氧化槐定堿通過Akt/Nrf2/HO-1和Akt/GSK3β信號通路對急性腎損傷的保護作用

王文文，張 敏

(1浙江大學醫學院附屬婦產科醫院病理科,杭州 310006;2浙江中醫藥大學附屬第一醫院病理科;*通訊作者,E-mail:bzwangwenwen@126.com)

氧化槐定堿通過Akt/Nrf2/HO-1和Akt/GSK3β信號通路對急性腎損傷的保護作用

王文文1*，張 敏2

(1浙江大學醫學院附屬婦產科醫院病理科,杭州 310006;2浙江中醫藥大學附屬第一醫院病理科;*通訊作者,E-mail:bzwangwenwen@126.com)

目的 探討氧化槐定堿(oxysophoridine,OSR)對小鼠腎缺血再灌注(ischemia/reperfusion,I/R)誘導的急性腎損傷的保護作用及其機制。 方法 C57/BL6小鼠30只隨機分為假手術組(sham組)、模型組(I/R組)和給藥組(I/R+OSR組)。I/R+OSR組預給藥5 d[250 mg/(kg·d),腹腔注射],sham組和I/R組給予相同體積的生理鹽水。第5天給藥后1 h開始利用左側腎切除、右側腎蒂缺血30 min再灌注24 h的方法構建缺血再灌注損傷模型。下腔靜脈取血并離心取血清,用自動生化儀檢測肌酐和尿素氮,試劑盒檢測丙二醛的生成及超氧化物歧化酶(superoxide dismutase,SOD)和過氧化氫酶(catalase,CAT)的活性,ELISA法檢測腫瘤壞死因子-α(TNF-α)和白細胞介素-1β(IL-1β),HE染色法檢測腎臟組織形態學,Western blot 法檢測蛋白激酶B(Akt)、磷酸化Akt(p-Akt)、細胞內核因子E2 相關因子2(Nrf2)、血紅素加氧酶(HO-1)、糖原合酶激酶-3β(GSK3β)、磷酸化GSK3β(p-GSK3β)、半胱天冬酶-3(pro-caspase-3)、活化的半胱天冬酶-3(cleaved caspase-3)、B淋巴細胞瘤-2(Bcl-2)的表達。 結果 與sham組比較,I/R組小鼠肌酐、尿素氮、丙二醛、TNF-α和IL-1β顯著升高(P<0.05),SOD、CAT的活性顯著降低(P<0.05),p-Akt、Nrf2、HO-1、p-GSK3β、Bcl-2、pro-caspase-3蛋白表達顯著降低(P<0.05),而促凋亡蛋白cleaved caspase-3表達顯著升高(P<0.05)。與I/R組比較,OSR組小鼠肌酐、尿素氮、丙二醛、TNF-α和IL-1β顯著降低(P<0.05),SOD、CAT的活性顯著增加(P<0.05),p-Akt、Nrf2、HO-1、p-GSK3β、Bcl-2、pro-caspase-3蛋白表達顯著升高(P<0.05),促凋亡蛋白cleaved caspase-3表達顯著降低(P<0.05)。與sham組比較,HE染色結果顯示I/R組腎損傷顯著加重,而給藥后可顯著改善急性腎損傷。 結論 OSR通過Akt/Nrf2/HO-1和Akt/GSK3β信號通路及抗炎和抗凋亡相關信號通路機制對腎缺血再灌注誘導的急性腎損傷起到保護作用。

氧化槐定堿; 腎缺血再灌注; 細胞內核因子E2相關因子2; 血紅素加氧酶; 糖原合酶激酶-3β

急性腎損傷(acute kidney injury,AKI)是一種以腎功能突然下降為特征的綜合征[1, 2],它會導致體內肌酐、尿素氮和其他生化廢物的蓄積及電解質和酸堿穩態和含氮廢物保留的失衡[3, 4]。對于住院病人來說,AKI是一種常見的和嚴重的并發癥[5, 6],有研究報道,普通住院患者中AKI的發病率約為5%,并且在世界范圍內每年約200萬人死于此疾病[7, 8]。關于AKI的機制研究報道已有很多,但是目前還沒有很好的方法來預防或治療AKI[9],AKI的信號分子機制目前尚不完全清楚,進一步探究AKI的病理生理機制是至關重要的。

