高糖應(yīng)激促脂肪變性肝細(xì)胞凋亡的線粒體機(jī)制*

唐 輝， 肖仔君， 蔣鑫煒， 郭紅輝△

(1韶關(guān)學(xué)院英東食品科學(xué)與工程學(xué)院，廣東 韶關(guān) 512005；2中山大學(xué)公共衛(wèi)生學(xué)院，廣東 廣州 510080)

高糖應(yīng)激促脂肪變性肝細(xì)胞凋亡的線粒體機(jī)制*

唐 輝1， 肖仔君1， 蔣鑫煒2， 郭紅輝1△

(1韶關(guān)學(xué)院英東食品科學(xué)與工程學(xué)院，廣東 韶關(guān) 512005；2中山大學(xué)公共衛(wèi)生學(xué)院，廣東 廣州 510080)

目的： 探討高糖應(yīng)激對脂肪變性肝細(xì)胞凋亡的作用及其可能機(jī)制。方法: C57BL/6J小鼠飼喂高脂飼料6周后，采用肝臟原位灌注技術(shù)分離得到脂肪變性原代肝細(xì)胞，在含有35 mmol/L葡萄糖的高糖DMEM培養(yǎng)基中孵育12 h，以正常DMEM培養(yǎng)基(添加30 mmol/L甘露醇)孵育的細(xì)胞作為對照，觀察高糖處理對脂肪變性肝細(xì)胞活力、線粒體膜電位、凋亡蛋白酶caspase活性及凋亡相關(guān)信號通路的影響。結(jié)果: 高糖應(yīng)激使脂肪變性肝細(xì)胞活力下降，凋亡顯著增加，而等滲甘露醇處理的對照細(xì)胞沒有明顯變化。高糖組細(xì)胞出現(xiàn)較為嚴(yán)重的線粒體去極化，導(dǎo)致線粒體膜電位降低，細(xì)胞色素C釋放增多。線粒體介導(dǎo)凋亡關(guān)鍵酶caspase-9和caspase-3活性在高糖組有顯著升高，抑制凋亡因子Bcl-2和Bcl-xL表達(dá)量明顯降低，促凋亡蛋白Bax水平顯著升高，而感受外源性凋亡信號的caspase-8的活性沒有明顯變化。結(jié)論: 高糖應(yīng)激會導(dǎo)致脂肪變性肝細(xì)胞線粒體膜電位下降，啟動(dòng)線粒體介導(dǎo)的內(nèi)源性凋亡途徑，引起肝細(xì)胞凋亡。這可能是高血糖加速非酒精性脂肪性肝病病程進(jìn)展的一個(gè)重要原因。

肝細(xì)胞； 線粒體； 高糖應(yīng)激； 細(xì)胞凋亡； 非酒精性脂肪性肝病

非酒精性脂肪性肝病(non-alcoholic fatty liver disease，NAFLD)是目前最為常見的慢性肝病，包括單純性脂肪肝、脂肪性肝炎(non-alcoholic steatohepatitis，NASH)、NASH相關(guān)肝纖維化和肝硬化等4個(gè)病程階段。單純性脂肪肝患者往往無機(jī)體不適癥狀，預(yù)后良好，而一旦進(jìn)展為NASH，就會出現(xiàn)不可逆的肝損傷，如肝細(xì)胞氣球樣變性、炎癥甚至凋亡，加速病程進(jìn)展[1]。最近的研究表明，糖耐量異常所致的空腹血糖水平升高是單純性脂肪肝向NASH轉(zhuǎn)變的一個(gè)危險(xiǎn)因素[2]，然而其中的病理生理機(jī)制尚未明確。高血糖會促進(jìn)肝細(xì)胞內(nèi)的脂質(zhì)合成，干擾脂肪酸進(jìn)行β氧化，可能引起線粒體功能障礙和細(xì)胞凋亡[3]。本研究首先利用高脂飲食喂養(yǎng)建立NAFLD小鼠模型，分離原代脂肪變性肝細(xì)胞，觀察體外高糖孵育對其活力、線粒體功能和凋亡相關(guān)信號通路的影響，以期明確高糖應(yīng)激引起脂肪變性肝細(xì)胞凋亡，促進(jìn)單純性脂肪肝向NASH進(jìn)展的作用機(jī)制，為NAFLD的早期干預(yù)提供理論依據(jù)。

