耿雅麗，田平，羅燕文，華燦楓，陶詩(shī)煜，田靖，倪迎冬

(南京農(nóng)業(yè)大學(xué)，農(nóng)業(yè)部動(dòng)物生理生化重點(diǎn)開(kāi)放實(shí)驗(yàn)室，江蘇 南京 210095)

高精料對(duì)泌乳奶山羊瘤胃上皮氧化應(yīng)激和膽固醇代謝的影響

耿雅麗，田平，羅燕文，華燦楓，陶詩(shī)煜，田靖，倪迎冬*

(南京農(nóng)業(yè)大學(xué)，農(nóng)業(yè)部動(dòng)物生理生化重點(diǎn)開(kāi)放實(shí)驗(yàn)室，江蘇 南京 210095)

為了研究長(zhǎng)期或短期飼喂高精料日糧對(duì)泌乳期奶山羊瘤胃上皮組織氧化應(yīng)激和膽固醇代謝的影響，實(shí)驗(yàn)選用17只健康的經(jīng)產(chǎn)泌乳中期關(guān)中奶山羊，隨機(jī)分為3組：飼喂低精料組(對(duì)照組，LC，n=5);長(zhǎng)期飼喂高精料組(HL,n=7)，19周飼喂期;短期飼喂高精料組(HS，n=5)，4周飼喂期。實(shí)驗(yàn)結(jié)束后采集瘤胃組織，用PBS反復(fù)清洗，于液氮中速凍后置于-80 ℃冰箱保存。結(jié)果顯示，與對(duì)照組相比，HL和HS組山羊瘤胃上皮組織中細(xì)胞周期相關(guān)基因CDK2和CDK4 mRNA表達(dá)水平顯著升高(Plt;0.05)，且HS組p-ERK1/2蛋白表達(dá)顯著升高(Plt;0.05)，但GPR41和GPR43蛋白表達(dá)無(wú)顯著變化(Pgt;0.05)；HL和HS組山羊瘤胃上皮組織促細(xì)胞凋亡基因Casepase9 mRNA表達(dá)顯著升高(Plt;0.05)，HS組抗凋亡Bcl-2/BaxmRNA表達(dá)比例呈下降趨勢(shì)(0.05lt;Plt;0.1)；HS組瘤胃上皮組織中抗氧化酶CAT和總抗氧化能力T-AOC顯著升高(Plt;0.05)，HL組CAT含量和SOD酶活性顯著升高(Plt;0.05)，但總抗氧化能力T-AOC無(wú)顯著差異(Pgt;0.05)；HS組瘤胃上皮組織中膽固醇含量下降達(dá)顯著水平(Plt;0.05)，且HL組呈下降趨勢(shì)(0.05lt;Plt;0.1)，膽固醇酰基轉(zhuǎn)移酶ACAT1和ACAT2基因表達(dá)顯著上調(diào)(Plt;0.05)，HS組瘤胃上皮組織中膽固醇的含量與ACAT1和ACAT2 mRNA的表達(dá)呈顯著的負(fù)強(qiáng)相關(guān)(P=0.0067,r=-0.8208;P=0.0100,r=-0.7980)。與對(duì)照組比較，HS和HL組瘤胃上皮組織中VFA轉(zhuǎn)運(yùn)相關(guān)基因AE2、DRA、NHE2、NHE3和νH+ATPasemRNA表達(dá)顯著升高(Plt;0.05),NHE2蛋白表達(dá)升高但未達(dá)顯著差異水平(Pgt;0.05)。與HL組相比，HS組山羊瘤胃上皮組織中總抗氧化能力T-AOC顯著升高(Plt;0.05)；且膽固醇含量顯著下降(Plt;0.05)。以上結(jié)果說(shuō)明，短期飼喂高精料日糧可加快泌乳期奶山羊瘤胃上皮組織的更新，提高組織總抗氧化能力；而長(zhǎng)期飼喂高精料日糧未引起細(xì)胞增殖相關(guān)蛋白和總抗氧化能力的顯著變化；飼喂高精料日糧可以加快瘤胃上皮組織對(duì)VFA的轉(zhuǎn)運(yùn)。此外，飼喂高精料日糧引起瘤胃上皮組織內(nèi)膽固醇含量降低，膽固醇酰基轉(zhuǎn)移酶基因表達(dá)的顯著上調(diào)，提示其對(duì)瘤胃上皮組織更新的潛在影響。