腎缺血/再灌注(ischemia reperfusion,I/R)損傷是AKI的一種常見原因。腎缺血再灌注損傷機制復雜,目前文獻報道的包括ATP缺失、細胞內Ca2+堆積、ROS、促炎細胞因子、細胞凋亡信號通路等導致的內皮功能障礙和腎小管細胞損傷[10, 11]。缺血再灌注產生的大量ROS,進而導致腎小管上皮細胞的損傷和凋亡,是造成腎損傷及病理反應的主要原因[12, 13]。氧化槐定堿(oxysophoridine,OSR)是從寧夏特色豆科槐屬植物苦豆子中提取的主要生物堿之一,其化學結構式是由兩個哌啶環組成。一些文獻報道氧化槐定堿具有抗氧化應激、抗炎、抗凋亡和神經保護作用[14-17],然而在腎缺血再灌注損傷中的保護作用以及作用機制尚未有研究報道。本實驗通過單側腎缺血再灌注模型建立小鼠急性腎損傷模型,探討氧化槐定堿對腎缺血再灌注損傷的保護作用及其機制。

1 材料和方法

1.1 實驗動物

12周齡SPF級健康雄性C57BL/6小鼠,體質量25-28 g,由浙江大學動物中心提供(訂購與上海斯萊克實驗動物有限責任公司,動物合格證號:SCXK(滬)2007-0005)。

1.2 主要試劑

氧化槐定堿(OSR),北京寰宇科創生物科技發展有限公司提供, 純度98%,使用時用0.9%的生理鹽水配成所需濃度。丙二醛(MDA)檢測試劑盒、總過氧化物歧化酶(SOD)活性檢測試劑盒、過氧化氫酶(CAT)檢測試劑盒和BCA蛋白定量試劑盒購買于上海碧云天生物技術有限公司;小鼠腫瘤壞死因子α(TNF-α)和白細胞介素1β (IL-1β)ELISA試劑盒購買于美國R&D公司。抗體 p-Akt、Akt、p-GSK3β、GSK3β購買于CST抗體公司;Nrf2、HO-1、Pro-caspase-3、Cleaved caspase-3、Bcl-2、β-actin,購買于abcam抗體公司。

1.3 分組及給藥

為了研究OSR對腎臟缺血再灌注誘導的急性腎損傷的保護作用及相關機制,本實驗設計如下:先將C57BL/6小鼠30只隨機分為3組(每組10只): sham組、I/R組、OSR組(250 mg/kg[15-17],腹腔注射給藥)。OSR組造模前預防性腹腔注射給藥5 d,每天1次,缺血前1 h再給藥1次。sham組和I/R組給予相同體積的生理鹽水。用自動生化儀檢測血清中肌酐和尿素氮的變化,用試劑盒檢測丙二醛含量變化、SOD和CAT的酶活性變化。用ELISA試劑盒檢測組織中TNF-α 和IL-1β表達變化。用Wes-tern blot 方法檢測Akt/Nrf2/HO-1和Akt/GSK3β信號通路相關蛋白指標的變化。

1.4 腎缺血再灌注損傷模型的制備

1.5 檢測肌酐、尿素氮、丙二醛、SOD、CAT、TNF-α和IL-1β

小鼠頸椎脫臼處死前,經由下腔靜脈取血,室溫靜置1-2 h,然后于3 000 r/min、4 ℃條件下離心15 min,取上清即為血清。利用自動生化儀檢測血清中的肌酐和尿素氮。按照丙二醛檢測試劑盒說明書操作檢測血清中丙二醛的含量。

將腎組織勻漿,用BCA試劑盒進行蛋白定量,取出一部分勻漿液分別用總SOD活性檢測試劑盒和CAT活性檢測試劑盒檢測組織中SOD和CAT含量。再取出一部分勻漿液分別用小鼠TNF-α 和小鼠IL-1β ELISA試劑盒檢測組織中TNF-α 和IL-1β表達變化,剩余的部分用Western blot 法檢測相關蛋白的變化。

1.6 蘇木精-伊紅(Hematoxylin-Eosin,HE)染色

處死小鼠取出腎臟,用4%的多聚甲醛溶液固定,用石蠟包埋,切成4 μm的腎切片然后按照HE染色的流程進行染色,并在顯微鏡下觀察腎臟切片的形態學變化。

1.7 蛋白提取定量和蛋白印跡分析(Western blot)

