梁倩++唐偉++劉文博++王威
[摘要]目的 探討辛伐他汀對高膽固醇血癥大鼠β-淀粉樣前體蛋白(APP)、β分泌酶(BACE)蛋白表達(dá)的影響,為阿爾茨海默?。ˋD)的預(yù)防與治療提供理論依據(jù)。方法 32只大鼠(3月齡)隨機分為普通飼料喂養(yǎng)組(n=16)和高膽固醇飼料喂養(yǎng)組(n=16)。將高脂飼料喂養(yǎng)鼠隨機分為高脂飼料安慰劑組(n=8)、高脂飼料藥物組(n=8),并開始灌胃給藥,高脂飼料藥物組給予辛伐他汀2 mg/(kg·d)灌胃,高脂飼料安慰劑組給同體積安慰劑(蒸餾水)灌胃,灌胃期間繼續(xù)喂高脂飼料。普通飼料喂養(yǎng)組隨機分為普通飼料安慰劑組(n=8)和普通飼料藥物組(n=8),分別給予同體積安慰劑(蒸餾水)和辛伐他汀2 mg/(kg·d)灌胃,灌胃期間繼續(xù)喂普通飼料。8周后APP、BACE蛋白的表達(dá)。結(jié)果 高脂飼料藥物組大鼠前額皮層、海馬區(qū)APP、BACE的蛋白表達(dá)較高脂飼料安慰劑組減少(P<0.05),高脂飼料藥物組與普通飼料安慰劑組、普通飼料藥物組之間無顯著性差異(P>0.05)。結(jié)論 辛伐他汀可能會影響高膽固醇血癥大鼠前額皮層、海馬區(qū)APP、BACE的蛋白表達(dá),從而對Aβ1-42的生成代謝產(chǎn)生影響。
[關(guān)鍵詞]辛伐他??;高膽固醇血癥;海馬;皮層;β-淀粉樣前體蛋白;β分泌酶;阿爾茨海默病
[中圖分類號] R749.1 [文獻(xiàn)標(biāo)識碼] A [文章編號] 1674-4721(2017)06(b)-0008-05
[Abstract]Objective To explore the efficacy of Simvastatin on the expression of APP and BACE in rats with hypercholesterolemia,providing theoretical basis for the prevention and treatment in Alzheimer′s Disease.Methods The 32 rats used in this experiment were 3 months old and randomly divided into normal diet group (n=16) and high cholesterol diet groups (n=16).The high diet groups were randomly divided into high lipid feed the placebo group (n=8) and high-fat feed drug group (n=8).And then medicine was filled into their stomach:the high fat diet drugs group were given simvastatin 2 mg/(kg·d),the high fat diet placebo group placebo (distilled water) by the same volume,the high fat forage during lavage.Normal feed group were randomly divided into normal feed placebo group and normal feed drug group,respectively to the same volume with the placebo (distilled water) and simvastatin 2 mg/(kg·d) to fill the stomach,the normal fat forage during lavage,with behavior evaluation after 8 weeks.Immune histochemical method to determine the brain′s protein expression with APP,BACE.Results The result of APP,BACE protein expression in the prefrontal cortex and the hippocampus with immunohistochemical detection:the protein expression of APP and BACE in the prefrontal cortexis and the hippocampus was decreased in high fat feed drug group than the normal feed placebo group (P<0.05),there was no significant difference in the normal feed placebo group,the normal feed drug group and the high fat feed drug group (P>0.05).Conclusion Simvastatin may affect the protein expression of APP,BACE in the prefrontal cortex and the hippocampus by the hypercholesterolemia rats,which have an impact on the metabolism of Aβ1-42.
