曲婉秋,唐曉琳,王秀武
遼寧師范大學(xué)生命科學(xué)學(xué)院,大連 116029
殼寡糖螯合鉻對(duì)糖尿病小鼠降血糖作用的研究
曲婉秋,唐曉琳,王秀武*
遼寧師范大學(xué)生命科學(xué)學(xué)院,大連 116029
探討殼寡糖螯合鉻(Cos-Cr)的降血糖作用。選擇發(fā)育相近的健康雄性小鼠10只為正常對(duì)照組,糖尿病小鼠30只隨機(jī)分為糖尿病對(duì)照組、殼寡糖組(Cos)和螯合鉻組(Cos-Cr),每組各10只,實(shí)驗(yàn)期4 w,測(cè)定小鼠血糖值。殼寡糖螯合鉻可以降低糖尿病小鼠血糖,增加體重,增加脾臟指數(shù),明顯緩解糖尿病小鼠饑餓和煩渴的癥狀。肝臟損傷明顯減輕。同時(shí)觀察肝組織病理變化。殼寡糖螯合鉻對(duì)糖尿病小鼠有降糖,改善糖尿病癥狀的作用。
殼寡糖螯合鉻;四氧嘧啶;糖尿病;血糖;肝臟;脾臟
鉻(chromium)是人和動(dòng)物體中脂類、蛋白質(zhì)和碳水化合物正常代謝所必須的微量元素之一[1]。補(bǔ)鉻對(duì)治療包括I型糖尿病、II型糖尿病、妊娠糖尿病和類固醇引起的糖尿病都有效[2-7]。美國(guó)醫(yī)學(xué)家Mertz和Schwarz早在1957年就發(fā)現(xiàn)并證實(shí)Gr3+是葡萄糖耐受因子(GTF)中的活性因子。無機(jī)鉻的吸收率很低為0.4%~3%[8],而有機(jī)鉻的吸收率可達(dá)10% ~25%[9]。殼寡糖是殼聚糖的降解產(chǎn)物,大量研究發(fā)現(xiàn)它具有免疫調(diào)節(jié)[10]、抗腫瘤[11]及降血糖[12]等功能。目前,國(guó)內(nèi)外對(duì)殼寡糖用于糖尿病治療[13-18]的相關(guān)研究不斷增多。有研究對(duì)分子量為1200 Da的殼寡糖及其鉻配合物[19]進(jìn)行了研究,本實(shí)驗(yàn)首次以分子量為1000 Da的六碳?xì)す烟呛蜔o機(jī)鉻(CrCl3)為原料螯合制備成易于吸收的有機(jī)鉻形式,目的在于產(chǎn)生協(xié)同作用,提高降糖活性,并進(jìn)一步探討了其對(duì)糖尿病小鼠的降糖作用。殼寡糖螯合鉻可用于開發(fā)新型的保健食品、醫(yī)藥,具有廣闊的應(yīng)用和開發(fā)前景。
1.1 材料
1.1.1 殼寡糖螯合鉻
選擇平均分子量為1000 Da,脫乙酰度>90%的殼寡糖(大連化物所1805室)和三氯化鉻為原料,按本研究室的方法制備成殼寡糖螯合鉻。
1.1.2 試驗(yàn)動(dòng)物
昆明種小鼠,雄性,體重為20±2 g,購(gòu)自大連醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心。
1.1.3 試劑和儀器
四氧嘧啶、苦味酸,購(gòu)自Sigma公司;其余試劑均為國(guó)產(chǎn)分析純?cè)噭?血糖儀,德國(guó)羅氏公司。
1.2 方法
1.2.1 殼寡糖螯合鉻的制備
