細胞浸潤加重,P-IRE1α、IRE1α、P-JNK、MCP-1蛋白表達上調(diào),IRE1α mRNA、MCP-1mRNA表達水平上調(diào);與DM組相比,DM+V組腎臟病理炎癥細胞浸潤減輕,P-IRE1α、IRE1α、P-JNK、MCP-1蛋白表達下調(diào),IRE1α mRNA、MCP-1 mRNA表達水平下調(diào)。3組間JNK mRNA及蛋白表達無明顯差異。 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)揮腎臟保護作用。
【關(guān)鍵詞】內(nèi)質(zhì)網(wǎng)應(yīng)激;炎癥反應(yīng);糖尿病腎?。焕i沙坦
糖尿病腎病(diabetic nephropathy,DN)是糖尿病的微血管并發(fā)癥,也是導致終末期腎病(end-stage renal disease, ESRD)最常見的基礎(chǔ)疾病之一。其發(fā)病機制復雜,近年來,代謝性炎癥與糖尿病腎病的關(guān)系越來越受到重視。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,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胞趨化因子MCP-1表達的影響,探討其抑制炎癥反應(yīng)的相關(guān)機制。
1材料與方法
1.1材料
1.1.1實驗試劑
鏈脲佐菌素(streptozotocin,STZ)購于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抗體均購于美國Santa Cruz公司,兔抗鼠MCP-1抗體購于北京博奧森公司,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實驗動物
SPF級SD雄性大鼠,體重190~210 g,購自北京維通利華實驗動物有限公司【SCXK(京)2011-0011】,飼養(yǎng)于北京中醫(yī)藥大學東直門醫(yī)院屏障環(huán)境動物室【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,隨機分為對照組(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,測定血糖≥16.7 mmol/L,尿糖+++~尿糖++++者確定為糖尿病大鼠。對照組血糖4~6 mmol/L 左右。所有大鼠試驗期間不用外源性胰島素,避免胰島素干擾實驗過程。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。實驗期間動物自由進食,整個實驗過程中DM、DM+V組各有1只大鼠死亡,各有1只大鼠血糖未達到模型標準,予以剔除。最終共30只完成實驗,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, 用于測定24 h尿蛋白定量;下腔靜脈取血3 mL,用于測定血肌酐、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)學改變及炎細胞浸潤情況。
1.2.3免疫組織化學檢測
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)源性過氧化物酶,抗原熱修復。正常山羊血清封閉。一抗 P-IRE1α、IRE1α、P-JNK、JNK( 1∶100) 稀釋,二抗為生物素化山羊抗兔IgG,PBS 替代一抗作為陰性對照,DAB 顯微鏡控制下顯色,蘇木素復染。
1.2.4Westen blot檢測蛋白表達
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℃過夜, 洗膜后加辣根過氧化物酶標記的羊抗兔抗體(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Western條帶進行定量分析, 確定雜交條帶的吸光度值。
1.2.5實時熒光定量PCR(FQ-PCR)檢測mRNA表達
按照Trizol 試劑盒說明書提取各組大鼠腎組織總RNA,并參照逆轉(zhuǎn)錄擴增試劑盒操作程序進行cDNA 合成。采用2-△△CT法進行數(shù)據(jù)的相對定量分析。擴增條件為: 94℃預(yù)變性10 min,活化Tag 酶,94℃ 15 s, 60℃ 60 s,45 個循環(huán)結(jié)束,每個cDNA 樣品在96孔板均設(shè)置3個復孔,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)計學分析
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,但差異無統(tǒng)計學意義(P>0.05)(見表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改變
光鏡下Con組腎組織未見明顯病理改變,DM組大鼠腎小球明顯肥大,腎小球細胞外基質(zhì)略增生,毛細血管袢明顯擴張,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。(見圖1)。
