生大豆及过量蛋白日粮致小鼠消化器官氧化应激的研究
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摘要
氧自由基和人类大部分疾病有关,如糖尿病、肥胖、肠炎、心血管疾病等。在慢性应激状态下,其对消化系统的氧化损伤不易察觉,危害性最大。消化系统是机体内营养素的消化、吸收和代谢的主要场所,因此过量的氧自由基极易诱发消化系统疾病。本课题选用KM和C57BL/6J小鼠为试验对象,研究日粮蛋白水平及种类和大豆胰蛋白酶抑制因子对动物消化系统氧化还原状态及对胰腺功能的影响。
1.生大豆致小鼠消化器官氧化应激及胰腺损伤研究
选用108只体重12-14g的雄性KM种小鼠,随机分为9组:组1,基础日粮;组2,基础日粮+30mg/kg半胱胺;组3,基础日粮+60mg/kg半胱胺;组4,低水平大豆胰蛋白酶抑制因子日粮;组5,低水平大豆胰蛋白酶抑制因子日粮+30mg/kg半胱胺;组6,低水平大豆胰蛋白酶抑制因子日粮+60mg/kg半胱胺;组7,高水平大豆胰蛋白酶抑制因子日粮;组8,高水平大豆胰蛋白酶抑制因子日粮+30mg/kg半胱胺;组9,高水平大豆胰蛋白酶抑制因子日粮+60mg/kg半胱胺。饲养14天后屠宰,分别测定:血浆、十二指肠、肝脏和胰腺中氧化和抗氧化指标,组织器官重及脏器指数,以及血浆、十二指肠和胰腺中消化酶活性,以研究大豆胰蛋白酶抑制因子对胰腺损伤机理是否与自由基有关。结果表明:与对照组比较,低水平和高水平大豆胰蛋白酶抑制因子日粮能显著增加小鼠血浆、十二指肠、肝脏和胰腺中氧化指标含量(超氧阴离子和MDA),显著降低抗氧化指标水平(P＜0.05);同时还能引起胰腺肥大,使血浆、十二指肠和胰腺中胰蛋白酶、淀粉酶和脂肪酶活性显著下降,而且这些影响与日粮中大豆胰蛋白酶抑制因子含量呈剂量依赖关系。添加半胱胺后,氧化和抗氧化不平衡被改善,而且对胰腺的影响也被减轻。上述结果表明,大豆胰蛋白酶抑制因子对胰腺损伤与自由基产生有关。
2.过量蛋白日粮对小鼠消化器官氧化还原状态的影响
试验选用72只体重12-13g C57BL/6J雄性小鼠,随机分为6组,组1,对照日粮组(酪蛋白);组2,过量蛋白日粮组(酪蛋白);组3,过量蛋白日粮组(酪蛋白)+60mg/kg半胱胺;组4,对照日粮组(大豆蛋白);组5,过量蛋白日粮组(大豆蛋白);组6,过量蛋白日粮组(大豆蛋白)+60mg/kg半胱胺。对照日粮为基础日粮。试验期14天。再取108只小鼠,随机分为9组。第一组为采食前即0h,其余八组为采食后(酪蛋白和大豆蛋白)0.5h、1h、1.5h和2h。通过测定小鼠十二指肠、肝脏和胰腺中氧化和抗氧化水平,器官重及脏器指数,血、空肠、十二指肠、肝脏和胰腺ROS水平,以及大豆蛋白和酪蛋白水解物体外清除DPPH和·OH自由基作用,研究日粮蛋白水平和种类对小鼠消化器官氧化还原状态的影响及过量蛋白日粮对ROS动态变化的影响。结果表明,过量蛋白日粮显著地增加了小鼠十二指肠、肝脏和胰腺中超氧阴离子和MDA含量,且使体内SOD、GSH-Px和CAT和Na~+K~+-ATP酶活性以及GSH含量显著地降低(P＜0.05)。体内试验表明与酪蛋白组相比,高水平大豆蛋白组的氧化水平要低以及抗氧化要高。体外模拟消化道水解过程,发现大豆蛋白酶解产物清除DPPH和羟自由基的作用高于酪蛋白水解产物,高水平大豆蛋白对机体的氧化应激小于酪蛋白。过量蛋白日粮添加半胱胺后,体内氧化应激被减轻,但其氧化水平仍高于对照组。采食过量蛋白日粮后,随时间延长体内ROS水平呈动态变化,且两种蛋白质的整体ROS水平在1.0h时最高。综上所述,过量蛋白能够破坏机体氧化和抗氧化平衡,诱导氧化应激的产生。
3.过量蛋白日粮对小鼠胰腺功能与氧化损伤研究
