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      延齡草總皂苷通過(guò)調(diào)控GRP78/IRE1α/TRAF2/JNK信號(hào)通路抑制內(nèi)質(zhì)網(wǎng)應(yīng)激而減輕PSCI大鼠海馬神經(jīng)元損傷*

      2023-03-10 05:55:04王剛楊麗君楊丹段壬澤趙方毓陳顯兵
      中國(guó)病理生理雜志 2023年2期
      關(guān)鍵詞:內(nèi)質(zhì)網(wǎng)象限海馬

      王剛, 楊麗君, 楊丹, 段壬澤, 趙方毓, 陳顯兵

      延齡草總皂苷通過(guò)調(diào)控GRP78/IRE1α/TRAF2/JNK信號(hào)通路抑制內(nèi)質(zhì)網(wǎng)應(yīng)激而減輕PSCI大鼠海馬神經(jīng)元損傷*

      王剛, 楊麗君, 楊丹, 段壬澤, 趙方毓, 陳顯兵△

      (湖北民族大學(xué)附屬民大醫(yī)院病理科,湖北民族大學(xué)醫(yī)學(xué)部,湖北 恩施 445000)

      探討延齡草總皂苷(TST)對(duì)卒中后認(rèn)知障礙(PSCI)大鼠海馬神經(jīng)元的保護(hù)作用及分子機(jī)制。將采用改良Zea Longa線栓法造模成功的大鼠隨機(jī)分為模型(model)組、TST(100 mg/kg)組和鹽酸多奈哌齊(DON; 0.45 mg/kg)組,另設(shè)假手術(shù)(sham)組,每組10只,連續(xù)給藥4周。采用Morris水迷宮實(shí)驗(yàn)檢測(cè)大鼠學(xué)習(xí)記憶能力;TTC染色檢測(cè)大鼠腦梗死體積變化,HE、Nissl和TUNEL染色觀察大鼠海馬組織神經(jīng)元病理變化;免疫組化及Western blot檢測(cè)葡萄糖調(diào)節(jié)蛋白78(GRP78)、肌醇需求酶1α(IRE1α)、腫瘤壞死因子受體相關(guān)因子2(TRAF2)、磷酸化c-Jun氨基末端激酶(p-JNK)、胱天蛋白酶12(caspase-12)、Bax和Bcl-2的蛋白水平。與sham組相比,model組大鼠逃避潛伏期顯著延長(zhǎng),穿越平臺(tái)次數(shù)及目標(biāo)象限停留時(shí)間顯著減少(<0.01);大鼠腦梗死體積顯著增大,神經(jīng)元尼氏小體數(shù)量顯著減少,凋亡細(xì)胞顯著增多;海馬組織中GRP78、IRE1α、TRAF2、p-JNK、caspase-12和Bax蛋白水平顯著升高,Bcl-2蛋白水平顯著降低(<0.01)。與model組比較,TST組及DON組大鼠逃避潛伏期顯著縮短,穿越平臺(tái)次數(shù)及目標(biāo)象限停留時(shí)間顯著增加(<0.01);大鼠腦梗死體積顯著縮小,神經(jīng)元尼氏小體數(shù)量顯著增多,凋亡細(xì)胞顯著減少;GRP78、IRE1α、TRAF2、p-JNK、caspase-12和Bax蛋白水平顯著降低,Bcl-2蛋白水平顯著升高(<0.01)。TST對(duì)PSCI大鼠海馬神經(jīng)元具有保護(hù)作用,其機(jī)制可能與減輕內(nèi)質(zhì)網(wǎng)應(yīng)激、減少神經(jīng)元凋亡并抑制GRP78/IRE1α/TRAF2/JNK信號(hào)通路有關(guān)。

      延齡草總皂苷;卒中后認(rèn)知障礙;內(nèi)質(zhì)網(wǎng)應(yīng)激;細(xì)胞凋亡;GRP78/IRE1α/TRAF2/JNK信號(hào)通路

      卒中后認(rèn)知障礙(post-stroke cognitive impairment, PSCI)是指由卒中引起的從輕度認(rèn)知障礙到癡呆的一系列綜合征,是世界范圍內(nèi)卒中后發(fā)病率和死亡率的主要來(lái)源。PSCI包括兩種不同程度的認(rèn)知損害,即卒中后認(rèn)知障礙非癡呆(post-stroke cognitive impairment with no dementia, PSCIND)和卒中后癡呆(post-stroke dementia, PSD)。研究表明,19.3%的卒中患者在中風(fēng)后10年內(nèi)發(fā)展為癡呆癥[1]。因此,針對(duì)PSCI早期干預(yù)治療,阻止PSCI向PSD發(fā)展,是亟待解決的問題。

