促皮質(zhì)激素釋放激素對魚類攝食的抑制作用

齊錦雯 吳源冰 王書瑤 唐 妮 李志瓊

(四川農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院，成都 611130)

魚類通過攝食為自身機(jī)體提供營養(yǎng)和能量，促進(jìn)生長發(fā)育[1]。攝食調(diào)控復(fù)雜且精細(xì)，各種外周食欲相關(guān)物理或化學(xué)信號通過神經(jīng)系統(tǒng)或體液傳遞途徑產(chǎn)生饑餓或飽腹信號到達(dá)中樞神經(jīng)系統(tǒng)調(diào)節(jié)攝食[2]。促皮質(zhì)激素釋放激素(corticotropin releasing factor，CRF)作為中樞調(diào)控的飽腹感信號因子(厭食欲因子)，其成熟肽含有41個(gè)氨基酸，可激活下丘腦-垂體-腎上腺(hypothalamus-hypophysis-interrenal，HPI)軸，引起垂體釋放促腎上腺皮質(zhì)激素(adrenocorticotropic hormone，ATCH)，影響動(dòng)物的攝食行為[3-4]。CRF已成為動(dòng)物攝食和能量代謝領(lǐng)域的研究熱點(diǎn)之一，目前在哺乳動(dòng)物上關(guān)于CRF的攝食調(diào)控有大量的研究報(bào)道，而魚類上較少。因此本文依據(jù)CRF在哺乳動(dòng)物和部分魚類中的研究現(xiàn)狀，闡述了CRF的發(fā)現(xiàn)歷史、分子結(jié)構(gòu)、對魚類攝食的調(diào)控作用及其機(jī)制，重點(diǎn)綜述了CRF作為外源多肽通過不同方式注射或作為內(nèi)源調(diào)節(jié)子通過不同方式處理對魚類攝食的影響，以及CRF與受體、皮質(zhì)醇和其他食欲因子之間的互作。這將為今后魚類攝食調(diào)控和生長的研究以及生產(chǎn)提供理論依據(jù)。

1 CRF的發(fā)現(xiàn)歷史和結(jié)構(gòu)

1955年Saffran等[5]體外培養(yǎng)大鼠垂體，通過下丘腦提取液刺激顯示ACTH的釋放增加，故此命名為CRF。直至1981年Vale等[4]從綿羊下丘腦處提取純化出含41個(gè)氨基酸殘基的CRF多肽。在此基礎(chǔ)上，1983年Furutani等[6]進(jìn)一步驗(yàn)證了CRF基因的結(jié)構(gòu)。CRF主要位于下丘腦處，可刺激垂體分泌ACTH。Vandenborne等[7]依據(jù)哺乳動(dòng)物和魚類的報(bào)道以及在家雞上的鑒定，總結(jié)了CRF基因結(jié)構(gòu)，它含2個(gè)外顯子和1個(gè)內(nèi)含子，外顯子1含5′非編碼區(qū)，外顯子2包含編碼區(qū)和3′非編碼區(qū)(圖1)。魚類上CRF基因cDNA長為0.9～1.0 kbp，編碼區(qū)一般為0.4～0.5 kbp。CRF編碼區(qū)編碼的氨基酸結(jié)構(gòu)包括了疏水信號肽，中間1段未證明功能的保守肽，以及C端含41個(gè)氨基酸殘基的成熟肽，激活C末端的2個(gè)位點(diǎn)(酰胺基團(tuán))可使CRF具有活性。在白亞口魚(Catostomuscommersonii)[8]、金魚(Carassiusauratus)[9]、紅大馬哈魚(Oncorhynchusnerka)[10]、虹鱒(Oncorhynchusmykiss)[11]和鯉魚(Cyprinuscarpio)[12]上發(fā)現(xiàn)CRF基因含2個(gè)亞型(CRF1和CRF2)，而羅非魚(Oreochromismossambicus)[13]、比目魚(Platichthysflesus)[14]、斑馬魚(Barchydanioreriovar)[15]和齊口裂腹魚(Schizothoraxprenanti)[16]上CRF基因無亞型。

prohormone:激素原；Mature peptide:成熟肽；Exon 1:外顯子1；Exon 2:外顯子2；Intro:內(nèi)含子；5′URT:5′非編碼區(qū) 5′ noncoding region；3′URT:3′非編碼區(qū) 3′noncoding region；CDS:編碼區(qū)序列 coding sequence。

圖1CRF基因結(jié)構(gòu)

Fig.1 The structure ofCRFgene[7]