高精料；瘤胃上皮；氧化應(yīng)激；膽固醇；山羊

為提高反芻動(dòng)物的生產(chǎn)性能，常給泌乳反芻動(dòng)物飼喂高精料日糧來(lái)滿足其高產(chǎn)乳的能量需求。然而，高精料日糧易引起瘤胃異常發(fā)酵，有害代謝產(chǎn)物蓄積，導(dǎo)致亞急性瘤胃酸中毒(subacute ruminal acids, SARA)，甚至誘發(fā)乳房炎、蹄葉炎和子宮內(nèi)膜炎等臨床常見(jiàn)疾病[1]。瘤胃是反芻動(dòng)物特有的器官，并且是反芻動(dòng)物消化代謝和營(yíng)養(yǎng)吸收最重要的場(chǎng)所之一。瘤胃上皮吸收瘤胃發(fā)酵產(chǎn)生的能量、營(yíng)養(yǎng)物質(zhì)，其生長(zhǎng)發(fā)育情況直接影響反芻動(dòng)物的生產(chǎn)性能[2]。在正常生理狀態(tài)下，反芻動(dòng)物瘤胃上皮組織產(chǎn)生自由基，同時(shí)也產(chǎn)生或從食物中直接獲得抵抗自由基的抗氧化物質(zhì)，以抵抗自由基對(duì)細(xì)胞的氧化損傷，抗氧化指標(biāo)能反映瘤胃上皮組織自由基代謝的變化。G蛋白偶聯(lián)受體是一種與三聚體G蛋白偶聯(lián)的細(xì)胞表面受體。GPR41和GPR43是目前已知的兩種特異性短鏈脂肪酸受體[3]，在瘤胃上皮組織中廣泛表達(dá)[4]。細(xì)胞外信號(hào)調(diào)節(jié)激酶(ERK)分為ERK1和ERK2，統(tǒng)稱為ERK1/2。ERK1/2被激活后磷酸化進(jìn)入細(xì)胞核作用于E1k-1，c-myc，c-fos，c-jun，ATF和AP-1等轉(zhuǎn)錄因子，促進(jìn)與細(xì)胞增殖和分化相關(guān)基因的轉(zhuǎn)錄與表達(dá)。有研究顯示，GPR41和43與ERK共同參與對(duì)細(xì)胞增殖、分化及發(fā)育的調(diào)節(jié)過(guò)程[5-6]。瘤胃發(fā)酵的主要產(chǎn)物是揮發(fā)性脂肪酸(volatile fatty acid, VFA)，主要包括乙酸，丙酸和丁酸。VFAs經(jīng)瘤胃上皮吸收可以滿足反芻動(dòng)物50%～70%的能量需要[7]。瘤胃內(nèi)的VFA主要以自由擴(kuò)散和載體轉(zhuǎn)運(yùn)的方式被吸收，在上皮細(xì)胞中介導(dǎo)VFA轉(zhuǎn)運(yùn)的載體主要包括鈉氫交換蛋白(Na+/H+exchanger, NHE)、單羧酸轉(zhuǎn)運(yùn)蛋白(monocarboxylate transporter, MCT)和陰離子交換蛋白(anion exchanger protein, AE)等[8-9]。

膽固醇是細(xì)胞膜的重要組成成分，也是合成膽汁酸和多種甾醇類激素的前體，在動(dòng)物機(jī)體中具有重要的生理功能。有研究發(fā)現(xiàn)，細(xì)胞內(nèi)膽固醇或其代謝物與DNA合成激發(fā)有關(guān)，其合成途徑中的許多產(chǎn)物也對(duì)維持細(xì)胞正常功能起十分重要的作用，包括膜結(jié)構(gòu)與功能的完整性、細(xì)胞信號(hào)傳導(dǎo)、某些蛋白質(zhì)合成，以及細(xì)胞周期進(jìn)程的調(diào)控等[10]。正常情況下細(xì)胞通過(guò)自身合成獲得內(nèi)源性膽固醇, 通過(guò)攝取低密度脂蛋白(low density lipoprotein, LDL)獲取外源性膽固醇, 同時(shí)也不斷向細(xì)胞外排出膽固醇, 使細(xì)胞膽固醇處于嚴(yán)格的動(dòng)態(tài)平衡之中, 膽固醇過(guò)高或過(guò)低都將影響細(xì)胞的功能[11]。反芻動(dòng)物瘤胃上皮細(xì)胞利用瘤胃內(nèi)的VFAs合成膽固醇，避免了VFAs在瘤胃內(nèi)的蓄積，降低瘤胃酸中毒的風(fēng)險(xiǎn)。已有研究表明，SARA易感牛和SARA耐受牛的瘤胃上皮吸收VFA的速率無(wú)明顯差異，但與SARA易感牛相比，SARA耐受牛瘤胃上皮細(xì)胞膽固醇合成相關(guān)基因的表達(dá)水平顯著升高[12]，且膽固醇的含量與反芻動(dòng)物瘤胃上皮組織的通透性、炎癥及增殖密切相關(guān)[13-14]。