[Key words]Simvastatin;Hypercholesterolemia;The hippocampus;Cortex;APP;BACE;Alzheimer′s Disease
阿爾茨海默?。ˋD)是多發(fā)于老年人的神經(jīng)系統(tǒng)變性疾病,臨床以進(jìn)行性記憶、認(rèn)知障礙及行為異常為特征[1]。隨著社會老齡化迅速發(fā)展,AD的發(fā)病率急劇上升,嚴(yán)重影響老人的生活質(zhì)量,并給家庭和社會帶來沉重的經(jīng)濟(jì)負(fù)擔(dān)。由于AD的病因復(fù)雜,臨床治療效果并不理想,對其發(fā)病機制的研究是防治AD發(fā)生發(fā)展的重要手段[2-3]。流行病學(xué)調(diào)查發(fā)現(xiàn)高血脂與AD發(fā)生相關(guān)[4-5]。動物實驗顯示β淀粉樣蛋白(Aβ)的形成或降解與血中脂類代謝紊亂相關(guān)[6]。為此,降脂治療能否調(diào)控Aβ形成,能否減緩AD發(fā)生發(fā)展等問題成為當(dāng)前關(guān)注的熱點。β分泌酶(BACE)是β-分泌酶的一種,是生成Aβ所必須的酶[7-8]。它能在體內(nèi)作用于淀粉樣前體蛋白(amyloid precursor protein,APP),是Aβ產(chǎn)生的初始酶,也是限速酶,被認(rèn)為是防治AD的主要藥物靶點[9]。本研究通過給予高脂血癥大鼠辛伐他汀治療,觀察BACE及APP蛋白表達(dá)的變化,初步說明辛伐他汀對AD的治療作用及其機制,現(xiàn)報道如下。
1材料與方法
1.1實驗動物
Wistar大鼠(160~180 g),3月齡,SPF級,由大連醫(yī)科大學(xué)實驗動物中心提供,許可證號:SCXK2015-0002,經(jīng)實驗動物倫理委員會批準(zhǔn),并進(jìn)行保種、繁衍后代,飼料營養(yǎng)充足,飲用無菌水,無菌處理2次/周,鼠籠為小刨花木材墊料籠,溫度20~25℃,濕度50%~60%。高膽固醇飼料由北京華阜康生物科技股份有限公司提供,高膽固醇飼料由普通鼠飼料88.7%、膽固醇1%、豬油10%和膽酸鈉0.3%組成。辛伐他汀片(舒降之,20 mg)由杭州默沙東制藥有限公司提供,國藥準(zhǔn)字H19990366。
1.2動物模型的建立及分組給藥
所用動物為32只大鼠,空腹12 h后取尾血,測定建模前血脂水平,然后隨機分為普通飼料喂養(yǎng)組和高膽固醇飼料喂養(yǎng)組,每組16只,喂養(yǎng)2周后測定血脂水平,血脂明顯升高且與普通飼料喂養(yǎng)組比較差異有顯著性時,認(rèn)為造模成功,再次分組,將高脂飼料喂養(yǎng)大鼠隨機分為高脂飼料安慰劑組、高脂飼料藥物組,每組8只,高脂飼料藥物組給予辛伐他汀2 mg/(kg·d)灌胃,高脂飼料安慰劑組給同體積安慰劑(蒸餾水)灌胃,灌胃期間繼續(xù)喂高脂飼料。將普通飼料喂養(yǎng)大鼠隨機分為普通飼料安慰劑組、普通飼料藥物組,每組8只,普通飼料藥物組給予辛伐他汀2 mg/(kg·d)灌胃,普通飼料安慰劑組給同體積安慰劑(蒸餾水)灌胃,灌胃期間繼續(xù)喂普通飼料。8周后再取尾血,測定血清膽固醇和低密度脂蛋白膽固醇,并取材。
1.3取材
每組8只大鼠用4%水合氯醛以10 ml/kg的劑量腹腔注射麻醉后開胸,充分暴露心臟,將針頭插入左心室找至主動脈,剪開右心耳,用100 ml生理鹽水灌流,直至流出的液體透明清澈無血液,然后用120 ml 4%多聚甲醛灌流固定,直至大鼠四肢及尾部僵硬為灌流固定充分,然后開顱、取腦,取出腦組織放置于4%多聚甲醛中固定,用于免疫組化標(biāo)本制備。
1.4免疫組化檢測小鼠APP、BACE表達(dá)水平