2 CRF調(diào)控魚類攝食

依據(jù)結(jié)構(gòu)和分布，CRF在魚類的食欲調(diào)控內(nèi)分泌系統(tǒng)中起著重要的調(diào)控作用。通過CRF作為外源多肽通過不同方式注射探討其對魚類攝食影響，或通過不同處理下魚類內(nèi)源CRF基因的表達(dá)水平和攝食的變化探究CRF的調(diào)控機(jī)制，結(jié)果顯示CRF可調(diào)控魚類攝食。

2.1 外源性CRF調(diào)控魚類攝食

2.1.1 中樞注射CRF調(diào)控魚類攝食

對哺乳動(dòng)物中樞(腦室)注射CRF探究其攝食量的變化有研究報(bào)道，結(jié)果均證明CRF通過中樞注射可抑制哺乳動(dòng)物攝食[17-18]。在魚類上，中樞注射CRF引起的攝食調(diào)控作用目前僅限金魚。De Pedro等[19]對金魚饑餓處理24 h后中樞注射CRF，注射后2 h攝食量減少，提示CRF可能通過中樞系統(tǒng)抑制金魚攝食。Berinier等[20]在金魚上證實(shí)CRF受體拮抗劑αh-CRF(9-41)預(yù)處理可使CRF注射效果逆轉(zhuǎn)。綜上，中樞注射CRF對金魚有抑制攝食的作用，而通過αh-CRF(9-41)預(yù)處理可使中樞CRF的作用效果逆轉(zhuǎn)。

2.1.2 外周注射CRF調(diào)控魚類攝食

現(xiàn)有少量研究表明在哺乳動(dòng)物和魚類上，外周注射CRF攝食量無顯著變化，但是有下降趨勢[19，21]。在魚類上，De Pedro等[19]對金魚腹腔注射CRF，攝食量呈下降趨勢，提示腹腔注射CRF可能影響金魚的攝食量。以上試驗(yàn)為只持續(xù)1 d的單次外周注射，無長期外周注射的報(bào)道，無法明確長期外周注射CRF對魚類攝食是否抑制。

雖然外周注射CRF對攝食調(diào)控的報(bào)道極少，但是外周CRF及CRF相關(guān)肽分布于嚙齒動(dòng)物的胃腸道系統(tǒng)中有報(bào)道，報(bào)道顯示CRF及CRF相關(guān)肽少量分布于外周組織中[22-23]。Taché等[24]認(rèn)為通過對嚙齒動(dòng)物的胃腸道直接或間接刺激，可使其外周CRF及CRF相關(guān)肽的水平上升，從而抑制嚙齒動(dòng)物攝食，提示嚙齒動(dòng)物上的外周CRF及CRF相關(guān)肽在應(yīng)激引起相關(guān)腸動(dòng)力改變過程中有重要的調(diào)控作用。在魚類上，Pepels等[25]檢測出應(yīng)激處理的尼羅羅非魚(Oreochromisniloticus)的血液中存在CRF，提示CRF可能也存在于魚類機(jī)體外周組織中。

2.2 內(nèi)源性CRF調(diào)控魚類攝食

2.2.1 不同攝食狀況對CRF的調(diào)控

在嚙齒動(dòng)物上，禁食后CRF基因表達(dá)水平降低[26]。而在魚類上目前報(bào)道僅限于金魚和齊口裂腹魚。Maruyama等[27]對金魚分3組試驗(yàn)7 d，結(jié)果顯示與正常投喂組相比，禁食組的腦CRF基因的表達(dá)水平呈下降趨勢，但差異不顯著，而過量投喂組則顯著上升。Wang等[16]對齊口裂腹魚短期(1 d)禁食處理，顯示下丘腦CRF基因表達(dá)水平無顯著變化，而長期(7 d)禁食后，下丘腦CRF基因表達(dá)水平顯著下降，復(fù)投喂第9天則回升。綜上，長期禁食可使魚類CRF作為厭食欲因子抑制魚類攝食。

2.2.2 環(huán)境因子對CRF的調(diào)控

影響魚類生長發(fā)育的所棲息的環(huán)境因子主要包括氧飽和度、氨氣含量和滲透性能。氧氣飽和程度越低、氨氣增多以及滲透壓上升可抑制魚類攝食，CRF基因表達(dá)水平上升，提示CRF在不同外界環(huán)境處理下可作為厭食欲因子調(diào)控魚類攝食。

