羅舒文 胡璇 鄭銘 徐雪雪 劉雪明 仲向前 張鈺 陳國(guó)宏 徐琪
摘 要 為闡明禽類在高血糖情況下未表現(xiàn)出任何臨床癥狀的內(nèi)在原因,以雞、家兔為研究對(duì)象,利用ELISA、高效液相色譜、液相色譜質(zhì)譜檢測(cè)等技術(shù)對(duì)兩者葡萄糖調(diào)節(jié)生理差異進(jìn)行比較。結(jié)果顯示,ELISA檢測(cè)雞的糖化白蛋白顯著高于兔,而糖化血紅蛋白無顯著性差異;雞血清果糖胺含量與兔無顯著性差異; 雞血清終末糖基化產(chǎn)物含量極顯著低于兔;高效液相色譜檢測(cè)結(jié)果顯示,雞的?;撬岷亢陀坞x氨基酸含量顯著高于兔;液相色譜質(zhì)譜檢測(cè)結(jié)果顯示,雞的甲基乙二醛含量與家兔無顯著性差異。以上研究結(jié)果表明,雞無慢性高血糖所表現(xiàn)出的有害效應(yīng)的主要原因是?;撬岷筒糠钟坞x氨基酸作為羰基清除劑降低果糖胺及甲基乙二醛的含量,從而導(dǎo)致糖基化終末產(chǎn)物的降低。
關(guān)鍵詞 雞;兔;血糖調(diào)節(jié);糖基化終末產(chǎn)物;糖化蛋白;?;撬幔患谆叶?/p>
就糖代謝而言,禽類是脊椎動(dòng)物的一個(gè)獨(dú)特的類別。因其有著明顯的胰島素抵抗[1],且血糖濃度很高,為哺乳動(dòng)物的2倍左右[2-3]。這種血糖濃度長(zhǎng)期處于哺乳動(dòng)物的病理水平,但卻無明顯的糖尿病并發(fā)癥狀,是一種很好的研究糖尿病疾病的動(dòng)物模型[4]。導(dǎo)致糖尿病并發(fā)癥的主要因素之一是葡萄糖美拉德反應(yīng),美拉德反應(yīng)實(shí)際上是糖的羰基與氨基酸、磷脂和核酸等生物分子的胺之間的多重反應(yīng)。而糖與生物分子這兩種化合物是生命必須的,且必須在細(xì)胞和生物體中共存,因此美拉德反應(yīng)是不可避免的[5]。此外,由于美拉德過程的自發(fā)性、隨機(jī)性和不可控性,且反應(yīng)產(chǎn)物通常是有毒的,因此會(huì)顯著導(dǎo)致糖尿病并發(fā)癥和機(jī)體的衰老。簡(jiǎn)單來說,美拉德反應(yīng)會(huì)導(dǎo)致形成多種產(chǎn)物,稱為晚期糖基化終末產(chǎn)物(AGEs)。而AGEs對(duì)細(xì)胞和生物體功能有著致命傷害,是一類與葡萄糖毒性和糖尿病并發(fā)癥相關(guān)的異常葡萄糖代謝物[6]。例如,AGEs通過蛋白質(zhì)交聯(lián)、基質(zhì)成分修飾、血小板聚集、內(nèi)皮功能障礙增加[7]、血管松弛缺陷和脂蛋白代謝異常加速動(dòng)脈粥樣硬化[8];AGEs通過誘導(dǎo)氧化應(yīng)激和炎癥反應(yīng),導(dǎo)致腎損傷或纖維化[9],加速腎臟功能障礙[10]等。綜上所述,了解AGEs及其前體物的發(fā)生發(fā)展對(duì)于研究糖尿病并發(fā)癥的內(nèi)在機(jī)理有著重要作用。
Szwergold等[11]在前期研究發(fā)現(xiàn)禽類可作為II型糖尿病無病理模型的突破點(diǎn)在于低濃度的AGEs,這可能是由于禽類有著抗糖基化的特性,但并無數(shù)據(jù)支撐[12]。故本研究通過檢測(cè)并比較禽類(雞)與哺乳動(dòng)物(兔)之間的糖尿病并發(fā)癥相關(guān)指標(biāo),對(duì)雞和兔血糖調(diào)節(jié)生理差異進(jìn)行比較,以期探索禽類高血糖卻無臨床癥狀的內(nèi)在原因,為控制人類的糖尿病并發(fā)癥等問題提供新思路。
1 材料與方法
1.1 試驗(yàn)動(dòng)物與樣品采集
試驗(yàn)動(dòng)物分別選用來自宿遷養(yǎng)雞場(chǎng)、侯氏花鳥店的40 d體質(zhì)量在2.5 kg左右的雞(羅斯308)和150 d體質(zhì)量在3 kg左右的家兔(新西蘭兔),于揚(yáng)州大學(xué)文匯路校區(qū)的養(yǎng)雞房和養(yǎng)兔房進(jìn)行飼喂,試驗(yàn)時(shí)間為2021年3月20日至5月2日。雞與家兔分別重復(fù)6只,其中雞采用地面平養(yǎng),自由采食和飲水;兔采用層疊式籠養(yǎng),乳頭飲水器飲水,自由采食。肉雞與家兔飼料由公司提供,二者基礎(chǔ)飼料配方及營(yíng)養(yǎng)水平分別見表1和表2。試驗(yàn)動(dòng)物食入日糧中均無額外糖分添加。