石蠟切片置60℃烤箱烘烤2 h;常規(guī)脫蠟至水,蒸餾水洗2 min;微波修復(fù)抗原20 min;3% H2O2溶液阻斷過氧化物酶,10 min;PBS洗10 min×3次;滴加Aβ1-42(1∶100),4℃過夜;于冰箱中取出切片后室溫靜止20 min,PBS洗10 min×3次;加二抗,37℃孵育50 min,PBS洗10 min×3次;DAB顯色2~3 min;復(fù)染、透明后封片,顯微鏡觀察。
1.5統(tǒng)計學(xué)方法
采用SPSS 17.0統(tǒng)計學(xué)軟件進(jìn)行數(shù)據(jù)分析,計量資料數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,多樣本均數(shù)比較采用多因素方差分析,組間兩兩比較采用LSD-t檢驗;以P<0.05為差異有統(tǒng)計學(xué)意義。
2結(jié)果
2.1辛伐他汀對高膽固醇血癥大鼠血脂的影響
共納入Wistar大鼠32只,所有大鼠均正常成功發(fā)育,進(jìn)入結(jié)果分析,各組小鼠喂養(yǎng)共10周后,通過檢測血清膽固醇提示:高脂飼料安慰劑組大鼠血清膽固醇(CHO)、低密度脂蛋白膽固醇(LDL-C)較高脂飼料藥物組大鼠明顯升高(P<0.05),辛伐他汀能明顯降低血清CHO和LDL-C水平。普通飼料安慰劑組、普通飼料藥物組和高脂飼料藥物組三者間無顯著差異(P>0.05)(圖1)。
2.2各組大鼠前額皮質(zhì)和海馬區(qū)APP的蛋白表達(dá)結(jié)果
免疫組化結(jié)果如圖2所示,APP蛋白主要表達(dá)于細(xì)胞胞漿、細(xì)胞突起和神經(jīng)纖維。計數(shù)APP表達(dá)陽性細(xì)胞數(shù)結(jié)果提示,高脂飼料藥物組大鼠前額皮層、海馬區(qū)APP的蛋白表達(dá)較高脂飼料安慰劑組減少(P<0.05),高脂飼料藥物組與普通飼料安慰劑組、普通飼料藥物組之間無顯著性差異(P>0.05)(圖2、3,表1)。
2.3各組大鼠前額皮質(zhì)和海馬區(qū)BACE的蛋白表達(dá)結(jié)果
從免疫組化結(jié)果可以看出,BACE蛋白著色部位主要位于在胞漿、細(xì)胞突起和神經(jīng)纖維,與APP蛋白定位較為一致(圖3)。計數(shù)BACE表達(dá)陽性細(xì)胞數(shù)結(jié)果提示,高脂飼料藥物組大鼠前額皮層、海馬區(qū)BACE的蛋白表達(dá)較高脂飼料安慰劑組減少(P<0.05),高脂飼料藥物組與普通飼料安慰劑組、普通飼料藥物組之間無顯著性差異(P>0.05)(圖4、5,表2)。
3討論
老年斑是AD腦內(nèi)特征性的變化,是確診AD的重要依據(jù)[10]。Aβ是老年斑的主要成分[11]。有學(xué)者認(rèn)為AD是一個漸進(jìn)性的過程,這一過程中伴隨神經(jīng)功能退化,同時研究還指出Aβ 的異常聚集事AD病理過程的起始因素,發(fā)生在AD病理過程的早期[12]。隨著病情的進(jìn)展,Aβ沉積過程伴隨神經(jīng)毒性物質(zhì)的積聚并能觸發(fā)一系列級聯(lián)變化,進(jìn)而促進(jìn)AD病理過程的發(fā)展。隨著對AD認(rèn)識的深入,人們發(fā)現(xiàn)APP蛋白與Aβ生成密切相關(guān)[13]。