短期缺氧下可使CRF在魚類上發(fā)揮厭食欲因子的作用，而長期缺氧下，CRF的厭食欲調(diào)節(jié)作用降低。Bernier等[28]使虹鱒處于50%或35%氧飽和度下24 h，結(jié)果顯示前腦CRF基因表達(dá)水平和攝食量均下降，并與缺氧處理程度呈正相關(guān)，進(jìn)一步在長期(72 h)缺氧處理后，前腦CRF基因表達(dá)水平下降幅度及其厭食欲調(diào)節(jié)作用呈降低的趨勢。

在氨氣處理中，盡管Wood[29]認(rèn)為低水平的外源性氨氣對魚類攝食無顯著影響，但是Ortega等[30]長期增加水中的氨氣可引起虹鱒攝食量呈劑量依賴性減少，CRF基因表達(dá)水平增加。Craig等[31]發(fā)現(xiàn)和淡水區(qū)域的相比，海水區(qū)域的虹鱒腦CRF表達(dá)水平的上升，攝食量下降，提示滲透壓上升CRF可作為厭食欲因子。

3 CRF調(diào)控魚類攝食的作用機(jī)制

CRF調(diào)控動(dòng)物包括魚類的攝食作用機(jī)制復(fù)雜，主要包括以下3種：1)CRF與受體結(jié)合發(fā)揮食欲調(diào)控作用；2)CRF通過HPI軸激活下游皮質(zhì)醇的釋放調(diào)控?cái)z食；3)CRF通過和其他食欲調(diào)節(jié)子結(jié)合直接或間接調(diào)控?cái)z食(圖2)。這3種機(jī)制之間也可相互作用共同影響CRF。

CRF:促皮質(zhì)激素釋放激素 corticoliberin；CRFR1:促皮質(zhì)激素釋放激素受體1 corticoliberin receptor 1；CRFR2:促皮質(zhì)激素釋放激素受體2 corticoliberin receptor；POMC：阿黑皮素原 proopiomelanocortin；CART:可卡因和安非他明調(diào)節(jié)轉(zhuǎn)錄子 Cocaine and amphetamine regulation transcripton；NPY：神經(jīng)肽Y neuropeptide Y；AgRP:刺鼠相關(guān)蛋白 agouti-related protein；α-MSH:α-促黑激素 α melanocyte stimulaing hormone；MC4R:黑皮素4受體 melanocortin 4 receptor；Ghrelin:饑餓素；Food intake:采食。實(shí)線箭頭表示促進(jìn)作用 solid arrow indicated promotion effect；虛線箭頭表示抑制作用 dashed arrow indicated inhibition effect。

圖2CRF調(diào)控?cái)z食機(jī)制

Fig.2 The mechanisms of CRF regulation on the feeding

3.1 CRF與受體結(jié)合抑制魚類攝食

哺乳動(dòng)物上，CRF可直接和CRF受體2(CRFR2)結(jié)合發(fā)揮食欲抑制作用，但是與CRF受體1(CRFR1)結(jié)合對攝食調(diào)控?zé)o影響。而魚類上，CRF系統(tǒng)可能存在不同通路調(diào)控魚類攝食。Vaughan等[32]報(bào)道CRF相關(guān)肽硬骨魚緊張肽Ⅰ(urotensin Ⅰ，UⅠ)和CRF均可與CRFR2親和發(fā)揮抑制攝食的作用，并且親和力度同等。Bernier等[33]認(rèn)為金魚上中樞注射UⅠ，和中樞注射CRF組相比，更有效地減少機(jī)體攝食量，提示了在魚類上CRF和UⅠ無選擇性親和CRFR2發(fā)揮厭食欲作用，或者CRF可能與CRFR1或CRFR2親和調(diào)節(jié)魚類攝食。

CRF與受體結(jié)合的區(qū)域主要位于下丘腦視前核(NPO)和結(jié)節(jié)外側(cè)核(NLT)[34]。而Arai等[35]在鯰魚(Silurusasotus)腦干中也發(fā)現(xiàn)CRFR1少量存在，提示中樞系統(tǒng)CRF可通過參與調(diào)節(jié)胃腸運(yùn)動(dòng)的腦干神經(jīng)元回路影響攝食。Cardoso等[36]在河豚(Fugurubripes)腸道發(fā)現(xiàn)微量CRFR1，Martínez等[37]對小鼠外周注射CRF，均證明胃腸道系統(tǒng)存在微量CRF受體，并與CRF結(jié)合刺激腸動(dòng)力變化調(diào)控機(jī)體攝食。

