張?zhí)煺? 譚曉華 吳翠嬌
[摘要] 目的 通過觀察內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)因子在缺血再灌注以及缺血后處理(IPO)腎臟中的表達,探討IPO對缺血再灌注損傷的保護作用及機制。
方法 將36只雄性SD大鼠隨機分為假手術(shù)組(Sham組)、缺血再灌注組(IR組)和IPO組。在給予各組大鼠相應(yīng)處理后24 h,采集大鼠下腔靜脈血,檢測血清中肌酐和尿素氮的濃度,評估腎功能;采集腎臟標(biāo)本進行蘇木精-伊紅(HE)染色,觀察組織形態(tài)變化;采用免疫組織化學(xué)法和Western blotting法檢測Cleaved Caspase3蛋白在腎近端小管上皮細(xì)胞中的表達;采用免疫熒光法檢測凋亡因子GRP78和CHOP及上皮標(biāo)志物E-Cadherin蛋白在腎近端小管上皮細(xì)胞內(nèi)的表達。
結(jié)果 Sham組血清肌酐和尿素氮濃度在正常范圍內(nèi),IR組的濃度明顯高于Sham組,IPO組的濃度明顯低于IR組(F=202.726、496.013,P<0.05)。與Sham組相比,IR組的腎組織損傷明顯,近端小管上皮細(xì)胞腫脹,管腔內(nèi)可見脫落的上皮細(xì)胞,血管球內(nèi)毛細(xì)血管嚴(yán)重淤血;而IPO組比IR組腎小管上皮細(xì)胞腫脹輕,管腔內(nèi)少見脫落的上皮細(xì)胞,腎小管損傷程度低,血管球內(nèi)毛細(xì)血管輕度淤血。IR組近端小管上皮細(xì)胞中Cleaved Caspase3蛋白陽性表達率明顯高于Sham組,而IPO組陽性表達率明顯低于IR組(F=195.804,P<0.05)。Sham組近端小管上皮細(xì)胞中GRP78和CHOP蛋白熒光信號極低,E-Cad-
herin蛋白熒光信號較強,顯示正常生理狀態(tài);IR組GRP78和CHOP蛋白熒光信號明顯高于Sham組,E-Cadherin蛋白熒光信號明顯低于Sham組;IPO組GRP78和CHOP蛋白熒光信號比IR組稍低,E-Cadherin蛋白熒光信號較IR組稍高。
結(jié)論 缺血再灌注可引起急性腎損傷,影響腎功能。IPO可能通過減輕內(nèi)質(zhì)網(wǎng)應(yīng)激強度,從而減少損傷后的細(xì)胞凋亡,減輕腎臟缺血再灌注所致的損傷,達到保護腎臟的目的。
[關(guān)鍵詞] 缺血后處理;再灌注損傷;腎;內(nèi)質(zhì)網(wǎng)應(yīng)激;細(xì)胞凋亡
[中圖分類號] R361;R329.25
[文獻標(biāo)志碼] A
[文章編號] 2096-5532(2021)05-0646-07
doi:10.11712/jms.2096-5532.2021.57.167
[開放科學(xué)(資源服務(wù))標(biāo)識碼(OSID)]
[網(wǎng)絡(luò)出版] https://kns.cnki.net/kcms/detail/37.1517.r.20210909.1524.006.html;2021-09-09 17:25:02
PROTECTIVE EFFECT OF ISCHEMIC POSTCONDITIONING AGAINST RENAL ISCHEMIA-REPERFUSION INJURY BY INHI-
BITING ENDOPLASMIC RETICULUM STRESS AND RELATED MECHANISM
ZHANG Tianzhen, TAN Xiaohua, WU Cuijiao
(Department of Human Anatomy, Histology and Embryology, School of Basic Medicine, Qingdao University, Qingdao 266071, China)
[ABSTRACT] Objective To investigate the protective effect and mechanism of ischemic postconditioning (IPO) against ischemia-reperfusion injury by observing the expression of endoplasmic reticulum stress-related factors in the kidney after ischemia/reperfusion and IPO.
Methods A total of 36 male Sprague-Dawley rats were randomly divided into sham-operation group (Sham group), ischemia/reperfusion group (IR group), and IPO group. At 24 h after corresponding treatment, blood samples were collected from the inferior vena cava to measure the serum concentrations of creatinine and urea nitrogen and evaluate renal function; HE staining was performed for renal specimens to observe histomorphological changes; immunohistochemistry and Western blotting were used to measure the protein expression of cleaved caspase-3 in proximal tubular epithelial cells; immunofluorescence assay was used to measure the protein expression of the apoptotic factors GRP78 and CHOP and the epithelial marker E-Cadherin in proximal tubular epithelial cells.
