高 景 齊智利

(華中農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院，武漢 430070)

反芻動(dòng)物通過瘤胃微生物發(fā)酵飼糧中的碳水化合物產(chǎn)生大量的乙酸、丙酸、丁酸等短鏈脂肪酸(short chain fatty acid,SCFA)并通過瘤胃上皮吸收。研究發(fā)現(xiàn)，一部分SCFA經(jīng)瘤胃上皮吸收進(jìn)入血液，在肝臟中糖異生生成葡萄糖供能，另一部分在瘤胃上皮細(xì)胞內(nèi)發(fā)生代謝，生成酮體、膽固醇。瘤胃上皮是SCFA吸收的主要場所，具有分層結(jié)構(gòu)，從瘤胃內(nèi)腔由外到內(nèi)可以分為角質(zhì)層、顆粒層、棘皮層以及基底層[1-2]。SCFA在瘤胃內(nèi)腔有2種存在形式，分別為解離狀態(tài)和未解離狀態(tài)。不同類型以及存在形式的SCFA，其轉(zhuǎn)運(yùn)吸收到瘤胃上皮細(xì)胞的方式和特點(diǎn)也不一樣。SCFA的轉(zhuǎn)運(yùn)是被動(dòng)擴(kuò)散與特異性載體轉(zhuǎn)運(yùn)共存[3-4]。而SCFA作為一種弱酸，不論在瘤胃內(nèi)腔里解離出H+還是進(jìn)入瘤胃上皮細(xì)胞后解離出H+，均會引起酸化，將會激活細(xì)胞膜上的相關(guān)轉(zhuǎn)運(yùn)載體如Na+/H+交換體(Na+/H+exchanger,NHE)，將細(xì)胞內(nèi)H+轉(zhuǎn)運(yùn)出細(xì)胞。SCFA在進(jìn)入瘤胃上皮細(xì)胞后進(jìn)行代謝，代謝率超過50%，其中以丁酸的代謝率最高。SCFA代謝途徑包括膽固醇合成和酮體合成，這2種合成途徑的前體物3-羥基3-甲基戊二酰輔酶A(3-hydroxy-3-methylglutaryl-CoA,HMG-CoA)是由SCFA經(jīng)過一系列反應(yīng)生成的。酮體生成后經(jīng)單羧酸鹽/H+共轉(zhuǎn)運(yùn)蛋白(monocarboxylate/H+co-transporters,MCT)轉(zhuǎn)運(yùn)到血液，為外周組織提供能量，當(dāng)生酮作用過強(qiáng)時(shí)，會導(dǎo)致反芻動(dòng)物血液中酮體濃度過高，嚴(yán)重時(shí)可引起酮病。而膽固醇積累過多則會導(dǎo)致細(xì)胞炎癥和氧化應(yīng)激等，進(jìn)而影響機(jī)體整體能量供應(yīng)，造成炎癥反應(yīng)。因此研究SCFA在瘤胃上皮的轉(zhuǎn)運(yùn)吸收對深入了解瘤胃動(dòng)態(tài)及構(gòu)建營養(yǎng)調(diào)控模型有重要指導(dǎo)意義。