將提取定量的蛋白樣本用十二烷基磺酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)分析,根據蛋白分子量大小選擇10%-13.5%的分離膠進行電泳[20, 21]。簡單來說,取等量蛋白樣品進行電泳,然后轉移至PVDF膜上,用5%的脫脂牛奶封閉后,根據抗體說明書按不同的比例稀釋一抗抗體并放于4 ℃搖床過夜孵育。洗膜后加入1 ∶5 000稀釋的二抗,將條帶放于室溫搖床孵育60 min,最后ECL化學發光法曝光并觀察結果。以β-actin和LaminB1分別作為胞漿蛋白和核蛋白的內參蛋白,通過計算目標蛋白與內參蛋白的灰度比值進行半定量分析統計。

1.8 統計學分析

2 結果

2.1 OSR對腎缺血再灌注損傷后腎功能指標的影響

與sham組比較,I/R組和I/R+OSR組小鼠血清中肌酐和尿素氮顯著升高(P<0.01);與I/R組比較,I/R+OSR組可明顯降低血清中肌酐和尿素氮的含量(P<0.05,見表1)。

組別n肌酐(μmol/L)尿素氮(mmol/L)sham組10 20.3±3.5 11.6±0.8I/R組8 168.4±6.4** 50.2±2.6**I/R+OSR組10 121.0±5.1**# 34.7±2.5**#

與sham組比較,**P<0.01;與I/R組比較,#P<0.05

2.2 OSR對腎缺血再灌注損傷后脂質過氧化的影響

與sham組比較,I/R組小鼠血清中丙二醛含量顯著增加(P<0.05);與I/R組比較,OSR組可明顯減少丙二醛的增加(P<0.05,見圖1)。

2.3 OSR對腎缺血再灌注損傷后SOD和CAT酶活性的影響

與sham組比較,I/R組腎組織中SOD和CAT顯著降低(P<0.05);與I/R組比較,OSR組可部分逆轉SOD和CAT的減少,并且有顯著性差異(P<0.05,見圖2)。

2.4 OSR對缺血再灌注后腎組織促炎性細胞因子表達的影響

ELISA結果表明,TNF-α和IL-1β蛋白的表達被OSR藥物的干預所抑制,從而起到抗炎的作用。與sham組比較,I/R組小鼠腎組織中TNF-α 和IL-1β表達明顯增加(P<0.05);與I/R組比較,OSR組可明顯逆轉TNF-α 和IL-1β表達的上調,且差異有統計學意義(P<0.05,見圖3)。

2.5 OSR對I/R損傷后組織形態學的影響

sham組腎小管上皮細胞結構清晰,基底膜完整;I/R組在皮質區以及外髓質區出現不同程度的腎小管損傷,包括腎小管上皮細胞腫脹變性,出現核溶解、碎裂,細胞脫落,基底膜裸露或不完整以及較多的炎性細胞浸潤;OSR可以明顯減輕IR誘導的腎組織損傷(見圖4)。

與sham組比較,*P<0.05;與I/R組比較,#P<0.05圖2 腎缺血再灌注后SOD和CAT活性變化Figure 2 Changes of SOD and CAT activity after renal ischemia reperfusion and OSR treatment

與sham組比較,*P<0.05;與I/R組比較,#P<0.05圖3 腎缺血再灌注后促炎因子TNF-α和IL-1β表達變化Figure 3 Changes of proinflammatory factor TNF-α and IL-1β expression in mice after renal ischemia reperfusion and OSR treatment

圖4 小鼠腎缺血再灌注后腎臟組織形態學變化 (HE染色,×200)Figure 4 The morphological changes of renal tissue in mice after renal ischemia reperfusion (HE,×200)

2.6 OSR對I/R損傷后Akt/Nrf2/HO-1和Akt/GSK3β通路蛋白表達的影響

與sham組相比,腎缺血再灌注損傷顯著降低了胞漿內p-Akt (見圖5A和B)、 HO-1 (見圖5E和F)、p-GSK3β (見圖5G和H)以及核內Nrf-2 (見圖5C和D)的表達。預給藥OSR后可以部分逆轉p-Akt、HO-1、p-GSK3β及Nrf2蛋白的下調。

2.7 OSR對腎缺血再灌注損傷后凋亡相關蛋白信號通路的影響

研究結果顯示,與sham組比較,I/R損傷后Pro-caspase-3和Bcl-2明顯下調,cleaved caspase-3明顯上調,進而誘發了腎組織細胞凋亡。OSR可以明顯降低cleaved caspase-3的激活,改善Pro-caspase-3和Bcl-2的下調(見圖6),從而起到保護缺血再灌注誘導的急性腎損傷的作用。