Results The serum concentrations of creatinine and urea nitrogen were within the normal range in the Sham group; the IR group had significantly higher concentrations than the Sham group, and the IPO group had significantly lower concentrations than the IR group (F=202.726,496.013;P<0.05). Compared with the Sham group, the IR group had obvious renal tissue injury, swelling of proximal tubular epithelial cells, exfoliated epithelial cells in the lumen of the proximal tubule, and severe congestion of capillaries in the glomus, and compared with the IR group, the IPO group had a lower degree of swelling of renal tubular epithelial cells, a smaller amount of exfoliated epithelial cells in the lumen, a lower degree of renal tubular injury, and mild congestion of capillaries in the glomus. The IR
group had a significantly higher positive rate of cleaved caspase-3protein in proximal tubular epithelial cells than the Sham group, and the IPO group had a significantly lower positive rate than the IR group (F=195.804,P<0.05). The Sham group had extremely low intensities of the fluorescence signals of GRP78 and CHOP proteins and a relatively strong intensity of the fluorescence signal of E-Cadherin protein in proximal tubular epithelial cells, which showed a normal physiological state; compared with the Sham group, the IR group had significantly higher intensities of the fluorescence signals of GRP78 and CHOP and a significantly lower intensity of the fluorescence signal of E-Cadherin; compared with the IR group, the IPO group had slightly lower intensities of the fluorescence signals of GRP78 and CHOP and a slightly higher intensity of the fluorescence signal of E-Cadherin.
Conclusion Ischemia/reperfusion can cause acute kidney injury and affect renal function. IPO protects the kidney possibly by alleviating endoplasmic reticulum stress, reducing cell apoptosis after injury, and alleviating the injury caused by renal ischemia/reperfusion.