材 料 和 方 法

1 動(dòng)物和細(xì)胞

SPF級C57BL/ 6J小鼠，雄性，6～8周齡，18～20 g，由廣東省醫(yī)學(xué)實(shí)驗(yàn)動(dòng)物中心提供，飼養(yǎng)在中山大學(xué)公共衛(wèi)生學(xué)院SPF動(dòng)物房[12 h晝夜交替，溫度(22 ± 2)℃]。喂飼小鼠脂肪供能比為45%的高脂飼料(江蘇美迪森生物醫(yī)藥有限公司)，6周后，采用改良兩步原位灌流法分離原代肝細(xì)胞[4]，油紅O染色觀察細(xì)胞內(nèi)脂肪積聚情況，臺盼藍(lán)染色確認(rèn)細(xì)胞存活率≥95%，按2.5×105密度接種于6孔培養(yǎng)板，低糖DMEM(葡萄糖濃度5 mmol/L)培養(yǎng)基、37 ℃、5% CO2條件下培養(yǎng)24 h后進(jìn)入實(shí)驗(yàn)，細(xì)胞分為3組：對照(control)組，低糖DMEM繼續(xù)培養(yǎng)；甘露醇(mannitol)組，DMEM培養(yǎng)基加入30 mmol/L甘露醇，與高糖組的培養(yǎng)基滲透勢相當(dāng)；高糖(high-glucose, HG)組，培養(yǎng)基葡萄糖濃度調(diào)高至35 mmol/L。各組細(xì)胞繼續(xù)培養(yǎng)12 h。

2 主要試劑

低糖DMEM細(xì)胞培養(yǎng)基、小牛血清和抗生素混合物購自廣州英韋創(chuàng)津生物科技有限公司；I 抗購自Santa Cruz和CST；II抗由武漢博士德生物工程有限公司提供；熒光探針JC-1和Hoechst 33258購自Molecular Probes；胰酶、膠原蛋白酶Ⅰ、臺盼藍(lán)購自Sigma；MTT細(xì)胞活力檢測試劑盒、乳酸脫氫酶(lactate dehydrogenase，LDH)細(xì)胞毒性檢測試劑盒和BCA蛋白濃度檢測試劑盒購自上海碧云天生物技術(shù)有限公司；其它試劑均為進(jìn)口分裝或國產(chǎn)分析純。

3 主要方法

3.1 細(xì)胞活力和毒性測定 將細(xì)胞移入96孔板，每孔加入MTT(5.0 g/L)50 μL、置入培養(yǎng)箱孵育4 h，離心棄上清，加入二甲基亞砜200 μL，酶標(biāo)儀測定570 nm處各孔吸光度值，評價(jià)細(xì)胞存活率。細(xì)胞處理期間釋放的乳酸脫氫酶作為細(xì)胞毒性的標(biāo)志，將細(xì)胞培養(yǎng)板500×g離心5 min，取10 μL到96孔酶標(biāo)板，加入反應(yīng)液25 ℃避光反應(yīng)30 min，終止反應(yīng)，在酶標(biāo)儀490 nm處讀數(shù)。以低糖組細(xì)胞作為參照，計(jì)算其它2組的細(xì)胞相對存活率和毒性。