试验选取36只体重12-13g的C57BL/6J雄性小鼠,随机分成3个处理组,分别饲喂对照日粮、过量蛋白日粮和过量蛋白日粮+60mg/kg半胱胺。试验期14天。通过测定小鼠器官重及脏器指数,胰腺中蛋白质、DNA和RNA浓度,血浆和胰腺中氧化和抗氧化水平、、消化酶活性以及生长抑素和胰岛素水平,研究过量蛋白日粮对胰腺自由基内外分泌功能的影响。结果表明:过量蛋白导致血浆和胰腺中超氧阴离子(225%和481.82%)和MDA(91%和185.19%)显著升高,SOD、GSH-Px活性和T-AOC显著下降(P＜0.05),脏器中胰腺重影响较大,表现为显著增加,蛋白质、DNA和RNA浓度显著提高,消化酶活性被显著降低,腺泡分泌能力下降,而胰岛细胞分泌功能被提高,表现为生长抑素和胰岛素水平显著提高(P＜0.05)。自由基清除剂半胱胺可以改善上述症状。这些结果证明过量蛋白引发胰腺自由基水平增加,引发氧化损伤,影响胰腺内外分泌功能。
4.过量蛋白日粮对小鼠肠基因表达的影响
试验选取24只体重12-13g的C57BL/6J雄性小鼠,随机分成2个处理组,分别饲喂对照日粮和过量蛋白日粮。试验期14天。利用基因芯片研究过量蛋白日粮对小鼠肠基因表达的影响。结果表明,过量蛋白日粮使小鼠肠大量基因表达发生了改变,其中引起上调的基因数为714,下调的基因数为458,而且从生物过程、分子功能和细胞成分三个方面广泛影响基因表达,它们主要与氧化还原、代谢、运输、免疫应答、防御反应和信号传导有关。利用MAPFinder分析GenMAPPs数据库中与过量蛋白响应基因显著相关的MAPP,结果发现Permute P值小于0.05的MAPP有63个,其中Adjusted P小于0.05的MAPP有3个:B细胞受体信号通路、蛋白质组氨酸激酶活性、线粒体肉毒碱棕榈酸酰转移酶。过量蛋白日粮引起Apo E、Gpx2、Cyba、Noxo1、Cat和Sod1等氧和活性氧族代谢相关基因表达上调,其中Cat的FC=2.143547,上调抑制性调节激素-生长抑素SS(FC=1.5157)。引起谷胱甘肽代谢以及谷胱甘肽转移酶相关基因表达,包括Gpx2、Gsta1/Gsta2、Gstm1、Gstm2等。此外,过量蛋白日粮对氨基酸、蛋白质、脂质和糖代谢相关基因的表达产生广泛影响。
5.过量蛋白日粮对小鼠肝脏基因表达的影响
试验选取24只体重12-13g的C57BL/6J雄性小鼠,随机分成2个处理组,分别饲喂对照日粮和过量蛋白日粮。试验期14天。利用基因芯片研究过量蛋白日粮对小鼠肝脏基因表达的影响。结果表明,过量蛋白日粮使小鼠肝脏大量基因表达发生了改变,其中引起上调的基因数为381,下调的基因数为300,而且从生物过程、分子功能和细胞成分三个方面广泛影响基因表达,它们主要与氧化还原、代谢、运输、免疫应答、防御反应和信号传导有关。利用MAPFinder分析GenMAPPs数据库中与过量蛋白响应基因显著相关的MAPP,结果发现Permute P值小于0.05的MAPP有73个,其中Adjusted P小于0.05的MAPP有7个:胆固醇生物合成、固醇生物合成、生物碱生物合成。过量蛋白日粮引起大部分氧和活性氧族代谢相关基因表达上调,而下调的基因只有Scd1、Mt2和Sod1三个,其中Scd1表达量相对较高,FC=-4.92458。过量蛋白影响包括Gpx2、Gpx1、Gpx4、Gstt2、Gstt1、Gstt3、Gsta4、Gstp1等谷胱甘肽代谢以及谷胱甘肽转移酶以及氨基酸、蛋白质、脂质和糖代谢相关基因的表达。
Oxygen free radical is related to many diseases,for example,diabetes,obesity, inflammatory bowel,cardiovascular disease and so on.Oxidatie damage of digestive system is very serious under the condition of chronic stress..Digestive system includes digestive,absorbtive and metabolic organs.Excessive free radicals can induce deseases of digestive system.The aim of our experiment was to study the effect of soybean trypsin inhibitor and the kind and level of dietary protein on digestive system.