[KEY WORDS] ischemic postconditioning; reperfusion injury; kidney; endoplasmic reticulum stress; apoptosis
腎缺血再灌注是指腎臟外科手術(shù)或腎實質(zhì)性損傷會阻斷腎臟血流造成缺血低氧,給予再灌注后,腎臟的血流和功能恢復(fù)。再灌注經(jīng)常伴隨著再氧化,會加重前期缺血低氧的組織損傷,造成再灌注損傷。在臨床上,腎缺血再灌注損傷是導(dǎo)致急性腎損傷的主要原因之一[1-2]。急性腎損傷后近端小管持續(xù)的炎癥狀態(tài)等病理改變會導(dǎo)致腎臟纖維化的發(fā)生,從而增加慢性腎臟病和終末期腎病的風(fēng)險[3]。缺血再灌注損傷的病理生理過程復(fù)雜,涉及腎微血管病變、線粒體功能障礙、自噬、炎癥、內(nèi)質(zhì)網(wǎng)應(yīng)激等多個方面[4-6]。其中內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)細(xì)胞凋亡,是除了死亡受體和線粒體功能障礙凋亡途徑外,新發(fā)現(xiàn)的第三種細(xì)胞凋亡途徑,也是缺血再灌注所致腎損傷的細(xì)胞反應(yīng)。已有研究結(jié)果證明,在腎缺血再灌注損傷后,內(nèi)質(zhì)網(wǎng)應(yīng)激的強度增強,細(xì)胞凋亡增加,組織損傷加重[7-8]。減輕再灌注損傷后內(nèi)質(zhì)網(wǎng)應(yīng)激強度或減少其持續(xù)時間,以降低器官組織結(jié)構(gòu)損傷和功能障礙,成為研究熱點。有研究通過給予腎臟缺血前處理(IPC)證明,IPC對缺血再灌注導(dǎo)致的腎損傷具有保護作用[9]。但由于器官缺血時間的不可預(yù)知性,IPC處理具有明顯的臨床局限。另研究表明,缺血后處理(IPO)可以減輕缺血再灌注器官(心、肝、腎、腸、胃)的損傷[10]。主要認(rèn)為IPO通過減輕炎癥反應(yīng)、氧化應(yīng)激和細(xì)胞自噬等機制,來減輕缺血再灌注引發(fā)的腎損傷[11-13]。但是有關(guān)缺血再灌注損傷后IPO影響內(nèi)質(zhì)網(wǎng)應(yīng)激進而影響組織損傷的研究甚少。本實驗通過建立動物模型,運用形態(tài)學(xué)、免疫組織化學(xué)、免疫熒光等技術(shù),檢測重要的內(nèi)質(zhì)網(wǎng)應(yīng)激凋亡因子的表達,來進一步探討IPO如何影響內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)的細(xì)胞凋亡,從而減輕缺血再灌注損傷。
1 材料與方法
1.1 實驗材料
1.1.1 實驗動物 健康雄性SD大鼠36只,體質(zhì)量180~200 g,由青島大學(xué)實驗動物中心提供。
1.1.2 主要試劑 免疫顯色試劑盒購自福州邁新生物技術(shù)開發(fā)公司;TUNEL染色試劑盒購自德國曼海姆公司;抗體GRP78、CHOP、E-Cadherin均購自英國Abcam公司;抗體Cleaved Caspase3購自美國Cell Signaling Technology公司;第二抗體購自索萊寶生物科技有限公司。
1.2 實驗方法
1.2.1 動物分組及處理 將36只大鼠隨機分為假手術(shù)組(Sham組)、缺血再灌注組(IR組)和IPO組,每組12只。IR組參照TAN等[14]的研究建立腎缺血再灌注模型。術(shù)前大鼠禁食12 h,可自由飲水。術(shù)時大鼠腹腔注射100 g/L水合氯醛5 mL/kg,麻醉后仰臥位將大鼠固定于鼠臺上,開腹暴露左、右腎,切除右腎,鈍性分離左腎,暴露左腎動脈,用無創(chuàng)動脈夾夾住左腎動脈,觀察腎臟顏色由鮮紅色變?yōu)榘瞪创_認(rèn)腎臟血流阻斷,為缺血狀態(tài)。夾閉45 min后松開動脈夾,觀察腎臟顏色由暗紅恢復(fù)成鮮紅色即確認(rèn)為血流恢復(fù),縫合腹部切口。
大鼠蘇醒后放回鼠籠,予正常飲食,24 h后IR模型制備成功。IPO組在左腎動脈夾閉45 min后,松開動脈夾3 min,再夾閉左腎動脈30 s,松開30 s,共6次,其余操作同IR組。Sham組只行開腹并切除右腎,游離左腎,分離出左腎動脈不夾閉,切口用生理鹽水紗布覆蓋,45 min后關(guān)腹。建模24 h后,麻醉大鼠,開腹,用注射器采集下腔靜脈血2 mL,置于EP管中,放冰上暫存。然后摘取右腎,處死大鼠。
