脂肪酸影響草魚肝細(xì)胞脂質(zhì)蓄積狀態(tài)及誘導(dǎo)其凋亡的離體研究

李雪賢 孫 健 吉 紅 陳昊杰

(西北農(nóng)林科技大學(xué)動(dòng)物科技學(xué)院, 楊凌 712100)

脂肪酸影響草魚肝細(xì)胞脂質(zhì)蓄積狀態(tài)及誘導(dǎo)其凋亡的離體研究

李雪賢 孫 健 吉 紅 陳昊杰

(西北農(nóng)林科技大學(xué)動(dòng)物科技學(xué)院, 楊凌 712100)

為探究外源脂肪酸對(duì)草魚肝細(xì)胞脂質(zhì)代謝及健康狀況的影響及其機(jī)理, 體外培養(yǎng)草魚肝細(xì)胞, 并采用不同濃度(0—1 mmol/L)油酸(Oleic acid)進(jìn)行細(xì)胞孵育, 噻唑蘭比色法(Methyl thiazolte trazoliu, MTT)和油紅O染色提取法檢測(cè)肝細(xì)胞活力及脂質(zhì)蓄積狀況, BODIBY和DAPI染色法觀察肝細(xì)胞脂滴及細(xì)胞核情況, 流式細(xì)胞術(shù)檢測(cè)肝細(xì)胞凋亡率變化, Real-time qPCR檢測(cè)脂質(zhì)合成標(biāo)志基因過(guò)氧化物酶體增殖物激活受體γ (Peroxidase proliferation activated receptor, PPARγ)和CCAAT/增強(qiáng)子結(jié)合蛋白α (CCAAT/enhancer binding protein alpha, C/EBPα)、凋亡相關(guān)基因Caspase家族等的表達(dá)情況。結(jié)果顯示, 隨著油酸處理濃度的增加, 肝細(xì)胞活力和細(xì)胞內(nèi)脂質(zhì)積累呈現(xiàn)先上升后下降的趨勢(shì), 分別在0.4和0.6 mmol/L時(shí)達(dá)到最大值(P＜0.05); 肝細(xì)胞凋亡率則先下降后上升, 在0.4 mmol/L油酸處理時(shí)最低, 1 mmol/L油酸處理時(shí)最高(P＜0.05); 此外, 0.4 mmol/L油酸處理抑制了肝細(xì)胞Caspase-3b和Caspase-9基因的表達(dá), 上調(diào)Bcl-2/Bax mRNA比值(P＜0.05), 而0.8 mmol/L油酸處理顯著促進(jìn)Caspase-3b、Caspase-8、Caspase-9及凋亡誘導(dǎo)因子(Apoptosis inducing factor, AIF)基因的表達(dá), 下調(diào)Bcl-2/Bax的mRNA比值(P＜0.05)。研究表明, 一定濃度的脂肪酸可增強(qiáng)草魚肝細(xì)胞活力, 促進(jìn)胞內(nèi)脂質(zhì)積累, 抑制細(xì)胞凋亡, 而脂肪酸濃度過(guò)高則抑制肝細(xì)胞活力并誘導(dǎo)肝細(xì)胞凋亡, 其作用與脂肪酸影響脂質(zhì)代謝及凋亡基因的表達(dá)有關(guān)。