稱取殼寡糖和氯化鉻用蒸餾水溶解配制成殼寡糖3%溶液,氯化鉻1%溶液,靜止20 min,將兩種液體等體積混合,用氫氧化鈉調(diào)節(jié)pH值為9,靜止20 min,加入3倍體積的無水乙醇,經(jīng)40 min的螯合后,抽濾,并用蒸餾水沖洗沉淀物1-5次,除去殘留的濾液,將沉淀物置于70℃的烘箱中5~6 h除去水分后得到墨綠色粉末狀固體即為殼寡糖螯合鉻。
1.2.2 四氧嘧啶糖尿病小鼠模型建立
選擇體重相近(20±2 g),昆明種雄性小鼠75只,自由采食基礎(chǔ)日糧1 w,隨機(jī)選取10只為正常對(duì)照組,其余小鼠禁食16 h后,用生理鹽水配制1%四氧嘧啶按100 mg/kg·bw進(jìn)行腹腔注射,正常對(duì)照組注射等劑量的生理鹽水,1 h后恢復(fù)正常采食和飲水。次日,用同樣方法二次建模。3 d后斷尾取血,測(cè)量血糖值,血糖值在13.8 mmol/L以上,認(rèn)為是造模成功。
1.2.3 實(shí)驗(yàn)設(shè)計(jì)及實(shí)驗(yàn)過程
將合格實(shí)驗(yàn)動(dòng)物分為正常對(duì)照組、糖尿病對(duì)照組、殼寡糖組和螯合鉻組,每組各10只,共計(jì)40只。實(shí)驗(yàn)期4 w。正常對(duì)照組和糖尿病對(duì)照組飼喂基礎(chǔ)日糧,殼寡糖組和螯合鉻組是在基礎(chǔ)日糧中分別添加殼寡糖和殼寡糖螯合鉻0.7 g/kg。每日上午9時(shí)測(cè)定各組小鼠的攝食量和飲水量,每周測(cè)定一次小鼠的體重,同時(shí)尾部采血,測(cè)量血糖值。
實(shí)驗(yàn)4 w結(jié)束時(shí),將各組小鼠脫臼處死,立即分離肝臟和脾臟,并用生理鹽水清洗,擦干后脾臟稱重并計(jì)算脾指數(shù),肝臟拍照,用Bouin液固定肝臟進(jìn)行H.E.染色,作病理組織學(xué)光鏡檢查。
1.2.4 數(shù)據(jù)處理
2.1 殼寡糖螯合鉻對(duì)糖尿病小鼠體重的影響
實(shí)驗(yàn)開始時(shí),各組小鼠的體重差異不明顯。實(shí)驗(yàn)期間,糖尿病組小鼠隨實(shí)驗(yàn)時(shí)間的延長(zhǎng)體重逐漸下降(P<0.05)。殼寡糖組和螯合鉻組小鼠體重試驗(yàn)期各周均比糖尿病組增加,而螯合鉻組的小鼠體重隨實(shí)驗(yàn)時(shí)間的延長(zhǎng)體重增加顯著(P<0.05)。結(jié)果如表1所示。
表1 殼寡糖螯合鉻對(duì)糖尿病小鼠體重的影響(g,±s)Table 1 Effect of chitooligosaccharide-chromium(Ⅲ)complex on body weight of diabetic mice(g,±s)
表1 殼寡糖螯合鉻對(duì)糖尿病小鼠體重的影響(g,±s)Table 1 Effect of chitooligosaccharide-chromium(Ⅲ)complex on body weight of diabetic mice(g,±s)
注:n=10;與正常組比較,*P<0.05;**P<0.01。與模型組比較,▲▲P<0.01。下同。Note:n=10;Compare with normal control,*P<0.05;**P<0.01.Compare with diabetes control,▲▲P<0.01.The same below.