1.2.2 腎功能指標(biāo)檢測 將裝有大鼠下腔靜脈血的EP管在4 ℃下以4 000 r/min離心10 min,用加樣器收集上層血清成分(不要吸到下層血細(xì)胞),將收集到的血清做好標(biāo)記后置-80 ℃冰箱保存。最后送醫(yī)院檢驗科使用半自動生化分析儀檢測血清肌酐和尿素氮濃度。
1.2.3 腎組織形態(tài)學(xué)觀察 以40 g/L的中性甲醛溶液固定腎組織48 h,常規(guī)石蠟包埋切片,蘇木精-伊紅(HE)染色,光學(xué)顯微鏡下觀察組織形態(tài)。腎小管損傷評分[15]:切片HE染色后在病變嚴(yán)重處采用Paller法對腎小管損傷程度進行評分,即每個高倍視野隨機選擇10個有病變的腎小管區(qū)域按100個腎小管計分。計分標(biāo)準(zhǔn):腎小管明顯擴張、細(xì)胞扁平1分,刷狀緣損傷或脫落1分或2分,管型2分,腎小管管腔內(nèi)有脫落的、壞死的細(xì)胞以及未成管型或細(xì)胞碎片1分。
1.2.4 免疫組織化學(xué)方法檢測腎組織中Cleaved Caspase3蛋白表達 石蠟切片經(jīng)二甲苯脫蠟及乙醇梯度水化后,用微波爐進行抗原修復(fù)(檸檬酸鈉,pH值6.0),加Cleaved Caspase3抗體(1∶200)4 ℃過夜孵育。次日,室溫復(fù)溫40 min,以PBS洗片,加二抗(羊抗兔)37 ℃孵育1 h,以PBS洗片后進行DAB染色,水洗終止染色后用蘇木精復(fù)染12 s。最后采用乙醇梯度脫水,二甲苯透明,中性樹膠封片,在光鏡下觀察。
1.2.5 Western blotting法檢測腎組織中Cleaved Caspase3蛋白表達 制作腎組織勻漿后提取蛋白。分別制作100、120 g/L的SDS-PAGE分離膠和50 g/L的濃縮膠,在膠板上按照25 μg的加樣量上樣,電泳1 h,然后電轉(zhuǎn)50 min。電轉(zhuǎn)轉(zhuǎn)膜后以50 g/L
脫脂奶粉封閉3 h,然后加Cleaved Caspase3抗體(1∶1 000)4 ℃過夜孵育。次日,以1×TBST洗PVDF膜后,再加入二抗孵育2 h。以1×TBST洗PVDF膜后,在膜上加顯影液用顯影儀顯影。然后用Image J軟件分析條帶的灰度值,以β-actin為內(nèi)參照,計算蛋白表達量。蛋白表達量=目的蛋白灰度值/β-actin蛋白灰度值。
1.2.6 TUNEL染色檢測腎組織細(xì)胞凋亡 常規(guī)石蠟包埋制作腎組織切片(5 μm),切片經(jīng)二甲苯脫蠟及梯度乙醇水化后,用PBS洗滌3次,每次5 min。用濾紙小心吸去載玻片上組織周圍的多余液體,立即在切片上加2滴TdT酶緩沖液,室溫放置5 min。用濾紙小心吸去切片周圍的多余液體,立即在切片上滴加54 μL TdT酶反應(yīng)液,置濕盒中于37 ℃反應(yīng)1 h。將切片置于染色缸中,加入已預(yù)熱到37 ℃的洗滌與終止反應(yīng)緩沖液,于37 ℃保溫30 min,每10 min將載玻片輕輕提起和放下1次,使液體輕微攪動。組織切片用PBS洗滌3次,每次5 min。直接在切片上滴加2滴過氧化物酶標(biāo)記的抗地高辛抗體,于濕盒中室溫反應(yīng)30 min。用PBS洗3次,每次5 min。在組織切片上直接滴加新鮮配制的0.5 g/L的DAB溶液,室溫顯色5 min。用蒸餾水洗4次,前3次每次1 min,最后1次5 min。于室溫下用甲基綠復(fù)染10 min。用蒸餾水洗3次,前2次將載玻片提起放下10次,最后1次靜置30 s。依同樣方法再用正丁醇洗3次,用二甲苯脫水。封片、干燥后在光學(xué)顯微鏡下取5個不同的視野,分別計數(shù)總的細(xì)胞數(shù)和凋亡細(xì)胞數(shù),計算細(xì)胞凋亡率(凋亡細(xì)胞數(shù)/總的細(xì)胞數(shù))。
1.2.7 免疫熒光檢測GRP78、CHOP和E-Cadherin蛋白表達 切片經(jīng)二甲苯脫蠟及乙醇梯度水化后,用微波爐進行抗原修復(fù)(檸檬酸鈉,pH值6.0),加一抗4 ℃過夜孵育。次日,室溫復(fù)溫30 min,以PBS洗片后加熒光二抗孵育1 h(此后實驗步驟避光操作),以DIPA染色10 min,用防淬滅熒光劑封片,在激光共聚焦顯微鏡下觀察并隨機采集圖片。
1.3 統(tǒng)計學(xué)處理
采用SPSS 25.0軟件進行數(shù)據(jù)統(tǒng)計分析。計量資料數(shù)據(jù)以±s表示,多組比較采用單因素方差分析,組間兩兩比較采用LSD-t檢驗。以P<0.05表示差異有統(tǒng)計學(xué)意義。
