黃志堅(jiān)，羅 剛，陳騰騰，江和基，曾新斌

(福建農(nóng)林大學(xué)動(dòng)物科學(xué)學(xué)院 福建省動(dòng)物藥物工程實(shí)驗(yàn)室，福州 350002)

牡蠣粗多糖對(duì)脂多糖刺激仔豬NF-κB信號(hào)通路相關(guān)基因轉(zhuǎn)錄的影響

黃志堅(jiān)，羅 剛，陳騰騰，江和基，曾新斌

(福建農(nóng)林大學(xué)動(dòng)物科學(xué)學(xué)院 福建省動(dòng)物藥物工程實(shí)驗(yàn)室，福州 350002)

旨在初步探討牡蠣粗多糖對(duì)免疫應(yīng)激仔豬NF-κB信號(hào)通路相關(guān)基因的影響。選取30頭28日齡左右的杜長(zhǎng)大三元閹公仔豬，分為5個(gè)處理組，即空白對(duì)照組(Ⅰ)、免疫應(yīng)激對(duì)照組(Ⅱ)以及牡蠣粗多糖低(Ⅲ)、中(Ⅳ)、高(Ⅴ)劑量組，每組6頭豬。Ⅰ、Ⅱ組飼喂基礎(chǔ)日糧，Ⅲ、Ⅳ、Ⅴ組分別飼喂含0.5%、0.8%、1.2%牡蠣粗多糖的基礎(chǔ)日糧。試驗(yàn)飼養(yǎng)30 d后，Ⅱ、Ⅲ、Ⅳ、Ⅴ組腹腔注射100 μg·kg-1LPS，Ⅰ組注射等量的生理鹽水。各組于注射后3 h剖檢取肝、脾、腎上腺、淋巴結(jié)和胸腺等組織，檢測(cè)TLR-4、p300和p50的相對(duì)轉(zhuǎn)錄水平。結(jié)果：(1)與Ⅰ組相比，Ⅱ組p300相對(duì)轉(zhuǎn)錄水平在淋巴結(jié)中極顯著性降低，其他指標(biāo)在各器官中極顯著升高(P<0.01)。Ⅲ組TLR-4在淋巴結(jié)中，p50在腎上腺中和p300在肝、腎上腺及淋巴結(jié)中的相對(duì)轉(zhuǎn)錄水平均極顯著降低(P<0.01)；p50在肝、胸腺和脾中，p300在胸腺和脾中相對(duì)轉(zhuǎn)錄水平極顯著升高(P<0.01)。Ⅳ組TLR-4在淋巴結(jié)和脾中，p300在肝、腎上腺和淋巴結(jié)中相對(duì)轉(zhuǎn)錄水平顯著降低(P<0.05或P<0.01)；TLR-4在肝、腎上腺和胸腺中，p50在肝和胸腺中相對(duì)轉(zhuǎn)錄水平均顯著升高(P<0.05或P<0.01)。Ⅴ組TLR-4在脾，p300在肝、淋巴結(jié)、胸腺和腎上腺中相對(duì)轉(zhuǎn)錄水平顯著降低(P<0.05或P<0.01)；TLR4在肝、腎上腺和淋巴結(jié)中，p50在肝、胸腺和腎上腺相對(duì)轉(zhuǎn)錄水平顯著升高(P<0.05或P<0.01)。(2)與Ⅱ組相比，除Ⅴ組TLR-4在淋巴結(jié)中的差異不顯著外，Ⅲ、Ⅳ、Ⅴ組TLR-4的相對(duì)轉(zhuǎn)錄水平在各器官中均顯著降低(P<0.05或P<0.01)；p50的相對(duì)轉(zhuǎn)錄水平在腎上腺、淋巴結(jié)和脾中極顯著降低(P<0.01)，Ⅲ組p50的相對(duì)轉(zhuǎn)錄水平在肝中顯著降低(P<0.01)，在胸腺中極顯著升高(P<0.01)，Ⅴ組p50的相對(duì)轉(zhuǎn)錄水平在胸腺中差異極顯著降低(P<0.01)；p300的相對(duì)轉(zhuǎn)錄水平在肝、腎上腺、胸腺和脾中極顯著降低(P<0.01)；在淋巴結(jié)中，Ⅳ組p300的相對(duì)轉(zhuǎn)錄水平極顯著升高(P<0.01)。牡蠣粗多糖可以緩解免疫應(yīng)激仔豬器官中NF-κB信號(hào)通路相關(guān)基因的轉(zhuǎn)錄水平。