1 瘤胃上皮形態(tài)與功能

瘤胃上皮有吸收、代謝SCFA以及保護(hù)瘤胃等重要生理功能。瘤胃上皮具有分層結(jié)構(gòu)，從瘤胃內(nèi)腔表面開始由外到內(nèi)按照細(xì)胞分布可分為4層，分別是角質(zhì)層、顆粒層、棘皮層及基底層?；讓蛹?xì)胞富含線粒體，顆粒層和棘皮層細(xì)胞位于中間，之間界限不明顯，其中棘皮層細(xì)胞也含有少量線粒體，因此基底層和棘皮層是瘤胃上皮SCFA代謝的主要場所。顆粒層細(xì)胞之間緊密間隙連接，角質(zhì)層位于最外面，細(xì)胞高度角質(zhì)化，起到了屏障保護(hù)作用，可作為對瘤胃內(nèi)物理環(huán)境的防御屏障[5-8]。角質(zhì)層細(xì)胞層數(shù)受到SCFA的調(diào)控。當(dāng)飼糧精粗比上升時(shí)，丙酸/乙酸上升，SCFA濃度上升，瘤胃液pH下降，角質(zhì)層細(xì)胞層數(shù)可增加至15層。反之，角質(zhì)層細(xì)胞層數(shù)可下降至4層。瘤胃上皮遍布葉狀乳頭，牛葉狀乳頭長度可達(dá)10～15 mm，大大增加了SCFA吸收表面積[9]。葉狀乳頭作為瘤胃吸收營養(yǎng)物質(zhì)的主要結(jié)構(gòu)，其分布密度和大小影響SCFA的吸收。有研究表明，高蛋白質(zhì)和高能量濃度攝入能提高血清胰島素樣生長因子1(IGF-1)濃度，在其與受體結(jié)合后激活下游Ras/Raf/絲裂原活化的細(xì)胞外信號調(diào)節(jié)激酶(MEK)/細(xì)胞外調(diào)節(jié)蛋白激酶(ERK)信號通路，上調(diào)細(xì)胞周期蛋白D1(cyclin D1)表達(dá)，促進(jìn)瘤胃上皮細(xì)胞增殖，增加其對SCFA的吸收[10-11]。Yazdi等[12-13]則發(fā)現(xiàn)熱應(yīng)激能增加乳頭高度。因此，瘤胃上皮形態(tài)結(jié)構(gòu)與SCFA的吸收處在動(dòng)態(tài)平衡的調(diào)節(jié)之中，兩者相互協(xié)調(diào)，維持瘤胃內(nèi)環(huán)境的穩(wěn)態(tài)。

2 瘤胃上皮SCFA吸收

SCFA在瘤胃內(nèi)腔和胞內(nèi)之間存在濃度差，但并不是簡單的順濃度梯度的擴(kuò)散，其在瘤胃內(nèi)腔內(nèi)有解離和非解離2種形式，以非解離SCFA為主，因此導(dǎo)致其吸收轉(zhuǎn)運(yùn)方式存在差異[14-15]。體外試驗(yàn)發(fā)現(xiàn)丁酸在無濃度梯度下呈現(xiàn)出最高凈吸收速率，而乙酸和丙酸的凈吸收率較低[16]。在SCFA的吸收轉(zhuǎn)運(yùn)中，被動(dòng)擴(kuò)散與特異性載體運(yùn)輸共存。NHE是膜上重要的轉(zhuǎn)運(yùn)Na+和H+的載體。未解離SCFA經(jīng)被動(dòng)擴(kuò)散進(jìn)入細(xì)胞后解離出H+，調(diào)節(jié)pH,引起胞質(zhì)酸化，使NHE表達(dá)上調(diào)，增加Na+吸收速率，將Na+轉(zhuǎn)入胞內(nèi)，H+轉(zhuǎn)出瘤胃內(nèi)腔，因此瘤胃上皮細(xì)胞吸收轉(zhuǎn)運(yùn)SCFA時(shí)引起胞質(zhì)酸化與NHE的活性增強(qiáng)存在功能性偶聯(lián)[17-18]。在牛瘤胃上皮細(xì)胞的NHE家族主要是NHE1、NHE2、NHE3和NHE8，而NHE1和NHE3分布在山羊及綿羊的瘤胃上皮細(xì)胞[19-21]。研究表明，NHE1維持鄰近顆粒層的細(xì)胞外pH，NHE1敲除動(dòng)物局部細(xì)胞外pH低于正常值[22]。因此NHE1的存在對于維持瘤胃液pH有重要作用。而MCT是介導(dǎo)瘤胃SCFA、酮體、乳酸等轉(zhuǎn)運(yùn)吸收的特異性載體。在牛瘤胃上皮細(xì)胞表達(dá)的是MCT1和MCT2。通過熒光染色將MCT1定位在瘤胃上皮的基底層，其負(fù)責(zé)將胞內(nèi)解離的SCFA，乳酸鹽和酮體共轉(zhuǎn)運(yùn)到血液中去除上皮細(xì)胞中的H+，防止因酮體及乳酸鹽過度積累引起的胞質(zhì)酸化[23-24]。