兩者飼養(yǎng)至體質(zhì)量相近且均處正常生理狀態(tài)時(shí),對(duì)雞和家兔利用脫氫酶電化學(xué)法使用羅氏血糖儀(羅氏AccuChekperforma卓越精采型,美國(guó)羅氏公司)進(jìn)行空腹血糖(禁食8 h以上,不禁水)和餐后血糖(餐后2 h以內(nèi))水平檢測(cè);分別對(duì)雞進(jìn)行翅靜脈采血、家兔耳緣靜脈采血,血清采集步驟:采血于促凝管,靜置30 min,3 500 r/min離心10 min后分裝至1.5 mL? EP管, -80 ℃保存,備用;全血及血漿采血至含肝素鈉的抗凝管中,全血保存于-20 ℃,備用。血漿立即3 500? ?r/min離心10 min,隨后分裝至1.5 mL EP管, ?-80 ℃保存,備用。
1.2 糖化白蛋白、糖化血紅蛋白檢測(cè)
新鮮血液利用Elisa試劑盒(江蘇科特生物科技有限公司提供)分別測(cè)定糖化白蛋白、糖化血紅蛋白含量。
1.3 甲基乙二醛檢測(cè)
將分離的血漿樣品經(jīng)0.22 μm有機(jī)濾膜,然后用Agilent 1290-6470液相色譜質(zhì)譜儀測(cè)定甲基乙二醛含量。牛磺酸液相色譜質(zhì)譜條件如下:色譜柱,Agilent C18柱(2.1 mm×100 mm,1.8 μm);柱溫, ?35? ℃,流速,0.3 mL/min;采集模式,ESI+;母離子,195;子離子,127、115;進(jìn)樣量,2 μL;流動(dòng)相為A 0.1% 甲酸水溶液,B乙腈,流動(dòng)相梯度見表3。甲基乙二醛含量按公式計(jì)算:
W=【(C-C0)×V×N】/m
式中:W表示試樣中甲基乙二醛含量,單位為μg/L;C表示試樣測(cè)定液中甲基乙二醛的質(zhì)量濃度,單位μg/L;V表示定容體積,單位mL;N表示稀釋倍數(shù);m表示試樣的取樣量,單位為 mL。
1.4 果糖胺、糖基化終末產(chǎn)物檢測(cè)
采用低速離心分離血清,利用Elisa試劑盒(江蘇科特生物科技有限公司提供)分別測(cè)定果糖胺、糖基化終末產(chǎn)物含量。
1.5 牛磺酸、游離氨基酸檢測(cè)
將分離的血漿樣品經(jīng)0.45 μm微孔濾膜,然后用Agilent 1260液相色譜儀測(cè)定。?;撬岷?7 個(gè)游離氨基酸標(biāo)準(zhǔn)品分別由廣州佳途科技股份有限公司和上海源葉生物科技公司提供。?;撬嵋合嗌V條件如下:色譜柱,C18 SHISEIDO柱(4.6 mm×250 mm×5 μm);檢測(cè)器,F(xiàn)LD檢測(cè)器;柱溫,30? ℃;進(jìn)樣量,10 μL;波長(zhǎng),激發(fā)波長(zhǎng)330 nm,發(fā)射波長(zhǎng)530 nm;流動(dòng)相,A為乙酸鈉緩沖液,B為乙腈,流動(dòng)相A∶流動(dòng)相B=70∶30。
游離氨基酸液相色譜條件如下:色譜柱,C18 SHISEIDO柱(4.6 mm×250 mm×5 μm);柱溫,40? ℃;進(jìn)樣量,10 μL;波長(zhǎng),254 nm;流動(dòng)相,A為0.1 mol/L無水乙酸鈉+乙腈=97+3,混勻后調(diào)pH至6.5(31.815 g乙酸鈉+3 880 mL水+120 mL乙腈),B為乙腈+水=80+20;流動(dòng)相梯度見表4。
1.6 數(shù)據(jù)處理與分析
采用Excel 2019建立數(shù)據(jù)庫(kù),利用SPSS? ?25.0軟件對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析,采用單因素方差分析進(jìn)行顯著性檢驗(yàn),以P<0.05表示差異有統(tǒng)計(jì)學(xué)意義。用作圖軟件GraphPad Prism 8.0進(jìn)行制圖,數(shù)據(jù)以“平均值±標(biāo)準(zhǔn)差”的形式表示。