2 結(jié)? 果
2.1 各組腎功能生化指標(biāo)比較
Sham組血清肌酐和尿素氮濃度在正常范圍內(nèi),IR組的濃度明顯高于Sham組,IPO組的濃度明顯低于IR組(F=202.726、496.013,P<0.05)。見表1。
各組血清肌酐和尿素氮比較,F(xiàn)=202.76、496.013,P<0.05。
2.2 腎組織的形態(tài)學(xué)觀察
光鏡下可見,Sham組皮質(zhì)迷路深部的腎小體較大,腎小體內(nèi)的血管球呈現(xiàn)大量毛細(xì)血管切面,腎小囊包繞血管球外,中間有腎小囊腔相隔;近端小管切面多,管壁厚,管腔小而不規(guī)則,上皮細(xì)胞為立方形或錐形,細(xì)胞體較大、分界不清,腔面有刷狀緣,胞質(zhì)呈強嗜酸性,細(xì)胞核圓形,位于近基底部;遠端小管切面少,管壁薄,管腔大而規(guī)則,上皮細(xì)胞為立方形,細(xì)胞體較小、分界清楚,游離面無刷狀緣,胞質(zhì)呈弱嗜酸性,細(xì)胞核圓形,位于中央。IR組病理改變明顯,血管球和腎小囊變形,血管球內(nèi)毛細(xì)血管嚴(yán)重淤血,腎小囊腔變窄或消失;近端小管改變明顯,上皮變厚,管腔變窄,上皮細(xì)胞腫脹,細(xì)胞內(nèi)出現(xiàn)較多空泡變性,腔面刷狀緣消失,小管腔內(nèi)出現(xiàn)壞死細(xì)胞碎片,間質(zhì)水腫,小血管淤血。IPO組腎組織改變比IR組明顯減輕,腎小管上皮細(xì)胞輕度水腫,有少量小的空泡,腎小管管腔內(nèi)可見少量脫落的上皮細(xì)胞,管周可見少量淤血;血管球內(nèi)毛細(xì)血管輕度淤血,腎小囊腔部分狹窄,間質(zhì)充血。提示IPO可以有效減輕缺血再灌注損傷后的腎損害。見圖1A。
Sham組、IR組和IPO組腎小管損傷評分分別為(0.10±0.32)、(5.20±1.48)和(1.90±0.99)分(n=12)。IR組腎小管損傷程度明顯高于Sham組,IPO組明顯低于IR組,差異有統(tǒng)計學(xué)意義(F=61.439,P<0.01)。
2.3 各組Cleaved Caspase3蛋白表達比較
免疫組織化學(xué)染色法檢測結(jié)果顯示,Sham組腎組織近端小管上皮細(xì)胞胞質(zhì)可見極少陽性著色;IR組近端小管上皮細(xì)胞胞質(zhì)出現(xiàn)大量棕褐色著色,表明Cleaved Caspase3蛋白在IR組呈強陽性表達,提示Cleaved Caspase3蛋白激活,細(xì)胞凋亡嚴(yán)重;IPO組近端小管上皮細(xì)胞胞質(zhì)有少量黃棕色著色,表明Cleaved Caspase3蛋白在IPO組陽性表達較低,提示Cleaved Caspase3蛋白部分激活,細(xì)胞凋亡減少。見圖1B。
Western blotting檢測結(jié)果顯示,Sham組、IR組和IPO組Cleaved Caspase3蛋白的相對表達量分別為0.12±0.01、1.27±0.11和0.32±0.02(n=12)。IR組Cleaved Caspase3蛋白表達量明顯高于Sham組,IPO組明顯低于IR組,差異有統(tǒng)計學(xué)意義(F=439.230,P<0.01)。表明IPO可以通過抑制腎小管上皮細(xì)胞凋亡來減輕腎損傷。見圖2。
2.4 TUNEL染色檢測細(xì)胞凋亡
TUNEL染色陽性定位于近端小管上皮細(xì)胞胞核內(nèi)。Sham組近端小管上皮細(xì)胞胞核內(nèi)有極少棕褐色顆粒,幾乎沒有陽性表達;IR組近端小管可見大量的上皮細(xì)胞胞核呈深棕色,陽性表達明顯多于Sham組;IPO組近端小管有少量的上皮細(xì)胞胞核染色陽性。見圖1C。
Sham組、IR組和IPO組細(xì)胞凋亡率分別為0.07±0.01、0.50±0.09和0.15±0.01(n=12)。IR組細(xì)胞凋亡率明顯高于Sham組,IPO組明顯低于IR組,差異有統(tǒng)計學(xué)意義(F=92.818,P<0.01)。表明IPO通過減少腎小管細(xì)胞凋亡來保護腎臟免受缺血再灌注損傷。
2.5 免疫熒光檢測GRP78、CHOP和E-Cadherin蛋白表達