3.2 細(xì)胞凋亡檢測 利用能夠直接和凋亡細(xì)胞DNA結(jié)合的熒光染料Hoechst 33258染色區(qū)分正常細(xì)胞和凋亡細(xì)胞。將培養(yǎng)板中的細(xì)胞用甲醇/丙酮溶液固定5 min，PBS緩沖液清洗3遍，加入10 μmol/L Hoechst 33258染色30 min，在TE2000熒光顯微鏡(Nikon)下(激發(fā)光350 nm，吸收光460 nm)觀察細(xì)胞核染色質(zhì)形態(tài)。凋亡細(xì)胞染色質(zhì)高度濃縮，發(fā)出較強(qiáng)的藍(lán)色熒光。隨機(jī)統(tǒng)計(jì)5個(gè)視野內(nèi)凋亡細(xì)胞占總細(xì)胞的比例。

3.3 線粒體膜電位檢測 采用JC-1熒光探針在細(xì)胞內(nèi)的紅/綠熒光強(qiáng)度比例來衡量線粒體去極化程度[5]。細(xì)胞培養(yǎng)基內(nèi)加入2.0 mg/L JC-1在37 ℃孵育30 min，熒光顯微鏡下觀察拍照，并在熒光酶標(biāo)儀分別測定紅色熒光(激發(fā)光580 nm，吸收光590 nm)和綠色熒光(激發(fā)光510 nm，吸收光527 nm)強(qiáng)度，計(jì)算二者的比值表示線粒體膜電位的變化情況。

3.4 細(xì)胞凋亡蛋白酶活性測定 采用Abcam提供的酶法試劑盒分別測定細(xì)胞內(nèi)3種含半胱氨酸的天冬氨酸蛋白水解酶(caspase-3、-8和-9)的活性。取50 μL細(xì)胞裂解液，加入5 μL相應(yīng)四肽標(biāo)記底物和50 μL反應(yīng)液于96孔酶標(biāo)板，37 ℃避光孵育30 min，405 nm處測定對硝基苯胺的生成量來表示酶活性大小，同時(shí)測定細(xì)胞裂解液蛋白質(zhì)濃度校準(zhǔn)酶活性。

3.5 Western blotting實(shí)驗(yàn) 利用Enzo Life Sciences的試劑盒分別提取細(xì)胞質(zhì)和線粒體蛋白質(zhì)，調(diào)整蛋白質(zhì)濃度后進(jìn)行電泳、轉(zhuǎn)膜、封閉、孵育I抗和II抗，使用Santa Cruz光化學(xué)發(fā)光試劑顯影，β-actin和細(xì)胞色素C氧化酶Ⅳ(cytochrome C oxidase Ⅳ,CoxⅣ)分別作為內(nèi)參照，Quantity One軟件分析蛋白條帶灰度值表示蛋白質(zhì)表達(dá)量。

4 統(tǒng)計(jì)學(xué)處理

用SPSS 19.0統(tǒng)計(jì)軟件進(jìn)行分析。數(shù)據(jù)均采用均數(shù)±標(biāo)準(zhǔn)誤(mean±SEM)表示，多組間比較采用單因素方差分析(one-way ANOVA)，組間兩兩比較采用SNK-q檢驗(yàn)。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1 高糖對脂肪變性肝細(xì)胞活力的影響

油紅O染色結(jié)果顯示，與普通飼料喂養(yǎng)小鼠肝細(xì)胞相比，高脂喂養(yǎng)小鼠肝細(xì)胞體積增大，胞內(nèi)有較多脂滴積聚，符合NAFLD單純性脂肪肝階段的病理特征。高脂喂養(yǎng)小鼠分離的肝細(xì)胞在經(jīng)過高糖培養(yǎng)基孵育12 h后，其細(xì)胞活力較生理濃度葡萄糖培養(yǎng)基孵育的對照組細(xì)胞有明顯下降，且細(xì)胞釋放的LDH增多了1.6倍。甘露醇組細(xì)胞的活力與對照組沒有顯著差異，LDH的釋放也沒有增多，說明高糖帶給細(xì)胞的毒性不是由于培養(yǎng)基高滲透勢造成的，見圖1。

Figure 1.High glucose-induced cytotoxicity in primary hepatocytes isolated from high-fat diet fed mice. Oil red O staining for hepatocytes isolated from chow (A) or high-fat (B) diet fed mice (×200). The cell viability (C) and LDH release (D) were assessed. Mean±SEM.n=4.*P<0.05vscontrol group.