免疫應(yīng)激；牡蠣粗多糖；p50；p300；TLR-4

動(dòng)物免疫應(yīng)激是指病原微生物、疫苗或異源蛋白質(zhì)等抗原通過刺激動(dòng)物機(jī)體，激活免疫系統(tǒng)，使后者產(chǎn)生了免疫應(yīng)答，并導(dǎo)致動(dòng)物群體中少數(shù)動(dòng)物采食量下降，生長(zhǎng)緩慢的一種普遍現(xiàn)象。目前，主要通過腹腔或靜脈注射脂多糖，制造模型來研究免疫應(yīng)激[1-5]。石君霞等[6]研究表明，注射脂多糖，誘導(dǎo)免疫應(yīng)激，可以使PPAR-γ表達(dá)量升高，前期試驗(yàn)也表明，牡蠣粗多糖能夠緩解脂多糖引起的免疫應(yīng)激，具有抗應(yīng)激的作用，且其作用與PPAR-γ有關(guān)[7]。PPAR-γ在細(xì)胞上的受體是TLR-4(Toll-like receptors 4)，PPAR-γ與TLR-4結(jié)合能激活NF-κB信號(hào)通路[8]。作者在前期試驗(yàn)基礎(chǔ)上，研究牡蠣粗多糖抗免疫應(yīng)激功能與信號(hào)通路NF-κB的關(guān)系。

1 材料與方法

1.1 動(dòng)物分組與設(shè)計(jì)

選取30頭日齡在(28±1)d的斷奶的閹杜長(zhǎng)大三元公仔豬，平均體重在(9.91±1.38)kg，按體重相近的原則，隨機(jī)分為空白對(duì)照組，應(yīng)激對(duì)照組，牡蠣粗多糖高、中、低劑量組，每組6個(gè)重復(fù)，每個(gè)重復(fù)1頭豬?？瞻讓?duì)照組和免疫應(yīng)激對(duì)照組飼喂基礎(chǔ)日糧(表1)，牡蠣粗多糖高(Ⅴ)、中(Ⅳ)、低(Ⅲ)劑量組分別飼喂含有1.2%、0.8%、0.5%牡蠣粗多糖的基礎(chǔ)日糧，飼喂周期為30 d。試驗(yàn)前12 h斷水?dāng)嗔希囼?yàn)各組腹腔注射100 μg·kg-1BW LPS，空白對(duì)照組注射等量生理鹽水。于注射3 h后剖檢，取胸腺、腸道淋巴結(jié)、腎上腺、脾、肝測(cè)定TLR-4、NF-κB 和p300的相對(duì)轉(zhuǎn)錄水平。

表1 斷奶仔豬基礎(chǔ)日糧配方

Table 1 Weaned basal diet recipes

項(xiàng)目Item810教槽料810Creepfeed811乳豬料811Sucklingpigfeed原料Ingredient一級(jí)玉米/%Corn40.449.8面粉/%Farina99液體飼料油/%Liquidfeedoil3.82豆粕/%Soybeanmeal9.221.3發(fā)酵豆粕/%Fermentedsoybeanmeal6.55進(jìn)口蒸汽魚粉/%Inletsteamfishmeal(CP65)4.23低灰分血漿蛋白粉/%Lowashplasmaproteinpowder40乳清粉/%Wheypowder110葡萄糖/%Glucose22蔗糖/%Sucrose22教槽預(yù)混料/%Creeppremix80乳豬預(yù)混料/%Sucklingpigpremix06營(yíng)養(yǎng)水平Nutrientlevels粗蛋白質(zhì)/%Crudeprotein19.5819.52粗纖維/%Crudefibre2.042.82粗脂肪/%Crudefat6.044.72鈣/%Calcium0.780.75總磷/%Totalphoaphonium0.50.54有效磷/%Availablephosphorus0.40.41干物質(zhì)/%Drymatter87.3687.53消化能/(kJ·kg-1)Digestiveenergy14759.5214268.82賴氨酸/%Lysine1.51.33錳/(mg·kg-1)Manganese45.7445.33鋅/(mg·kg-1)Zn2168.972133.39銅/(mg·kg-1)Cu134.17133.6鐵/(mg·kg-1)Fe302.24295.51硒/(mg·kg-1)Se0.50.47鈷/(mg·kg-1)Co0.310.31維生素A/萬IUVA10192.210165.63維生素D3/萬IUVD31009.511006.88維生素E/(mg·kg-1)VE76.7875.04

