丁敏,李春君,邢云芝,于倩,王鵬華,于德民
專題研究·內(nèi)分泌疾病
GLP-1Ra減少高糖誘導(dǎo)的β細胞凋亡作用機制探討
丁敏,李春君,邢云芝,于倩,王鵬華,于德民△
目的探討胰高血糖素樣肽-1(GLP-1)受體激動劑(GLP-1Ra)減少高糖誘導(dǎo)的β細胞凋亡作用的可能機制。方法正常對照(N,普通飼料喂養(yǎng))組、2型糖尿?。═2DM)組和GLP-1 Ra組[利拉魯肽200 μg/(kg·d)]大鼠干預(yù)12周。比較各組大鼠造模前、造模后給藥前(0周)及12周末血糖水平。高壓液相色譜分析法測定糖化血紅蛋白(HbA1c);全自動生化分析儀測定天冬氨酸轉(zhuǎn)氨酶(AST)、肌酐(CR)及尿素氮(BUN)等;TUNEL染色觀察胰島細胞凋亡情況;免疫組化法測定cleaved caspase 3;DCFH-DA熒光探針檢測胰島活性氧簇(ROS);免疫組織化學(xué)法檢測NADPH氧化酶(NOX)催化亞基(NOX2)。結(jié)果12周時,GLP-1Ra組的血糖、HbA1c、總膽固醇(TC)及低密度脂蛋白膽固醇(LDL-c)水平均低于T2DM組(P<0.05);GLP-1Ra組胰島細胞凋亡率和cleaved caspase 3水平較T2DM組下降(P<0.05);應(yīng)用Apocynin抑制前,GLP-1Ra組胰島ROS水平明顯低于T2DM組,并與N組差異無統(tǒng)計學(xué)意義(P>0.05),應(yīng)用Apocynin抑制后,各組間差異均無統(tǒng)計學(xué)意義(P>0.05)。GLP-1Ra組胰島NOX2水平較T2DM組下降(P<0.05)。結(jié)論GLP-1Ra能抑制糖尿病大鼠β細胞的凋亡,抑制NOX2來源的ROS產(chǎn)生可能是重要的潛在機制之一。
糖尿病,2型;疾病模型,動物;胰高糖素樣肽-1;NOX2;氧化應(yīng)激;凋亡;利拉魯肽;活性氧簇
抑制β細胞凋亡是預(yù)防和減緩2型糖尿病(T2DM)進展的根本,胰高血糖素樣肽-1(GLP-1)受體激動劑(GLP-1Ra)——利拉魯肽可顯著改善2型糖尿病患者胰島β細胞功能已獲得共識,但其具體作用機制尚未明確。研究證實,高糖、高脂導(dǎo)致的氧化應(yīng)激與β細胞凋亡密切相關(guān),而GLP-1能降低氧化應(yīng)激誘導(dǎo)的β細胞凋亡[1]。但GLP-1抑制β細胞活性氧簇(ROS)產(chǎn)生的機制尚不完全清楚。近年有研究證實,NADPH氧化酶(NOX)催化亞基(NOX2)是β細胞產(chǎn)生ROS的重要來源,抑制NOX2可減少ROS產(chǎn)生及β細胞凋亡[2],且它能通過作用于NOX2-ROS軸發(fā)揮對心肌細胞的保護作用[3]。但GLP-1 Ra是否通過抑制β細胞NOX2水平最終達到已知的抑制ROS產(chǎn)生和減少β細胞凋亡的作用,目前尚少見相關(guān)研究。本研究旨在對該假設(shè)進行初步探討,為尋找延緩糖尿病患者胰島β細胞凋亡的治療方法提供參考。
1.1 材料8周齡清潔級SD大鼠購自北京華阜康生物科技股份有限公司。血糖試紙及血糖儀購自上海羅氏制藥有限公司。利拉魯肽由諾和諾德中國制藥有限公司贊助。鏈脲佐菌素(STZ)購自美國Sigma公司。通用性免疫組化試劑盒購自美國Proteintech公司。微量注射器購自上海醫(yī)用制品有限公司。RPMI 1640培養(yǎng)基、胎牛血清購自北京Solarbio科學(xué)技術(shù)公司;抗NOX2/gp91phox及cleaved caspase 3抗體購自美國Abcam公司。ROS檢測試劑盒購自北京泛博生物化學(xué)有限公司;一步法TUNEL細胞凋亡檢測試劑盒購自碧云天生物技術(shù)公司。
1.2 方法