2 結(jié)果與分析
2.1 雞與家兔的血糖、糖化白蛋白、糖化血紅蛋白含量比較
對(duì)雞和家兔的血糖(BG)、糖化白蛋白(GA)和糖化血紅蛋白(HbA1c)進(jìn)行測(cè)定。結(jié)果顯示(圖1),雞和家兔的平均空腹血糖為11.283 mmol/L和5.617 mmol/L,平均餐后血糖為 ?11.983 mmol/L和7.267 mmol/L,雞的餐前餐后血糖無顯著變化,而家兔的血糖顯著增加(P< ?0.05);雞的餐前餐后血糖均顯著高于家兔(P<0.01)。糖化白蛋白測(cè)定結(jié)果顯示,雞GA的含量極顯著高于家兔(P<0.01)。而糖化血紅蛋白測(cè)定結(jié)果顯示,雞與家兔的HbA1c含量無顯著差異(P>0.05)。
2.2 雞與家兔的果糖胺、糖基化終末產(chǎn)物含量比較
通過ELISA法對(duì)雞與家兔的
從這4個(gè)指標(biāo)的檢測(cè)結(jié)果可以看出,雞的血糖在顯著高于兔的前提下,雞HbA1c的含量與家兔并無顯著差異,GA和FA的含量略高于家兔,但總AGEs的含量雞卻顯著低于家兔。說明雞的血糖濃度雖然很高,致使檢測(cè)短期糖基化的兩個(gè)蛋白指標(biāo)高于家兔,但長(zhǎng)期監(jiān)測(cè)的HbA1c指標(biāo)并無顯著差異。從另一角度而言,這意味著雞的高血糖濃度可能并不會(huì)導(dǎo)致蛋白長(zhǎng)期受糖基化的影響,這與Jessica等[29]的結(jié)果相一致。但糖化蛋白濃度究竟是如何隨著時(shí)間的增長(zhǎng)而降低的,其中原因不得而知,還需進(jìn)一步研究。
3.2 雞與家兔AGEs的含量與MG濃度差異密切相關(guān)
前人研究發(fā)現(xiàn)AGEs受體(RAGE)在AGEs的命運(yùn)中扮演著消極的角色。AGEs與其結(jié)合會(huì)激活機(jī)體的應(yīng)激反應(yīng),導(dǎo)致炎癥和細(xì)胞功能障礙[30],因此RAGE基因被認(rèn)為是糖尿病并發(fā)癥發(fā)生的重要因素之一[11]。近年來研究發(fā)現(xiàn)禽類缺少RAGE基因[31],這是解釋它們能成功應(yīng)對(duì)高血糖并發(fā)癥的能力之一。但從結(jié)果中可以看出,高血糖雞的AGEs比家兔正常血糖下的AGEs還要低許多,說明缺少RAGE基因并非關(guān)鍵因素,因此尋找致使雞血清低AGEs濃度的原因更為重要。
鑒于以上結(jié)果,本研究另外檢測(cè)了兩物種的甲基乙二醛含量。甲基乙二醛(MG)的產(chǎn)生與前面提到的蛋白糖基化相互關(guān)聯(lián),并相互影響:血糖濃度升高導(dǎo)致糖酵解生成MG[32];另一方面,葡萄糖通過互變異構(gòu)生成果糖,果糖分解代謝后形成α-氧醛,MG即為其中之一[18]。MG是生成AGEs的重要前體物質(zhì),MG及其兩個(gè)相鄰的羰基可以直接形成AGE[33]。Brownlee等[6]發(fā)現(xiàn)在培養(yǎng)牛主動(dòng)脈內(nèi)皮細(xì)胞時(shí)發(fā)現(xiàn),AGE含量的增加是由于甲基乙二醇產(chǎn)量的增加。此外據(jù)估計(jì),MG的反應(yīng)活性約是葡萄糖的20 000倍[34],且對(duì)細(xì)胞有著高度的毒性[35]。所以,動(dòng)物體內(nèi)MG的含量對(duì)糖尿病患者是否產(chǎn)生并發(fā)癥發(fā)揮著重要作用?;蛟S,雞的AGEs的低含量是由于MG含量本就不高導(dǎo)致。但通過檢測(cè)發(fā)現(xiàn),雞與家兔的MG并無顯著差異,可能是由于雞體內(nèi)有某個(gè)物質(zhì)或某些物質(zhì)在MG后續(xù)形成AGEs的過程中起了調(diào)節(jié)作用。
3.3 雞與家兔?;撬岷筒糠钟坞x氨基酸含量 ?比較