1.2 材料

LPS(大腸桿菌血清型O55：B5，Sigma公司)；Trizol，Prime Script?RT reagent Kit Perfect Real Time(TaKaRa Biotechnology Co.，Ltd)；熒光試劑盒(北京百泰克生物技術(shù)有限公司)；瓊脂糖，牡蠣粗多糖(作者實(shí)驗(yàn)室制備[9])。

1.3TLR-4、p300和p50基因熒光定量PCR檢測(cè)引物

根據(jù)GenBank中收錄號(hào)為NM_001113039.1的野豬TLR-4基因序列、收錄號(hào)為KC316024.1的野豬NF-κBp50亞基基因序列和收錄號(hào)為XM_001929213.2的野豬p300亞基基因序列，利用Primer5.0 引物設(shè)計(jì)軟件各設(shè)計(jì)引物一對(duì)，并由上海生工生物工程技術(shù)服務(wù)有限公司合成。序列見表2。

表2 熒光定量PCR檢測(cè)引物序列

Table 2 Primer pairs for Fluorescence quantitative PCR detection

檢測(cè)基因Gene引物Primer引物序列Primersequence片段大小/bpSizeTLR-4SenseprimersAnti-senseprimers5'-ACAGAGCCGATGGTGTATCTTT-3'5'-AGCAGGGACTTCTCCAACTTCT-3'121NF-κBp50SenseprimersAnti-senseprimers5'-GGTTATTGTTCAGTTGGTCACA-3'5'-GTCATTCGTGCTTCCAGTGTT-3'192p300SenseprimersAnti-senseprimers5'-CTTCCCAGCCTCAAACTACAAT-3'5'-GCATCTTTCTTCCACACTCTGT-3'108

1.4 熒光定量PCR反應(yīng)

采用Bioteke Power 2×SYBR Real-time PCR Premixture 熒光定量試劑盒，用CFX-96 型Real-time PCR擴(kuò)增儀對(duì)反轉(zhuǎn)錄產(chǎn)物進(jìn)行擴(kuò)增。TLR-4、p50和p300的相對(duì)轉(zhuǎn)錄水平用2-ΔΔCt計(jì)算，用SPSS軟件進(jìn)行分析。

2 結(jié) 果

2.1 不同器官TLR-4的相對(duì)轉(zhuǎn)錄水平

圖1顯示，與空白對(duì)照組相比，應(yīng)激對(duì)照組TLR-4 mRNA的相對(duì)轉(zhuǎn)錄水平在肝、腎上腺、淋巴結(jié)、胸腺和脾都極顯著性升高(P<0.01)；牡蠣粗多糖低劑量組淋巴結(jié)中TLR-4的相對(duì)轉(zhuǎn)錄水平極顯著降低(P<0.01)，而在肝、腎上腺、胸腺和脾TLR-4 mRNA的相對(duì)轉(zhuǎn)錄水平差異不顯著(P>0.05)；牡蠣粗多糖中劑量組TLR-4 mRNA的相對(duì)轉(zhuǎn)錄水平在肝顯著升高(P<0.05)，在腎上腺和胸腺極顯著升高(P<0.01)，在淋巴結(jié)中顯著性降低(P<0.05)，在脾極顯著降低(P<0.01)；牡蠣粗多糖高劑量組TLR-4 mRNA的相對(duì)轉(zhuǎn)錄水平在肝、腎上腺和淋巴結(jié)中極顯著升高(P<0.01)，在脾極顯著降低(P<0.01)，在胸腺差異不顯著(P>0.05)。與應(yīng)激對(duì)照組相比，除了牡蠣粗多糖高劑量組TLR-4 mRNA的相對(duì)轉(zhuǎn)錄水平在淋巴結(jié)差異不顯著(P>0.05)，牡蠣粗多糖組TLR-4 mRNA的相對(duì)轉(zhuǎn)錄水平在各器官均極顯著降低(P<0.01)，牡蠣粗多糖高劑量組在肝顯著降低(P<0.05)。由此可得出，牡蠣粗多糖對(duì)LPS刺激導(dǎo)致各組織TLR-4 mRNA相對(duì)轉(zhuǎn)錄水平顯著性升高有一定的緩解作用，且添加劑量在0.5%左右效果更佳。