      內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulum stress, ERS)是指細(xì)胞受到各種應(yīng)激因素的影響,使內(nèi)質(zhì)網(wǎng)腔內(nèi)出現(xiàn)錯(cuò)誤折疊和未折疊蛋白質(zhì)積聚、Ca2+平衡紊亂,引起細(xì)胞內(nèi)穩(wěn)態(tài)失衡的狀態(tài)[2-3]。而未折疊蛋白反應(yīng)(unfolded protein response, UPR)是指ER應(yīng)激發(fā)生時(shí),細(xì)胞為促進(jìn)蛋白質(zhì)正確折疊,或降解錯(cuò)誤折疊蛋白質(zhì)而產(chǎn)生的應(yīng)激反應(yīng)[4]。PSCI的發(fā)病機(jī)制復(fù)雜,有缺血再灌注損傷、氧化應(yīng)激、炎癥反應(yīng)、自噬等機(jī)制[5-6]。研究顯示,ERS在PSCI發(fā)展過(guò)程中起重要作用,適當(dāng)?shù)腅RS有利于去除錯(cuò)誤折疊的蛋白質(zhì),對(duì)疾病的病理過(guò)程起到保護(hù)作用[7]。但是,在嚴(yán)重或長(zhǎng)期內(nèi)質(zhì)網(wǎng)應(yīng)激的病理?xiàng)l件下,內(nèi)質(zhì)網(wǎng)的生物學(xué)功能受損,引發(fā)海馬神經(jīng)元凋亡,從而導(dǎo)致認(rèn)知功能下降[8-9]。目前,能夠引起細(xì)胞凋亡的信號(hào)轉(zhuǎn)導(dǎo)途徑共有三種:死亡受體信號(hào)途徑、線粒體途徑以及內(nèi)質(zhì)網(wǎng)應(yīng)激途徑[10-11]。而在內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的凋亡途徑中,JNK和caspase-12的激活起著重要作用[12-13]。

      延齡草(Maxim, TTM)又名頭頂一顆珠,為湖北省恩施地區(qū)珍貴的土家藥材,該藥味甘、性平,有小毒。前期研究發(fā)現(xiàn),其提取物TTM總皂苷(total saponins from TTM, TST)具有抗氧化、延緩衰老和神經(jīng)保護(hù)作用,但其作用效果和分子機(jī)制仍有待深入研究[14-15]。因此,本項(xiàng)工作采用改良線栓法行大腦中動(dòng)脈栓塞術(shù)制備PSCI大鼠模型,進(jìn)一步探討其對(duì)PSCI大鼠學(xué)習(xí)記憶能力及海馬神經(jīng)元的保護(hù)機(jī)制。

      材料和方法

      1 實(shí)驗(yàn)動(dòng)物

      SPF級(jí)雄性SD大鼠60只,2月齡,體質(zhì)量(200±20) g,由三峽大學(xué)實(shí)驗(yàn)動(dòng)物中心提供,許可證號(hào):SCXK(鄂)2017-0012。動(dòng)物飼養(yǎng)在本校清潔級(jí)動(dòng)物房,溫度控制在23~25 ℃,相對(duì)濕度控制在50%~60%,常規(guī)適應(yīng)性喂養(yǎng)1周后開始實(shí)驗(yàn)。

      2 藥品、試劑與儀器

      頭頂一顆珠采自恩施本地,經(jīng)專家鑒定為百合科延齡草屬植物延齡草的干燥根莖。潔凈藥材后,經(jīng)烘干粉碎置于圓底燒瓶?jī)?nèi),加入適量75%乙醇浸泡萃取。將提取后的藥液過(guò)濾并濃縮干燥,得延齡草提取物,加入雙蒸水將提取物完全溶解,采用飽和正丁醇再次萃取,減壓濃縮干燥,檢測(cè)提取物中總皂苷含量為14.72 mg/g生藥。鹽酸多奈哌齊(donepezil hydrochloride, DON)片(批號(hào):0000001782,浙江華海藥業(yè)股份有限公司)。

      TTC染液(批號(hào):G3005,Solarbio);TUNEL凋亡試劑盒(批號(hào):E-CK-A321,武漢伊萊瑞特生物科技股份有限公司);免疫組化試劑盒(批號(hào):2201269710A,福州邁新生物技術(shù)開發(fā)有限公司);葡萄糖調(diào)節(jié)蛋白78(glucose-regulated protein 78, GRP78)抗體、肌醇需求酶1α(inositol-requiring enzyme 1α, IRE1α)抗體、HRP標(biāo)記的山羊抗兔IgG和HRP標(biāo)記的山羊抗小鼠IgG(批號(hào)分別為ab21685、ab37073、ab6721和ab6789;Abcam);腫瘤壞死因子受體相關(guān)因子2(tumor necrosis factor receptor-associated factor 2, TRAF2)、磷酸化c-Jun氨基末端激酶(phosphorylated c-Jun N-terminal kinase, p-JNK)和胱天蛋白酶12(caspase-12)抗體(批號(hào)分別為:ML02846、WL01813、WL03268,萬(wàn)類生物技術(shù)有限公司);Bcl-2和Bax抗體(批號(hào)分別為A19693和A19684,ABclonal);β-actin抗體(批號(hào):66009-1-Ig,武漢三鷹生物技術(shù)有限公司)。

      1510型酶標(biāo)儀(Thermo Scientific);水迷宮系統(tǒng)(Top Scan 2.00);Western Blot電泳及轉(zhuǎn)膜全套裝置(Bio-Rad)。