脂肪酸; 肝細(xì)胞; 凋亡; 草魚

脂肪酸作為重要的營(yíng)養(yǎng)物質(zhì)和信號(hào)分子, 對(duì)魚類的生長(zhǎng)、發(fā)育和繁殖具有多方面的作用[1]。機(jī)體內(nèi)的游離脂肪酸(FFA)一方面經(jīng)吸收、轉(zhuǎn)運(yùn)至貯存場(chǎng)所, 形成甘油三酯(TAG), 另一方面可以分解、代謝供應(yīng)機(jī)體的能量需求[2]。肝臟是魚體內(nèi)最主要的代謝器官, 參與體內(nèi)的消化、代謝、解毒及免疫等多種功能, 也是魚類脂肪合成的主要場(chǎng)所, 其在魚類脂代謝過(guò)程中起著重要的調(diào)節(jié)作用[3,4]。肝臟是脂質(zhì)代謝的主要場(chǎng)所, 當(dāng)肝臟內(nèi)積聚的FFA超過(guò)合成和分解代謝需要時(shí), 細(xì)胞內(nèi)FFA動(dòng)態(tài)平衡被打破, TAG在肝細(xì)胞內(nèi)大量?jī)?chǔ)存, 導(dǎo)致肝細(xì)胞脂肪變性, 使肝臟對(duì)炎癥反應(yīng)和各種損傷因素的敏感性增高[5—7]。過(guò)量的脂質(zhì)沉積還會(huì)導(dǎo)致細(xì)胞線粒體功能障礙, 造成脂毒性, 致使細(xì)胞的損傷和凋亡[8—10]。已有研究發(fā)現(xiàn), 脂肪肝病變的大黃魚肝臟中FFA含量顯著上升, 說(shuō)明過(guò)量的FFA進(jìn)入肝臟, 造成大黃魚肝臟脂質(zhì)積累[11]。這種因能量攝入過(guò)多導(dǎo)致肝細(xì)胞內(nèi)過(guò)量蓄積脂質(zhì)的肝病稱為魚類營(yíng)養(yǎng)性脂肪肝[12]。盡管已有較多報(bào)道探討了脂質(zhì)對(duì)魚類肝臟脂質(zhì)代謝及健康狀況的影響[13—16], 但直接研究脂肪酸對(duì)肝細(xì)胞凋亡作用的報(bào)道還比較有限, 相關(guān)研究有待進(jìn)一步深入。

為了深入系統(tǒng)地探討脂肪酸對(duì)草魚肝細(xì)胞脂質(zhì)代謝及凋亡特性的影響及其作用機(jī)制, 本研究在離體條件下, 以油酸為代表, 研究不同濃度脂肪酸處理情況下草魚肝細(xì)胞活力、脂質(zhì)蓄積和細(xì)胞凋亡的狀況, 為魚類營(yíng)養(yǎng)性脂肪肝的發(fā)病機(jī)制及防控對(duì)策的研究提供參考資料。

1 材料與方法

1.1 主要試劑

胎牛血清、M199培養(yǎng)基及無(wú)脂肪酸牛血清白蛋白購(gòu)自Gibco公司, 油紅O、MTT、BODIPY、DAPI和油酸均購(gòu)自Sigma公司, 碳酸氫鈉、無(wú)水乙醇等化學(xué)試劑均為國(guó)產(chǎn)分析純。Trizol、反轉(zhuǎn)錄試劑盒及熒光定量試劑盒購(gòu)自TaKaRa公司, Annexin V-FITC/PI試劑盒購(gòu)自北京全式金生物公司。油酸用無(wú)水乙醇配制成0.1 mol/L貯存液, 保存于-20℃,使用前用2%無(wú)脂肪酸牛血清白蛋白的M199培養(yǎng)基將脂肪酸稀釋成0.01 mol/L。

1.2 草魚肝細(xì)胞的培養(yǎng)

草魚肝細(xì)胞(L8824)購(gòu)自中國(guó)典型培養(yǎng)物保藏中心(China Center for Type Culture Collection, CCTCC)。將肝細(xì)胞置于10%胎牛血清的M199培養(yǎng)液中, 并于28℃、5% CO2飽和濕度培養(yǎng)箱中培養(yǎng)。隔天換液, 待細(xì)胞生長(zhǎng)至匯合后, 設(shè)置處理組和對(duì)照組。對(duì)照組使用10%胎牛血清的M199培養(yǎng)液培養(yǎng), 處理組使用含不同濃度油酸的培養(yǎng)液培養(yǎng)。

1.3 MTT檢測(cè)不同濃度油酸對(duì)肝細(xì)胞活力影響

草魚肝細(xì)胞均勻接種于96孔板, 使用含不同濃度油酸(0、0.2、0.4、0.6、0.8和1 mmol/L)的培養(yǎng)液培養(yǎng)肝細(xì)胞24h后, 每孔加入20 μL的MTT (5 mg/mL),于28℃、5% CO2條件下繼續(xù)培養(yǎng)4h, 吸棄培養(yǎng)液后, 用PBS洗1次后, 每孔加入150 μL二甲基亞砜(Dimethyl sulfoxide, DMSO), 28℃孵育10min, 溶解細(xì)胞中的甲臢。使用酶標(biāo)儀于490 nm波長(zhǎng)處測(cè)定吸光度值, 每組6個(gè)重復(fù)。