?;撬嵊?8世紀(jì)首次從膽汁中被分離出,是動(dòng)物機(jī)體內(nèi)的條件性必需氨基酸[36]。它含有磺酸鹽,但并無羧基,主要通過肝臟中的半胱氨酸的氧化及脫羧合成[37]。作為一種體內(nèi)的氧化劑,?;撬峥梢詫?duì)多種細(xì)胞功能進(jìn)行調(diào)節(jié),包括膽汁酸結(jié)合、防止由于氧化損傷引起的膜通透性變化等[38]。有研究報(bào)道,糖尿病患者的體內(nèi)?;撬崴匠掷m(xù)下降,而適當(dāng)補(bǔ)充?;撬嵩谝葝u素依賴、胰島素抵抗[39]及糖尿病并發(fā)癥方面有顯著效果[40]。而本研究發(fā)現(xiàn),對(duì)于體質(zhì)量相近的雞和家兔,雞的?;撬岷匡@著高于家兔。這或許是雞AGEs濃度低的關(guān)鍵:一方面,對(duì)于糖基化蛋白,例如糖化白蛋白過高的后果是紅細(xì)胞脆性增加,而盡管雞的糖化白蛋白含量較高,但有?;撬釋?duì)細(xì)胞的形態(tài)和功能進(jìn)行調(diào)節(jié),才導(dǎo)致雞盡管血糖高但并無明顯的病理現(xiàn)象;另一方面,牛磺酸較其他氨基酸而言具有與醛基較高的反應(yīng)活性[41],而醛基是AGEs合成所必須的,意味著牛磺酸可以直接抑制AGEs的形成。
此外已有研究表明,游離氨基酸除了有抗氧化的作用外,還可以作為羰基清除劑[42]。Siahbalaei等[43]研究發(fā)現(xiàn)游離氨基酸尤其是精氨酸、谷氨酸具有良好的抑制葡萄糖氧化的能力。但在機(jī)體的血漿糖基化含量較高的情況下,游離氨基酸含量會(huì)減少,其抗氧化活性亦會(huì)降低[44]。結(jié)果發(fā)現(xiàn)與家兔相比,雞血漿中作為活性羰基清除劑的?;撬岷推渌坞x氨基酸的濃度顯著更高 ?(P<0.01)。家兔與雞的甲基乙二醛差異并不顯著,而游離氨基酸含量的檢測(cè)結(jié)果可以解釋這一原因:可能是由于進(jìn)入血漿的MG會(huì)被精氨酸或其他清除劑中和。
4 結(jié) 論
本研究發(fā)現(xiàn)雞有慢性高血糖卻無任何臨床癥狀的原因可能是?;撬岷筒糠钟坞x氨基酸作為羰基清除劑降低了果糖胺及甲基乙二醛的含量,從而導(dǎo)致糖基化終末產(chǎn)物的降低,抑制了其對(duì)生物機(jī)體的損害。這為探明禽類高血糖耐受生理特性提供了新的科學(xué)數(shù)據(jù),也為人類防治Ⅱ型糖尿病及其并發(fā)癥提供了新思路。
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Comparison of Physiological Differences in Blood Glucose Regulation between Chickens and Rabbits
LUO Shuwen1, HU Xuan1, ZHENG Ming1, XU Xuexue1, LIU Xueming1, ZHONG Xiangqian2, ZHANG Yu1, CHEN Guohong1? and?? XU Qi1
Abstract The fasting blood glucose concentrations of birds are about twice of themammals, and the birds have significant insulin resistance, but there is no significant diabetic complications, the reasons for the absense are unclear.In order to elucidate the intrinsic reasons for no clinical signs of hyperglycaemia inthe poultry, this study was conducted to compare the physiological differences in glucose regulation between chickens and rabbits using ELISA, high performance liquid chromatography and liquid