Sham組GRP78和CHOP蛋白熒光信號極弱,E-Cadherin蛋白熒光信號強,尤其是近端小管上皮細(xì)胞E-Cadherin蛋白表達較強。IR組GRP78和CHOP蛋白熒光信號強,在近端小管上皮細(xì)胞中呈強陽性表達;E-Cadherin蛋白熒光信號弱,在近端小管上皮細(xì)胞的信號強度明顯低于Sham組。IPO組的GRP78和CHOP蛋白熒光信號較IR組弱,較Sham組強;E-Cadherin蛋白的熒光信號介于IR組和Sham組之間。另外,在IPO組還觀察到少量遠端小管上皮細(xì)胞E-Cadherin蛋白熒光信號較強。上述結(jié)果表明,IR組近端小管上皮細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激強度大且細(xì)胞凋亡數(shù)量多,而IPO可以減輕腎小管細(xì)胞凋亡。見圖3、4。
3 討? 論
本實驗通過建立大鼠腎缺血再灌注模型,并給予IPO,探討IPO對缺血再灌注損傷的保護作用及機制。為保證動物實驗?zāi)P偷挠行裕瑢Ω鹘M腎功能指標(biāo)(血清肌酐和尿素氮)及腎組織形態(tài)的變化進行了觀察,結(jié)果顯示,血清肌酐和尿素氮的濃度在各組的表達變化明顯,IR組明顯高于Sham組,IPO組明顯低于IR組,提示大鼠再灌注損傷后腎小球濾過功能嚴(yán)重下降,IPO可使濾過功能得以改善。光鏡下觀察和腎小管損傷評分分析顯示,相對于正常組織形態(tài)的Sham組,IR組近端小管上皮細(xì)胞腫脹明顯,出現(xiàn)大量空泡,并有大量上皮細(xì)胞脫落到管腔內(nèi),腎小管損傷嚴(yán)重;IPO組近端小管上皮細(xì)胞腫脹輕微,空泡變性較少,腎小管損傷程度比IR組輕。上述結(jié)果表明動物實驗建模成功,IPO可以減少腎小管上皮細(xì)胞凋亡,減輕腎組織損傷。
缺血再灌注損傷可致細(xì)胞內(nèi)許多信號轉(zhuǎn)導(dǎo)通路的異常激活,出現(xiàn)級聯(lián)反應(yīng),導(dǎo)致細(xì)胞功能障礙和凋亡的發(fā)生。Caspase家族是細(xì)胞凋亡過程中最重要的蛋白酶。其中Caspase12定位于內(nèi)質(zhì)網(wǎng),當(dāng)內(nèi)質(zhì)網(wǎng)過多蛋白積聚時內(nèi)質(zhì)網(wǎng)可激活Caspase12,活化Caspase12可進一步剪切Caspase3[16]。Caspase3為Caspase家族的核心成員,是調(diào)節(jié)細(xì)胞凋亡的關(guān)鍵蛋白酶,因此也被稱為“死亡蛋白酶”,是目前已知細(xì)胞凋亡最后執(zhí)行因子,在腫瘤、缺血再灌注損傷等許多疾病的發(fā)生發(fā)展中發(fā)揮重要作用[17]。Cleaved Caspase3是Caspase3活化過程中經(jīng)剪切產(chǎn)生的活性片段,其表達水平可以反映Caspase3的活性和細(xì)胞凋亡情況[18]。
本實驗對Cleaved Caspase3蛋白進行免疫組織化學(xué)和Western blotting檢測,并采用TUNEL染色對細(xì)胞凋亡的情況進行分析。3種技術(shù)檢測得到的結(jié)果一致。Sham組近端小管上皮細(xì)胞胞質(zhì)有極少Cleaved Caspase3蛋白陽性表達,黃色顆粒極少,TUNEL染色陽性表達也非常弱,屬于正常生理性的細(xì)胞凋亡;IR組大量近端小管上皮細(xì)胞胞質(zhì)中Cleaved Caspase3蛋白高表達,棕褐色顆粒在近端小管上皮細(xì)胞胞質(zhì)分布廣泛,TUNEL染色呈強陽性表達;IPO組近端小管上皮細(xì)胞胞質(zhì)中有少量Cleaved Caspase3蛋白陽性表達,褐色顆粒稀少,表達量介于Sham組和IR組之間,TUNEL染色有少量陽性表達。表明腎臟缺血再灌注后發(fā)生損傷,Caspase3被激活,啟動細(xì)胞凋亡,使近端小管上皮細(xì)胞大量凋亡;但IPO抑制了Caspase3的激活,減弱了缺血再灌注后腎小管上皮細(xì)胞的凋亡,對缺血再灌注損傷具有保護作用。