組別Group實(shí)驗(yàn)前Before experiment試驗(yàn)期間Period experiment 1 w 2 w 3 w 4 w正常組Normal control 27.21±3.39 32.03±2.73 34.32±2.52 36.11±0.98 37.19±3.07模型組 Diabetes control 27.63±1.09 27.55±2.17*26.80±2.60**26.02±1.24**25.72±1.09**?dú)す烟墙MCOS 27.57±1.28 27.57±2.11*27.08±2.09**28.80±2.72**30.24±1.63**▲▲螯合鉻組COS-Cr 27.77±2.31 27.65±3.22*28.12±4.41*30.17±4.27*33.58±4.08▲▲
2.2 殼寡糖螯合鉻對(duì)小鼠攝食量、飲水量的影響
實(shí)驗(yàn)開始時(shí),各組小鼠的攝食量、飲水量差異不明顯。實(shí)驗(yàn)開始后糖尿病組小鼠攝食量、飲水量不斷增加,殼寡糖組和螯合鉻組隨實(shí)驗(yàn)時(shí)間的延長(zhǎng)小鼠采食量、飲水量逐漸下降,自實(shí)驗(yàn)開始后第3 w螯合鉻組小鼠采食量顯著減少(P<0.05),飲水量也顯著較少(P<0.05),螯合鉻組比殼寡糖組的飲水量減少趨勢(shì)明顯。結(jié)果如表2~3所示。
表2 殼寡糖螯合鉻對(duì)糖尿病小鼠攝食量的影響(g/d,±s)Table 2 Effect of chitooligosaccharide-chromium(Ⅲ)complex on the content of food intake of diabetic mice(g/d,±s))
表2 殼寡糖螯合鉻對(duì)糖尿病小鼠攝食量的影響(g/d,±s)Table 2 Effect of chitooligosaccharide-chromium(Ⅲ)complex on the content of food intake of diabetic mice(g/d,±s))
注:與模型組比較,▲P<0.05。下同。Note:Compare with diabetes control,▲P<0.05.The same below.
組別Group實(shí)驗(yàn)前Before experiment試驗(yàn)期間Period experiment 1 w 2 w 3 w 4 w正常組Normal control 7.14±0.34 7.20±0.45 7.26±0.36 7.48±0.50 7.60±0.59模型組Diabetes control 7.02±0.42 9.03±0.35**9.70±0.39**9.84±0.55**9.96±0.48**?dú)す烟墙MCOS 7.19±0.68 9.49±0.97**9.28±0.72**8.94±0.56**8.61±0.49**▲螯合鉻組COS-Cr 7.14±0.47 9.49±1.20**9.00±0.86**8.48±0.73*▲8.20±0.50▲▲
表3 殼寡糖螯合鉻對(duì)糖尿病小鼠飲水量的影響(mL/d,±s)Table 3 Effect of chitooligosaccharide-chromium(Ⅲ)complex on the content of water intake of diabetic mice(mL/d,±s)
表3 殼寡糖螯合鉻對(duì)糖尿病小鼠飲水量的影響(mL/d,±s)Table 3 Effect of chitooligosaccharide-chromium(Ⅲ)complex on the content of water intake of diabetic mice(mL/d,±s)
組別Group實(shí)驗(yàn)前Before experiment試驗(yàn)期間Period experiment 1w 2w 3w 4w正常組Normal control 6.43±0.36 6.90±0.20 6.93±0.45 6.95±0.33 7.10±0.47模型組 Diabetes control 6.53±0.31 22.27±0.63**22.44±0.77**22.67±0.62**22.88±0.81**?dú)す烟墙MCOS 6.45±0.34 22.28±1.10**21.15±0.72**20.02±0.78**▲▲19.52±1.00**▲▲螯合鉻組COS-Cr 6.37±0.23 22.23±1.50**20.70±2.20**19.35±2.40**▲▲18.57±2.20**▲▲
2.3 殼寡糖螯合鉻對(duì)糖尿病小鼠血糖的影響