2.3免疫組織化學檢測 P-IRE1α、P-JNK、MCP-1蛋白在腎臟的定位表達
P-IRE1α、P-JNK、MCP-1在Con組大鼠腎皮質(zhì)均有表達,陽性細胞為細胞核呈棕黃色,在100 倍視野中連續(xù)不重疊的計數(shù)100 個腎小管的細胞總數(shù)和陽性細胞數(shù)及50 個腎小球的陽性細胞數(shù),以腎小管細胞的陽性率和單個腎小球切面的陽性細胞數(shù)作為比較指標。P-IRE1α在Con組弱表達,主要位于遠端小管和集合管上皮細胞胞質(zhì)及胞核內(nèi);DM組表達明顯增多,腎小球及腎小管胞質(zhì)胞核均明顯表達。P-JNK在Con組弱表達,主要位于近端及遠端腎小管上皮細胞胞質(zhì)內(nèi);DM組表達明顯增多,主要位于腎小球系膜細胞及腎小管上皮細胞內(nèi),以腎小管上皮細胞表達明顯,部分上皮細胞腎小球呈強陽性。MCP-1在Con組表達極弱; DM組表達明顯增強,尤以遠曲小管胞質(zhì)最為明顯,部分腎小球內(nèi)細胞胞質(zhì)也呈強陽性表達。DM+V組P-IRE1α、P-JNK、MCP-1表達部位與DM組大致相同,程度介于Con組、DM組之間(見圖2)。
注: A為Con組,腎組織未見明顯病理改變;B為DM組,腎小球明顯肥大,腎小球系膜細胞輕度增生,腎小管上皮細胞腫脹,可見空泡變性;C為DM+V組,腎小球及腎小管病變相對于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蛋白陽性表達,黑色箭頭指示腎小管目標蛋白陽性表達?! D2 各組大鼠腎皮質(zhì)免疫組化P-IRE1α、P-JNK及MCP-1蛋白表達變化(×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IRE1α、P-IRE1α、P-JNK、JNK、MCP-1蛋白表達情況
JNK表達在各組均無明顯差異。與Con組相比,DM組IRE1α、P-IRE1α、P-JNK、MCP-1表達增高(P<0.01)。與DM組相比,DM+V組IRE1α、P-IRE1α、P-JNK、MCP-1表達降低(P<0.01)(見圖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 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法檢測腎組織mRNA 的表達情況
6周末,Con組大鼠腎組織IRE1α、JNK、MCP-1mRNA 僅有微量表達。與Con組比較,DM組IRE1α、MCP-1 mRNA表達明顯增高,差異有統(tǒng)計學意義(P<0.01),JNK mRNA表達亦有增高,差異無統(tǒng)計學意義(P>0.05); 與DM組相比,DM+V組IRE1α、MCP-1mRNA表達量明顯降低(P<0.01,P<0.05), JNK mRNA表達差異無統(tǒng)計學意義(P>0.05)(見圖4)。
注:與Con組相比,** P<0.01;與DM組相比,##P<0.01,#P<0.05?! D4 各組大鼠腎組織IRE1α、JNK和MCP-1mRNA的表達變化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)病機制除蛋白激酶C(PKC)學說、氧化應(yīng)激(ROS)學說、細胞因子學說及遺傳分子學說外[2],炎癥學說目前倍受關(guān)注。微炎癥反應(yīng)和隨后的細胞外基質(zhì)擴張是上述機制在糖尿病腎病中進展的共同途徑,炎癥通路在糖尿病腎病的進展中發(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,這從湖南省從嚴治黨、依法治理方面就可見一斑。比如,記者近日在湖南省高級人民法院采訪,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。被告某村委會三年時間里在原告田某(某鎮(zhèn)郵電局的一名臨聘人員)處訂閱報紙,三年共欠原告報刊費1409元,并向原告田某出具了一張條據(jù)。后來,該村村委會換屆,原告多次找被告某村委會催要此款,被告某村委會卻以“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求法院判令被告某村委會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,從而導致腎臟局部ANGII的濃度增加[4]。體外實驗已發(fā)現(xiàn),ANGII可誘導腎小管細胞的NF-κB的激活,進而引起炎癥反應(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(中性粒細胞趨化因子1)呈下降趨勢。腎臟 MCP-1 的增加是糖尿病腎病患者腎臟損傷的顯著特點,是導致單核巨噬細胞浸潤腎組織的重要因素[7]。本實驗研究中我們也發(fā)現(xiàn),DM組大鼠模型6周時腎組織中的MCP-1表達量增高,同時炎性細胞浸潤明顯。DM+V組,MCP-1蛋白表達水平下調(diào),同時炎癥細胞浸潤減輕,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胞聚集,抑制炎癥反應(yīng)的作用。