Ⅰ.空白對(duì)照組；Ⅱ.應(yīng)激對(duì)照組；Ⅲ.牡蠣粗多糖低劑量組；Ⅳ.牡蠣粗多糖中劑量組；Ⅴ.牡蠣粗多糖高劑量組。下圖同Ⅰ.Control；Ⅱ.Stress control；Ⅲ.Low-dose group；Ⅳ.Medial-dose group；Ⅴ.High-dose group.The same as below圖1 TLR-4在免疫器官中 mRNA的相對(duì)轉(zhuǎn)錄水平Fig.1 mRNA expression of TLR-4 in immune organs

2.2 不同器官NF-κBp50的相對(duì)轉(zhuǎn)錄水平

從圖2中可知，與空白對(duì)照組相比，應(yīng)激對(duì)照組p50 mRNA的相對(duì)轉(zhuǎn)錄水平在肝、腎上腺、淋巴結(jié)、胸腺和脾都極顯著升高(P<0.01)；牡蠣粗多糖低劑量組肝、胸腺和脾p50 mRNA的相對(duì)轉(zhuǎn)錄水平極顯著升高(P<0.01)，在腎上腺極顯著降低(P<0.01)，在淋巴結(jié)中差異不顯著(P>0.05)；牡蠣粗多糖中劑量組肝和胸腺中p50 mRNA的相對(duì)轉(zhuǎn)錄水平極顯著升高(P<0.01)，在腎上腺、淋巴結(jié)和脾中差異不顯著(P>0.05)；牡蠣粗多糖高劑量組p50 mRNA的相對(duì)轉(zhuǎn)錄水平在肝和胸腺極顯著升高(P<0.01)，在腎上腺顯著性升高(P<0.05)，而在淋巴結(jié)和脾差異不顯著(P>0.05)。與應(yīng)激對(duì)照組相比，牡蠣粗多糖組p50 mRNA的相對(duì)轉(zhuǎn)錄水平在腎上腺、淋巴結(jié)和脾極顯著降低(P<0.01)，低、中劑量添加組p50 mRNA的相對(duì)轉(zhuǎn)錄水平在肝顯極著降低(P<0.01)，在胸腺極顯著升高(P<0.01)，高劑量組p50 mRNA的相對(duì)轉(zhuǎn)錄水平在肝差異不顯著(P>0.05)，在胸腺差異極顯著性降低。由圖2可知，牡蠣粗多糖對(duì)LPS刺激導(dǎo)致肝、腎上腺、淋巴結(jié)和脾p50 mRNA相對(duì)轉(zhuǎn)錄水平顯著性升高有一定的緩解作用，且添加劑量在0.8%左右效果更佳，而對(duì)胸腺中p50 mRNA相對(duì)轉(zhuǎn)錄水平顯著性升高無緩解作用。

圖2 p50在免疫器官中 mRNA的相對(duì)轉(zhuǎn)錄水平Fig.2 mRNA expression of p50 in immune organs