反芻動(dòng)物飼喂高精料易使瘤胃內(nèi)微生物菌群發(fā)生改變，瘤胃代謝紊亂，VFA總量增加，瘤胃液pH降低，脂多糖(lipopolysaccharide, LPS)含量升高等[15-16]，這會(huì)導(dǎo)致瘤胃乳頭變大數(shù)量增多，同時(shí)瘤胃上皮的轉(zhuǎn)運(yùn)功能也受影響。目前，有關(guān)高精料日糧對(duì)瘤胃上皮功能的影響研究多集中于短期實(shí)驗(yàn)，而長(zhǎng)期飼喂的影響研究尚未見(jiàn)報(bào)道。因此本研究以泌乳期奶山羊?yàn)槟Ｐ?，旨在研究長(zhǎng)期飼喂高精料對(duì)瘤胃上皮組織VFA轉(zhuǎn)運(yùn)和膽固醇代謝的影響及相關(guān)調(diào)節(jié)機(jī)制。

1 材料與方法

1.1試劑與儀器

試劑：TRIzol Reagent(購(gòu)自上海英俊生物技術(shù)有限公司)；SYBR Premix(購(gòu)自Biotool公司)。

儀器：Ex TaqTM(購(gòu)自Takara 公司)；冷凍離心機(jī)(Allegra TM64R,BECKMAN COULTERTM,USA);組織勻漿器；PCR儀(STRATAGENE,USA)；酶標(biāo)儀(Synergy2，Biotek，USA)；NanoDrop TM1000 (Thermo Scientific，USA)。

1.2試驗(yàn)動(dòng)物與飼養(yǎng)

試驗(yàn)于2015年4月27日開(kāi)始選用健康的經(jīng)產(chǎn)泌乳期關(guān)中奶山羊17只，體重與泌乳量相近，隨機(jī)分為低精料日糧組(精粗比35∶65，n=10)和長(zhǎng)期飼喂高精料日糧組(精粗比65∶35,n=7)。飼喂15周后，將對(duì)照組羊分為2組，一組繼續(xù)飼喂低精料日糧作為實(shí)驗(yàn)對(duì)照組(LC,精粗比35∶65,n=5)，另一組飼喂高精料日糧，飼喂期為4周，作為高精料日糧短期飼喂組(HS,精粗比65∶35,n=5)。長(zhǎng)期飼喂高精料日糧組飼喂期為19周(HL,精粗比65∶35,n=7)。

1.3樣品采集、處理與分析測(cè)定

1.3.1樣品的采集 采取瘤胃組織，迅速用磷酸緩沖鹽溶液(phosphate buffer saline, PBS)反復(fù)清洗，剪碎后立即置于液氮中速凍保存，采樣結(jié)束后放于-80 ℃冰箱保存。

1.3.2樣品總RNA的提取和cDNA的制備 稱取100 mg左右的瘤胃組織，用Trizol方法提取總的RNA，每個(gè)樣品每次取2 μL，用Nano Drop分光光度計(jì)檢測(cè)RNA濃度(260/280=1.8～2.0)，2%瓊脂糖凝膠電泳驗(yàn)證RNA質(zhì)量，-70 ℃保存?zhèn)溆?。反轉(zhuǎn)錄酶體系及Tap酶為Promega產(chǎn)品。引物采用Primer 5.0軟件設(shè)計(jì)，由上海捷瑞生物工程有限公司合成，引物見(jiàn)表1。PCR的反應(yīng)條件為95 ℃預(yù)變性3 min, 95 ℃變性30 s, 64 ℃退火20 s, 72 ℃延伸20 s,共40個(gè)循環(huán)。實(shí)時(shí)熒光定量分析采用2-△△CT法，以內(nèi)標(biāo)基因GAPDH為參照，計(jì)算目的基因轉(zhuǎn)錄的相對(duì)量，通過(guò)以下公式計(jì)算出每一個(gè)樣本的△△CT值，最后每一個(gè)樣本值以2-△△CT表示，x表示任意一個(gè)樣本，公式如下：△△CT=(CT.目的基因-CT.內(nèi)參基因)x-(CT.目的基因-CT.內(nèi)參基因)control。