本實驗采用免疫熒光技術(shù)檢測了內(nèi)質(zhì)網(wǎng)應(yīng)激調(diào)節(jié)因子GRP78和CHOP蛋白以及上皮標(biāo)志物E-Cadherin蛋白的表達。GRP78是一種內(nèi)質(zhì)網(wǎng)上的多功能鈣結(jié)合蛋白,在內(nèi)質(zhì)網(wǎng)蛋白質(zhì)合成、內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài)維持、細(xì)胞信號控制、細(xì)胞生存等多方面起著關(guān)鍵作用[19]。在應(yīng)激過程中,作為內(nèi)質(zhì)網(wǎng)分子伴侶及鈣結(jié)合蛋白,過表達的GRP78可與錯誤折疊或未折疊蛋白的疏水性殘基端以及鈣離子結(jié)合,減少內(nèi)質(zhì)網(wǎng)上錯誤折疊或未折疊蛋白的積累并維持鈣離子平衡,恢復(fù)內(nèi)質(zhì)網(wǎng)生理功能;此外,GRP78作為內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志蛋白,可與內(nèi)質(zhì)網(wǎng)應(yīng)激激活的促凋亡受體結(jié)合,抑制其信號轉(zhuǎn)導(dǎo),從而保護細(xì)胞,維持機體內(nèi)環(huán)境穩(wěn)態(tài)[20]。CHOP作為核轉(zhuǎn)錄因子可抑制抗凋亡基因Bcl-2表達,促進Bim、PUMA、TRB3等多種凋亡相關(guān)基因表達,誘導(dǎo)細(xì)胞凋亡[21]。E-Cadherin是鈣黏附蛋白分子家族中跨膜蛋白亞型的一種,主要分布于上皮組織中,對維持上皮細(xì)胞的形態(tài)和結(jié)構(gòu)完整性起著重要作用。作為上皮細(xì)胞間彼此連接的主要蛋白,E-Cadherin在正常細(xì)胞組織中保持高表達,從而維持細(xì)胞之間的聚集功能;若其低表達,則表明細(xì)胞的完整性受損[22]。本實驗觀察到,Sham組近端小管上皮細(xì)胞中幾乎沒有GRP78和CHOP蛋白熒光信號,而E-Cadherin蛋白熒光信號較強;IR組近端小管上皮細(xì)胞中GRP78和CHOP蛋白熒光信號增強,而E-Cadherin蛋白熒光信號減弱;IPO組近端小管上皮細(xì)胞中GRP78、CHOP和E-Cadherin蛋白的熒光信號強度介于Sham組和IR組之間。上述結(jié)果表明,缺血再灌注損傷后,內(nèi)質(zhì)網(wǎng)應(yīng)激發(fā)生并處于持續(xù)應(yīng)激狀態(tài),細(xì)胞黏著連接受損嚴(yán)重;IPO后內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)減弱,上皮細(xì)胞間的連接增強。
綜上所述,缺血再灌注可以引起內(nèi)質(zhì)網(wǎng)應(yīng)激因子GRP78、CHOP在腎小管上皮細(xì)胞中高表達,激活Cleaved Caspase3,誘導(dǎo)上皮細(xì)胞大量凋亡,從而導(dǎo)致急性腎損傷,使腎功能受損;IPO能夠抑制缺血再灌注所致的內(nèi)質(zhì)網(wǎng)應(yīng)激因子的表達,阻止細(xì)胞凋亡,減輕腎損傷。IPO作用的發(fā)揮與抑制內(nèi)質(zhì)網(wǎng)應(yīng)激強度有關(guān),IPO可通過降低內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng),穩(wěn)定腎小管上皮細(xì)胞結(jié)構(gòu),減少細(xì)胞凋亡,從而減輕因缺血再灌注損傷造成的腎小管上皮細(xì)胞功能障礙或喪失,減輕缺血再灌注后腎組織損傷。
[參考文獻]
[1]SERVIDDIO G, ROMANO A D, GESUALDO L, et al. Postconditioning is an effective strategy to reduce renal ischaemia/reperfusion injury[J].? Nephrology Dialysis Transplantation, 2008,23(5):1504-1512.
[2]DA COSTA M F B, LIBRIO A B, TELES F, et al. Red propolis ameliorates ischemic-reperfusion acute kidney injury[J].? Phymedicine, 2015,22(9):787-795.
[3]CHEN H, WANG L, XING B Z, et al. Ischemic postconditioning attenuates inflammation in rats following renal ischemia and reperfusion injury[J].? Experimental and Therapeutic Medicine, 2015,10(2):513-518.