2.3 不同器官p300的相對(duì)轉(zhuǎn)錄水平

從圖3可知，與空白對(duì)照組相比，應(yīng)激對(duì)照組p300 mRNA的相對(duì)轉(zhuǎn)錄水平在腎上腺、肝、胸腺和脾極顯著升高(P<0.01)，在淋巴結(jié)極顯著降低(P<0.01)；牡蠣粗多糖低劑量組p300 mRNA的相對(duì)轉(zhuǎn)錄水平在肝、腎上腺和淋巴結(jié)極顯著降低(P<0.01)，在胸腺中顯著升高(P<0.05)，在脾極顯著升高(P<0.01)；牡蠣粗多糖中劑量組p300 mRNA的相對(duì)轉(zhuǎn)錄水平在肝、腎上腺和淋巴結(jié)極顯著降低(P<0.01)，在胸腺和脾差異不顯著(P>0.05)；牡蠣粗多糖高劑量組p300 mRNA的相對(duì)轉(zhuǎn)錄水平在肝、淋巴結(jié)和胸腺極顯著降低(P<0.01)，在腎上腺顯著降低(P<0.05)，在脾差異不顯著(P>0.05)。與應(yīng)激對(duì)照組相比，牡蠣粗多糖添加組p300 mRNA的相對(duì)轉(zhuǎn)錄水平在肝、腎上腺、胸腺和脾極顯著降低(P<0.01)；在淋巴結(jié)，牡蠣粗多糖低、高劑量組p300 mRNA的相對(duì)轉(zhuǎn)錄水平差異不顯著(P>0.05)，中劑量組極顯著升高(P<0.01)。由此可知，牡蠣粗多糖對(duì)LPS刺激導(dǎo)致肝、腎上腺、胸腺和脾p300 mRNA相對(duì)轉(zhuǎn)錄水平顯著升高及淋巴結(jié)中p300 mRNA相對(duì)轉(zhuǎn)錄水平顯著降低有一定的緩解作用，且添加劑量在0.8%左右效果更佳。

圖3 p300在免疫器官中 mRNA的相對(duì)轉(zhuǎn)錄水平Fig.3 mRNA expression of p300 in immune organs

3 討 論

3.1 不同器官TLR-4的相對(duì)轉(zhuǎn)錄水平

肝是機(jī)體內(nèi)先天性免疫的淋巴器官，被譽(yù)為“消化道與其他組織器官的過濾器”[10]，同時(shí)也是機(jī)體內(nèi)重要的解毒器官，故可以通過檢測(cè)肝的一些指標(biāo)，判斷動(dòng)物機(jī)體的免疫應(yīng)答狀態(tài)。近年來，用LPS刺激動(dòng)物機(jī)體，經(jīng)TLR4信號(hào)途徑造成肝損傷模型，已廣泛應(yīng)用于基礎(chǔ)性研究[11-14]。H.An等[15]研究表明脂多糖刺激未成熟的樹突狀細(xì)胞，可以導(dǎo)致TLR-4 mRNA顯著上升；任大賓等[16]利用RT-PCR和Western blot研究了LPS注射前后肝內(nèi)CD14和TLR-4的表達(dá)量，結(jié)果表明注射LPS后肝內(nèi)TLR-4的表達(dá)量顯著提高；萬幸等[17]研究也表明LPS刺激后能夠顯著提高肺、肝和脾中TLR-4的相對(duì)轉(zhuǎn)錄水平。本試驗(yàn)表明，LPS刺激后TLR-4 mRNA在肝、腎上腺、淋巴結(jié)、胸腺和脾中的相對(duì)轉(zhuǎn)錄水平極顯著升高，提示LPS腹腔注射斷奶仔豬，激活機(jī)體內(nèi)的免疫系統(tǒng)的機(jī)制與TLR4信號(hào)途徑相關(guān)，與上述研究結(jié)果基本一致，也進(jìn)一步證實(shí)了劉玉蘭等[18]的研究結(jié)果，即LPS刺激后在腎上腺、脾和胸腺中TLR-4 mRNA相對(duì)轉(zhuǎn)錄水平顯著升高。

多糖的免疫調(diào)節(jié)作用與TLR4介導(dǎo)的信號(hào)通路有關(guān)，如靈芝多糖通過TLR4/TLR2介導(dǎo)的信號(hào)通路，引起鼠B細(xì)胞產(chǎn)生抗體[19]；紅花多糖通過TLR4激活NF-κB信號(hào)通透，誘導(dǎo)巨噬細(xì)胞分泌細(xì)胞因子[8]，豬苓多糖通過TLR4的介導(dǎo)能激活巨噬細(xì)胞[20]。研究表明，牡蠣多糖具有免疫調(diào)節(jié)作用，但對(duì)其免疫調(diào)節(jié)機(jī)制的研究比較少。本研究結(jié)果表明，牡蠣粗多糖添加組TLR-4 mRNA相對(duì)轉(zhuǎn)錄水平在各器官中極顯著低于應(yīng)激對(duì)照組，說明牡蠣粗多糖可以緩解由LPS刺激引起的TLR-4 mRNA升高，一定程度上說明牡蠣粗多糖緩解免疫應(yīng)激的機(jī)制，即一方面通過降低PPAR-γ的表達(dá)，從而減少炎性細(xì)胞因子的釋放來緩解免疫應(yīng)激，另一方面通過降低TLR-4的表達(dá)，進(jìn)而減少細(xì)菌或LPS侵襲細(xì)胞所必須的受體，降低細(xì)菌或LPS的入侵。