1.3.3組織總蛋白提取 蛋白酶抑制劑混合片(Roche，4693132001)購(gòu)于上海羅氏；BCA蛋白測(cè)定試劑盒(Thermo，23225)購(gòu)于生興;NC膜(PALL，T91375)購(gòu)于巴傲得；發(fā)光檢測(cè)試劑盒(Thermo，NC15080或34076)購(gòu)于生興。

表1 目的基因引物序列Table 1 The primer sequences of the target genes

F:上游引物 Forward primer; R:下游引物 Reverse primer.GAPDH: 甘油醛-3-磷酸脫氫酶 Glyceraldehyde 3 phosphate dehydrogenase;CDK1: 細(xì)胞周期蛋白依賴性激酶 1 Cyclin-dependent kinase 1;CDK2: 細(xì)胞周期蛋白依賴性激酶 2 Cyclin-dependent kinase 2;CDK4: 細(xì)胞周期蛋白依賴性激酶 4 Cyclin-dependent kinase 4;CDK6: 細(xì)胞周期蛋白依賴性激酶 6 Cyclin-dependent kinase 6;DRA: SCFA-/HCO3-轉(zhuǎn)運(yùn)載體 Downregulated in adenoma;PAT1: 陰離子轉(zhuǎn)運(yùn)載體 1 Putative anion transporter, isform 1;AE2: 陰離子轉(zhuǎn)運(yùn)載體 2 Anion exchanger 2;NHE1: Na+/H+交換蛋白 1 Na+/H+exchanger 1;NHE2: Na+/H+交換蛋白 2 Na+/H+exchanger 2;NHE3: Na+/H+交換蛋白 3 Na+/H+exchanger 3;νH+ATPase: 空泡型H+ATP酶 Vacuolar H+ATPase subunit B;HMGCR: 3-羥-3-甲戊二酰輔酶A還原酶 3-hydroxy-3-methylglutaryl-CoA reductase;LDLR: 低密度脂蛋白受體 Low density lipoprotein receptor;ACAT1: 乙酰輔酶a膽固醇?；D(zhuǎn)移酶 1 Acetyl-CoA cholesterol acyltransferase 1;ACAT2: 乙酰輔酶a膽固醇酰基轉(zhuǎn)移酶 2 Acetoacetyl-CoA thiolase 2;ABCA1:ATP結(jié)合盒轉(zhuǎn)運(yùn)蛋白 1 ATP-binding cassette transporters 1;Casepase9:含半胱氨酸的天冬氨酸蛋白水解酶9 Cysteinyl aspartate specific proteinase 9;Bcl-2: B細(xì)胞淋巴瘤2 B-cell lymphoma 2;Bax: B細(xì)胞淋巴瘤2相關(guān)X蛋白 Bcl-2-associated X protein.

從-80 ℃冰箱取出瘤胃組織，稱取100 mg左右，按1∶10(w∶v)加入冰浴的RIPA總蛋白裂解液；用勻漿器勻漿組織冰上靜置10 min后，12000 r/min離心，4 ℃，20 min，取上清。用BCA試劑盒測(cè)定蛋白濃度，將蛋白統(tǒng)一用蛋白裂解液稀釋至合適的濃度，變性，-80 ℃冰箱儲(chǔ)存。上樣量60 μg，分離膠10%，濃縮膠4%，電泳，轉(zhuǎn)印至硝酸纖維膜上，之后室溫封閉2 h，接著一抗4 ℃孵育過(guò)夜，1×TBST洗膜5 min 6次，再用二抗室溫孵育2 h，1×TBST洗膜5 min 6次，最后，用pierce發(fā)光試劑盒和Versa DocTM imaging system成像系統(tǒng)，檢測(cè)蛋白條帶，并用Quantity One software軟件進(jìn)行數(shù)據(jù)統(tǒng)計(jì)分析。

1.3.4抗氧化能力的測(cè)定 所有瘤胃上皮組織抗氧化能力測(cè)定試劑盒均購(gòu)自南京建成生物工程研究所,包括總抗氧化能力(total anti-oxidative capacity, T-AOC)測(cè)定試劑盒(貨號(hào)：A015)；超氧化物歧化酶(superoxide dismutase, SOD)測(cè)定試劑盒(貨號(hào)：A001-3)；過(guò)氧化氫酶(catalase, CAT)測(cè)定試劑盒(貨號(hào)：A007-1)。

1.3.5總膽固醇的測(cè)定 瘤胃上皮組織總膽固醇的測(cè)定采用組織細(xì)胞甘油三酯酶法測(cè)定試劑盒(貨號(hào)：E1013)，購(gòu)自北京普利萊基因技術(shù)有限公司。