chromatography mass spectrometry detection techniques.The results showed that glycosylated albumin was higher in chickens than in rabbits, while glycosylated? haemoglobin was not significantly different; serum fructosamine levels inchickens were not significantly different from those in rabbits; and serum levels of advanced glycation end-products were significantly lower in chickens than in rabbits .The results of high performance liquid chromatography showed that the taurine content and free amino acid content of chickens were extremely significantly higher than those of rabbits; the results of liquid chromatography mass spectrometry showed that the methylglyoxal content of chickens was not significantly different from that of rabbits.In conclusion, the main reason forchickens to exhibite deleterious effects under the no chronic hyperglycaemiais that taurine and some free amino acids act as carbonyl scavengers to reduce the levels of fructosamine and methylglyoxal, and? decrease the advanced glycation end-products.
Key words Chicken; Rabbit; Glucose regulation; Glycosylation end products; Glycated proteins; Taurine; Methylglyoxal
Received2022-09-29Returned 2022-11-17
Foundation item Jiangsu Modern Agricultural (Broiler) Industry Technology System (No.JATS [2022]163).
First author LUO? Shuwen, female, doctoral student. Research area:chicken genetic breeding and? ?reproduction. E-mail:santifay@163.com
Corresponding?? author XU Qi, male, Ph.D, professor. Research area:poultry genetics and breeding. E-mail: xuqi@yzu.edu.cn
(責(zé)任編輯:顧玉蘭 Responsible editor:GU Yulan)
收稿日期:2022-09-29 修回日期:2022-11-17
基金項(xiàng)目:江蘇現(xiàn)代農(nóng)業(yè)(肉雞)產(chǎn)業(yè)技術(shù)體系(JATS[2022]163)。
第一作者:羅舒文,女,博士研究生,研究方向?yàn)殡u的遺傳育種與繁殖。E-mail:santifay@163.com
通信作者:徐 琪,男,博士,教授,研究方向?yàn)榧仪葸z傳育種。E-mail:xuqi@yzu.edu.cn