1.The effect of raw soybean on redox state of the peptic and gland alveolus function of pancreas in mice
The objective of the this study was to investigate the effect of STI on redox state of the digestive system and gland alveolus function of pancreas in mice.A total of 108 male,KM mice weighing 12-14g,have been used for the experiments.The One hundred and eight were randomly divided into nine groups of 12 each:group 1,basic diet;group 2,basic diet supplemented with 30mg/kg cysteamine;group 3,basic diet supplemented with 60mg/kg cysteamine;group 4,basic diet containing low level of STI;group 5,basic diet containing low level of STI supplemented with 30mg/kg cysteamine;group6,basic diet containing low level of STI supplemented with 60mg/kg;group 7,basic diet containing high level of STI;group 8,basic diet containing low level of STI supplemented with 30mg/kg cysteamine;group 9,basic diet containing low level of STI supplemented with 60mg/kg cysteamine.Mice were fed for two weeks.The effect of STI on the redox state and gland alveolus of pancreas in were evaluated by determination of oxidative and antioxidative indexes in plasma and the digestive system,and activities of amylase,lipase and trypsin.The results show higher content of oxidative index STI and lower level of antioxidant index were found in plasma and duodenum,liver and pancreas in the basic diet containing STI(P＜0.05);STI caused pancreas pancrease hypertrophy,and significantly lowed activities of amylase,lipase and trypsin in plasma,duodenum and pancreas.These result was dependent-level of STI.A balance between oxidatant and antioxidant was improved and lesion of pancreas was lessened by cysteamine.These results strongly suggest that damage of STI on pancreas was related to production of free radicals.
2.The effect of dietary protein origin and level on the redox status in peptic of mice
To study the effects of dietary protein kind(soybean protein or casein) and level on a balance between free radicals production and antioxidant level in digestive organs of mice and the dynamic change of ROS level.A total of 72 male,C57BL/6J mice weighing 12-13g,have been used for the experiments.The seventy-two were randomly divided into six groups of 12 each:group 1,control diet containing casein; group 2,excessive protein diet containing casein;group 3,excessive protein diet containing casein supplemented with 60mg/kg cysteamine;group 4,control diet containing soybean;group 5,excessive protein diet containing soybean;group 6, excessive protein diet containing soybean supplemented with 60mg/kg cysteamine. Mice were fed for two weeks.In addition,the one hundred and eight mice were randomly divided into nine groups of 12 each:group 1,before intake,that is,0h;other eight groups were 0.5h,1.0h 1.5h and 2.0h after intake(casein and soybean protein). Oxidative and antioxidative levels in digestive system,organs weight and viscera indexes in mice,ROS level in blood,jejunum,duodenum,liver and pancreas,and the scavenging effect of soybean and casein hydrolysate on DPPH and·OH in vitro were determined.The results show that ingestion of excessive protein markedly increased contents of superoxide anion and MDA and decreased activities of SOD,GSH-Px, CAT and Na~+K~+-ATPase,and content of GSH in duodenum,liver and pancreas of mice(P＜0.05).Levels of oxidative parameters were lower and antioxidant capacity of both enzyme and non-enzyme was higher in mice fed with soybean protein than those fed with casein in vivo.The scavenging capacity of soybean hydrolysate on DPPH and·OH was stronger than that of casein hydrolysate in vitro.In groups fed excessive protein diets supplemented with cysteamine,oxidative stress was mitigated;however, oxidative parameters levels were still higher than those of control diet-fed groups. ROS level showed the dynamic change with time after intake,and the whole level of ROS was highest at 1.0h.Based on above results,it was concluded that a balance between oxidant and antioxidant was destroyed,furthermore,oxidative stress was induced by excessive protein diet.
3.The study of excessive protein diet on pancreas function and oxidative damage
To estimate the effect of excessive protein diet on free radicals and the pancreas function in mice,thirty-six mice with an average weight of 12-13g were randomly divided into three groups of twelve each.The mice received control diet,excessive protein diet and excessive protein diet supplemented with 60mg/kg cysteamine, respectively,for 14 days.Organs weight and viscera indexes,protein,DNA and RNA concentration in pancreas,oxidative and antioxidative levels,digestive enzyme activity,and level of somatostatin and insulin in plasma and pancreas in mice were determined.The results show that ingestion of excessive protein markedly increased contents of superoxide anion(225%and 481.82%) and MDA(91%and 185.19%) and decreased activities of SOD,GSH-Px,and T-AOC(P＜0.05).Pancreas weight and concentration of protein,DNA and RNA were significantly increased.But capacity of islet cell secretion was enhanced,that is,levels of somatostatin and insulin were enhanced(P＜0.05).Cysteamine improved the above symptom.These results show excessive protein diet might cause the increase of free radicals levels and oxidative damage,and influence pancreas function.