1.4數(shù)據(jù)統(tǒng)計(jì)與分析

采用SPSS 17.0軟件對(duì)試驗(yàn)數(shù)據(jù)進(jìn)行ANOVA方差分析，結(jié)果用平均值±標(biāo)準(zhǔn)誤(mean±SEM)表示，Plt;0.05表示差異顯著，Plt;0.01表示差異極顯著。

2 結(jié)果與分析

2.1飼喂高精料日糧對(duì)山羊瘤胃上皮細(xì)胞周期與功能狀態(tài)的影響

如圖1所示，與對(duì)照組相比，HL和HS組山羊瘤胃上皮組織細(xì)胞增殖相關(guān)基因CDK2和CDK4 mRNA表達(dá)顯著升高(Plt;0.05)(圖1A)，HS組ERK1/2蛋白磷酸化水平顯著升高(Plt;0.05)，但各組間GPR41和GPR43蛋白表達(dá)無(wú)顯著變化(圖1B)。

圖1 高精料日糧對(duì)瘤胃上皮細(xì)胞周期相關(guān)基因與蛋白表達(dá)的影響 Fig.1 Effects of high concentrate diets on cell cycle of ruminal epithelium A: 細(xì)胞周期相關(guān)基因mRNA of the cell cycle; B: 增殖相關(guān)蛋白Protein of the proliferation。LC：低精料對(duì)照組Low concentrate diet control group; HL：高精料長(zhǎng)期組Long-term feeding high concentrate diet group; HS：高精料短期組Short-term feeding high concentrate diet group。不同小寫字母表示差異顯著(Plt;0.05)，相同或無(wú)字母表示差異不顯著(Pgt;0.05)，#表示有趨勢(shì)(0.05lt;Plt;0.1)。下同。Different small letters mean significant difference (Plt;0.05), and the same or no letter mean no significant difference (Pgt;0.05)，# means tendency (0.05lt;Plt;0.1). The same below.

2.2飼喂高精料日糧對(duì)山羊瘤胃上皮細(xì)胞凋亡和抗氧化能力的影響

由圖2可知，與對(duì)照組相比，HL和HS組山羊瘤胃上皮組織細(xì)胞凋亡相關(guān)基因Caspase9 mRNA表達(dá)水平顯著升高(Plt;0.05)，HS組Bcl-2/Bax基因表達(dá)比例呈下降趨勢(shì)(P=0.08)(圖2A);HS組瘤胃上皮組織中抗氧化酶CAT和總抗氧化能力T-AOC顯著升高(Plt;0.05)，HL組CAT含量和SOD酶活性顯著升高(Plt;0.05)，但總抗氧化能力T-AOC無(wú)顯著差異(Pgt;0.05)(圖2B～D)。與HL組相比，HS組山羊瘤胃上皮組織中總抗氧化能力T-AOC顯著升高(Plt;0.05)(圖2D)。

2.3飼喂高精料日糧對(duì)山羊瘤胃上皮組織膽固醇代謝的影響

由圖3可知，與對(duì)照組相比，HS組山羊瘤胃上皮組織中膽固醇含量顯著下降(Plt;0.05)(圖3A)，HL組膽固醇含量呈下降趨勢(shì)(P=0.06),與HL組相比，HS組山羊瘤胃上皮組織中膽固醇含量顯著下降(Plt;0.05)(圖3A)；HL和HS組瘤胃上皮組織中膽固醇代謝相關(guān)基因ACAT1和ACAT2 mRNA表達(dá)顯著升高(Plt;0.05)(圖3B)；且HS組瘤胃上皮組織中膽固醇的含量與ACAT1和ACAT2 mRNA的表達(dá)呈顯著的負(fù)強(qiáng)相關(guān)(P=0.0067,r=-0.8208;P=0.0100,r=-0.7980)(圖3C)。

圖2 高精料日糧對(duì)山羊瘤胃上皮組織細(xì)胞凋亡和抗氧化能力的影響 Fig.2 Effects of high concentrate diets on cell apoptosis and antioxidant ability of ruminal epithelium

圖3 高精料日糧對(duì)山羊瘤胃上皮組織中膽固醇代謝的影響Fig.3 Effects of high concentrate diets on the cholesterol metabolism in ruminal epithelium