左邊一列為各蛋白的Western blot檢測結果；右邊一列為相應的蛋白Western blot結果的統計分析;與sham組比較,*P<0.05;與I/R組比較,#P<0.05圖5 腎缺血再灌注后Akt/Nrf2/HO-1和Akt/GSK3β信號通路相關蛋白質表達變化Figure 5 Changes of protein expression of Akt/Nrf2/HO-1 and Akt/GSK3β signaling pathways after renal ischemia reperfusion

與sham組比較,*P<0.05;與I/R組比較,#P<0.05圖6 腎缺血再灌注后凋亡相關蛋白質表達變化Figure 6 Changes of protein expression of apoptosis-related proteins after renal ischemia reperfusion

3 討論

AKI疾病對病人有很高的致死率[22]。AKI的主要病因包括腎缺血、缺氧或腎毒性等[23, 24]。I/R損傷是AKI的一種常見原因。I/R損傷會引起腎臟ROS的過多生成和內源性抗氧化物的減少[25]。I/R損傷的病理生理機制包括內皮細胞功能障礙、ROS過多產生、促炎癥細胞因子的釋放、凋亡信號通路的激活等[26-28]。AKI的發病機制至今仍未被完全闡明,但是很多研究證明,減輕氧化應激、凋亡和炎性反應,對I/R損傷有著重要保護作用。有研究表明可以通過Nrf2/ARE/HO-1信號通路的激活,進而抵抗氧化應激,減少I/R損傷導致的腎小管凋亡,起到保護腎臟的作用[12, 29];也可以經由抗炎作用和抗凋亡信號通路起到保護腎臟的作用[29-31]。

本研究結果顯示,I/R損傷后腎功能指標肌酐和尿素氮水平顯著升高,而與IR組比較,OSR藥物組可以明顯改善腎臟功能指標。I/R損傷后腎組織SOD、CAT酶活性減弱,表明腎臟自身的自由基清除能力下降,從而導致氧化應激激活,氧化應激激活后一方面可以誘導脂質過氧化導致丙二醛在體內蓄積,另一方面也會誘導凋亡信號通路的激活,從而導致腎損傷。OSR給藥明顯改善了酶活性,減弱了氧化應激的激活,減輕了腎組織損傷。腎臟I/R損傷后促炎因子TNF-α和IL-β也大量釋放,過多的炎癥因子的釋放也會誘發腎損傷,而OSR組可以減少促炎癥因子的釋放,改善腎損傷。HE染色形態學顯示,I/R損傷后腎組織的小管損傷壞死增加,而OSR預給藥減輕了腎小管的損傷和壞死。

細胞保護自身抵抗傷害性刺激的內源性防御機制對于細胞的存活尤為重要。顯而易見, Keap1/Nrf2/HO-1信號通路在氧化應激的病理生理反應中是一個關鍵的調控因子,已經成為細胞抗氧化應激的主要防御機制[27, 32]。PI3K/Akt信號通路在調節細胞生長和存活中起著重要的作用,并且Akt的激活可以減輕I/R誘導的急性腎損傷[33, 34]。PI3K/Akt信號通路也可以調節Nrf2的活性,Akt的磷酸化可以增強下游Nrf2的活性,從而促進細胞的內源性抗氧化作用[35]。給藥后Akt,p-Akt,Nrf2 和HO-1的蛋白表達證明OSR可以通過Akt/Nrf2/HO-1信號通路起到保護腎臟的作用。Akt通過抑制凋亡而促進細胞存活是經由磷酸化和失活它的一些靶標,其中包括GSK3β[36]。本研究結果顯示I/R損傷后Akt的磷酸化下調,導致下游GSK3β磷酸化也下調,而OSR給藥后可以逆轉這種下調,表明OSR可以通過Akt/GSK3β信號通路起到保護腎臟的作用。

Pro-caspase-3的激活形式cleaved caspase-3是一種重要的促凋亡蛋白。Bcl-2家族蛋白通過誘導凋亡或抗凋亡調控著細胞的命運,Bcl-2被認為是一個重要的抗凋亡蛋白。研究結果顯示I/R損傷后Bcl-2下調,cleaved caspase-3上調,凋亡增加,而OSR給藥后可以下調cleaved caspase-3,上調Bcl-2,從而起到保護腎臟的作用。