1.4 油紅O染色提取法測(cè)定脂質(zhì)含量

草魚肝細(xì)胞均勻接種于96孔板, 使用上述不同濃度油酸培養(yǎng)細(xì)胞24h后, 棄去培養(yǎng)基, 每孔用PBS洗2次, 加入濃度為10%的甲醛固定液固定30min, PBS洗2次, 加入新鮮配制的油紅O染液浸染15min, PBS清洗2次, 于倒置顯微鏡下觀察細(xì)胞內(nèi)脂滴的染色情況, 并拍照記錄。隨后向每孔加入150 μL的異丙醇萃取, 于490 nm波長(zhǎng)處測(cè)定吸光度值, 每組6個(gè)重復(fù)。

1.5 BODIPY染色法和DAPI染色法觀察肝細(xì)胞內(nèi)脂滴和細(xì)胞核變化

草魚肝細(xì)胞均勻接種于48孔板, 給予上述不同濃度油酸處理24h。棄去培養(yǎng)基, 每孔加入0.5 mL固定液, 固定30min。吸棄固定液, PBS洗兩次, 同時(shí)加入BODIPY染色液和DAPI染色液染色15min。吸棄染色液, PBS洗兩次, 每次5min。加入少量PBS (防止過(guò)干細(xì)胞皺縮), 熒光顯微鏡下觀察并照相。

1.6 流式細(xì)胞術(shù)檢測(cè)不同濃度油酸對(duì)肝細(xì)胞凋亡的影響

草魚肝細(xì)胞均勻接種于25 cm2培養(yǎng)瓶中, 使用含不同濃度油酸的培養(yǎng)基處理細(xì)胞24h, 收集細(xì)胞,用預(yù)冷的PBS洗滌細(xì)胞, 并以100 μL冷結(jié)合緩沖液懸浮細(xì)胞, 加入10 μL Annexin V-FITC染液和10 μL PI染液混勻室溫孵育15min, 再加入400 μL結(jié)合緩沖液, 流式細(xì)胞儀檢測(cè)細(xì)胞凋亡情況, 每組3個(gè)重復(fù),根據(jù)陰性對(duì)照設(shè)定適當(dāng)?shù)闹本€門。

1.7 Real-time PCR檢測(cè)相關(guān)基因的mRNA的表達(dá)

草魚肝細(xì)胞均勻接種于24孔板, 分別使用含0.4和0.8 mmol/L油酸培養(yǎng)基處理細(xì)胞24h后, 棄去培養(yǎng)基, PBS清洗2次, 提取總RNA, 并反轉(zhuǎn)錄。采用CFX-96實(shí)時(shí)定量PCR檢測(cè)系統(tǒng)(Bio-Rad, USA)檢測(cè)相關(guān)基因的mRNA表達(dá)水平。20 μL反應(yīng)體系,包括: 上下游引物(10 mmol/μL)各0.6 μL、cDNA 1 μL、2×SYBR Premix Ex TaqTMII 10 μL、滅菌雙蒸水加至20 μL。反應(yīng)條件: 95℃, 10s; 95℃, 15s, 57℃, 15s; 40個(gè)循環(huán)。根據(jù)擴(kuò)增曲線得到的Ct值, 計(jì)算出目標(biāo)基因與內(nèi)參基因 β-actin 的比值2-ΔΔCt, 并以此計(jì)算出試驗(yàn)組目標(biāo)基因相對(duì)于對(duì)照組目標(biāo)基因的表達(dá)倍數(shù)2-ΔΔCt, 從而制作相對(duì)定量的圖表, 每組3個(gè)重復(fù)?；驒z測(cè)實(shí)時(shí)定量引物見表 1。

1.8 統(tǒng)計(jì)分析

所有數(shù)據(jù)均以平均值±標(biāo)準(zhǔn)差(Mean±SD)表示。采用SPSS18.0軟件中的單因素方差分析(One-way ANOVA)以及鄧肯(Duncan)多重比較對(duì)所得數(shù)據(jù)進(jìn)行顯著性檢驗(yàn)分析。當(dāng)P＜0.05, 認(rèn)為差異顯著。

2 結(jié)果

2.1 油酸處理對(duì)草魚肝細(xì)胞活力的影響

從圖 1可以看出, 隨著油酸處理濃度的升高, 肝細(xì)胞活力先升高后降低(P＜0.05), 且油酸處理濃度為0.6 mmol/L時(shí), 肝細(xì)胞活力最高(P＜0.05), 但是當(dāng)油酸濃度上升至0.8 mmol/L時(shí), 肝細(xì)胞活力顯著下降, 但相比于對(duì)照組仍有促進(jìn)作用(P＜0.05), 而當(dāng)油酸濃度達(dá)到1 mmol/L時(shí), 肝細(xì)胞活力被抑制(P＜0.05)。