      3 主要方法

      3.1大腦中動(dòng)脈閉塞(middle cerebral artery occlusion, MCAO)大鼠模型的制備60只SD大鼠隨機(jī)分為造模組(50只)和假手術(shù)(sham)組(10只)。造模組參照文獻(xiàn)[16]采用改良Zea Longa線栓法制備MCAO大鼠模型;sham組僅切開皮膚,鈍性分離肌肉及筋膜,分離出左側(cè)頸總動(dòng)脈后即縫合切口。手術(shù)后將動(dòng)物置于放有清潔墊料的飼養(yǎng)盒內(nèi)并維持大鼠體溫,待大鼠蘇醒后進(jìn)行行為學(xué)評(píng)分,1~3分為造模成功。其中造模組50只大鼠術(shù)后共死亡13只,sham組無(wú)死亡,剩余存活造模組大鼠進(jìn)行神經(jīng)功能評(píng)分,納入評(píng)分標(biāo)準(zhǔn)的造模大鼠一共30只。

      3.2MCAO模型的評(píng)價(jià)與動(dòng)物的分組干預(yù)按照Z(yǔ)ea Longa 5分法對(duì)造模后1、3、7、14和28 d大鼠神經(jīng)功能進(jìn)行評(píng)分,并將30只造模成功大鼠隨機(jī)分為模型(model)組(10只)、TST組(10只)和DON組(10只),連同上述sham組(10只)大鼠進(jìn)行實(shí)驗(yàn)。根據(jù)動(dòng)物與人體間等效劑量確定各組大鼠的給藥量,TST組給予100 mg/kg TST進(jìn)行灌胃,DON組給予0.45 mg/kg DON進(jìn)行灌胃,sham及model組給予等體積的雙蒸水灌胃,各組均連續(xù)灌胃28 d。

      3.3Morris水迷宮評(píng)價(jià)大鼠學(xué)習(xí)記憶能力給藥結(jié)束當(dāng)天對(duì)大鼠進(jìn)行定位航行實(shí)驗(yàn),共持續(xù)5 d,提前將實(shí)驗(yàn)大鼠搬進(jìn)水迷宮實(shí)驗(yàn)室以適應(yīng)新環(huán)境,系統(tǒng)自動(dòng)將圓形水面分為4個(gè)象限,求生平臺(tái)放在第3象限略低于水面0.6~1.0 cm處,大鼠面向池壁分別沿第1、2、4象限放入,系統(tǒng)自動(dòng)記錄60 s內(nèi)大鼠尋找平臺(tái)所需時(shí)間,即逃避潛伏期;第6天休息;第7天上午進(jìn)行正式定位航行實(shí)驗(yàn),將大鼠沿第1象限放入求得尋找平臺(tái)所需時(shí)間,下午進(jìn)行空間探索實(shí)驗(yàn),將平臺(tái)撤去,大鼠沿第1象限放入,記錄60 s內(nèi)大鼠穿越平臺(tái)次數(shù)及目標(biāo)象限停留時(shí)間比,并進(jìn)行統(tǒng)計(jì)分析。

      3.4腦組織灌注及海馬病理形態(tài)學(xué)觀察給藥7 d后每組隨機(jī)挑選出3只大鼠進(jìn)行TTC染色,以10%水合氯醛按3 mL/kg腹腔注射麻醉大鼠,將大腦完整剝?nèi)〕龊螅?20 ℃冰箱冷凍20 min,沿冠狀位切1 mm厚片,加入適量TTC染液覆蓋腦組織,放入37 ℃恒溫箱中20 min,期間注意翻面,拍照分析;給藥28 d后每組取3只大鼠進(jìn)行灌注固定,大鼠麻醉后,經(jīng)心臟灌注固定,斷頭取全腦,沿大腦視交叉處做4~5 mm冠狀切片,將其置于4%多聚甲醛中固定48 h。常規(guī)脫水、石蠟包埋后,行4 μm石蠟切片,進(jìn)行HE、Nissl和TUNEL染色,切片掃描并觀察腦組織形態(tài)改變情況。

      3.5免疫組化測(cè)定GRP78、IRE1α、TRAF2、p-JNK和caspase-12蛋白在海馬CA1區(qū)的表達(dá)定位腦組織被制成3 μm石蠟切片后,置于75 ℃攤片機(jī)中烘烤2 h,二甲苯脫蠟3次,每次10 min,無(wú)水乙醇浸泡3次,每次5 min,純水沖洗2 min,置于檸檬酸鈉配成的修復(fù)液中,水浴修復(fù)20 min。自然冷卻室溫后,組化筆圈起組織,PBS沖洗3次,每次3 min。嚴(yán)格按照免疫組化試劑盒說(shuō)明操作,滴加A液(內(nèi)源性過(guò)氧化物酶阻斷劑)反應(yīng)10 min;PBS沖洗3次,除去PBS,滴加B液(非特異染色阻斷劑)反應(yīng)10 min,滴加稀釋好的GRP78抗體(1∶1 000)、IRE1α抗體(1∶500)、TRAF2抗體(1∶200)、p-JNK抗體(1∶200)和caspase-12抗體(1∶200),室溫孵育1 h;PBS沖洗3次,除去PBS,滴加C液(生物素標(biāo)記的羊抗兔IgG聚合物),反應(yīng)10 min;PBS沖洗3次,除去PBS,滴加D液(辣根過(guò)氧化物酶)反應(yīng)10 min;PBS沖洗3次,除去PBS,滴加DAB顯色液避光顯色2 min,PBS沖洗3次,置于蘇木素中復(fù)染1 min,水洗1 min,分化30 s,水洗1 min,返藍(lán)15 s,水洗1 min,無(wú)水乙醇2 min,二甲苯2 min,中性樹膠濕封,切片掃描分析。