3 瘤胃上皮細(xì)胞SCFA代謝

瘤胃上皮細(xì)胞內(nèi)代謝活躍，研究發(fā)現(xiàn)，從瘤胃內(nèi)腔到血液中約75%的丙酸和95%的丁酸在瘤胃上皮細(xì)胞內(nèi)被代謝掉[34]。瘤胃上皮細(xì)胞不依賴于葡萄糖、酮體以及谷氨酰胺等供能，而是氧化終端發(fā)酵產(chǎn)物SCFA來獲取大部分能量。而在SCFA中，丁酸的代謝率最高，因此丁酸將是主要代謝底物[35]。被瘤胃上皮攝取后，SCFA的代謝由?；o酶A合成酶家族加入輔酶A酯生成乙酰輔酶A開始[36]，然后3-羥基3-甲基戊二酰輔酶A合成酶(3-hydroxy-3-methylglutaryl-CoA synthase,HMGCS)將乙酰輔酶A轉(zhuǎn)化為HMG-CoA。HMG-CoA是瘤胃上皮的中心代謝物，合成酮體和膽固醇的前體物，分布于線粒體和細(xì)胞質(zhì)中[37-39]。HMGCS有2個(gè)亞型：位于細(xì)胞質(zhì)內(nèi)的HMGCS1和專一性位于線粒體中HMGCS2。HMGCS2調(diào)控瘤胃上皮細(xì)胞酮體合成，是反應(yīng)限速酶[40]。De Rosa等[41]發(fā)現(xiàn)25和50 μmol/L的二十二碳六烯酸(docosahexaenoic acid,DHA)、二十碳五烯酸(eicosapentaenoic acid,EPA)、花生四烯酸(arachidonic acid,AA)均能在轉(zhuǎn)錄和翻譯水平上調(diào)HMGCS2的表達(dá)，25 mmol/L果糖和胰島素處理人肝腫瘤細(xì)胞(HepG2)24 h則降低mRNA和蛋白質(zhì)表達(dá)量。多不飽和脂肪酸(polyunsaturated fatty acids,PUFA)能通過與過氧化物酶體增殖激活受體α(peroxisome proliferator-activated receptor α,PPARα)結(jié)合調(diào)控脂肪從頭合成、脂肪酸氧化等多

種代謝途徑[42]。而HMGCS2啟動(dòng)子區(qū)域含有過氧化物酶體增殖反應(yīng)元件(peroxisome proliferator response elemen,PPARE)，其與PPARα結(jié)合后啟動(dòng)HMGCS2的轉(zhuǎn)錄[43-44]。研究發(fā)現(xiàn)，當(dāng)PPARαmRNA表達(dá)上調(diào)時(shí)，HMGCS2 mRNA表達(dá)量也隨之增加[45]。因此，我們推測多PUFA可能是通過直接結(jié)合PPARα等核受體上調(diào)HMGCS2表達(dá)來調(diào)節(jié)酮體合成。反芻動(dòng)物酮體生成的主要部位包括瘤胃和肝臟。在線粒體中的酮體合成過程如圖1所示。當(dāng)瘤胃上皮生酮作用過強(qiáng)，SCFA代謝紊亂，造成高血酮癥，最終引起酮病，將會嚴(yán)重危害動(dòng)物健康[48]。因此進(jìn)一步研究瘤胃上皮細(xì)胞酮體合成調(diào)控機(jī)制對預(yù)防酮病有重要意義。