APP是一種跨膜蛋白質(zhì),由一條較長的細(xì)胞外N端節(jié)段與一條較短的細(xì)胞內(nèi)C端節(jié)段組成,在不同的分泌酶作用下,APP裂解成不同的多肽產(chǎn)物,生理條件下這些多肽產(chǎn)物可以被細(xì)胞清除,在病理條件下Aβ生成增多,且清除不完全,逐漸沉積最終形成老年斑[14]。隨著生活水平的提高,高脂飲食成為了老年斑形成的重要因素,也因此成為了人們研究AD發(fā)病的熱點[15]。有研究報道膽固醇增多能夠加強BACE的活性,從而增強APPβ裂解途徑的作用,導(dǎo)致Aβ 1-40與Aβ 1-42多肽生成增多,造成Aβ沉積[16]。參與APP切割的關(guān)鍵酶(α、β、γ分泌酶)因其疏水性而必須在一個富含脂肪的環(huán)境中才具有生物活性,膜區(qū)的脂筏為APP和分泌酶提供了一個活性平臺。在高膽固醇含量的脂筏環(huán)境中,APP更傾向于產(chǎn)生Aβ的代謝途徑:BACE活性顯著提高,β位點的切割增加,Aβ生產(chǎn)增多,從而形成大量老年斑,而在低膽固醇含量的脂筏環(huán)境中,APP更傾向于不產(chǎn)生Aβ的代謝途徑:α分泌酶活性提高,Aβ產(chǎn)生減少[17-18]。
為了進(jìn)一步探討控制高脂能否影響APP、BACE的表達(dá),本研究首先通過給予大鼠高脂飲食構(gòu)建高膽固醇大鼠,通過檢測大鼠血清膽固醇,低密度脂蛋白膽固醇含量提示高脂飲食成功構(gòu)建高膽固醇大鼠模型。近年大量研究均證實了他汀藥物在大鼠AD模型上對腦Aβ沉積物的作用,結(jié)果顯示阿托伐汀可以顯著減弱Aβ在動物模型中的沉積[19-20]。那么,辛伐他汀有效改善高膽固醇血癥大鼠的Aβ1-42是通過何種機制實現(xiàn),是否與APP、BACE有關(guān)?本研究通過免疫組化方法檢測大鼠前額皮層、海馬區(qū)APP蛋白與BACE蛋白的表達(dá),結(jié)果提示高脂飼料藥物組大鼠前額皮層、海馬區(qū)APP、BACE的蛋白表達(dá)較高脂飼料安慰劑組表達(dá)明顯減少,二者之間比較差異明顯,具有統(tǒng)計學(xué)意義(P<0.05),說明辛伐他汀能夠降低腦內(nèi)APP、BACE的蛋白表達(dá)有關(guān)。本研究結(jié)果提示辛伐他汀可能通過抑制前額皮質(zhì)與海馬區(qū)的APP及BACE蛋白的表達(dá),進(jìn)而影響Aβ1-42的表達(dá)來影響高脂血癥大鼠向AD的發(fā)展。
綜上所述,本研究結(jié)果提示了辛伐他汀能夠有效控制高脂血癥,同時能夠抑制Aβ1-42調(diào)控因子APP與BACE蛋白的表達(dá),這一作用機制提示辛伐他汀可能作為今后控制AD進(jìn)展的潛在治療藥物。由于本實驗尚存在不足,進(jìn)一步證實辛伐他汀的治療AD的作用及其深入的機制尚需要更多的實驗來證實。
[參考文獻(xiàn)]
[1]Mary Ann A DeMichele-Sweet Ph D,Robert A.Genetics of psychosis in Alzheimer disease[J].Curr Genet Med Rep,2014, 2(1):30-38.
[2]Cervellati C,Wood PL,Romani A,et al.Oxidative challenge in Alzheimer′s disease:state of knowledge and future needs [J].Amer Fed Clin Res,2016,64(1):21-32.
[3]Simic G,Babic Leko M,Wray S,et al.Tau Protein Hyperphosphorylation and Aggregation in Alzheimer′s Disease and Other Tauopathies,and Possible Neuroprotective Strategies[J].Biomolecules,2016,6(6):1-28.
[4]Ricciarelli R,Canepa E,Marengo B,et al.Cholesterol and Alzheimer′s disease:a still poorly understood correlation[J].IUBMB life,2012,12(64):931-935.