2.4飼喂高精料日糧對(duì)山羊瘤胃上皮組織VFA 轉(zhuǎn)運(yùn)相關(guān)基因和蛋白表達(dá)的影響

如圖4所示，與對(duì)照LC組相比，HL和HS組山羊瘤胃上皮組織中VFA轉(zhuǎn)運(yùn)相關(guān)基因AE2、DRA、NHE2、NHE3和νH+ATPasemRNA表達(dá)水平均顯著升高(Plt;0.05)(圖4A)，但NHE2蛋白表達(dá)無(wú)顯著差異(圖4B)。

3 討論

3.1飼喂高精料日糧對(duì)山羊瘤胃上皮細(xì)胞周期與功能狀態(tài)的影響

瘤胃上皮細(xì)胞數(shù)量增多是通過(guò)加快細(xì)胞的有絲分裂來(lái)完成的，細(xì)胞從一次有絲分裂結(jié)束開(kāi)始到下一次有絲分裂完成所經(jīng)歷的整個(gè)有序過(guò)程稱為細(xì)胞周期。細(xì)胞周期受兩個(gè)蛋白家族的調(diào)控：細(xì)胞周期蛋白(Cyclin)家族和細(xì)胞周期蛋白依賴性激酶(cyclin-dependent kinase，CDK)家族。Cyclin D/CDK4/6和Cyclin E/CDK2復(fù)合物調(diào)控細(xì)胞從G1進(jìn)入S期[19]。有研究表明，山羊日糧精料含量從10%提高到35%時(shí)，瘤胃上皮中CDK和Cyclin基因mRNA的表達(dá)水平顯著升高[18]。同樣，本實(shí)驗(yàn)結(jié)果顯示，飼喂高精料日糧顯著上調(diào)瘤胃上皮組織中CDK2和CDK4 mRNA的表達(dá)。GPR41和GPR43是VFAs特異性受體，其中丙酸和丁酸與GPR41和GPR43結(jié)合的能力強(qiáng)于乙酸。GPR41可以通過(guò)組蛋白乙?；饔谜{(diào)節(jié)細(xì)胞的增殖、凋亡以及細(xì)胞周期，丙酸可以激活ERK通路參與調(diào)節(jié)細(xì)胞活力[20]。本研究結(jié)果顯示，65%精料飼喂泌乳期奶山羊4或19周，瘤胃液中乙酸、丙酸、丁酸和VFA的總量沒(méi)有發(fā)生顯著變化[21]，且瘤胃上皮組織的VFAs特異性受體GPR41和GPR43未被激活。HS組磷酸化ERK1/2蛋白表達(dá)水平顯著升高，提示短期飼喂高精料日糧可促進(jìn)瘤胃上皮組織細(xì)胞的增殖，但其增殖可能不是G蛋白偶聯(lián)通路介導(dǎo)的。

機(jī)體都具有清除體內(nèi)生物活性物質(zhì)的抗氧化防御系統(tǒng)[22]，機(jī)體防御系統(tǒng)的抗氧化能力的強(qiáng)弱與健康程度存在著密切聯(lián)系。季節(jié)和日糧均可影響山羊瘤胃組織的抗氧化能力[23]。血液和組織中SOD、GSH-PX(谷胱甘肽過(guò)氧化物酶，glutathione peroxidase)、CAT、MDA和T-AOC是反映機(jī)體氧化/抗氧化狀態(tài)的重要參數(shù)?？寡趸瘎┠苡行д{(diào)控機(jī)體的氧化應(yīng)激與細(xì)胞凋亡，保障機(jī)體細(xì)胞內(nèi)外環(huán)境的穩(wěn)定和生理機(jī)能正常[24]。而在細(xì)胞凋亡過(guò)程中，Bcl-2是細(xì)胞凋亡的負(fù)調(diào)控因子，可保護(hù)細(xì)胞免于凋亡；Bax是促凋亡因子，Bcl-2/Bax比值可反映細(xì)胞凋亡狀態(tài)。本研究結(jié)果表明，短期飼喂高精料日糧可提高山羊瘤胃上皮組織的抗氧化能力，同時(shí)伴隨細(xì)胞凋亡的增加，提示更高的細(xì)胞更新速率，這與其高泌乳性能相一致。然而，長(zhǎng)期飼喂高精料對(duì)瘤胃上皮組織的細(xì)胞凋亡和總抗氧化能力無(wú)顯著影響，其原因可能與機(jī)體應(yīng)激狀態(tài)的改變有關(guān)。高精料因其快速發(fā)酵引起瘤胃內(nèi)VFAs累積，導(dǎo)致SARA的發(fā)生。從而推測(cè)，SARA長(zhǎng)期存在威脅機(jī)體健康，并最終降低泌乳反芻動(dòng)物的生產(chǎn)性能。