1.2.1 T2DM大鼠模型的構(gòu)建及分組36只SD大鼠適應(yīng)性飼養(yǎng)1周,采用隨機數(shù)字表法隨機分為3組,每組12只。隨機血糖>11.1 mmol/L為造模成功。對照(N)組普通飼料喂養(yǎng),剩余大鼠高糖高脂飲食喂養(yǎng)8周,致胰島素抵抗,第9周應(yīng)用小劑量STZ(30 mg/kg)尾靜脈注射,1周內(nèi)2次,2只大鼠血糖未達到成模標準予以剔除,成模率91.7%。造模成功后1周,22只大鼠根據(jù)血糖水平再隨機分為T2DM組及GLP-1Ra組,每組11只。N組及T2DM組給予注射用水10 μL/d頸后皮下注射,2組均連續(xù)給予12周;GLP-1Ra組給予頸后皮下注射100 μg/(kg·d)GLP-1Ra 1周后,劑量增加至200 μg/(kg·d),連續(xù)給予11周。大鼠均分籠飼養(yǎng),自由進食進水,并根據(jù)墊料情況每1~2 d更換墊料,保持籠內(nèi)干燥。至實驗結(jié)束,N組大鼠死亡1只,T2DM組死亡3只,GLP-1Ra組死亡2只。
1.2.2 血糖及生化指標監(jiān)測各組大鼠每2周禁食12 h后應(yīng)用羅氏血糖儀剪尾法測定血糖水平。比較各組大鼠造模前、造模后給藥前(0周)及12周末血糖水平。12周末處死大鼠后取血,采用高壓液相色譜分析法測定糖化血紅蛋白(HbA1c),全自動生化分析儀測定丙氨酸轉(zhuǎn)氨酶(ALT)、肌酐(CR)、尿素氮(BUN)、總膽固醇(TC)、三酰甘油(TG)、高密度脂蛋白膽固醇(HDL-c)和低密度脂蛋白膽固醇(LDL-c)水平。
1.2.3 胰腺組織取材及組織切片制備第12周末處死大鼠。迅速留取胰腺組織,取近胰頭的1/2置于10%多聚甲醛溶液中進行固定,24 h后常規(guī)脫水透明,石蠟包埋,用于制備免疫組織化學(xué)切片。
1.2.4 大鼠胰島的消化、提取與培養(yǎng)大鼠禁食12 h后用10%的水合氯醛腹腔注射麻醉。固定大鼠,用75%的乙醇消毒皮膚,取正中切口,逐層分離皮膚、肌肉,并暴露腹腔臟器。分離膽總管,于近肝門處以1號絲線結(jié)扎,并于近十二指腸乳頭端放置1號絲線備用。破心放血處死大鼠。用0.45 mm的留置針由十二指腸乳頭進針向膽總管內(nèi)穿刺,視體積而定向胰腺內(nèi)灌注約8~10 mL 1 g/L的Ⅴ型膠原酶,至胰腺完全膨脹。迅速完整切取胰腺,盡量剔除血管及脂肪組織,置于50 mL離心管中,(37±1)℃水浴17 min。冰浴下迅速倒入4℃的Hanks液約40~50 mL,終止消化。在15 mL的離心管中劇烈震蕩胰腺組織,至呈泥沙狀,轉(zhuǎn)移至50 mL離心管中,冰浴10 min,待其靜置分層,吸棄上層液體,加入40~50 mL Hanks液。重復(fù)2次。將所得消化物置于平皿上,體視顯微鏡下手工挑揀形狀規(guī)則的圓形胰島,放入含11.1 mmol/L葡萄糖的RPMI 1640培養(yǎng)基中,37℃含5%CO2的溫箱孵育備用。
1.2.5 胰島細胞凋亡檢測和cleaved caspase 3水平測定(1)胰島細胞凋亡檢測。按一步法TUNEL細胞凋亡檢測試劑盒說明書檢測胰島細胞凋亡,凋亡細胞核固縮,呈棕黃色。(2)胰島cleaved caspase 3水平測定。用3%H2O2進行內(nèi)源性過氧化物酶的封閉,用微波加熱抗原修復(fù),10%的山羊血清孵育,滴加一抗(濃度1∶200),4℃室溫過夜。PBS緩沖液沖洗后滴加二抗,DAB顯色,蘇木素復(fù)染,常規(guī)封片。每只大鼠選擇3張胰腺病理圖片,每張隨機取5個視野用Image-Pro Plus 6.0圖像分析系統(tǒng)拍攝圖片,并測定平均光密度(OD)值。
1.2.6 胰島ROS水平測定摘取的胰島在RPMI 1640培養(yǎng)基中孵育1 h,3組每只大鼠各取60個胰島,分裝入2個5 mL Ep管(每管30個胰島)中,1 000×g離心10 s后棄上清液,然后加入新培養(yǎng)基。3組任選一個Ep管加入100 mg/L的Apocynin 200 μL,放入37℃溫箱孵育3 h,按說明書裝載DCFH-DA探針15 min。另一組直接加入稀釋的DCFH-DA放入,37℃溫箱孵育15 min。溫育結(jié)束后吸棄上清液,用緩沖液洗滌2次,應(yīng)用熒光顯微鏡觀察(藍光激發(fā)綠光),并拍攝圖片。每組隨機取5個視野,顯色結(jié)果采用Image-Pro
Plus 6.0圖像分析系統(tǒng)測定ROS水平。比較各組Apocynin干預(yù)(抑制)前、后的ROS水平。
1.2.7 胰島NOX2免疫組織化學(xué)染色具體步驟同cleaved caspase 3水平測定,一抗?jié)舛?∶150。陽性為棕黃色顆粒,位于胞漿。
