羅舒文 胡璇 鄭銘 徐雪雪 劉雪明 仲向前 張鈺 陳國宏 徐琪



摘 要 為闡明禽類在高血糖情況下未表現出任何臨床癥狀的內在原因,以雞、家兔為研究對象,利用ELISA、高效液相色譜、液相色譜質譜檢測等技術對兩者葡萄糖調節生理差異進行比較。結果顯示,ELISA檢測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物含量極顯著低于兔;高效液相色譜檢測結果顯示,雞的牛磺酸含量和游離氨基酸含量顯著高于兔;液相色譜質譜檢測結果顯示,雞的甲基乙二醛含量與家兔無顯著性差異。以上研究結果表明,雞無慢性高血糖所表現出的有害效應的主要原因是牛磺酸和部分游離氨基酸作為羰基清除劑降低果糖胺及甲基乙二醛的含量,從而導致糖基化終末產物的降低。
關鍵詞 雞;兔;血糖調節;糖基化終末產物;糖化蛋白;牛磺酸;甲基乙二醛
就糖代謝而言,禽類是脊椎動物的一個獨特的類別。因其有著明顯的胰島素抵抗[1],且血糖濃度很高,為哺乳動物的2倍左右[2-3]。這種血糖濃度長期處于哺乳動物的病理水平,但卻無明顯的糖尿病并發癥狀,是一種很好的研究糖尿病疾病的動物模型[4]。導致糖尿病并發癥的主要因素之一是葡萄糖美拉德反應,美拉德反應實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。而糖與生物分子這兩種化合物是生命必須的,且必須在細胞和生物體中共存,因此美拉德反應是不可避免的[5]。此外,由于美拉德過程的自發性、隨機性和不可控性,且反應產物通常是有毒的,因此會顯著導致糖尿病并發癥和機體的衰老。簡單來說,美拉德反應會導致形成多種產物,稱為晚期糖基化終末產物(AGEs)。而AGEs對細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癥相關的異常葡萄糖代謝物[6]。例如,AGEs通過蛋白質交聯、基質成分修飾、血小板聚集、內皮功能障礙增加[7]、血管松弛缺陷和脂蛋白代謝異常加速動脈粥樣硬化[8];AGEs通過誘導氧化應激和炎癥反應,導致腎損傷或纖維化[9],加速腎臟功能障礙[10]等。綜上所述,了解AGEs及其前體物的發生發展對于研究糖尿病并發癥的內在機理有著重要作用。
Szwergold等[11]在前期研究發現禽類可作為II型糖尿病無病理模型的突破點在于低濃度的AGEs,這可能是由于禽類有著抗糖基化的特性,但并無數據支撐[12]。故本研究通過檢測并比較禽類(雞)與哺乳動物(兔)之間的糖尿病并發癥相關指標,對雞和兔血糖調節生理差異進行比較,以期探索禽類高血糖卻無臨床癥狀的內在原因,為控制人類的糖尿病并發癥等問題提供新思路。
1 材料與方法
1.1 試驗動物與樣品采集
試驗動物分別選用來自宿遷養雞場、侯氏花鳥店的40 d體質量在2.5 kg左右的雞(羅斯308)和150 d體質量在3 kg左右的家兔(新西蘭兔),于揚州大學文匯路校區的養雞房和養兔房進行飼喂,試驗時間為2021年3月20日至5月2日。雞與家兔分別重復6只,其中雞采用地面平養,自由采食和飲水;兔采用層疊式籠養,乳頭飲水器飲水,自由采食。肉雞與家兔飼料由公司提供,二者基礎飼料配方及營養水平分別見表1和表2。試驗動物食入日糧中均無額外糖分添加。
兩者飼養至體質量相近且均處正常生理狀態時,對雞和家兔利用脫氫酶電化學法使用羅氏血糖儀(羅氏AccuChekperforma卓越精采型,美國羅氏公司)進行空腹血糖(禁食8 h以上,不禁水)和餐后血糖(餐后2 h以內)水平檢測;分別對雞進行翅靜脈采血、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
新鮮血液利用Elisa試劑盒(江蘇科特生物科技有限公司提供)分別測定糖化白蛋白、糖化血紅蛋白含量。
1.3 甲基乙二醛檢測
將分離的血漿樣品經0.22 μm有機濾膜,然后用Agilent 1290-6470液相色譜質譜儀測定甲基乙二醛含量。牛磺酸液相色譜質譜條件如下: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;進樣量,2 μL;流動相為A 0.1% 甲酸水溶液,B乙腈,流動相梯度見表3。甲基乙二醛含量按公式計算:
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表示試樣測定液中甲基乙二醛的質量濃度,單位μg/L;V表示定容體積,單位mL;N表示稀釋倍數;m表示試樣的取樣量,單位為 mL。
1.4 果糖胺、糖基化終末產物檢測
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)分別測定果糖胺、糖基化終末產物含量。
1.5 牛磺酸、游離氨基酸檢測
將分離的血漿樣品經0.45 μm微孔濾膜,然后用Agilent 1260液相色譜儀測定。牛磺酸和17 個游離氨基酸標準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。牛磺酸液相色譜條件如下:色譜柱,C18 SHISEIDO柱(4.6 mm×250 mm×5 μm);檢測器,FLD檢測器;柱溫,30? ℃;進樣量,10 μL;波長,激發波長330 nm,發射波長530 nm;流動相,A為乙酸鈉緩沖液,B為乙腈,流動相A∶流動相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? ℃;進樣量,10 μL;波長,254 nm;流動相,A為0.1 mol/L無水乙酸鈉+乙腈=97+3,混勻后調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;流動相梯度見表4。