圖1 高糖對脂肪變性肝細(xì)胞的影響

2 高糖促進(jìn)細(xì)胞凋亡

為明確高糖能否引起脂肪變性肝細(xì)胞凋亡，我們利用Hoechst 33258對細(xì)胞核進(jìn)行了染色。高糖組出現(xiàn)了較多的染色質(zhì)著色亮斑。統(tǒng)計(jì)結(jié)果顯示，高糖組細(xì)胞的凋亡率顯著高于對照組和甘露醇組(P<0.05)，見圖2。

3 高糖對細(xì)胞線粒體膜電位的影響

正常細(xì)胞線粒體膜電位較高，結(jié)合的JC-1探針發(fā)出紅色熒光，當(dāng)線粒體膜去極化以后，電位降低，綠色熒光增強(qiáng)[5]。合并的熒光圖顯示，高糖組細(xì)胞的紅色熒光要弱于對照組和甘露醇組。進(jìn)一步用熒光酶標(biāo)儀測定結(jié)果確認(rèn)，高糖處理使細(xì)胞線粒體紅色熒光與綠色熒光的比值顯著降低(P<0.05)，說明線粒體膜電位降低，膜的完整性受到破壞，見圖3。

4 高糖對細(xì)胞凋亡蛋白酶活性的影響

各組細(xì)胞感受外源性凋亡信號的caspase-8活性相當(dāng)，而內(nèi)源性凋亡途徑，即線粒體介導(dǎo)凋亡關(guān)鍵酶caspase-9和caspase-3活性在高糖組有顯著升高，見圖4。

5 高糖對線粒體介導(dǎo)細(xì)胞凋亡相關(guān)信號通路的影響

利用Western blotting 技術(shù)檢測細(xì)胞胞漿和線粒體內(nèi)的細(xì)胞色素C相對濃度，可見高糖組細(xì)胞線粒體內(nèi)細(xì)胞色素C的濃度僅相當(dāng)于對照組的39%，而在細(xì)胞質(zhì)中則有顯著升高(P<0.01)，提示由于線粒體膜完整性受到破壞，線粒體大量的細(xì)胞色素C釋放到細(xì)胞質(zhì)。與對照細(xì)胞相比，高糖組細(xì)胞胞漿內(nèi)促凋亡蛋白Bax的表達(dá)量顯著升高(P<0.01)，相應(yīng)的2個(gè)抑制凋亡蛋白Bcl-2和Bcl-xL的表達(dá)量明顯降低,見圖5。

Figure 2.High glucose-induced apoptosis in primary hepatocytes. A: the cells were stained with Hoechst 33258 and visualized on a fluorescent microscope to observe chromatin condensation(×200); B: the apoptotic cells were counted based on morphology, and the number of apoptotic cells was expressed as percentage of total cells. Mean±SEM.n=3.*P<0.05vscontrol group.

圖2 高糖誘導(dǎo)脂肪變性肝細(xì)胞凋亡

Figure 3.High glucose-induced mitochondrial membrane depolarization in primary hepatocytes. A: the cells were stained with JC-1 and visualized on an inverted fluorescence microscope (×200). Red fluorescence of JC-1 dimers was present in the cell areas with high mitochondrial membrane potential, while green fluorescence of JC-1 monomers was prevalent in the cell areas with low mitochondrial membrane potential. B: the ratio of red/green fluorescence intensity. Mean±SEM.n=3.*P<0.05vscontrol group.