3.2 不同器官NF-κBp50亞基的相對(duì)轉(zhuǎn)錄水平

R.Sen等首先發(fā)現(xiàn)核因子κB(nuclear factor-kappa B，NF-κB)是一種能與免疫球蛋白κ鏈增強(qiáng)子上特異性序列結(jié)合，轉(zhuǎn)錄免疫球蛋白上的輕鏈基因的核蛋白因子[21]。它廣泛的存在于真核細(xì)胞中，是由兩個(gè)來自Rel家族的亞基組成的二聚體蛋白。Rel家族成員在N末端有300個(gè)相同的氨基酸序列，稱為Rel同源區(qū)，而NF-κB活性形式主要是由p50和p65兩個(gè)亞基組成的異源二聚體。動(dòng)物機(jī)體在正常情況下，NF-κB與IκB結(jié)合，存在于細(xì)胞質(zhì)中，其信號(hào)通路被抑制。當(dāng)動(dòng)物機(jī)體受到病毒、細(xì)菌感染或者外界刺激時(shí)，會(huì)激活細(xì)胞表面相應(yīng)的受體，例如Toll樣受體，導(dǎo)致NF-κB信號(hào)通路上游激酶IκK磷酸化，磷酸化的IκK進(jìn)一步將IκB磷酸化而降解，致使NF-κB與IκB分離后DNA結(jié)合位點(diǎn)暴露，轉(zhuǎn)位入核以后與靶基因上啟動(dòng)子或增強(qiáng)子結(jié)合，激活相應(yīng)基因的轉(zhuǎn)錄表達(dá)，這些基因中包括IL-1β、IL-6和TNF-α。前面研究顯示，LPS腹腔刺激后血清中炎性細(xì)胞因子水平顯著性升高，可能與NF-κB信號(hào)通路激活有關(guān)，為證實(shí)NF-κB信號(hào)通路是否激活，研究各組織中p50基因的mRNA表達(dá)水平有一定意義。

國(guó)內(nèi)外對(duì)NF-κB在各器官內(nèi)的表達(dá)水平的研究相對(duì)較少。研究表明[22-23]，脂多糖刺激可以導(dǎo)致肝和免疫器官中NF-κB表達(dá)量顯著性升高。湯夏冰等[24]研究也表明，LPS刺激喉癌細(xì)胞可以導(dǎo)致NF-κB蛋白表達(dá)量顯著提高。王建春等[25]在脂多糖刺激的急性肺損傷模型的研究中發(fā)現(xiàn)肺組織細(xì)胞中NF-κB的表達(dá)量顯著升高，且在刺激前后由細(xì)胞質(zhì)轉(zhuǎn)移到細(xì)胞核中而活化。本試驗(yàn)研究表明，注射脂多糖后斷奶仔豬肝、腎上腺、淋巴結(jié)、胸腺和脾中p50 mRNA相對(duì)轉(zhuǎn)錄水平顯著性升高，與以上研究結(jié)果基本相似。劉玉蘭[18]研究也顯示，脂多糖刺激斷奶仔豬，可以導(dǎo)致腎上腺、脾和胸腺中NF-κBp65 mRNA的相對(duì)轉(zhuǎn)錄水平顯著性升高，說明脂多糖腹腔注射可以激活NF-κB的信號(hào)通路。