[4]ZU K A, BONVENTRE J V. Acute kidney injury[J].? Annual Review of Medicine, 2016,67:293-307.
[5]彭顯月,梁國標(biāo). 自噬在腎缺血再灌注損傷中作用機制的研究進展[J].? 醫(yī)學(xué)研究雜志, 2020,49(7):9-11,16.
[6]李爽,包宇實. 缺血再灌注急性腎損傷機制研究進展[J].? 醫(yī)學(xué)綜述, 2020,26(19):3848-3853.
[7]KUZNETSOV G, BUSH K T, ZHANG P L, et al. Perturbations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia[J].? PNAS, 1996,93(16):8584-8589.
[8]TOTH A, NICKSON P, MANDL A, et al. Endoplasmic reticulum stress as a novel therapeutic target in heart diseases[J].? Cardiovascular & Hematological Disorders Drug Targets, 2007,7(3):205-218.
[9]XIE Y, JIANG D F, XIAO J, et al. Ischemic preconditioning attenuates ischemia/reperfusion-induced kidney injury by activating autophagy via the SGK1 signaling pathway[J].? Cell Death & Disease, 2018,9(3):338.
[10]馬小波,張建福. 缺血后處理對再灌注器官保護作用的研究進展[J].? 徐州醫(yī)學(xué)院學(xué)報, 2009,29(11):778-783.
[11]CHEN R, ZENG Z, ZHANG Y Y, et al. Ischemic postconditioning attenuates acute kidney injury following intestinal ischemia-reperfusion through Nrf2-regulated autophagy, anti-oxidation, and anti-inflammation in mice[J].? FASEB Journal, 2020,34(7):8887-8901.
[12]TIAN Y, SHU J, HUANG R Z, et al. Protective effect of renal ischemic postconditioning in renal ischemic-reperfusion injury[J].? Translational Andrology and Urology, 2020,9(3):1356-1365.
[13]GAO S M, ZHU Y, LI H B, et al. Remote ischemic postconditioning protects against renal ischemia/reperfusion injury by activation of T-LAK-cell-originated protein kinase (TOPK)/PTEN/Akt signaling pathway mediated anti-oxidation and anti-inflammation[J].? International Immunopharmacology, 2016,38:395-401.
[14]TAN X H, YIN R L, CHEN Y, et al. Postconditioning atte-
nuates renal ischemia-reperfusion injury by mobilization of stem cells[J].? Journal of Nephrology, 2015,28(3):289-298.
[15]董文斌,冉隆瑞,馮志強,等. 白細(xì)胞參與新生鼠窒息后腎損傷的作用及機制研究[J]. 中國危重病急救醫(yī)學(xué), 2000(4):208-210.
[16]XU Y, GUO M, JIANG W, et al. Endoplasmic reticulum stress and its effects on renal tubular cells apoptosis in ischemic acute kidney injury[J].? Renal Failure, 2016,38(5):831-837.
[17]KANG S J, SANCHEZ I, JING N S, et al. Dissociation between neurodegeneration and caspase-11-mediated activation of caspase-1 and caspase-3 in a mouse model of amyotrophic la-
teral sclerosis[J].? The Journal of Neuroscience, 2003,23(13):5455-5460.
[18]GOWN A M, WILLINGHAM M C. Improved detection of apoptotic cells in archival paraffin sections: immunohistoche-
mistry using antibodies to cleaved caspase 3[J].? Journal of Histochemistry & Cytochemistry, 2002,50(4):449-454.
[19]LUO B, LEE A S. The critical roles of endoplasmic reticulum chaperones and unfolded protein response in tumorigenesis and anticancer therapies[J].? Oncogene, 2013,32(7):805-818.
[20]GRKOVIC S, OREILLY V C, HAN S, et al. IGFBP-3 binds GRP78, stimulates autophagy and promotes the survival of breast cancer cells exposed to adverse microenvironments[J].? Oncogene, 2013,32(19):2412-2420.
[21]ARIYASU D, YOSHIDA H, HASEGAWA Y. Endoplasmic reticulum (ER) stress and endocrine disorders[J].? Internatio-
nal Journal of Molecular Sciences, 2017,18(2):382.
[22]丁丹丹,徐錦程. Survivin、 E-cadherin及N-cadherin與舌鱗狀細(xì)胞癌關(guān)系的研究進展[J].? 齊齊哈爾醫(yī)學(xué)院學(xué)報, 2020,41(8):997-999.
(本文編輯 馬偉平)