表 1 實(shí)時(shí)定量檢測(cè)引物列表Tab. 1 Primer used for quantitative real-time PCR

圖 1 不同濃度油酸對(duì)草魚肝細(xì)胞活力的影響(n=6Fig. 1 Viability of Ctenopharyngodon idellus hepatocytes cells after exposure to various doses of oleic acid (n=6,

2.2 油酸處理對(duì)草魚肝細(xì)胞脂質(zhì)蓄積的影響

油紅O染色及脂質(zhì)提取結(jié)果 對(duì)照組(圖 2A)細(xì)胞邊緣清晰, 呈多角形排列, 細(xì)胞間結(jié)合緊密, 無(wú)縫隙。油紅O染色顯示細(xì)胞內(nèi)少見紅色脂滴。而分別使用0.2、0.4和0.6 mmol/L濃度油酸處理后, 肝細(xì)胞多變?yōu)閳A形或橢圓形, 脂滴成環(huán)狀位于細(xì)胞膜內(nèi)側(cè), 將細(xì)胞核擠向一側(cè)(分別為圖 2B-D), 但是當(dāng)油酸處理濃度上升至0.8和1 mmol/L后, 肝細(xì)胞間結(jié)合不再緊密, 棱角不清, 細(xì)胞數(shù)量減少(圖 2E, 2F)。油紅O染色提取比色法(以O(shè)D值表示)定量分析細(xì)胞內(nèi)TAG的含量, 結(jié)果表明, 隨著油酸處理濃度的增加, 肝細(xì)胞TAG含量逐漸增加(P＜0.05), 且在油酸濃度達(dá)到0.4 mmol/L時(shí), 肝細(xì)胞TAG含量達(dá)到最大值(P＜0.05)。在此之后隨油酸的濃度的升高, 肝細(xì)胞TAG含量顯著降低(P＜0.05)(圖 3)。

BODIPY和DAPI染色相比于對(duì)照組(圖 4A),隨著油酸處理濃度的升高(0.2—0.6 mmol/L), 肝細(xì)胞數(shù)量逐漸增加, 脂滴增多變大, 肝細(xì)胞核為圓形或橢圓形, 呈現(xiàn)均勻的藍(lán)色(分別為圖 4B-D)。而當(dāng)油酸濃度達(dá)到0.8和1 mmol/L時(shí), 肝細(xì)胞數(shù)量下降,細(xì)胞內(nèi)脂滴減少, 細(xì)胞核變小, 染色質(zhì)凝集而呈現(xiàn)致密濃染, 甚至出現(xiàn)碎塊狀, 出現(xiàn)明顯的凋亡特征(圖 4E, 4F, 如圖中箭頭所示)。

2.3 油酸處理對(duì)草魚肝細(xì)胞凋亡率的影響

相比于對(duì)照組(圖 5A), 0.4 mmol/L濃度油酸處理的草魚肝細(xì)胞凋亡率顯著降低(圖 5C), 但在此之后, 隨著油酸處理濃度的增加, 肝細(xì)胞凋亡率顯著上升(P＜0.05)。

2.4 油酸處理對(duì)草魚肝細(xì)胞脂質(zhì)生成基因和凋亡相關(guān)基因表達(dá)的影響

圖 2 油紅O染色觀察油酸誘導(dǎo)24h后草魚肝細(xì)胞中的脂質(zhì)積累和細(xì)胞形態(tài)變化Fig. 2 Oil red O staining analysis of the accumulation of lipid droplets and morphological changes in C. idellus hepatocytes after 24 hours treatment with oleic acidA. 0 (×100); B. 0.2 mmol/L (×100); C. 0.4 mmol/L (×100); D. 0.6 mmol/L (×100); E. 0.8 mmol/L (×100); F. 1 mmol/L (×100)