1.6 數據處理與分析
采用Excel 2019建立數據庫,利用SPSS? ?25.0軟件對數據進行統計分析,采用單因素方差分析進行顯著性檢驗,以P<0.05表示差異有統計學意義。用作圖軟件GraphPad Prism 8.0進行制圖,數據以“平均值±標準差”的形式表示。
2 結果與分析
2.1 雞與家兔的血糖、糖化白蛋白、糖化血紅蛋白含量比較
對雞和家兔的血糖(BG)、糖化白蛋白(GA)和糖化血紅蛋白(HbA1c)進行測定。結果顯示(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)。糖化白蛋白測定結果顯示,雞GA的含量極顯著高于家兔(P<0.01)。而糖化血紅蛋白測定結果顯示,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、糖基化終末產物含量比較
通過ELISA法對雞與家兔的
從這4個指標的檢測結果可以看出,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,致使檢測短期糖基化的兩個蛋白指標高于家兔,但長期監測的HbA1c指標并無顯著差異。從另一角度而言,這意味著雞的高血糖濃度可能并不會導致蛋白長期受糖基化的影響,這與Jessica等[29]的結果相一致。但糖化蛋白濃度究竟是如何隨著時間的增長而降低的,其中原因不得而知,還需進一步研究。
3.2 雞與家兔AGEs的含量與MG濃度差異密切相關
前人研究發現AGEs受體(RAGE)在AGEs的命運中扮演著消極的角色。AGEs與其結合會激活機體的應激反應,導致炎癥和細胞功能障礙[30],因此RAGE基因被認為是糖尿病并發癥發生的重要因素之一[11]。近年來研究發現禽類缺少RAGE基因[31],這是解釋它們能成功應對高血糖并發癥的能力之一。但從結果中可以看出,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。
鑒于以上結果,本研究另外檢測了兩物種的甲基乙二醛含量。甲基乙二醛(MG)的產生與前面提到的蛋白糖基化相互關聯,并相互影響:血糖濃度升高導致糖酵解生成MG[32];另一方面,葡萄糖通過互變異構生成果糖,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,MG及其兩個相鄰的羰基可以直接形成AGE[33]。Brownlee等[6]發現在培養牛主動脈內皮細胞時發現,AGE含量的增加是由于甲基乙二醇產量的增加。此外據估計,MG的反應活性約是葡萄糖的20 000倍[34],且對細胞有著高度的毒性[35]。所以,動物體內MG的含量對糖尿病患者是否產生并發癥發揮著重要作用。或許,雞的AGEs的低含量是由于MG含量本就不高導致。但通過檢測發現,雞與家兔的MG并無顯著差異,可能是由于雞體內有某個物質或某些物質在MG后續形成AGEs的過程中起了調節作用。
3.3 雞與家兔牛磺酸和部分游離氨基酸含量 ?比較
牛磺酸于18世紀首次從膽汁中被分離出,是動物機體內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]。作為一種體內的氧化劑,牛磺酸可以對多種細胞功能進行調節,包括膽汁酸結合、防止由于氧化損傷引起的膜通透性變化等[38]。有研究報道,糖尿病患者的體內牛磺酸水平持續下降,而適當補充牛磺酸在胰島素依賴、胰島素抵抗[39]及糖尿病并發癥方面有顯著效果[40]。而本研究發現,對于體質量相近的雞和家兔,雞的牛磺酸含量顯著高于家兔。這或許是雞AGEs濃度低的關鍵:一方面,對于糖基化蛋白,例如糖化白蛋白過高的后果是紅細胞脆性增加,而盡管雞的糖化白蛋白含量較高,但有牛磺酸對細胞的形態和功能進行調節,才導致雞盡管血糖高但并無明顯的病理現象;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]研究發現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。但在機體的血漿糖基化含量較高的情況下,游離氨基酸含量會減少,其抗氧化活性亦會降低[44]。結果發現與家兔相比,雞血漿中作為活性羰基清除劑的牛磺酸和其他游離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結果可以解釋這一原因:可能是由于進入血漿的MG會被精氨酸或其他清除劑中和。
4 結 論
本研究發現雞有慢性高血糖卻無任何臨床癥狀的原因可能是牛磺酸和部分游離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,從而導致糖基化終末產物的降低,抑制了其對生物機體的損害。這為探明禽類高血糖耐受生理特性提供了新的科學數據,也為人類防治Ⅱ型糖尿病及其并發癥提供了新思路。
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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
基金項目:江蘇現代農業(肉雞)產業技術體系(JATS[2022]163)。
第一作者:羅舒文,女,博士研究生,研究方向為雞的遺傳育種與繁殖。E-mail:santifay@163.com
通信作者:徐 琪,男,博士,教授,研究方向為家禽遺傳育種。E-mail:xuqi@yzu.edu.cn