1.3 統(tǒng)計學(xué)方法釆用SPSS 17.0軟件進行統(tǒng)計分析。正態(tài)分布的計量數(shù)據(jù)采用均數(shù)±標準差表示,多組間均數(shù)比較用方差分析,組間多重比較采用LSD-t法;抑制前后相關(guān)指標比較用配對t檢驗。不符合正態(tài)分布時以M(P25,P75)表示,組間比較采用秩和檢驗。P<0.05為差異有統(tǒng)計學(xué)意義。
2.1 各組大鼠血糖變化情況比較各組造模前血糖水平差異無統(tǒng)計學(xué)意義;造模后0周和12周,T2DM組及GLP-1Ra組高于N組(P<0.05),且12周時GLP-1Ra組低于T2DM組(P<0.05)。T2DM組和GLP-1Ra組HbA1c均高于N組,GLP-1Ra組低于T2DM組(P<0.05)。見表1。
Tab.1Comparison of blood glucose levels before and after the experiment between three groups of rats表1 3組大鼠實驗前后血糖水平的比較
Tab.1Comparison of blood glucose levels before and after the experiment between three groups of rats表1 3組大鼠實驗前后血糖水平的比較
**P<0.01;a與N組比較,b與T2DM組比較,P<0.05
血糖(m m o l / L)組別N組T 2 D M組G L P -1 R a組F n 造模前5 . 3 8 ± 0 . 1 9 5 . 4 0 ± 0 . 6 0 5 . 4 5 ± 0 . 7 1 4 . 7 7 6 0周4 . 8 5 ± 0 . 2 0 1 9 . 2 9 ± 2 . 5 4 a 2 1 . 3 1 ± 1 . 5 2 a 1 3 7 . 7 6 6 * 1 2周5 . 0 0 ± 0 . 1 2 2 6 . 6 7 ± 0 . 7 4 a 2 1 . 5 8 ± 0 . 7 1 a b 1 8 3 4 . 2 7 8 * * H b A 1 c(%)2 . 5 0 ± 0 . 0 8 3 . 8 1 ± 0 . 1 5 a 2 . 7 3 ± 0 . 1 0 a b 1 4 8 . 5 7 1 * * 1 1 8 9
2.2 各組生化指標結(jié)果比較3組TC及LDL-c水平差異有統(tǒng)計學(xué)意義(P<0.05),其中GLP-1Ra組TC、LDL-c水平均低于T2DM組(P<0.05),見表2。
2.3 各組胰島細胞凋亡及cleaved caspase 3水平比較(1)細胞凋亡。凋亡的胰島細胞內(nèi)見棕黃色顆粒,位于細胞核(紅色箭頭所示),見圖1A。N組、T2DM組及GLP-1Ra組的平均OD值分別為0.225± 0.022、0.332±0.041及0.271±0.057(F=20.208,P<0.05),其中T2DM組較N組增多,GLP-1Ra較T2DM組減少,2組均高于N組(P<0.05)。(2) cleaved caspase 3水平。cleaved caspase 3陽性細胞內(nèi)存在棕黃色顆粒,位于胞漿,見圖1B。N組、T2DM組及GLP-1Ra組的平均OD值分別為0.747± 0.182、2.485±0.127及1.609±0.141(F=132.943,P<0.05),其中T2DM組高于N組和GLP-1Ra(P<0.05),但GLP-1組與N組差異無統(tǒng)計學(xué)意義。
2.4 GLP-1Ra對T2DM大鼠胰島ROS的影響(1)3組間抑制前后比較。抑制前,T2DM組胰島ROS水平高于N組和GLP-1Ra組(P<0.05),但GLP-1Ra組與N組間差異無統(tǒng)計學(xué)意義(P>0.05);抑制后,3組間ROS水平差異無統(tǒng)計學(xué)意義(P>0.05)。(2)各組內(nèi)抑制前后比較。N組及T2DM組抑制后的ROS水平均低于抑制前(P<0.05),GLP-1Ra組抑制前后ROS水平差異無統(tǒng)計學(xué)意義(P>0.05)。見表3。