根據(jù)以上試驗(yàn)結(jié)果選擇0.4和0.8 mmol/L濃度的油酸培養(yǎng)草魚肝細(xì)胞, 進(jìn)行相關(guān)基因mRNA水平的檢測(cè)。如圖 6所示, 兩種油酸濃度處理肝細(xì)胞24h后, PPARγ和C/EBPα基因的mRNA表達(dá)量均顯著上升(P＜0.05)。凋亡相關(guān)基因mRNA水平的檢測(cè)發(fā)現(xiàn), 與對(duì)照組相比, 0.4 mmol/L油酸抑制Caspase-3b和Caspase-9的mRNA表達(dá)(P＜0.05), 但對(duì)Caspase-8和AIF的mRNA表達(dá)并無(wú)影響。而當(dāng)0.8 mmol/L油酸處理肝細(xì)胞時(shí), Caspase-3b、Caspase-8、Caspase-9和AIF的mRNA表達(dá)均顯著上升(P＜0.05)。計(jì)算Bcl-2/Bax mRNA比值, 發(fā)現(xiàn)0.4 mmol/L油酸處理組的Bcl-2/Bax比值顯著高于對(duì)照組, 而0.8 mmol/L油酸處理組Bcl-2/Bax比值顯著低于對(duì)照組(P＜0.05)。

圖 3 不同濃度油酸處理后草魚肝細(xì)胞中甘油三酯含量(n = 6,±SD)Fig. 3 Triglyceride content in oleic acid -treated C. idellus hepatocytes (n=6,±SD)

3 討論

魚類營(yíng)養(yǎng)性脂肪肝是一種由于高能日糧的攝入, 肝細(xì)胞中TAG合成量超過(guò)機(jī)體需要量, 而在肝細(xì)胞內(nèi)過(guò)量蓄積脂質(zhì)為特征的營(yíng)養(yǎng)性疾病[12], 體內(nèi)循環(huán)的FFA在肝脂肪變性過(guò)程中起到了至關(guān)重要的作用[17]。正常生理狀態(tài)時(shí), 肝細(xì)胞內(nèi)脂肪酸首先在線粒體中進(jìn)行β氧化為機(jī)體提供能量, 多余的脂肪酸在內(nèi)質(zhì)網(wǎng)中合成脂蛋白或構(gòu)成細(xì)胞的結(jié)構(gòu)脂肪; 當(dāng)脂肪酸過(guò)量輸送至肝臟時(shí), 超過(guò)了肝細(xì)胞的處理能力, 導(dǎo)致脂肪酸的β氧化和脂蛋白合成障礙, TAG在肝細(xì)胞內(nèi)大量蓄積進(jìn)而導(dǎo)致細(xì)胞的脂肪變性[18, 19]。

目前, 對(duì)魚類營(yíng)養(yǎng)性脂肪肝的研究最常采用的是動(dòng)物模型, 鮮有采用細(xì)胞模型進(jìn)行研究的報(bào)道[20—22]。本研究采用不同濃度的油酸處理草魚肝細(xì)胞, 研究外源脂肪酸對(duì)草魚肝細(xì)胞活力, 脂質(zhì)蓄積和凋亡的影響。研究發(fā)現(xiàn), 隨著油酸處理濃度的增加, 肝細(xì)胞活力和脂質(zhì)含量均呈現(xiàn)先上升后下降的趨勢(shì)。已有研究指出, 油酸能夠促進(jìn)小鼠原代肝細(xì)胞內(nèi)TAG富集及脂滴的積累, 但對(duì)細(xì)胞凋亡的影響甚小[23]。Vinciguerra等[24]發(fā)現(xiàn)油酸的處理濃度在0.1 mmol/L以下時(shí), 并不能影響HepG2的活性, 反而促進(jìn)細(xì)胞的增殖; 當(dāng)油酸濃度升高到0.2 mmol/L時(shí), 細(xì)胞的活力顯著下降, Caspase-3酶活性增加。但也有研究表明, 0.2 mmol/L油酸并無(wú)細(xì)胞毒性, 也不會(huì)誘導(dǎo)細(xì)胞凋亡[25]。Gomez-Lechon等[26]等用油酸處理人原代肝細(xì)胞, 發(fā)現(xiàn)當(dāng)油酸濃度上升至2 mmol/L時(shí)對(duì)細(xì)胞活性仍沒有顯著影響。Maestre等[27]研究顯示, 油酸濃度超過(guò)0.5 mmol/L時(shí), Ins-1細(xì)胞有一半以上發(fā)生凋亡。而Ariel等[28]發(fā)現(xiàn), 使用1 mmol/L的油酸和棕櫚酸混合物處理HepG2細(xì)胞可以刺激TNF-α的表達(dá), 促進(jìn)肝細(xì)胞脂毒性, 誘導(dǎo)細(xì)胞的凋亡。本研究發(fā)現(xiàn), 油酸濃度在0.6 mmol/L以下時(shí), 有促進(jìn)細(xì)胞活力的作用; 而當(dāng)濃度上升至0.8 mmol/L時(shí), 細(xì)胞活力顯著降低(P＜0.05)。這可能是因?yàn)椴蒴~肝細(xì)胞可以吸收利用一定水平的脂肪酸, 而且低濃度的油酸可能通過(guò)激活A(yù)KT通路促進(jìn)細(xì)胞的增殖和TAG的累積; 而當(dāng)油酸的濃度超過(guò)了機(jī)體所能承受的閾值,就會(huì)造成細(xì)胞的損傷和凋亡[29]。