圖3 高糖使脂肪變性肝細(xì)胞線粒體膜電位降低

Figure 4.The effect of high-glucose treatment on enzymatic activities of caspases in primary hepatocytes. Aliquots of cell lysate (50 μg protein) were assayed forinvitrocaspase-3, -8, and -9 activity using DEVD-pNA, IETD-pNA, and LEHD-pNA as substrates respectively. Mean±SEM.n=3.*P<0.05vscontrol group.

圖4 高糖對細(xì)胞凋亡蛋白酶活性的影響

討 論

高血糖是糖尿病的典型病征，會引起腎病、視網(wǎng)膜病變和神經(jīng)損傷等多種并發(fā)癥。實(shí)際上，肝臟是糖代謝的主要器官，高糖引起的肝損傷也應(yīng)當(dāng)受到高度重視。Wanless等[6]對351名NAFLD患者的肝臟標(biāo)本進(jìn)行病理分析后發(fā)現(xiàn)，血糖正常組NASH比例為4.7%，而2型糖尿病組NASH比例升高至12.2%。對NASH患者的追蹤研究進(jìn)一步證實(shí)，長期高血糖會加速肝纖維化進(jìn)程[7]。Yin等[8]給高脂喂養(yǎng)小鼠每天腹腔注射葡萄糖誘發(fā)血糖波動(dòng)，發(fā)現(xiàn)會加劇小鼠肝臟炎癥反應(yīng)和肝細(xì)胞凋亡。之前也有關(guān)于高糖促使肝細(xì)胞凋亡的體外實(shí)驗(yàn)研究報(bào)道，但多是選用永生化的細(xì)胞系或普通飼料喂養(yǎng)小鼠的原代肝細(xì)胞，并且采用了更高的葡萄糖濃度(≥40 μmol/L)[9-10]。本研究利用高脂喂養(yǎng)小鼠的原代肝細(xì)胞作為實(shí)驗(yàn)?zāi)Ｐ?，采用較為接近糖尿病患者血糖水平的葡萄糖濃度，能夠更好地模仿體內(nèi)環(huán)境。對細(xì)胞進(jìn)行12 h高糖孵育處理，發(fā)現(xiàn)會引起線粒體功能障礙，天冬氨酸蛋白水解酶caspase-9和caspase-3活性顯著升高，導(dǎo)致細(xì)胞凋亡，提示脂肪變性細(xì)胞可能對高糖應(yīng)激更為敏感。為排除滲透勢對細(xì)胞的不利影響，我們設(shè)置了甘露醇處理對照，明確了高糖可以直接促進(jìn)脂肪變性肝細(xì)胞的凋亡。

Figure 5.The effects of high-glucose treatment on mitochondria-mediated apoptotic pathways in primary hepatocytes. A: cytosolic and mitochondrial proteins were probed with anti-cytochrome C antibody. β-actin and cytochrome C oxidase (Cox) IV were used as loading controls for the cytosolic and mitochondrial fractions, respectively. B: the cells were lysed and Bcl-2, Bcl-xL, and Bax expression were detected by Western blotting. Representative images and band intensity quantifications were shown. Mean±SEM.n=3.*P<0.05vscontrol group.