Tab.3Comparison of ROS levels of islets before and after treatment with Apocynin between three groups表3 各組Apocynin抑制前后胰島ROS水平的比較
Tab.3Comparison of ROS levels of islets before and after treatment with Apocynin between three groups表3 各組Apocynin抑制前后胰島ROS水平的比較
*P<0.05,**P<0.01;a與N組比較,b與T2DM組比較,P<0.05
n t 8 9 5 . 1 3 6 * 6 . 0 5 4 * 1 . 6 9 0 1 1組別N組T 2 D M組G L P -1 R a組F抑制前0 . 7 7 1 ± 0 . 2 2 8 1 . 2 5 7 ± 0 . 2 9 0 a 0 . 7 6 6 ± 0 . 1 2 0 b 1 1 . 0 8 7 * *抑制后0 . 3 3 1 ± 0 . 0 7 1 0 . 6 4 2 ± 0 . 2 1 0 0 . 6 0 5 ± 0 . 2 3 1 3 . 5 3 9
2.5 GLP-1Ra對T2DM大鼠胰島NOX2水平的影響各組大鼠胰島組織細胞膜及胞漿中均可見棕黃色的陽性顆粒,見圖2。N組、T2DM組及GLP-1Ra組NOX2的平均OD值分別為0.737±0.068、1.616± 0.101及1.020±0.258(F=26.036,P<0.05),其中T2DM組最高,GLP-1Ra組次之,N組最低(P<0.05)。
Tab.2Comparison of biochemical indicators between three groups of rats表2 各組大鼠生化指標的比較
研究證實,T2DM患者中β細胞的數(shù)量減少主要與β細胞的凋亡增加有關(guān)[4]。GLP-1Ra利拉魯肽為一種新的降糖藥物,加用治療能使HbAlc下降1.26%~1.36%,且無低血糖等明顯不良反應(yīng)[5-6]。本研究結(jié)果顯示,經(jīng)過12周GLP-1Ra治療,GLP-1Ra
組大鼠HbA1c水平較T2DM組下降約1.1%,顯示出其良好的降糖效果。另有研究顯示,GLP-1Ra在降血糖[7]的同時,可以減輕體質(zhì)量[8-9]、降低血脂及改善胰島功能等[10-11]。本研究結(jié)果顯示,T2DM組大鼠TC及LDL-c水平較N組明顯升高,而GLP-1Ra治療后兩者水平則明顯降低,提示GLP-1Ra除能直接降血糖外,還可以改善血脂代謝。
此外,本研究顯示,T2DM組較N組胰島細胞凋亡明顯升高,而經(jīng)GLP-1Ra治療后,胰島細胞的凋亡率明顯得到改善,這可能是GLP-1Ra降低血糖、改善胰島功能的主要機制。多項研究已經(jīng)證實,高糖及炎癥等誘導(dǎo)的氧化應(yīng)激過度激活是導(dǎo)致β細胞凋亡的重要原因[12-13],且是糖尿病及其并發(fā)癥的共同致病因素[14]。本研究顯示,T2DM組胰島ROS水平較N組升高,提示高糖狀態(tài)下氧化應(yīng)激被明顯激活,而給予GLP-1Ra處理后胰島內(nèi)ROS水平下降,且與N組差異無統(tǒng)計學(xué)意義,提示GLP-1Ra具有良好的抗氧化作用。近年來應(yīng)用Exendin-4(Ex-4,另一種GLP-1Ra)進行的相關(guān)研究結(jié)果已經(jīng)證實,Ex-4可以通過減少c-Jun氨基末端激酶(JNK)和糖原合成激酶3β(GSK3β)的激活減少氧化應(yīng)激誘導(dǎo)的β細胞凋亡[1]。但其抑制ROS產(chǎn)生的具體機制尚不明確。Syed等[15]研究證實,NOX2是糖尿病胰島ROS產(chǎn)生的重要來源。Xiang等[16]應(yīng)用基因敲除技術(shù)對NOX2-/-的C57BL/6大鼠進行相關(guān)研究顯示,NOX2敲除后能通過降低ROS水平和免疫反應(yīng),減弱STZ誘導(dǎo)的嚴重的高血糖,減少胰島細胞數(shù)量。另有研究證實,高糖可以激活I(lǐng)NS-1 832/13細胞Apocynin敏感的NOX2,使INS-1細胞ROS水平升高約2倍,而NOX2在高糖導(dǎo)致的β細胞ROS的產(chǎn)生中起重要的作用[17]。針對心肌細胞進行的研究顯示,GLP-1能通過作用于CML-NOX2-ROS軸抑制心臟的炎癥前狀態(tài),發(fā)揮對心肌細胞的保護作用[3],但在β細胞中的作用尚未明確。