圖 4 BODIPY和DAPI染色檢測(cè)油酸處理后草魚肝細(xì)胞脂滴和細(xì)胞核的變化(×200)Fig. 4 BODIPY和DAPI staining analysis of lipid droplets and nuclear change in C. idellus hepatocytes after treatment with oleic acid (×200)A. 0 (×200); B. 0.2 mmol/L (×200); C. 0.4 mmol/L (×200); D. 0.6 mmol/L (×200); E. 0.8 mmol/L (×200); F. 1 mmol/L (×200)

圖 5 Annexin V- FITC /PI流式細(xì)胞術(shù)檢測(cè)油酸誘導(dǎo)草魚肝細(xì)胞凋亡 (n=3,xˉ±SD)Fig. 5 Apoptosis of C. idellus hepatocytes were analyzed by flow cytometry with Annexin V-FITC and PI staining (n=3,xˉ±SD)四個(gè)象限：上左. 損傷及碎片；上右. 晚期凋亡細(xì)胞；下左. 正常細(xì)胞；下右. 早期凋亡細(xì)胞Four quadrants: UL. Injured cells; UR. Late apoptotic cells; LL. Viable cells; LR. Early apoptotic cells. A. 0; B. 0.2 mmol/L; C. 0.4 mmol/L; D. 0.6 mmol/L; E. 0.8 mmol/L; F. 1 mmol/L

眾所周知, 轉(zhuǎn)錄因子PPARγ在調(diào)節(jié)脂肪酸的攝取和貯存方面發(fā)揮著關(guān)鍵作用[30]。研究表明, 小鼠肝臟中特異性敲除PPARγ后, 可以免受肝臟脂肪變性的影響[31,32], 而過(guò)表達(dá)PPARγ后, 小鼠肝臟發(fā)生脂肪變性, 成脂基因和脂肪合成相關(guān)基因被激活[33]。C/EBPα被認(rèn)為是調(diào)節(jié)脂肪細(xì)胞分化的關(guān)鍵轉(zhuǎn)錄因子[34], 但有研究發(fā)現(xiàn)C/EBP家族在非酒精性脂肪肝(NAFLD)的形成中也發(fā)揮著重要的作用[35]。Zhang等[36]研究發(fā)現(xiàn)C/EBPα的基因和蛋白表達(dá)在NAFLD的早中期升高, 而在后期降低。同時(shí)有研究證明NAFLD模型中PPARγ和C/EBPα的mRNA表達(dá)均顯著升高,說(shuō)明PPARγ和C/EBPα對(duì)肝細(xì)胞脂質(zhì)蓄積過(guò)程具有重要的作用[37]。在本試驗(yàn)中, 0.4和0.8 mmol/L兩種濃度油酸處理時(shí), PPARγ和C/EBPα的基因表達(dá)量都顯著上調(diào), 表明外源性脂肪酸造成肝細(xì)胞脂質(zhì)積累很可能是通過(guò)促進(jìn)PPARγ和C/EBPα的表達(dá)實(shí)現(xiàn)的。

圖 6 Real-time PCR法測(cè)定油酸誘導(dǎo)后草魚肝細(xì)胞中相關(guān)基因的相對(duì)表達(dá)量(n=3,±SD)Fig. 6 The relative expression level of related genes in C. idellus hepatocytes after treatment with oleic acid were examined by Real-time PCR (n=3,±SD)