3.2 CRF激活HPI軸上的末端產(chǎn)物皮質(zhì)醇來調(diào)控魚類攝食

在HPI軸上，由下丘腦釋放的CRF刺激垂體釋放ACTH，進(jìn)一步激活腎上腺的糖皮質(zhì)激素皮質(zhì)醇直接調(diào)控魚類攝食，或通過和其他內(nèi)分泌調(diào)節(jié)途徑互作以及胃腸道營養(yǎng)吸收間接調(diào)節(jié)魚類攝食。

一些研究報(bào)道皮質(zhì)醇作為糖皮質(zhì)激素可抑制攝食。Gregory等[38]研究表明升高虹鱒血漿皮質(zhì)醇水平，可抑制其攝食量。而皮質(zhì)醇的增多可負(fù)反饋于CRF，使CRF引起的厭食欲的效果減小。進(jìn)一步有研究報(bào)道顯示，CRF可通過和其他內(nèi)分泌途徑互作調(diào)控皮質(zhì)醇對攝食的影響。Bernier等[39]在金魚上進(jìn)行適量且慢性增加血漿皮質(zhì)醇處理時(shí)，可使攝食量增加，前腦CRF基因表達(dá)水平降低，神經(jīng)肽Y(NPY)基因表達(dá)水平升高，進(jìn)一步增加皮質(zhì)醇濃度的分解可使CRF基因表達(dá)水平降低，而對NPY基因表達(dá)無影響，攝食量有下降趨勢。CRF引起糖皮質(zhì)激素皮質(zhì)醇的釋放，可間接通過腸道營養(yǎng)吸收來影響攝食。Ducouret等[40]研究表明，糖皮質(zhì)激素受體在魚類胃腸道中存在，Vllette等[41]發(fā)現(xiàn)皮質(zhì)醇對腸道鈉/鉀-ATP酶活性有提高作用。

3.3 CRF與其他食欲調(diào)節(jié)子結(jié)合調(diào)控魚類攝食

CRF與受體結(jié)合后可通過和中樞食欲調(diào)控神經(jīng)系統(tǒng)互作，或通過血液循環(huán)和外周食欲調(diào)節(jié)因子互作傳遞飽腹感信號。CRF與中樞食欲調(diào)控神經(jīng)系統(tǒng)調(diào)節(jié)因子包括下丘腦神經(jīng)元阿黑皮素原(POMC)/可卡因和安非他明調(diào)節(jié)轉(zhuǎn)錄子(CART)、NPY/刺鼠相關(guān)蛋白(AgRP)、POMC多肽加工處理的可調(diào)控動(dòng)物攝食的片段α-促黑激素(α-MSH)及黑皮素4受體(MC4R)相互作用調(diào)控魚類攝食。CRF與外周食欲調(diào)控因子包括胃腸道分泌的饑餓素(ghrelin)和胰腺分泌的胰島素三者之間互作調(diào)控魚類攝食。CRF也可與腦腸肽(apelin)互作調(diào)控魚類攝食。

3.3.1 CRF與POMC/CART或NPY/AgRP互作調(diào)控?cái)z食

在大鼠上通過體外和體內(nèi)試驗(yàn)說明通過CRF系統(tǒng)可上調(diào)POMC/CART參與厭食欲作用，并且POMC/CART通過CRF系統(tǒng)激活HPI軸[42]。而NPY/AgRP可下調(diào)CRF調(diào)節(jié)攝食，NPY/AgRP與CRF之間的互作也和糖皮質(zhì)激素緊密相關(guān)。Heinrichs等[43]對大鼠中樞共注射NPY和CRF受體拮抗劑αh-CRF(9-41)，注射部位為下丘腦室旁核區(qū)域，攝食量增加；隨后對通過糖皮質(zhì)激素地塞米松處理后的大鼠，中樞注射NPY，結(jié)果顯示室旁核的CRF基因表達(dá)下調(diào)，攝食量增加。在魚類上的報(bào)道還較缺乏，目前僅在金魚上有研究。在金魚上，Bernier等[39]緩慢適量增加血漿皮質(zhì)醇濃度，刺激NPY釋放，抑制CRF基因表達(dá)，攝食量增加。