本研究顯示,與N組相比,T2DM組的NOX2水平明顯升高,GLP-1Ra組較T2DM組下降,提示GLP-1Ra能減少高糖導(dǎo)致的胰島內(nèi)NOX2表達水平。同時,對各組大鼠摘取的胰島進行ROS水平的測定結(jié)果同樣顯示,T2DM組大鼠胰島的ROS水平較N組明顯升高,GLP-1Ra組則明顯降低,且與N組無差異。由于NOX2是胰島內(nèi)ROS產(chǎn)生的重要來源,由此推斷,GLP-1Ra治療對NOX2的抑制可能是GLP-1Ra組ROS水平下降的重要原因。進一步給予特異性NOX2抑制劑干預(yù)后結(jié)果顯示,N組及T2DM組ROS水平有明顯下降,且抑制后T2DM組與GLP-1Ra組水平差異無統(tǒng)計學(xué)意義,GLP-1Ra組抑制后ROS水平與抑制前差異無統(tǒng)計學(xué)意義,提示給予特異性NOX2抑制劑干預(yù)前,胰島內(nèi)的ROS已被GLP-1Ra明顯抑制,而且這些ROS主要為NOX2來源,因此繼續(xù)應(yīng)用NOX2抑制劑后才未顯示出進一步的明顯的降低。
綜上所述,GLP-1Ra可能通過抑制NOX2來源的ROS產(chǎn)生,從而減少β細胞凋亡,這為尋找延緩T2DM患者β細胞凋亡的方法提供了思路。
(圖1~2見插頁)
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(2015-09-14收稿 2015-10-30修回)
(本文編輯 陸榮展)
Glucagon-like peptide 1 receptor agonist protects high-glucose induced β cells apoptosis via inhibition of NOX2-dependent ROS production
DING Min,LI Chunjun,XING Yunzhi,YU Qian,WANG Penghua,YU Demin△
Metabolic Diseases Hospital&Tianjin Institute of Endocrinology,Tianjin Medical University/Ministry of Health Key Laboratory of Hormones and Development,Tianjin 300070,China△
ObjectiveTo investigate the possible mechanisms of glucagon-like peptide 1 receptor agonists(GLP-1Ra)protection against hyperglycemic induced beta cell apoptosis through depression of NOX2-dependent ROS production. MethodsThe rat model of type 2 diabetes(T2DM)was established by injecting small doses of streptozotocin(STZ)fol?lowed by 8-week high fat diet.The experimental animals were divided into three groups:normal control(N)group,diabetes(T2DM)group and GLP-1Ra group[treated with liraglutide 200 μg/(kg·d)for 12 weeks].The blood glucose levels were compared before and after modeling,before treatment and 12-week