細(xì)胞凋亡是多基因嚴(yán)密調(diào)控的結(jié)果。目前認(rèn)為, 細(xì)胞凋亡發(fā)生機(jī)制主要包括死亡受體信號(hào)轉(zhuǎn)導(dǎo)通路、線粒體信號(hào)通路和內(nèi)質(zhì)網(wǎng)途徑[38], 而在肝細(xì)胞凋亡中, 通常將內(nèi)質(zhì)網(wǎng)凋亡途徑包含在線粒體凋亡信號(hào)通路中[39]。Malhi等[40]發(fā)現(xiàn)過(guò)量的FFA能夠激活JNK依賴的線粒體凋亡途徑導(dǎo)致細(xì)胞色素C釋放和促凋亡蛋白Bcl-2激活, 誘發(fā)細(xì)胞凋亡。Feldstein等[41]認(rèn)為油酸處理肝細(xì)胞會(huì)使溶酶體失去穩(wěn)定性, 進(jìn)而激活NF-κB依賴的TNF-α表達(dá), 導(dǎo)致細(xì)胞的凋亡。在肝細(xì)胞凋亡過(guò)程中, Caspase家族也發(fā)揮著巨大的作用。本實(shí)驗(yàn)室研究發(fā)現(xiàn), 草魚的Caspase-3基因分為Caspase-3a (GenBank登錄號(hào)KP145001)和Caspase-3b (GenBank登錄號(hào)KP145002)兩種亞型,本試驗(yàn)檢測(cè)了兩種不同濃度油酸處理草魚肝細(xì)胞時(shí)這兩種基因亞型的表達(dá)狀況, 發(fā)現(xiàn)Caspase-3a和Caspase-3b的表達(dá)模式完全不同。高低油酸濃度處理的肝細(xì)胞中, Caspase-3a的mRNA表達(dá)量都顯著下降; 而Caspase-3b的mRNA表達(dá)量在低濃度油酸處理時(shí)顯著下降, 在高濃度油酸處理時(shí)顯著上升(P＜0.05)。同一基因兩種基因型的不同表達(dá), 說(shuō)明在FFA誘導(dǎo)的草魚肝細(xì)胞凋亡中, Caspase-3b發(fā)揮主要作用。Caspase-8和Caspase-9是凋亡的啟動(dòng)者, 二者活化后再激活下游的Caspase-3, 觸發(fā)凋亡的級(jí)聯(lián)反應(yīng), 導(dǎo)致細(xì)胞的凋亡[42]。凋亡誘導(dǎo)因子(AIF)可以引發(fā)不依賴于Caspase的細(xì)胞凋亡, 當(dāng)細(xì)胞受到凋亡刺激后, AIF釋放, 引起細(xì)胞核內(nèi)染色體凝聚、DNA片段化[43]。本研究發(fā)現(xiàn), 當(dāng)油酸處理濃度達(dá)到0.8 mmol/L時(shí), 肝細(xì)胞內(nèi)Caspase-8、Caspase-9和AIF基因的mRNA水平顯著上調(diào)(P＜0.05)。而0.4 mmol/L油酸處理組, Caspase-9的基因表達(dá)量甚至低于對(duì)照組, 說(shuō)明0.8 mmol/L的油酸已經(jīng)超過(guò)細(xì)胞對(duì)FFA的最大承受能力, 可能已造成細(xì)胞的損傷。細(xì)胞受到凋亡刺激時(shí), 抗凋亡蛋白活性被抑制, 并激活促凋亡蛋白活性, 導(dǎo)致促凋亡因子釋放[44]。Bcl-2/Bax mRNA的比值被稱作凋亡開關(guān), 當(dāng)二者比值升高時(shí),抑制細(xì)胞的凋亡; 比值降低時(shí), 促進(jìn)細(xì)胞的凋亡[45]。在本研究中, 0.4 mmol/L油酸處理肝細(xì)胞時(shí), Bcl-2/Bax比值上升, 顯示抗凋亡因子占優(yōu)勢(shì), 細(xì)胞的凋亡被抑制; 當(dāng)油酸濃度上升至0.8 mmol/L時(shí), Bcl-2/Bax mRNA比值顯著下降, 說(shuō)明促凋亡因子起主導(dǎo)作用, 細(xì)胞凋亡被激活。Shimabukuro等[46]也發(fā)現(xiàn)1 mmol/L的FFA混合物(油酸∶棕櫚酸2∶1)能抑制胰島B細(xì)胞Bcl-2的基因和蛋白表達(dá), 而添加瘦素可以使其恢復(fù)正常。而Maestre等[27]的研究發(fā)現(xiàn), 油酸孵育不僅使得INS-1細(xì)胞的Bcl-2蛋白表達(dá)下調(diào), 同時(shí)還會(huì)增加細(xì)胞色素C和AIF的釋放量。提示FFA對(duì)細(xì)胞的毒性可能是通過(guò)損傷線粒體進(jìn)行的[27]。