      3.6Western blot檢測(cè)海馬中相關(guān)蛋白表達(dá)大鼠麻醉后,斷頭取腦,冰上剝?nèi)∽髠?cè)海馬并標(biāo)記好放入-80 ℃?zhèn)溆?;稱取50 mg組織放入EP管中,加入裂解液和PMSF混合液(100∶1)500 μL,冰上剪碎、研磨、超聲、裂解,得到混懸液,10 080×離心15 min,吸取上清液,配平組織蛋白濃度。制備12% SDS-PAGE凝膠,依次進(jìn)行電泳、轉(zhuǎn)膜、封閉、洗膜;加入GRP78抗體(1∶1 000)、IRE1α抗體(1∶1 000)、TRAF2抗體(1∶1 000)、p-JNK抗體(1∶500)、caspase-12抗體(1∶1 000)、Bax抗體(1∶1 000)和Bcl-2抗體(1∶1 000),4 ℃冰箱孵育過(guò)夜;次日洗膜,Ⅱ抗室溫孵育1 h;洗膜,滴加ECL顯色液進(jìn)行曝光處理,采用GIS 1D圖像分析軟件進(jìn)行灰度值計(jì)算。

      4 統(tǒng)計(jì)學(xué)處理

      采用統(tǒng)計(jì)軟件SPSS 22.0進(jìn)行數(shù)據(jù)分析處理,實(shí)驗(yàn)數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示。多組間比較采用單因素方差分析(one-way ANOVA)。以<0.05為差異具有統(tǒng)計(jì)學(xué)意義。

      結(jié)果

      1 各組大鼠神經(jīng)功能評(píng)分的比較

      如圖1所示,術(shù)后model組中的大鼠神經(jīng)功能評(píng)分在前階段呈現(xiàn)出升高的趨勢(shì),3 d后出現(xiàn)降低的趨勢(shì),TST組與DON組在給藥兩周后神經(jīng)功能恢復(fù)顯著。與sham組比較,model組大鼠術(shù)后神經(jīng)功能評(píng)分顯著升高(<0.01);與model組比較,TST組及DON組神經(jīng)功能評(píng)分顯著降低(<0.01);TST組與DON組比較無(wú)統(tǒng)計(jì)學(xué)意義。

      Figure 1. Comparison of neurological function scores of the rats in each group. Mean±SD. n=7. **P<0.01 vs sham group; ##P<0.01 vs model group.

      2 TST對(duì)PSCI大鼠學(xué)習(xí)記憶能力的影響

      如圖2所示,sham組與給藥組大鼠路線清晰較短且表現(xiàn)出一定的目標(biāo)趨向性,model組大鼠路線雜亂且長(zhǎng)目標(biāo)趨向混亂。與sham組相比,model組大鼠逃避潛伏期時(shí)間顯著增加(<0.01),穿越平臺(tái)次數(shù)(<0.05)及目標(biāo)象限停留時(shí)間顯著減少(<0.01);與model組相比,TST組和DON組大鼠逃避潛伏期時(shí)間顯著減少(<0.01),穿越平臺(tái)次數(shù)(<0.05)及目標(biāo)象限停留時(shí)間顯著增加(<0.01),見圖3。

      Figure 2. Morris water maze swimming trajectories of the rats in each group.

      Figure 3. The results of Morris water maze experiment in the rats of each group. A: escape latencies in rats for 5 consecutive days; B: bar chart of escape latency of the rats in each group; C: the times of the rat crossing the platform within 60 s; D: the ratio of the time that the rats stay in the target quadrant. Mean±SD. n=7. **P<0.01 vs sham group; #P<0.05, ##P<0.01 vs model group.

      3 TST對(duì)各組大鼠腦梗死體積的影響

      如圖4所示,假手術(shù)組大鼠腦組織呈均勻紅色,無(wú)缺血梗死灶,其它各組大鼠腦組織均有不同程度的缺血梗死灶出現(xiàn)。與sham組相比,model組大鼠左側(cè)腦組織白色缺血梗死灶顯著增大,與model組相比,TST組及DON組大鼠左側(cè)腦組織白色缺血梗死灶顯著減小。

      Figure 4. The effect of TST on the volume of cerebral infarction in the rats of each group. In sham group, the brain tissue was uniformly red, with no ischemic infarction; in model group, the left brain tissue had obvious white ischemic infarction; the left infarction in TST group and DON group decreased to varying degrees.