圖5 高糖對線粒體介導(dǎo)細(xì)胞凋亡相關(guān)信號通路的影響

細(xì)胞凋亡的途徑主要包括外源性死亡受體途徑和內(nèi)源性線粒體細(xì)胞色素C釋放途徑，前者是通過胞外信號激活細(xì)胞內(nèi)的凋亡酶caspase-8，后者是指細(xì)胞應(yīng)激反應(yīng)或凋亡信號引起線粒體細(xì)胞色素C釋放，作為凋亡誘導(dǎo)因子，細(xì)胞色素C能與Apaf-1、caspase-9前體、ATP/dATP形成凋亡體，然后募集并激活caspase-3，進(jìn)而引發(fā)細(xì)胞凋亡[11]。本研究發(fā)現(xiàn)，高糖條件下細(xì)胞的去極化比例顯著升高，標(biāo)志著線粒體膜電位的下降，這與之前的研究報(bào)道相一致[5, 12]。此外，細(xì)胞線粒體向胞漿釋放的細(xì)胞色素C增多，caspase-3活性升高，下游的促凋亡蛋白Bax表達(dá)水平升高，而抑制凋亡蛋白Bcl-2和Bcl-xL的表達(dá)量明顯降低，caspase-8活性沒有明顯變化，說明高糖應(yīng)激是通過線粒體介導(dǎo)的內(nèi)源性凋亡途徑誘發(fā)脂肪變性肝細(xì)胞凋亡的。

綜上所述，高糖應(yīng)激會導(dǎo)致脂肪變性肝細(xì)胞線粒體膜電位下降，啟動(dòng)線粒體介導(dǎo)的內(nèi)源性凋亡途徑，引起肝細(xì)胞凋亡，線粒體功能障礙可能是高血糖加速NAFLD病程進(jìn)展的一個(gè)重要原因。

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(責(zé)任編輯： 陳妙玲， 余小慧)

Mitochondrial mechanism of hyperglycemia-induced apoptosis in primary mouse hepatocytes with steatosis

TANG Hui1, XIAO Zi-jun1, JIANG Xin-wei2, GUO Hong-hui1

(1HenryFokSchoolofFoodScienceandEngineering,ShaoguanUniversity,Shaoguan512005,China;2SchoolofPublicHealth,SunYat-senUniversity,Guangzhou510080,China.E-mail:guohh1999@163.com)

AIM: To investigate the role of high glucose in primary hepatocytes of mice fed with a high fat diet.METHODS: Male C57BL/6J mice were fed a high fat (45% of calories) dietadlibitumfor 6 weeks to induce hepatic steatosis. Primary hepatocytes were isolated from the mouse liver by the 2 step collagenase perfusion method. The cells were incubated in low glucose (5 mmol/L), low glucose plus mannitol (30 mmol/L), or high glucose (35 mmol/L) DMEM medium for 12 h. The cell viability, apoptosis, mitochondrial membrane potential, and caspase enzymatic activities were measured. Furthermore, proteins related to the stress-sensitive signaling pathway of regulating high glucose-induced apoptosis in primary hepatocytes were determined by Western blotting.RESULTS: Incubation with 35 mmol/L glucose resulted in a significant decrease in cell viability and an increase in apoptosis, whereas mannitol had no significant effect on the cell viability or apoptosis. A progressive depolarization of the mitochondria, an increase in cytosol cytochrome C and a dramatic decrease in mitochondrial cytochrome C in high-glucose stressed hepatocytes were observed. The enzymatic activities of caspase-9 and caspase-3, but not caspase-8, were significantly increased in high glucose-stressed hepatocytes (P<0.05). High glucose treatment suppressed the expression of Bcl-2 and Bcl-xL, while it increased the expression of the pro-apoptotic factor Bax.CONCLUSION: High glucose stress reduces mitochondrial membrane potential, initiates mitochondria-mediated apoptotic pathways and promotes apoptosis of hepatocytes with steatosis. This may be an important pathological mechanism of hyperglycemia-induced progression of nonalcoholic fatty liver disease.

Hepatocyte; Mitochondria; High glucose stress; Apoptosis; Nonalcoholic fatty liver disease

1000- 4718(2016)08- 1419- 06

2016- 01- 26

2016- 05- 23

國家自然科學(xué)基金資助項(xiàng)目(No. 81372994)；廣東省揚(yáng)帆計(jì)劃高層次人才項(xiàng)目(No. 201434015)

R363.2; R575

A

10.3969/j.issn.1000- 4718.2016.08.013

雜志網(wǎng)址： http://www.cjpp.net

△通訊作者 Tel: 0751-8120167; E-mail: guohh1999@163.com