[5]Dias HK,Brown CL,Polidori MC,et al.LDL-lipids from patients with hypercholesterolaemia and Alzheimer′s disease are inflammatory to microvascular endothelial cells:mitigation by statin intervention[J].Clin Sci,2015,129(12):1195-1206.
[6]Kuo PH,Lin CI,Chen YH,et al.A high-cholesterol diet enriched with polyphenols from Oriental plums(Prunus salicina) improves cognitive function and lowers brain cholesterol levels and neurodegenerative-related protein expression in mice[J].Brit J Nutr,2015,113(10):1550-1557.
[7]Mamada N,Tanokashira D,Hosaka A,et al.Amyloid beta-protein oligomers upregulate the beta-secretase,BACE1,through a post-translational mechanism involving its altered subcellular distribution in neurons[J].Mol Brain,2015,11(8):73-77.
[8]Shimizu H,Tosaki A,Kaneko K,et al.Crystal structure of an active form of BACE1,an enzyme responsible for amyloid beta protein production[J].Mol Cell Biol,2008,28(11):3663-3671.
[9]Devi L,Ohno M.Effects of BACE1 haploinsufficiency on APP processing and Abeta concentrations in male and female 5XFAD Alzheimer mice at different disease stages[J].Neuroscience,2015,307(8):128-137.
[10]Ohno-Matsui K.Parallel findings in age-related macular degeneration and Alzheimer′s disease[J].Prog Retin Eye Res,2011,30(4):217.
[11]Parsons CG,Ruitenberg M,F(xiàn)reitag CE,et al.Rammes G.MRZ-99030-A novel modulator of Abeta aggregation:I-Mechanism of action(MoA) underlying the potential neuroprotective treatment of Alzheimer′s disease,glaucoma and age-related macular degeneration(AMD)[J].Neuropharmacol,2015,92(21):158-169.
[12]Iaccarino HF,Singer AC,Martorell AJ,et al.Gamma frequency entrainment attenuates amyloid load and modifies microgli[J].Nature,2016,540(32):230-235.
[13]Ourdev D,F(xiàn)oroutanpay BV,Wang Y,et al.The Effect of Aβ1-42 Oligomers on APP Processing and Aβ1-40 Generation in Cultured U-373 Astrocytes[J].Neuro-degenerative diseases,2015,15(6):361-368.
[14]Dobrowolska JA,Michener MS,Wu G,et al.CNS amyloid-beta,soluble APP-alpha and-beta kinetics during BACE inhibition[J].Neurosci,2014,34(24):8336-8346.
[15]Tang Y,Peng Y,Liu J,et al.Early inflammation-associated factors blunt sterol regulatory element-binding proteins-1-mediated lipogenesis in high-fat diet-fed APP/PSEN1dE9 mouse model of Alzheimer′s diseas[J].J Neurochem,2015, 136(4):791-803.
[16]Cui W,Sun Y,Wang Z,et al.Activation of liver X receptor decreases BACE1 expression and activity by reducing membrane cholesterol levels[J].Neurochem Res,2011,36(10):1910-1921.
[17]Savage MJ,Holder DJ,Wu G,et al.Soluble BACE-1 Activity and sAβPPβ Concentrations in Alzheimer′s Disease and Age-Matched Healthy Control Cerebrospinal Fluid from the Alzheimer′s Disease Neuroimaging Initiative-1 Baseline Cohort[J].JAD,2015,46(2):431-440.
[18]Stromberg K,Eketjall S,Georgievska B,et al.Combining an amyloid-beta(Abeta)cleaving enzyme inhibitor with a gamma-secretase modulator results in an additive reduction of Abeta production [J].The FEBS journal,2015,282(1):65-73.
[19]DeKosky ST.Statin therapy in the treatment of Alzheimer disease:what is the rationale?[J].Am J Med,2005,12(12):48-53.
[20]Li G,Larson EB,Sonnen JA,et al.Statin therapy is associated with reduced neuropathologic changes of Alzheimer disease[J].Neurology,2008,69(9):878-885.
(收稿日期:2017-03-29 本文編輯:馬 越)