      4 TST對(duì)PSCI大鼠海馬組織病理變化的影響

      如圖5所示,sham組大鼠腦組織著色均勻,海馬CA1區(qū)神經(jīng)元飽滿豐富、排列整齊、數(shù)量較多,胞質(zhì)內(nèi)有豐富的尼氏體。皮質(zhì)區(qū)細(xì)胞排列緊密,細(xì)胞核著色清晰,未見明顯的壞死細(xì)胞及空泡化現(xiàn)象;與sham組相比,model組大鼠腦組織著色不均,皮質(zhì)區(qū)域著色較淺,海馬CA1區(qū)神經(jīng)元排列紊亂,胞質(zhì)中尼氏體明顯減少,出現(xiàn)核固縮現(xiàn)象。皮質(zhì)區(qū)出現(xiàn)大量壞死細(xì)胞及空泡化現(xiàn)象;與model組相比,TST組及DON組大鼠腦組織著色較為均勻,海馬CA1區(qū)神經(jīng)元較飽滿,胞質(zhì)內(nèi)尼氏體明顯增多,空泡化現(xiàn)象減少。皮質(zhì)區(qū)著色較深,細(xì)胞空泡化現(xiàn)象及壞死細(xì)胞明顯減少。

      Figure 5. The effect of TST on the pathological changes of brain tissue in PSCI rats (HE staining, scale bar=1 000 μm in HP, scale bar=50 μm CA1 and CA3; Nissl staining, scale bar=50 μm in CA1 and CA3). In sham group, the nerve cells were neatly arranged, complete and abundant, and there were abundant Nissl bodies in the cytoplasm. In model group, part of the cerebral cortex showed a loose mesh structure, the arrangement of nerve cells was disordered, the cells shrank, the cell number decreased, and the Nissl bodies in the cytoplasm decreased significantly. In TST group and DON group, the atrophic morphology of nerve cells was improved, the cell number increased, and the Nissl bodies in the cytoplasm increased significantly.

      5 TST對(duì)PSCI大鼠海馬神經(jīng)元凋亡的影響

      如圖6所示,圖中顯藍(lán)色為細(xì)胞核,綠色為凋亡標(biāo)記神經(jīng)元。與sham組相比,model組大鼠海馬CA1區(qū)綠色熒光強(qiáng)度明顯增強(qiáng),陽(yáng)性細(xì)胞數(shù)量明顯增多。與model組相比,TST組及DON組大鼠海馬CA1區(qū)綠色熒光強(qiáng)度明顯減弱,陽(yáng)性細(xì)胞數(shù)量明顯減少。

      Figure 6. The effect of TST on the apoptosis of hippocampal neurons in PSCI rats (scale bar=50 μm). In sham group, the green fluorescence intensity in the CA1 region of the hippocampus was weak, and the number of apoptotic cells was small. In model group, the green fluorescence intensity in the CA1 region of the hippocampus was significantly enhanced, and the number of apoptotic cells increased. The green fluorescence intensity in hippocampal CA1 area of TST group and DON group was significantly weakened, and the number of positive cells was significantly reduced.

      6 TST對(duì)大鼠海馬CA1區(qū)GRP78、IRE1α、TRAF2、p-JNK和caspase-12蛋白定位表達(dá)的影響

      如圖7所示,掃描圖像400倍放大后顯示,除IRE1α為細(xì)胞核表達(dá)外,其余蛋白均為胞質(zhì)表達(dá)。與sham組相比,model組大鼠海馬CA1區(qū)GRP78、IRE1α、TRAF2、p-JNK和caspase-12的陽(yáng)性細(xì)胞數(shù)量顯著增多;與model組相比,TST組及DON組GRP78、IRE1α、TRAF2、p-JNK和caspase-12的陽(yáng)性細(xì)胞數(shù)量顯著減少。

      Figure 7. The effect of TST on the protein expression and localization of GRP78, IRE1α, TRAF2, p-JNK and caspase-12 in the CA1 region of the rat hippocampus (scale bar=50 μm). In sham group, the numbers of positive cells for GRP78, IRE1α, TRAF2, p-JNK and caspase-12 in the CA1 region of the hippocampus were less. In model group, the numbers of positive cells of GRP78, IRE1α, TRAF2, p-JNK and caspase-12 in the CA1 region of the hippocampus increased significantly. In TST group and DON group, the numbers of positive cells for GRP78, IRE1α, TRAF2, p-JNK and caspase-12 were significantly reduced compared with model group.

      7 TST對(duì)大鼠海馬組織GRP78、IRE1α、TRAF2、p-JNK、caspase-12、Bax和Bcl-2蛋白表達(dá)的影響

      如圖8所示,與sham組相比,model組大鼠海馬組織GRP78、IRE1α、TRAF2、p-JNK、caspase-12和Bax的蛋白表達(dá)水平顯著升高,Bcl-2的蛋白表達(dá)水平顯著降低(<0.01);與model組相比,TST組及DON組大鼠海馬組織GRP78、IRE1α、TRAF2、p-JNK、caspase-12和Bax的蛋白表達(dá)水平顯著降低,Bcl-2的蛋白表達(dá)水平顯著升高(<0.01)。

      Figure 8. The effect of TST on the protein levels of GRP78, IRE1α, TRAF2, p-JNK, caspase-12, Bax and Bcl-2 in rat hippocampus. A: the protein levels of GRP78, IRE1α, TRAF2 and p-JNK; B: the protein levels of caspase-12, Bax and Bcl-2. Mean±SD. n=4. **P<0.01 vs sham group; ##P<0.01 vs model group.