實(shí)驗(yàn)開始時(shí),各組小鼠空腹血糖值差異不顯著。實(shí)驗(yàn)期間,糖尿病組小鼠空腹血糖一直維持在較高水平,而殼寡糖組和螯合鉻組的小鼠血糖水平在不斷下降,其中實(shí)驗(yàn)第2 w(P<0.05)和第4 w(P< 0.01)下降顯著,而螯合鉻組下降極顯著。實(shí)驗(yàn)4 w同1 w相比,殼寡糖組血糖平均值下降了1.99 mmoL/L,螯合鉻組血糖平均值下降了2.76 mmoL/ L。表明殼寡糖螯合鉻具有明顯降低糖尿病小鼠空腹血糖值的作用。結(jié)果如表4所示。
表4 殼寡糖螯合鉻對(duì)糖尿病小鼠血糖的影響(mmol/L·d,±s)Table 4 Effect of chitooligosaccharide-chromium(Ⅲ)complex on the content of blood glucose of diabetic mice(mmol/L·d,±s)
表4 殼寡糖螯合鉻對(duì)糖尿病小鼠血糖的影響(mmol/L·d,±s)Table 4 Effect of chitooligosaccharide-chromium(Ⅲ)complex on the content of blood glucose of diabetic mice(mmol/L·d,±s)
組別Group實(shí)驗(yàn)前Before experiment試驗(yàn)期間Period experiment 1w 2w 3w 4w正常組normal control 6.61±0.23 6.67±0.93 6.60±0.35 6.89±0.91 6.86±0.30模型組diabetes control 6.93±1.69 17.21±1.17**17.69±1.01**18.31±1.33**19.03±2.34**?dú)す烟墙MCOS 6.79±0.74 17.58±1.49**17.12±1.13**16.81±0.52**▲15.59±1.05**▲螯合鉻組COS-Cr 6.47±0.85 17.50±1.02**16.03±0.63**▲15.21±0.19**▲▲14.74±0.23**▲▲
2.4 殼寡糖螯合鉻對(duì)糖尿病小鼠脾指數(shù)的影響
與正常對(duì)照組相比,殼寡糖組和螯合組的脾臟指數(shù)都下降了,說明脾臟明顯萎縮,特異性免疫功能顯著下降。與糖尿病組相比,殼寡糖組和螯合鉻組的脾臟指數(shù)都上升了,而螯合鉻組上升的更多,表明經(jīng)飼喂殼寡糖和殼寡糖螯合鉻的小鼠,脾指數(shù)明顯提高。結(jié)果如表5所示。
表5 殼寡糖螯合鉻對(duì)糖尿病小鼠脾指數(shù)的影響(±s)Table 5 Effect of chitooligosaccharide-chromium(Ⅲ)complex on spleen index of diabetic mice(±s)
表5 殼寡糖螯合鉻對(duì)糖尿病小鼠脾指數(shù)的影響(±s)Table 5 Effect of chitooligosaccharide-chromium(Ⅲ)complex on spleen index of diabetic mice(±s)
組別Group脾臟質(zhì)量(mg) Spleen weight(mg)脾指數(shù)(g/mg) Spleen index(g/mg)正常組normal control 141.28±10.57 4.01±0.23模型組diabetes control 76.60±7.65 2.66±0.17**?dú)す烟墙MCOS 120.70±12.77 3.53±0.45▲螯合鉻組COS-Cr 129.80±12.30 3.99±0.20▲▲
2.5 殼寡糖螯合鉻對(duì)糖尿病小鼠肝臟損傷的修復(fù)
取各組小鼠的肝臟進(jìn)行觀察,正常對(duì)照組小鼠肝臟(A),表面平整光滑,肝葉清晰,顏色正常,質(zhì)地柔軟。糖尿病組小鼠肝臟(B),葉片嚴(yán)重粘連,不清晰,肝臟萎縮,重量減輕,質(zhì)地變硬。殼寡糖組小鼠肝臟(C)與糖尿病組小鼠相比肝小葉粘連情況好轉(zhuǎn),但是與殼寡糖螯合鉻組(D)相比,粘連情況嚴(yán)重些。殼寡糖螯合鉻組小鼠雖也有呈球狀樣變的趨勢(shì),但與糖尿病組相比,肝小葉粘連狀況明顯好轉(zhuǎn)。結(jié)果如圖1所示。
圖1 殼寡糖螯合鉻對(duì)糖尿病小鼠肝臟損傷的修復(fù)Fig.1 hepatic tissue after alloxan treatment and with the COS and Cos-Cr
2.6 組織病理學(xué)檢查