總之,本研究初步證明藥物OSR通過抗氧化應激、抗炎和抗凋亡起到保護腎臟的作用,其作用機制是通過Akt/Nrf2/HO-1和Akt/GSK3β信號通路起到改善腎功能的作用。生物體內抗氧化系統是一個復雜的分子網絡,涉及到的信號通路不止有Akt/Nrf2/HO-1和Akt/GSK3β信號通路;Nrf2的上游調控分子也不止Akt一個,也有其他的上游調控蛋白如ERK1/2和MAPK等;PI3K/Akt通路下游亦不止這些,是否還有其他的分子參與OSR對急性腎損傷的保護作用有待進一步研究。目前臨床上獲批的能治療AKI的藥物幾乎沒有[10],本研究證實OSR對急性腎損傷有保護作用,或許OSR可以作為潛在治療AKI的藥物,這對治療AKI藥物的基礎研發和臨床研究都有著很好的指導意義。

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ProtectiveeffectofoxysophoridinefromacutekidneyinjurybyAkt/Nrf2/HO-1andAkt/GSK3βsignalingpathways

WANG Wenwen1*,ZHANG Min2

(1DepartmentofPathology,Women’sHospital,SchoolofMedicine,ZhejiangUniversity,Hangzhou310006,China;2DepartmentofPathology,FirstAffiliatedHospitalofZhejiangChineseMedicalUniversity;*Correspondingauthor,E-mail:bzwangwenwen@126.com)

ObjectiveTo investigate the protective effects of oxysophoridine(OSR) from renal ischemia reperfusion-induced acute kidney injury in mice and its possible mechanism.MethodsC57/BL6 mice were randomly divided into 3 groups: sham-operated group(sham group), model group(I/R group), OSR group(I/R+OSR). The mice in OSR group were pretreated with OSR for 5 d before inducing the I/R injury model(250 mg/kg, once a day, intraperitoneal injection). The mice in sham group and I/R group were given the same volume of physiological saline. One hour after the last administration, the right renal pedicle was clamped for 30 min and left nephrectomy was performed to produce I/R injury model in mice. Blood samples were collected from the inferior vena cava and centrifuged. Serum creatinine and blood urea nitrogen were measured by an automatic biochemical analyzer. Malondialdehyde concentration and enzymatic activity of superoxide dismutase(SOD) and catalase(CAT) were measured by Kits respectively. Tumor necrosis factor alpha(TNF-α) and interleukin-1 beta(IL-1β) were detected by ELISA Kits. The histological structure changes of renal tissues were observed under microscope. The expression levels of protein kinase B(Akt), phosphorylated Akt(p-Akt), nuclear factor erythroid 2-related factor 2(Nrf2), heme oxygenase(HO-1), glycogen synthase kinase 3 beta(GSK3β), phosphorylation GSK3β(p-GSK3β), pro-caspase-3, cleaved caspase-3, B-cell lymphoma-2(Bcl-2) were detected by Western blot.ResultsCompared with sham group, serum creatinine, blood urea nitrogen, malondialdehyde, TNF-α and IL-1β increased significantly in I/R group(P<0.05), SOD and CAT decreased significantly in I/R group(P<0.05), and the expression of p-Akt, Nrf2, HO-1, p-GSK3β, Bcl-2, pro-caspase-3 reduced and cleaved caspase-3 increased significantly in I/R group(P<0.05). Compared with model group, serum creatinine, blood urea nitrogen, malondialdehyde, TNF-α and IL-1β decreased significantly in OSR group(P<0.05), SOD and CAT increased significantly in OSR group(P<0.05), and the expression of p-Akt, Nrf2, HO-1, p-GSK3β, Bcl-2, pro-caspase-3 increased and cleaved caspase-3 decreased significantly in OSR group(P<0.05). Compared with sham group, HE staining results showed severe kidney injury in I/R group, but the kidney damage was improved in OSR group.ConclusionOSR has the protective effect from the renal ischemia-reperfusion injury in mice and may be involved in anti-oxidative stress, anti-inflammation, and anti-apoptosis.

oxysophoridine; renal ischemia reperfusion; Nrf2; heme oxygenase(HO); glycogen synthase kinase 3 beta

R363,R692

A

1007-6611(2017)10-0992-07

10.13753/j.issn.1007-6611.2017.10.004

王文文,女, 1986-10生,碩士,住院醫師,E-mail:bzwangwenwen@126.com

2017-06-09