      討論

      缺血性腦卒中是一種急性腦血管疾病,其特征是大腦局部的血流突然中斷,導(dǎo)致局部產(chǎn)生缺血缺氧狀態(tài),認(rèn)知功能損害是其并發(fā)癥之一。研究顯示,ERS是導(dǎo)致神經(jīng)元損傷的關(guān)鍵因素之一,也被認(rèn)為是缺血性腦損傷干預(yù)的潛在治療靶點(diǎn)。腦缺血后,蛋白質(zhì)的錯(cuò)誤折疊和過(guò)度積累,以及缺血再灌注引起的ROS過(guò)度產(chǎn)生,會(huì)觸發(fā)ERS并增加神經(jīng)元凋亡,從而導(dǎo)致認(rèn)知功能的下降[17-18]。本研究采用水迷宮對(duì)各組大鼠的學(xué)習(xí)記憶能力進(jìn)行評(píng)價(jià),結(jié)果顯示,模型大鼠逃避潛伏期延長(zhǎng),尋找平臺(tái)的路徑雜亂且缺乏目標(biāo)趨向性;在給予TST藥物干預(yù)后,大鼠逃避潛伏期及尋找平臺(tái)路徑縮短,目標(biāo)趨向明確,這表明TST對(duì)于缺血性腦卒中引起的認(rèn)知功能障礙具有明顯的改善作用。

      內(nèi)質(zhì)網(wǎng)是最大的管狀網(wǎng)狀細(xì)胞器,在細(xì)胞生理過(guò)程中發(fā)揮重要作用,主要負(fù)責(zé)蛋白質(zhì)的合成和折疊、鈣的儲(chǔ)存和釋放、脂質(zhì)的合成和分配等功能[19]。研究表明,ERS在腦缺血病理生理學(xué)中起著至關(guān)重要的作用,輕微的ERS有助于提高細(xì)胞耐受性并恢復(fù)細(xì)胞穩(wěn)態(tài);然而,過(guò)度或長(zhǎng)期ERS會(huì)導(dǎo)致細(xì)胞的凋亡[20, 21]。本研究顯示,經(jīng)改良線栓法造模后的大鼠出現(xiàn)肢體及認(rèn)知功能損害,在經(jīng)TTC染色后病灶側(cè)出現(xiàn)缺血變白區(qū)域,初步判定大腦局部出現(xiàn)缺血缺氧的病理性損傷。進(jìn)一步采用HE及尼氏染色對(duì)大鼠左側(cè)半腦缺氧狀態(tài)下的病理微觀變化進(jìn)行分析,結(jié)果顯示,model組大鼠皮質(zhì)及海馬CA1區(qū)神經(jīng)元壞死凋亡現(xiàn)象最為顯著,神經(jīng)元出現(xiàn)排列疏松、核固縮及空泡化現(xiàn)象,且尼氏體數(shù)量顯著減少。在給予TST治療后,神經(jīng)元空泡化及核固縮現(xiàn)象顯著減輕,這說(shuō)明TST對(duì)于缺血缺氧造成的神經(jīng)元丟失及損傷具有抑制作用。TUNEL染色顯示,model組海馬CA1區(qū)綠色熒光強(qiáng)度明顯增強(qiáng),陽(yáng)性細(xì)胞明顯增多,而給予TST治療后其熒光強(qiáng)度明顯減弱,陽(yáng)性細(xì)胞數(shù)明顯減少,表明TST在一定程度上能夠抑制神經(jīng)元的凋亡,對(duì)神經(jīng)元起到一定保護(hù)作用。

      研究表明,GRP78/IRE1α/TRAF2/JNK通路已被廣泛應(yīng)用在調(diào)節(jié)內(nèi)質(zhì)網(wǎng)應(yīng)激引起的神經(jīng)細(xì)胞凋亡中,并且發(fā)揮重要作用[22-23]。當(dāng)細(xì)胞受到輕微應(yīng)激時(shí),ERS激活UPR使GRP78與IRE1α發(fā)生解離,此時(shí)IRE1α在內(nèi)質(zhì)網(wǎng)膜上發(fā)生二聚化和自磷酸化從而激活JNK,活化后的JNK可以下調(diào)抗凋亡蛋白Bcl-2來(lái)增強(qiáng)Bax依賴性凋亡[24]。然而,在長(zhǎng)期或嚴(yán)重的應(yīng)激下,IRE1α無(wú)法緩解ERS時(shí),UPR會(huì)激活凋亡信號(hào)通路,此時(shí)激活后的IRE1α募集TRAF2,TRAF2可以募集和激活凋亡信號(hào)調(diào)節(jié)蛋白1,最終激活JNK和caspase-12通路來(lái)促進(jìn)細(xì)胞凋亡,以維持內(nèi)環(huán)境的穩(wěn)定[25-27]。本研究顯示,model組大鼠海馬CA1區(qū)內(nèi)質(zhì)網(wǎng)相關(guān)蛋白棕色陽(yáng)性表達(dá)明顯,給予TST及DON治療后海馬CA1區(qū)棕色陽(yáng)性表達(dá)減弱;Western blot結(jié)果顯示,TST能夠下調(diào)海馬組織中GRP78、IRE1α、TRAF2、p-JNK、caspase-12和Bax的蛋白表達(dá),上調(diào)Bcl-2的蛋白表達(dá),這表明TST可能對(duì)GRP78/IRE1α/TRAF2/JNK信號(hào)通路起到抑制作用,從而對(duì)PSCI大鼠海馬神經(jīng)元起到一定保護(hù)作用。

      綜上所述,延齡草總皂苷可以改善PSCI大鼠的學(xué)習(xí)記憶能力,并能改善由內(nèi)質(zhì)網(wǎng)應(yīng)激引起的海馬神經(jīng)元損傷,其機(jī)制可能與其抑制GAP78/IRE1α/TRAF2/JNK信號(hào)通路,上調(diào)Bcl-2的蛋白表達(dá),下調(diào)caspase-12及Bax的蛋白表達(dá)有關(guān)。

      [1] Ivan CS, Seshadri S, Beiser A, et al. Dementia after stroke: the Framingham study[J]. Stroke, 2004, 35(6):1264-1268.