3.2飼喂高精料日糧對(duì)山羊瘤胃上皮組織膽固醇代謝的影響

膽固醇是細(xì)胞膜的重要組成成分，且是多種激素的合成前體物，絕大多數(shù)組織能合成膽固醇。正常情況下，組織中膽固醇的合成受到嚴(yán)格的調(diào)控，從而使膽固醇的含量不致過(guò)多蓄積或缺乏。反芻動(dòng)物瘤胃上皮組織利用瘤胃發(fā)酵產(chǎn)生的VFAs，合成膽固醇從而減少瘤胃VFA的蓄積。β-羥-β-甲基戊二酸單酰輔酶α還原酶(3-hydroxy-3-methyl-glutaryl-CoA reductase，HMGCR)是膽固醇合成的限速酶。低密度脂蛋白受體(low density lipoprotein receptor，LDLR)將膽固醇轉(zhuǎn)運(yùn)入細(xì)胞進(jìn)行生物轉(zhuǎn)化。ATP結(jié)合盒轉(zhuǎn)運(yùn)蛋白(ATP-binding cassette transporters，ABCA1)調(diào)節(jié)細(xì)胞內(nèi)的膽固醇流出細(xì)胞膜[25]。膽固醇?；D(zhuǎn)移酶(Acetyl-CoA cholesterol acyltransferase，ACAT)是細(xì)胞內(nèi)已知的唯一一個(gè)催化游離膽固醇與長(zhǎng)鏈脂肪酸連接形成膽固醇酯的酶[26-27]。干奶期奶牛采食高精料日糧后，瘤胃上皮組織中膽固醇代謝相關(guān)的基因表達(dá)呈動(dòng)態(tài)變化[28]。本研究結(jié)果表明,與LC組相比，HL和HS組瘤胃上皮組織中ACAT1和ACAT2基因表達(dá)顯著上調(diào)，組織中膽固醇含量顯著下降。瘤胃組織內(nèi)膽固醇含量的降低提示對(duì)其功能的潛在不利影響。

3.3飼喂高精料日糧對(duì)山羊瘤胃上皮組織VFA轉(zhuǎn)運(yùn)的影響

瘤胃上皮吸收VFA為動(dòng)物機(jī)體提供能量，同時(shí)伴有質(zhì)子的吸收保證了瘤胃內(nèi)環(huán)境和pH的穩(wěn)態(tài)。瘤胃上皮組織中NHE在Na+的吸收和維持瘤胃上皮細(xì)胞內(nèi)的pH起著重要的作用[29]。DRA，PAT1和AE2的主要功能是將碳酸氫鹽從上皮細(xì)胞運(yùn)出同時(shí)將游離的VFA轉(zhuǎn)運(yùn)進(jìn)入瘤胃上皮，在VFA吸收過(guò)程中起到中和酸的作用。飼喂一定比例的精料日糧既可增強(qiáng)瘤胃的發(fā)酵功能又可以增加VFA的吸收。高精料在瘤胃內(nèi)發(fā)酵產(chǎn)生大量的VFA，瘤胃上皮對(duì)VFA的吸收增加。為了避免瘤胃細(xì)胞內(nèi)因質(zhì)子過(guò)量積累而引起pH下降，NHE3的表達(dá)水平會(huì)升高，從而加快瘤胃上皮細(xì)胞的Na+和H+交換，將細(xì)胞內(nèi)過(guò)多的H+排出細(xì)胞[30]。本實(shí)驗(yàn)結(jié)果與此相一致，HL和HS組維持山羊瘤胃上皮細(xì)胞pH相關(guān)基因(NHE2,NHE3和νH+ATPase)的mRNA表達(dá)水平顯著升高，且與VFA轉(zhuǎn)運(yùn)相關(guān)基因(DRA,AE2)的mRNA表達(dá)水平顯著升高。結(jié)果提示，飼喂高精料日糧可以加快泌乳期奶山羊瘤胃上皮組織對(duì)VFA的轉(zhuǎn)運(yùn)，但與飼喂時(shí)間長(zhǎng)短無(wú)關(guān)。

4 結(jié)論

短期飼喂高精料日糧可加快泌乳期奶山羊瘤胃上皮組織的更新，提高組織氧化應(yīng)激能力；而長(zhǎng)期飼喂高精料日糧未引起細(xì)胞增殖相關(guān)蛋白和總抗氧化能力的顯著變化；飼喂高精料日糧可以加快瘤胃上皮組織對(duì)VFA的轉(zhuǎn)運(yùn)。此外，飼喂高精料日糧引起瘤胃上皮組織內(nèi)膽固醇含量降低，膽固醇?；D(zhuǎn)移酶基因表達(dá)顯著上調(diào)，提示其對(duì)瘤胃上皮組織更新的潛在影響。