綜上所述, 本研究發(fā)現(xiàn), 隨著油酸處理濃度的增加, 肝細(xì)胞脂質(zhì)蓄積量呈現(xiàn)一個(gè)先增加后降低的趨勢(shì), 脂質(zhì)合成基因的表達(dá)呈現(xiàn)相同的趨勢(shì)。且低濃度的油酸并不會(huì)造成肝細(xì)胞的凋亡, 反而促進(jìn)肝細(xì)胞增殖, 過(guò)高濃度的油酸則會(huì)導(dǎo)致細(xì)胞凋亡。這說(shuō)明草魚肝細(xì)胞可耐受一定水平的外源脂肪酸, 但脂肪酸劑量過(guò)高時(shí), 則會(huì)引起細(xì)胞功能紊亂, 導(dǎo)致細(xì)胞的凋亡, 這可能與過(guò)量的FFA促使草魚肝細(xì)胞中促凋亡因子Bcl-2和AIF被激活, 誘發(fā)Caspase家族的級(jí)聯(lián)反應(yīng), 同時(shí)抑制了抗凋亡因子Bax的活性密切相關(guān)。

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INFLUENCE OF FATTY ACIDS ON LIPID ACCUMULATION AND APOPTOSIS STATUS OF GRASS CARP CTENOPHARYNGODON IDELLUS HEPATOCYTE IN VITRO

LI Xue-Xian, SUN Jian, JI Hong and CHEN Hao-Jie
(College of Animal Science and Technology, Northwest A & F University, Yangling 712100, China)

To investigate the effects of fatty acids on hepatocyte lipid metabolism and health status of grass carp (Ctenopharyngodon idellus), the normal grass carp hepatocytes were cultured with different concentrations of oleic acid (0—1 mmol/L) for 24h to measure cell viability and lipids content by methylthiazolyl tetrazolium (MTT) assay and oil red O staining, respectively. The lipid droplets and nuclear of hepatocytes were observed with BODIPY and DAPI staining. Hepatocyte apoptosis was analyzed by flow cytometry. Real-time qPCR was performed to detect the mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein alpha (C/EBPα) and apoptosis related genes [Caspase3a, Caspase-3b, Caspase-8, Caspase-9, apoptosis inducing factor (AIF), Bcl-2, Bax]. The results showed that the cell viability and intracellular lipid accumulation of hepatocytes increased significantly after treatment with 0.6 and 0.4 mmol/L oleic acid (P＜0.05). The lowest hepatocytes apoptosis rate was in 0.4 mmol/L group and the highest was in 1 mmol/L oleic acid group (P＜0.05). The mRNA levels of Caspase-3b and Caspase-9 reduced significantly and the mRNA ration of Bcl-2/Bax was elevated by 0.4 mmol/L oleic acid (P＜0.05). Consistently, 0.4 mmol/L oleic acid significantly reduced caspase-3b, caspase-9 and AIF mRNA levels and increased the mRNA ratio of Bcl-2/Bax (P＜0.05) and these genes showed opposite expression pattern in hepatocytes treated with 0.8 mmol/L oleic acid (P＜0.05). These results demonstrated dosage-dependent role for fatty acids in grass carp hepatocytes viability and intracellular lipid accumulation via the regulation of the genes related to lipid metabolism and apoptosis.

Fatty Acids; Hepatocyte; Apoptosis; Ctenopharyngodon idellus

Q173

A

1000-3207(2017)01-0056-09

10.7541/2017.8

2015-12-11;

2016-04-27

國(guó)家自然科學(xué)基金項(xiàng)目(31372538)資助 [Supported by the National Natural Science Foundation of China (31372538)]

李雪賢(1990—), 女; 碩士研究生; 主要從事水生動(dòng)物營(yíng)養(yǎng)與飼料學(xué)研究。E-mail: lxx0663@163.com

吉紅(1967—), 男, 河南靈寶人; 博士生導(dǎo)師; 主要從事水生經(jīng)濟(jì)動(dòng)物營(yíng)養(yǎng)與飼料學(xué)研究。E-mail: jihong@nwsuaf.edu.cn