      [2] Jo F, Jo H, Hilzendeger AM, et al. Brain endoplasmic reticulum stress mechanistically distinguishes the saline-intake and hypertensive response to deoxycorticosterone acetate-salt[J]. Hypertension, 2015, 65(6):1341-1348.

      [3] Zhang XY, Yang SM, Zhang HP, et al. Endoplasmic reticulum stress mediates the arsenic trioxide-induced apoptosis in human hepatocellular carcinoma cells[J]. Int J Biochem Cell Biol, 2015, 68:158-165.

      [4] Smith HL, Mallucci GR. The unfolded protein response: mechanisms and therapy of neurodegeneration[J]. Brain, 2016, 139(Pt 8):2113-2121.

      [5] Hetz C, Saxena S. ER stress and the unfolded protein response in neurodegeneration[J]. Nat Rev Neurol, 2017, 13(8):477-491.

      [6]陳麗敏, 白艷杰, 王巖, 等. 線粒體質(zhì)量控制失調(diào)介導(dǎo)卒中后認(rèn)知障礙的研究進(jìn)展[J]. 中國(guó)病理生理雜志, 2022, 38(7):1320-1327.

      Chen LM, Bai YJ, Wang Y, et al. Progress in mitochondrial quality control disorder-mediated cognitive impairment after stroke[J]. Chin J Pathophysiol, 2022, 38(7):1320-1327.

      [7] Mohammed Thangameeran SI, Tsai ST, Hung HY, et al. A role for endoplasmic reticulum stress in intracerebral hemorrhage[J]. Cells, 2020, 9(3):750.

      [8] Xin Q, Ji B, Cheng B, et al. Endoplasmic reticulum stress in cerebral ischemia[J]. Neurochem Int, 2014, 68:18-27.

      [9] Chen L, Xia YF, Shen SF, et al. Syntaxin 17 inhibits ischemic neuronal injury by resuming autophagy flux and ameliorating endoplasmic reticulum stress[J]. Free Radic Biol Med, 2020, 160:319-333.

      [10] Ten V, Galkin A. Mechanism of mitochondrial complex I damage in brain ischemia/reperfusion injury: a hypothesis[J]. Mol Cell Neurosci, 2019, 100:103408.

      [11] Datta A, Sarmah D, Mounica L, et al. Cell death pathways in ischemic stroke and targeted pharmacotherapy[J]. Transl Stroke Res, 2020, 11(6):1185-1202.

      [12] Di Sano F, Ferraro E, Tufi R, et al. Endoplasmic reticulum stress induces apoptosis by an apoptosome-dependent but caspase 12-independent mechanism[J]. J Biol Chem, 2006, 281(5):2693-2700.

      [13] Szegezdi E, Logue SE, Gorman AM, et al. Mediators of endoplasmic reticulum stress-induced apoptosis[J]. EMBO Rep, 2006, 7(9):880-885.

      [14] Wang L, Du J, Zhao F, et al. Trillium tschonoskii maxim saponin mitigates D-galactose-induced brain aging of rats through rescuing dysfunctional autophagy mediated by Rheb-mTOR signal pathway[J]. Biomed Pharmacother, 2018, 98:516-522.

      [15] Chen XB, Wang ZL, Yang QY, et al. Diosgenin glucoside protects against spinal cord injury by regulating autophagy and alleviating apoptosis[J]. Int J Mol Sci, 2018, 19(8):2274.

      [16] Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats[J]. Stroke, 1989, 20(1):84-91.

      [17] Huang M, Li ZX, Chen J, et al. Extracts of bauhinia championiialleviate acute neuronal injury after ischemic reperfusion by improving endoplasmic reticulum stress-mediated neuronal apoptosis[J]. Curr Med Sci, 2022, 42(3):483-490.

      [18] Xin Q, Ji B, Cheng B, et al. Endoplasmic reticulum stress in cerebral ischemia[J]. Neurochem Int, 2014, 68:18-27.

      [19] Addinsall AB, Wright CR, Andrikopoulos S, et al. Emerging roles of endoplasmic reticulum-resident selenoproteins in the regulation of cellular stress responses and the implications for metabolic disease[J]. Biochem J, 2018, 475(6):1037-1057.

      [20] Wang L, Liu Y, Zhang X, et al. Endoplasmic reticulum stress and the unfolded protein response in cerebral ischemia/reperfusion injury[J]. Front Cell Neurosci, 2022, 16:864426.