研究表明，黃芪多糖能夠緩解創(chuàng)傷應(yīng)激引起的脾和胸腺淋巴細(xì)胞中NF-κB mRNA的過高表達(dá)[26]，本研究也表明，牡蠣粗多糖添加組脾p50 mRNA相對(duì)轉(zhuǎn)錄水平顯著性低于應(yīng)激對(duì)照組，胸腺p50 mRNA相對(duì)轉(zhuǎn)錄水平在低、中劑量顯著性高于應(yīng)激對(duì)照組，在高劑量組顯著性低于應(yīng)激對(duì)照組，說明添加量在1.2%左右時(shí)對(duì)胸腺有緩解作用，與上述研究結(jié)果基本一致。試驗(yàn)結(jié)果還表明，在腎上腺和淋巴結(jié)，牡蠣粗多糖添加組p50 mRNA相對(duì)轉(zhuǎn)錄水平極顯著性低于應(yīng)激對(duì)照組，肝中牡蠣粗多糖低、中劑量組顯著性低于應(yīng)激對(duì)照組，高劑量與應(yīng)激對(duì)照組差異不顯著，說明牡蠣粗多糖的添加劑量在0.5%～0.8%對(duì)腎上腺、淋巴結(jié)和肝的緩解效果較好。

3.3 不同器官p300的相對(duì)轉(zhuǎn)錄水平

染色質(zhì)是由核小體和DNA凝集在一塊形成的，而核小體是由組蛋白八聚體和連接組蛋白的H1或H5組成。基因在轉(zhuǎn)錄前，RNA聚合酶無法結(jié)合到染色質(zhì)上去進(jìn)行轉(zhuǎn)錄，需要借助轉(zhuǎn)錄共激活因子(如p300)與轉(zhuǎn)錄因子結(jié)合，通過對(duì)核小體上的組蛋白進(jìn)行修飾，使染色質(zhì)發(fā)生重組，基因調(diào)控區(qū)域和內(nèi)部核小體結(jié)構(gòu)發(fā)生改變，RNA聚合酶才容易與基因相應(yīng)部位結(jié)合，進(jìn)行轉(zhuǎn)錄[27]。

脂多糖刺激斷奶仔豬，與細(xì)胞膜上的受體TLR4結(jié)合，激活NF-κB通路，致使NF-κB在核內(nèi)與轉(zhuǎn)錄共激活因子p300結(jié)合，啟動(dòng)炎性細(xì)胞因子基因的轉(zhuǎn)錄表達(dá)。通過測(cè)量應(yīng)激仔豬各器官中p300 mRNA相對(duì)轉(zhuǎn)錄水平，可以一定程度上反映仔豬炎性因子的表達(dá)水平。國(guó)內(nèi)外研究表明，癌癥病人細(xì)胞中p300的表達(dá)量顯著上升[28-31]，LPS刺激可以導(dǎo)致單核細(xì)胞p300的表達(dá)[32]，細(xì)菌或病毒感染的動(dòng)物，體內(nèi)炎性細(xì)胞因子顯著提高[33-34]，本試驗(yàn)前期研究也表明，LPS刺激可以導(dǎo)致血清中IL-1β、IL-6和TNF-α的水平顯著性提高，說明LPS腹腔注射可以導(dǎo)致p300基因的廣泛表達(dá)，協(xié)助炎性因子的轉(zhuǎn)錄表達(dá)。本試驗(yàn)結(jié)果表明，LPS刺激后，肝、腎上腺、胸腺和脾p300 mRNA的相對(duì)轉(zhuǎn)錄水平顯著上升，牡蠣粗多糖添加組比應(yīng)激組顯著性降低，表示牡蠣粗多糖對(duì)LPS刺激引起的p300 mRNA表達(dá)量的上升有緩解作用，且中劑量組的緩解作用更好。而淋巴結(jié)中p300 mRNA 相對(duì)轉(zhuǎn)錄水平在應(yīng)激后顯著降低，原因可能與PPAR-γ的降低有關(guān)，具體原因有待進(jìn)一步研究。牡蠣粗多糖中劑量組淋巴結(jié)中p300 mRNA的相對(duì)轉(zhuǎn)錄水平顯著高于應(yīng)激對(duì)照組，說明對(duì)LPS刺激導(dǎo)致淋巴結(jié)中p300 mRNA的相對(duì)轉(zhuǎn)錄水平的降低有一定緩解作用。

4 結(jié) 論

牡蠣粗多糖可以緩解免疫應(yīng)激仔豬免疫器官中TLR-4、NF-κBp50 和p300的相對(duì)轉(zhuǎn)錄水平，且其緩解機(jī)制可能與NF-κB信號(hào)通路有關(guān)。