正常組(A)肝細(xì)胞索排列整齊,細(xì)胞形態(tài)正常。模型組(B)中央靜脈周圍肝細(xì)胞皺縮變形,部分壞死,炎細(xì)胞侵潤(rùn)。殼寡糖組(C)和螯合鉻組(D)也有一定的炎細(xì)胞侵潤(rùn)和肝細(xì)胞變形,與模型組(B)相比炎細(xì)胞侵潤(rùn)數(shù)量減少,肝細(xì)胞變形也減少。但螯合鉻組(D)要比殼寡糖組(C)修復(fù)效果更佳。
圖2 實(shí)驗(yàn)各組小鼠肝臟組織切片結(jié)果Fig.2 The tissue pathologic sections of any groups of mice liver(400×)
四氧嘧啶可以破壞胰島β細(xì)胞從而使動(dòng)物患糖尿?。?0]。本實(shí)驗(yàn)中注射飼養(yǎng)嘧啶小鼠血糖值大于13.8 mmol/L,說明造模成功。肝臟是胰島素作用的主要靶器官,胰島素特異性受體大量存在于肝細(xì)胞膜上,糖尿病病理過程中由氧化應(yīng)激產(chǎn)生的大量自由基損傷肝臟細(xì)胞膜,從而使肝細(xì)胞膜胰島素受體減少,敏感性降低,對(duì)血糖利用率下降[21]。本實(shí)驗(yàn)發(fā)現(xiàn)四氧嘧啶也可造成小鼠肝臟損傷,推測(cè)是四氧嘧啶在體內(nèi)產(chǎn)生大量自由基導(dǎo)致肝臟損傷。同時(shí)表明殼寡糖螯合鉻能夠有效的抑制四氧嘧啶引發(fā)小鼠肝臟自由基升高。殼寡糖降低血糖作用可能與其能作用于胰島素受體,使胰島素受體敏感性增強(qiáng),從而控制血糖升高有關(guān)[22]。鉻作為糖尿病耐量因子(GTF)的主要成分參與體內(nèi)糖脂代謝,并可通過硫氫鍵使胰島素結(jié)合到膜受體上,從而增加胰島素效應(yīng),并影響胰島素依賴系統(tǒng),抑制胰島素酶[23]。本實(shí)驗(yàn)表明殼寡糖螯合鉻能夠顯著降低糖尿病小鼠的血糖值,推測(cè)可能是殼寡糖和氯化鉻的協(xié)同作用增強(qiáng)了降糖效果,并能有效地改善糖尿病小鼠“三多一少”的癥狀,使體重增加,同時(shí)使增加脾指數(shù),肝臟損傷明顯減輕,具有廣闊的應(yīng)用前景。
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Hypoglycemic Effects of Chitooligosaccharide-chromium(Ⅲ) Complex on Diabetic Mice
QU Wan-qiu,TANG Xiao-lin,WANG Xiu-wu*
College of Life Sciences,Liaoning Normal University,Dalian 116029,China
To study the effects of chitooligosaccharide-chromium(Ⅲ)complex(Cos-Cr)on diabetic mice.Selecting ten male mice as the normal control group.Thirty diabetes mice were randomly divided into 3 groups,diabetes control group (n=10),Cos group(n=10),Cos-Cr group(n=10).The mice were dissected after 4 weeks,determinations of serum glucose concentration.Cos-Cr can significantly decrease fasting blood glucose level,increase body weight as well as index spleen.Symptoms of hunger and thirsty were improved obviously in diabetic mice.Cos-Cr has protective effects on alloxan-induced hepatic injury in mice.Hepatocytes lesion was alleviated markedly.Cos-Cr can decrease blood glucose concentration and improve diabetic symptoms in mice.
chitooligosaccharide-chromium(Ⅲ);alloxan;diabetic;blood glucose;liver;spleen
R151.2
A
1001-6880(2012)05-0605-05
2011-10-12 接受日期:2011-12-23
*通訊作者 E-mail:quwanqiu@163.com