然而對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所,當各種因素導致內(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)錯誤折疊及未折疊蛋白在腔內(nèi)聚集的狀態(tài)稱為內(nèi)質(zhì)網(wǎng)應(yīng)激。適度的ERS可以恢復ER及內(nèi)環(huán)境穩(wěn)態(tài),保持細胞活性,但當外界刺激因素持續(xù)存在時,過強或過長時間的ERS將最終導致細胞的炎癥反應(yīng)及凋亡[8]。腎組織系膜細胞內(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),保護腎臟的潛在機制。
IRE-1(肌醇依賴酶1)是定位于內(nèi)質(zhì)網(wǎng)膜上介導內(nèi)質(zhì)網(wǎng)信號的重要蛋白質(zhì)分子,與炎癥反應(yīng)的發(fā)生密切相關(guān)。生理條件下與內(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/或錯誤折疊的蛋白質(zhì)生成增加,可競爭結(jié)合GRP78,使其與IRE-1解離,促進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蛋白及核酸表達上調(diào),同時P-IRE1α蛋白量增加,提示DM大鼠腎臟6周時可能已發(fā)生ERS,6周時已有明顯炎癥反應(yīng),可能持續(xù)存在的ERS促進了炎癥反應(yīng)的發(fā)生。Cao等[11]研究發(fā)現(xiàn),在8周時DM大鼠腎臟中存在GRP78 表達的增加,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,但未探及對炎癥反應(yīng)的影響。本實驗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),進而減輕了相關(guān)的炎癥反應(yīng)。
JNK (c-Jun 氨基端激酶)是負責轉(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刺激后(如生長因子、環(huán)境因素、細胞因子等應(yīng)激),JNK被激活并轉(zhuǎn)運到細胞核,使核內(nèi)轉(zhuǎn)錄因子 c-Jun 的氨基末端磷酸化,進而激活c-Jun而增強其轉(zhuǎn)錄活性。進而引起下游炎癥因子(包括MCP-1)的激活[12]。因此,JNK 通路是介導巨噬細胞相關(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[13]。本實驗研究發(fā)現(xiàn)DM組、DM+V組大鼠腎臟JNK蛋白及核酸表達均無明顯變化,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導致的JNK磷酸化的激活占主要作用,JNK蛋白表達并不明顯。纈沙坦可能是抑制了ERS,減少了JNK蛋白的磷酸化,進而抑制了炎癥反應(yīng)。
對于ANGII受體抑制劑纈沙坦如何抑制了ERS,其作用機制尚不明確,可能與以下因素有關(guān):①ANGII具有強大的促進細胞增殖和肥大,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)的蛋白負荷,進而引起ERS;②ANGII可誘導系膜細胞活性氧自由基(Ros)的產(chǎn)生[15],及血管內(nèi)皮細胞細胞內(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類藥物有明顯的降低尿蛋白的作用,減少了腎小管上皮細胞對尿蛋白的重吸收,進而減輕腎小管上皮細胞的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)揮腎臟保護作用。
參考文獻
[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)病機制的研究進展 [J]. 中國醫(yī)藥導報, 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胞凋亡之間的關(guān)系. 中國應(yīng)用生理學雜志, 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)功能原理模型對研究者的重要指導作用。從自身實際研究的角度,談了自己選題、設(shè)計、實施……的體會。本文有助于讀者和研究人員今后的研究實踐。
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
【中圖分類號】Q95-33
【文獻標識碼】A
【文章編號】1005-4847(2015) 02-0132-06
[通訊作者]張承英,碩士生導師。Email: zhangchy1969@126.com
[作者簡介]陳凱(1986-),男,碩士研究生,研究方向:糖尿病腎病的發(fā)病機制及治療。Email: chenk.ly@163.com
[基金項目]國家自然基金面上資助項目(No.81273706)。