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(編輯 白永平)

Explore Mechanism of Oyster Crude Polysaccharide Alleviated Immune Stress on Weanling Piglets

HUANG Zhi-jian，LUO Gang，CHEN Teng-teng，JIANG He-ji，ZENG Xin-bin

(CollegeofAnimalScience，F(xiàn)ujianAgricultureandForestryUniversity/EngineeringLaboratoryofAnimalPharmaceuticalsofFujianProvince，F(xiàn)uzhou350002，China)

This study was designed to explore the impact of Oyster Polysaccharides on immune stress piglets of NF-κB signaling pathway-related genes A total of thirty Duroc × Landrace × Yorkshire castrated piglets of 28±1 d were randomly allocated into five groups，namely Blank control (Ⅰ)，Immunological stress control (Ⅱ) and Oyster polysaccharide Low (Ⅲ)，Medium (Ⅳ) and High (Ⅴ) dose group，with six replicates per group according to the principle of similar weight.Piglets were fed basal diet (Control) or 0.5%，0.8%，1.2% OPS (OPS Low，Medium and High dose group) for 30 days.The piglets were injected i.p with a dose ofEscherichiacoliLPS (100 μg·kg-1BW) except the Blank control which were injected with the same dose of normal saline.Three hours later，the liver，spleen，adrenal gland，lymph nodes and thymus were collected for detecting the relative transcription level ofTLR-4，p50 andp300.Results were as follows：(1) Compared with the GroupⅠ，p300 relative transcript levels of GroupⅡ were significantly decreased in Lymph Node，while the other indicators were significantly increased in various organs (P<0.01).The relative transcript levels ofTLR-4 in lymph nodes，p50 in adrenal gland andp300 in liver，adrenal gland and lymph nodes were significantly decreased (P<0.01) while the relative transcript levels ofp50 in adrenal gland andp300 in liver，adrenal gland and lymph node were significantly increased (P<0.01) in Group Ⅲ than GroupⅠ.The relative transcript levels ofTLR-4 in lymph nodes and spleen，p300 in liver，adrenal gland and lymph nodes were significantly decreased (P<0.05 orP<0.01) while the relative transcript levels ofTLR-4 in liver，adrenal gland and thymus andp50 in liver，thymus were significantly increased (P<0.05 orP<0.01) in Group Ⅳ than GroupⅠ.The relative transcript levels ofTLR-4 in spleen，p300 in liver，lymph nodes，thymus and adrenal gland were significantly decreased (P<0.05 orP<0.01) while the relative transcript levels ofTLR-4 in liver，adrenal gland and lymph nodes andp50 in liver，thymus and adrenal gland were significantly increased in Group Ⅴ than Group Ⅰ(P<0.05 orP<0.01).(2)Compared with the Group Ⅱ，the relative transcription ofTLR-4 levels were significantly decreased in these organs of Group Ⅲ，Ⅳ and Ⅴ (P<0.05 orP<0.01)other than in lymph node of Group Ⅴ (P>0.05).The relative transcription ofp50 levels were significantly decreased in adrenal glands，lymph glands and spleen of Group Ⅲ，Ⅳ，and Ⅴ(P<0.01)；and were significantly decreased in liver and significantly increased in thymus of Group Ⅲ(P<0.01)；and that were significantly increased in thymus of Group Ⅴ (P<0.01).The relative transcription ofp300 mRNA levels were significantly decreased in liver，adrenal glands，thymus and spleen of Group Ⅲ，Ⅳ，and Ⅴ(P<0.01)；and that were significantly increased in lymph nodes of Group Ⅳ (P<0.01).Oyster polysaccharides can relieve stress piglets transcription of NF-κB signaling pathway-related genes.

immunological stress；OPS；p50；p300；TLR-4

10.11843/j.issn.0366-6964.2015.06.021

2014-06-09

福建省科技廳農(nóng)業(yè)科技重點(diǎn)項(xiàng)目(2011N0001)

黃志堅(jiān)(1963-)，男，福建惠安人，教授，主要從事動(dòng)物疾病防治與保健研究，E-mail：huangzj1999@sina.com

S852.4

A

0366-6964(2015)06-1037-10