after treatment with GLP-1Ra.The level of glycosylated hemoglobin(HbA1c)was detected by high-pressure liquid chromatography.Automatic biochemical analyzer was used to de?tect levels of aspertate aminotransferase(AST),creatinine(CR)and urea nitrogen(BUN).The apoptotic rates of islets were determined by TUNEL method and cleaved caspase 3 was detected by immunohistochemistry.DCFH-DA fluorescent probe was used to detect reactive oxygen species(ROS)levels of islets.Levels of NADPH oxidase(NOX)catalytic subunit(NOX2)in islets were measured by immunohistochemistry.ResultsAt the end of the study,glycemic control(average blood glucose/ week and HbA1c)and lipid situation were improved significantly in the GLP-1Ra group than those of N group(P<0.05). TUNEL staining and displayed that β cell apoptotic and cleaved caspase 3 level were significantly decreased in GLP-1Ra group compared to those of T2DM group(P<0.05).ROS levels were significantly decreased in GLP-1Ra group than those of T2DM group before treatment with Apocynin,but no significant difference between GLP1-Ra group and N group(P>0.05). After application Apocynin for inhibition,there were no significant differences between three groups(P>0.05).The level of NOX2 was significantly lower in GLP-1Ra group compared to that of T2DM group(P<0.05).ConclusionGLP-1Ra can inhibit apoptosis of β cells in diabetes rat,and the depression of NOX2-dependent ROS may be one of the important underly?ing mechanisms.
diabetes,type 2;disease models,animal;Glucagon-like peptide-1;NOX2;oxidative stress;apoptosis;liraglutide;ROS
R587.1
A
10.11958/j.issn.0253-9896.2015.11.001
國家自然科學(xué)基金青年科學(xué)基金項目(81300663);天津市衛(wèi)生局科技基金(2013KZ098)
天津醫(yī)科大學(xué)代謝病醫(yī)院,衛(wèi)生部激素與發(fā)育重點實驗室(郵編300070)
丁敏(1981),女,主治醫(yī)師,主要從事糖尿病及其并發(fā)癥相關(guān)機制的研究
△通訊作者E-mail:yudemintij@126.com