表1 瘤胃上皮短鏈脂肪酸轉(zhuǎn)運(yùn)載體

除了作為酮體生成的底物之外，SCFA也可以在細(xì)胞質(zhì)和內(nèi)質(zhì)網(wǎng)中進(jìn)行膽固醇生物合成。膽固醇生物合成途徑的前部分發(fā)生在細(xì)胞質(zhì)中，SCFA在細(xì)胞質(zhì)中經(jīng)過一系列反應(yīng)生成HMG-CoA，HMG-CoA從細(xì)胞質(zhì)遷移到內(nèi)質(zhì)網(wǎng)上，因?yàn)閮?nèi)質(zhì)網(wǎng)上有羥甲基戊二酰輔酶還原酶(HMG-CoA reductase,HMGR)。遷移到內(nèi)質(zhì)網(wǎng)上的HMG-CoA被HMGR還原催化成甲羥戊酸(通常稱為甲羥戊酸途徑)[49-50]。HMGR是膽固醇生物合成的限速酶。然后甲羥戊酸脫羧轉(zhuǎn)化為類異戊二烯中間體，如法尼醇焦磷酸(farnesyl-PP,FPP)。異戊二烯中間體通過膜相關(guān)信號蛋白的附著，亞細(xì)胞定位和細(xì)胞內(nèi)運(yùn)輸來誘導(dǎo)細(xì)胞增殖，遷移和氧化應(yīng)激。膽固醇生物合成的最終分支點(diǎn)即角鯊烯合酶(FDPS)催化FPP生成角鯊烯，角鯊烯再轉(zhuǎn)化為羊毛甾醇，經(jīng)過一系列反應(yīng)最終生成為膽固醇[51]。膽固醇雖然是哺乳動(dòng)物細(xì)胞膜的主要成分，但當(dāng)細(xì)胞內(nèi)膽固醇及其代謝物(類異戊二烯)積累過多時(shí)會增大膜通透性，引發(fā)炎癥反應(yīng)[52]。研究發(fā)現(xiàn)，飼喂高精飼糧會增加瘤胃上皮通透性和炎癥[53-54]。因?yàn)楦呔暭Z促進(jìn)瘤胃發(fā)酵，增加SCFA濃度，促進(jìn)瘤胃上皮細(xì)胞膽固醇生物合成，增大細(xì)胞通透性，引發(fā)炎癥。Steele等[55]研究發(fā)現(xiàn)，當(dāng)持續(xù)1周飼喂高精飼糧時(shí)，瘤胃液SCFA濃度顯著增加，出現(xiàn)瘤胃酸中毒，牛膽固醇合成相關(guān)基因HMGS1、HMGRmRNA表達(dá)量上調(diào)，膽固醇濃度升高，引發(fā)炎癥。而隨著試驗(yàn)進(jìn)行到第3周時(shí)，SCFA濃度仍顯著高于正常值，但HMGS1、HMGRmRNA表達(dá)量顯著下降，抑制膽固醇合成，瘤胃酸中毒減緩。因此，瘤胃上皮內(nèi)膽固醇的合成是受到SCFA濃度和持續(xù)時(shí)間的共同調(diào)控。短期內(nèi)SCFA增加能促進(jìn)瘤胃上皮細(xì)胞膽固醇的合成，增加瘤胃上皮通透性，引發(fā)炎癥，而當(dāng)持續(xù)時(shí)間延長時(shí)，瘤胃上皮細(xì)胞則通過甾醇調(diào)節(jié)元件結(jié)合蛋白(sterol regulatory element binding protein,SREBP)通路來抑制膽固醇合成通路上相關(guān)酶的表達(dá)，抑制膽固醇合成，減緩炎癥和瘤胃酸中毒[56]。SREBP是調(diào)控牛肝和乳腺中的膽固醇和脂肪基因表達(dá)的轉(zhuǎn)錄因子家族[57]。?；蚪M編碼3個(gè)SREBP亞型，即SREBP-1a、SREBP-1c和SREBP-2，其中SREBP-2優(yōu)先激活膽固醇生物合成[58]。高膽固醇濃度時(shí)，在內(nèi)質(zhì)網(wǎng)的SREBP與SREBP切割活化蛋白(SREBP cleavage-activating protein，SCAP)結(jié)合，并從轉(zhuǎn)錄水平上抑制膽固醇合成相關(guān)基因表達(dá)(圖2)；低膽固醇濃度時(shí)，SREBP-SCAP復(fù)合物從內(nèi)質(zhì)網(wǎng)遷移到高爾基體，SREBP在發(fā)生蛋白剪切，釋放N端，啟動(dòng)核基因表達(dá)，膽固醇合成作用增強(qiáng)[59-60]。因此，為了維持瘤胃上皮細(xì)胞內(nèi)膽固醇濃度的穩(wěn)定，減緩膽固醇積累導(dǎo)致的瘤胃上皮通透性增加和炎癥，需要進(jìn)一步研究SCFA在瘤胃上皮細(xì)胞膽固醇合成的分子調(diào)控機(jī)制，為有效預(yù)防及緩解瘤胃酸中毒提供依據(jù)。