3.3.2 CRF與α-MSH和MC4R互作調(diào)控?cái)z食

CRF作為整合厭食欲信號的神經(jīng)多肽，整合了POMC多肽加工處理的可調(diào)控動(dòng)物攝食的片段α-MSH及其受體MC4R調(diào)控?cái)z食[44]。CRF與黑皮素系統(tǒng)之間的互作在魚類上目前僅在金魚中有報(bào)道。Matsuda等[45]對金魚中樞共注射α-MSH興奮劑(MT Ⅱ)和αh-CRF(9-41)，結(jié)果顯示MT Ⅱ引起的食欲抑制效果減小。然而，共注射CRF和MC4R拮抗劑HS024，發(fā)現(xiàn)對金魚攝食量無顯著影響。進(jìn)一步通過免疫組化分析檢測出CRF和MSH神經(jīng)元位于大腦處，其中α-MSH包含的神經(jīng)纖維末梢和下丘腦區(qū)域的CRF神經(jīng)元緊密結(jié)合。綜上，提示了在魚類上，α-MSH和MC4R通過CRF信號通路發(fā)揮厭食欲作用。

3.3.3 CRF與ghrelin和胰島素互作調(diào)控?cái)z食

通過CRF信號途徑可使外周食欲調(diào)節(jié)因子ghrelin抑制食欲，又因ghrelin參與糖代謝，所以CRF、ghrelin和胰島素之間互作對動(dòng)物攝食產(chǎn)生影響。ghrelin和CRF共同作用可使胰島素敏感性降低。Solomon等[46]對大鼠預(yù)處理靜脈注射0.5 mL ghrelin特異性抗體和非特異性抗體，然后對皮下組織單次注射生理鹽水、胰島素和2-脫氧葡萄糖，通過免疫組化方法檢測出，胰島素和ghrelin抗體共處理組的CRF陽性神經(jīng)元比胰島素處理組的更高，并且與無特異性抗體預(yù)處理組之間進(jìn)行比較，胰島素處理組和2-脫氧葡萄糖處理組的CRFc-fos(1種即刻早期基因)陽性神經(jīng)元均顯著高于生理鹽水對照組，而攝食量與對照組相比卻顯著增加。在CRF厭食欲信號中通過胰島素處理出現(xiàn)反常的現(xiàn)象，說明低血液葡萄糖應(yīng)激可能導(dǎo)致HPI軸激活補(bǔ)償葡萄糖水平，刺激少量ACTH和皮質(zhì)醇的生成，少量皮質(zhì)醇可刺激動(dòng)物攝食。

在魚類上，J?nsson等[47]對虹鱒幼魚分4組進(jìn)行中樞注射，ghrelin組顯著降低攝食量，αh-CRF(9-41)組對攝食量無顯著影響，共注射組的攝食量和ghrelin組比較有升高的趨勢，并與對照組的攝食量相當(dāng)。此結(jié)果提示ghrelin通過CRF系統(tǒng)抑制魚類攝食。

3.3.4 CRF與apelin互作調(diào)控?cái)z食

CRF與apelin對攝食調(diào)控的互作有少量報(bào)道。apelin可刺激CRF的釋放，并通過CRF系統(tǒng)抑制哺乳動(dòng)物攝食[48-49]。Lv等[49]禁食處理雄性小鼠24 h，分別中樞注射0.3、1.0和3.0 μg/kg apelin-13，對照組注射生理鹽水，檢測出試驗(yàn)組攝食量和對照組的比較呈劑量依賴性抑制；進(jìn)一步對小鼠分成4組，與注射生理鹽水對照組相比，αh-CRF(9-41)處理組的4 h攝食量無顯著變化，apelin-13處理組的累積攝食量則極顯著減少，而apelin-13和αh-CRF(9-41)共注射處理組與對照組比較攝食量無顯著差異，與apelin-13處理組比較攝食量顯著增加。CRF與腦腸肽apelin之間的互作目前在魚類上還未見報(bào)道。

4 小 結(jié)

魚類通過攝食為自身機(jī)體提供營養(yǎng)和能量，促進(jìn)其生長發(fā)育和繁殖。攝食調(diào)控復(fù)雜且精細(xì)，通過中樞或者外周的厭食欲因子和增食欲因子互作調(diào)控機(jī)體攝食。CRF作為重要的中樞調(diào)控厭食欲因子，是動(dòng)物攝食和能量代謝領(lǐng)域的研究熱點(diǎn)之一。目前，CRF關(guān)于攝食調(diào)控和機(jī)制的研究主要集中在哺乳動(dòng)物上，而魚類上研究少。由于魚類所處環(huán)境差異達(dá)，生理結(jié)構(gòu)和機(jī)能差異較大，所以未來應(yīng)在借鑒哺乳動(dòng)物研究結(jié)果的基礎(chǔ)上，深入探討CRF對不同魚類的攝食調(diào)節(jié)機(jī)制，為魚類攝食調(diào)控和生產(chǎn)應(yīng)用提供理論依據(jù)。

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