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Effectsoffeedinghighconcentratedietstolactatingdairygoatsonoxidativestressandcholesterolmetabolisminruminalepithelium

GENG Ya-Li, TIAN Ping, LUO Yan-Wen, HUA Can-Feng, TAO Shi-Yu, TIAN Jing, NI Ying-Dong*

Key Laboratory of Animal Physiology and Biochemistry, Ministry of Agriculture, Nanjing Agriculture University, Nanjing 210095, China

In order to investigate the effects of feeding high concentrate diets on the functional status of ruminal epithelia in terms of oxidative stress and cholesterol metabolism, seventeen lactating goats with similar body weight and milk production were randomly divided into three groups, including LC (n=5), HS (n=5) and HL (n=7) groups, which were fed by diets of concentrate∶forage of 35∶65 for whole experiment period (LC), concentrate∶forage of 65∶35 for 4 weeks (HS), and concentrate∶forage of 65∶35 for 19 weeks (HL), respectively. At the end of the experiment, ruminal epithelial tissues were sampled, promptly immersed in liquid nitrogen and then stored at -80 ℃ for analysis. The results showed that mRNA expression of theCDK2 andCDK4 genes related to cell cycle significantly increased in both HS and HL goats (Plt;0.05). The level of p-ERK1/2 proteins increased in HS but not in HL goats (Plt;0.05). However, GPR41 and 43 protein expression in the ruminal epithelium was not changed both in HS and HL compared to LC. The expression of theCasepase9 gene participating in cell apoptosis was significantly up-regulated in the HL and HS groups, while the anti-apoptotic index of the relative ratio ofBcl-2/BaxmRNA expression showed a tendency to decrease in the HS group compared to LC group (0.05lt;Plt; 0.1). In addition, the enzyme activity of SOD and CAT in the epithelium significantly increased in HL and HS goats compared to LC goats (Plt;0.05), whereas the total anti-oxidative capacity (T-AOC) increased only in HS goats (Plt;0.05). The level of cholesterol in ruminal epithelium significantly decreased in the HS group (Plt;0.01), and also showed a decreasing trend in the HL group (P=0.06). The enzymesACAT1 and 2, which may be involved in the regulation of cholesterol metabolism, significantly increased in HL and HS goats compared to LC goats (Plt;0.05). Correlation analysis revealed that cholesterol concentration in the ruminal epithelium was negatively correlated withACAT1 andACAT2 mRNA expression in the HS group (P=0.0067,r=-0.8208;P=0.0100,r=-0.7980). Compared with the LC group, mRNA expressions ofAE2,DRA,NHE2,NHE3andνH+ATPasegenes involving VFAs transportation significantly increased (Plt;0.05), while NHE2 protein expression was not altered both in HS and HL groups. Compared with the HL group, the HS group’s T-AOC significantly increased (Plt;0.05) and the level of cholesterol significantly decreased (Plt;0.05). These results indicate that feeding high concentrate diets over a short time period can improve the health status and functions of ruminal epithelium, while feeding high concentrate diets over a longer period might lessen these beneficial effects.

high concentrate diet; ruminal epithelium; oxidative stress; cholesterol; goat

10.11686/cyxb2017160http//cyxb.lzu.edu.cn

耿雅麗, 田平, 羅燕文, 華燦楓, 陶詩(shī)煜, 田靖, 倪迎冬. 高精料對(duì)泌乳奶山羊瘤胃上皮氧化應(yīng)激和膽固醇代謝的影響. 草業(yè)學(xué)報(bào), 2017, 26(11): 94-103.

GENG Ya-Li, TIAN Ping, LUO Yan-Wen, HUA Can-Feng, TAO Shi-Yu, TIAN Jing, NI Ying-Dong. Effects of feeding high concentrate diets to lactating dairy goats on oxidative stress and cholesterol metabolism in ruminal epithelium. Acta Prataculturae Sinica, 2017, 26(11): 94-103.

2017-03-31；改回日期：2017-06-14

國(guó)家重大基礎(chǔ)研究發(fā)展計(jì)劃 973 項(xiàng)目(No. 2011CB100802)資助。

耿雅麗(1992-)，女，河北衡水人，在讀碩士。E-mail:2604413221@qq.com

*通信作者Corresponding author. E-mail:niyingdong@njau.edu.cn