Acetyl-CoA：乙酰輔酶A acetylcoenzyme A；CoA：輔酶A coenzyme A；HMGCS1：3-羥基3-甲基戊二酰輔酶A合成酶1 3-hydroxy-3-methylglutaryl-CoA synthase 1；Acetoacetyl-CoA：乙酰乙酰輔酶A acetoacetylcoenzyme A；HMG-CoA：3-羥基3-甲基戊二酰輔酶A 3-hydroxy-3-methylglutaryl-CoA；HMGR：羥甲基戊二酰輔酶還原酶 HMG-CoA reductase；mevalonate：甲羥戊酸；squalene：角鯊烯；cholesterol：膽固醇；AE：陰離子交換劑 anion exchangers；NHE：Na+/H+交換體 Na+/H+exchanger；TCA：三羧酸循環(huán) tricarboxylic acid cycle；HMG-CoA layse：HMG-CoA裂解酶 3-hydroxy-3-methylglutaryl-CoA layse；BDH1：β-羥基丁酸脫氫酶 β-hydroxybutyrate dehydrogenase；βOHB：β-羥基丁酸β-hydroxybutyrate；NAD+：氧化型煙酰胺腺嘌呤二核苷酸oxidized form of nicotinamide adenine dinucleotide；NADH：還原型煙酰胺腺嘌呤二核苷酸 reduced form of nicotinamide adenine dinucleotide；AcAc：乙酰乙酸 acetoacetate；Acetone：丙酮。下圖同 The same as below。

圖1瘤胃上皮細(xì)胞內(nèi)SCFA的轉(zhuǎn)運(yùn)吸收及代謝(結(jié)合文獻(xiàn)[46-47]繪制)

Fig.1 Absorbtion and metabolism of SCFA in rumen epithelium cells (drawed based on references[46-47])

SCFA：短鏈脂肪酸 short chain fatty acid；NADPH:還原型煙酰胺腺嘌呤二核苷酸磷酸 reduced form of nicotinamide-adenine dinucleotide phosphate;Mevalonate-PP：甲羥戊酸焦磷酸 mevalonate pyrophosphate；Farnesyl-PP：法尼醇焦磷酸 farnesyl-pyrophosphate；squalene synthetase：角鯊烯合成酶；squalene epoxidase：角鯊烯環(huán)氧酶；lanosterol：羊毛甾醇；bile acid:膽汁酸；steroid hormone:固醇激素；vitamin D：維生素D；SREBP:甾醇調(diào)節(jié)元件結(jié)合蛋白 sterol regulatory element binding protein;SCAP：SREBP切割活化蛋白 SREBP cleavage-activating protein。

圖2瘤胃上皮細(xì)胞膽固醇生物合成途徑(修改自文獻(xiàn)[51])

Fig.2 Cholesterol biosynthesis pathway in rumen epithelium cell (modified from reference [51])

4 小結(jié)與展望

由于反芻動(dòng)物瘤胃發(fā)酵的特異性，SCFA是其主要能量供應(yīng)底物，因此SCFA在瘤胃上皮的吸收代謝與機(jī)體能量代謝、健康生長密切相關(guān)，深入研究并闡明機(jī)制有利于瘤胃健康的營養(yǎng)調(diào)控。目前關(guān)于瘤胃上皮細(xì)胞各種特異性載體蛋白的種類、作用機(jī)制進(jìn)行了大量的研究，但關(guān)于不同載體之間的相互作用以及不同生理狀態(tài)如熱應(yīng)激、瘤胃酸中毒等對載體的影響尚不清楚。關(guān)于SCFA在瘤胃上皮細(xì)胞的代謝轉(zhuǎn)化可以與肝臟代謝、血液循環(huán)等相結(jié)合做進(jìn)一步的研究，從整體上深入闡明瘤胃上皮SCFA的吸收代謝調(diào)控機(jī)理。

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