      [21] 蘭卓, 王歡, 何夕松, 等. 柚皮素對(duì)心肌缺血/再灌注損傷大鼠PI3K/AKT信號(hào)通路和內(nèi)質(zhì)網(wǎng)應(yīng)激及其相關(guān)凋亡通路的影響[J]. 中國(guó)病理生理雜志, 2021, 37(1):41-47.

      Lan Z, Wang H, He XS, et al. Effects of naringenin on PI3K/AKT signaling pathway and endoplasmic reticulum stress and its related apoptotic pathways in rats with myocardial ischemia/reperfusion injury[J]. Chin J Pathophysiol, 2021, 37(1):41-47.

      [22] Xu B, Xu J, Cai N, et al. Roflumilast prevents ischemic stroke-induced neuronal damage by restricting GSK3β-mediated oxidative stress and IRE1α/TRAF2/JNK pathway[J]. Free Radic Biol Med, 2021, 163:281-296.

      [23] Ji M, Niu S, Guo J, et al. Silencing RNF13 alleviates Parkinson's disease-like problems in mouse models by regulating the endoplasmic reticulum stress-mediated IRE1α- TRAF2-ASK1-JNK pathway[J]. J Mol Neurosci, 2020, 70(12):1977-1986.

      [24] Lei K, Davis RJ. JNK phosphorylation of Bim-related members of the Bcl2 family induces Bax-dependent apoptosis[J]. Proc Natl Acad Sci U S A, 2003, 100(5):2432-2437.

      [25] Hetz C, Papa FR. The unfolded protein response and cell fate control[J]. Mol Cell, 2018, 69(2):169-181.

      [26] Wang F, Weng H, Quon MJ, et al. Dominant negative FADD dissipates the proapoptotic signalosome of the unfolded protein response in diabetic embryopathy[J]. Am J Physiol Endocrinol Metab, 2015, 309(10):E861-E873.

      [27] Sch?nthal AH. Pharmacological targeting of endoplasmic reticulum stress signaling in cancer[J]. Biochem Pharmacol, 2013, 85(5):653-666.

      Total saponins fromMaxim attenuate hippocampal neuronal injury in PSCI rats by inhibiting endoplasmic reticulum stress via regulating GRP78/IRE1α/TRAF2/JNK signaling pathway

      WANG Gang, YANG Lijun, YANG Dan, DUAN Renze, ZHAO Fangyu, CHEN Xianbing△

      (,,,445000,)

      To investigate the protective effect of total saponins fromMaxim (TST) on hippocampal neurons in rats with post-stroke cognitive impairment (PSCI), and to explore the molecular mechanism.The rat PSCI model was successfully established by the modified Zea Longa suture method, and the rats were randomly divided into model group, TST (100 mg/kg) group, and donepezil hydrochloride (DON; 0.45 mg/kg) group. Another normal rats served as sham group. There were 10 animals in each group, and they were given continuous administration for 4 weeks. Morris water maze was used to detect the learning and memory ability of rats, TTC staining was used to detect the volume change of cerebral infarction in rats, and HE, Nissl and TUNEL staining was used to observe the pathological changes of neurons in the hippocampus of rats. The protein levels of glucose-regulated protein 78 (GRP78), inositol-requiring enzyme 1α (IRE1α), tumor necrosis factor receptor-associated factor 2 (TRAF2), phosphorylated c-Jun N-terminal kinase (p-JNK), caspase-12, Bax and Bcl-2 were detected by immunohistochemistry and Western blot.Compared with sham group, the escape latency of the rats in model group was significantly prolonged, and the number of platform crossings and the residence time in the target quadrant were significantly decreased (<0.01). The volume of cerebral infarction in rats was significantly increased, the number of neuronal Nissl bodies was significantly decreased, and the number of apoptotic cells was significantly increased. The protein levels of GRP78, IRE1α, TRAF2, p-JNK, caspase-12 and Bax in the hippocampus were significantly increased, and the expression of Bcl-2 was significantly decreased (<0.01). Compared with model group, the escape latency of the rats in TST group and DON group was significantly shortened, and the times of crossing the platform and the residence time of the target quadrant were significantly increased (<0.01). The cerebral infarction volume of the rats was significantly reduced, and the number of neuronal Nissl bodies was significantly increased. Apoptotic cells were significantly reduced. The protein levels of GRP78, IRE1α, TRAF2, p-JNK, caspase-12 and Bax were significantly decreased, and the protein expression of Bcl-2 was significantly increased (<0.01).TST has a protective effect on hippocampal neurons in PSCI rats, and its mechanism may be related to reducing endoplasmic reticulum stress and neuronal apoptosis and inhibiting GRP78/IRE1α/TRAF2/JNK signaling pathway.

      total saponins fromMaxim; post-stroke cognitive impairment; endoplasmic reticulum stress; apoptosis; GRP78/IRE1α/TRAF2/JNK signaling pathway

      R74; R363.2

      A

      10.3969/j.issn.1000-4718.2023.02.006

      1000-4718(2023)02-0241-09

      2022-09-30

      2022-12-16

      [基金項(xiàng)目]國(guó)家自然科學(xué)基金資助項(xiàng)目(No. 82260821);湖北民族大學(xué)博士啟動(dòng)基金項(xiàng)目(No. MD2020B014)

      Tel: 13517134626; E-mail: chenxianbing7612@163.com

      (責(zé)任編輯:林白霜,羅森)

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