4.The effect of excessive protein diet on gene expression of intestine in mice
A total of 24 C57BL/6J male mice of an average body weight of 12-13g were randomly allocated to two dietary treatments of twelve each:control diet and excessive protein diet.These animals were fed for two weeks.The effect of excessive protein diet on gene expression of intestine in mice was estimated by gene chip.The results show excessive protein diet made genes of 714 up-regulated and genes of 458 down-regulated,and influenced gene expresstion of intestine at biological process,molecule function and cell component,mainly including oxidoreduction,metabolism,transport,immune response,defense reaction and signal conduction.We found that there were 63 Permute P less than 0.05 and 3(B cell receptor signaling pathway,Protein histidine kinase activity, Mitochondrial-Carnitine-Palmitoyl transferase-CPT-System-BioCarta) Adjusted P less than 0.05 by analyzing GenMAPPs datacase.Excessive protein diet induced up-regulation of Apoe,Gpx2,Cyba,Noxo1,Cat and Scd1 and so on related oxygen and reactive oxygen species metabolism(Cat,FC=2.143547),and genes expression of glutathione metabolism and glutathione transferase including Gpx2、Gsta1/Gsta2、Gstm1、Gstm2 and so on.In addition,excessive protein diet had wide effect on gene expression of metabolism of amino acid,protein,lipid and glucose.Gene of somatostatin was up-regulated and FC of it was 1.515717.
5.The effect of excessive protein diet on gene expression of liver in mice
A total of 24 C57BL/6J male mice of an average body weight of 12-13g were randomly allocated to two dietary treatments of twelve each:control diet and excessive protein diet.These animals were fed for two weeks.The effect of excessive protein diet on gene expression of liver in mice was estimated by gene chip. The results show excessive protein diet made genes of 381 up-regulated and genes of 300 down-regulated,and influenced gene expresstion of intestine at biological process, molecule function and cell component,mainly including oxidoreduction,metabolism, transport,immune response,defense reaction and signal conduction.We found that there were 73 Permute P less than 0.05 and 7(Cholesterol Biosynthetic Process, Sterol Biosynthesis,Alkaloid BiosynthesisⅡ) Adjusted P less than 0.05 by analyzing GenMAPPs datacase.Excessive protein diet induced up-regulation of major of genes related oxygen and reactive oxygen species metabolism and down-regulation of three genes including Scd1(FC,-4.92458),Mt2,Sod1,and genes expression of glutathione metabolism and glutathione transferase including Gpx2、Gpx1、Gpx4、Gstt2、Gstt1、Gstt3、Gsta4、Gstp1 and so on.In addition,excessive protein diet had wide effect on gene expression of metabolism of amino acid,protein,lipid and glucose.
1.生大豆致小鼠消化器官氧化应激及胰腺损伤研究
选用108只体重12-14g的雄性KM种小鼠,随机分为9组:组1,基础日粮;组2,基础日粮+30mg/kg半胱胺;组3,基础日粮+60mg/kg半胱胺;组4,低水平大豆胰蛋白酶抑制因子日粮;组5,低水平大豆胰蛋白酶抑制因子日粮+30mg/kg半胱胺;组6,低水平大豆胰蛋白酶抑制因子日粮+60mg/kg半胱胺;组7,高水平大豆胰蛋白酶抑制因子日粮;组8,高水平大豆胰蛋白酶抑制因子日粮+30mg/kg半胱胺;组9,高水平大豆胰蛋白酶抑制因子日粮+60mg/kg半胱胺。饲养14天后屠宰,分别测定:血浆、十二指肠、肝脏和胰腺中氧化和抗氧化指标,组织器官重及脏器指数,以及血浆、十二指肠和胰腺中消化酶活性,以研究大豆胰蛋白酶抑制因子对胰腺损伤机理是否与自由基有关。结果表明:与对照组比较,低水平和高水平大豆胰蛋白酶抑制因子日粮能显著增加小鼠血浆、十二指肠、肝脏和胰腺中氧化指标含量(超氧阴离子和MDA),显著降低抗氧化指标水平(P＜0.05);同时还能引起胰腺肥大,使血浆、十二指肠和胰腺中胰蛋白酶、淀粉酶和脂肪酶活性显著下降,而且这些影响与日粮中大豆胰蛋白酶抑制因子含量呈剂量依赖关系。添加半胱胺后,氧化和抗氧化不平衡被改善,而且对胰腺的影响也被减轻。上述结果表明,大豆胰蛋白酶抑制因子对胰腺损伤与自由基产生有关。
2.过量蛋白日粮对小鼠消化器官氧化还原状态的影响
试验选用72只体重12-13g C57BL/6J雄性小鼠,随机分为6组,组1,对照日粮组(酪蛋白);组2,过量蛋白日粮组(酪蛋白);组3,过量蛋白日粮组(酪蛋白)+60mg/kg半胱胺;组4,对照日粮组(大豆蛋白);组5,过量蛋白日粮组(大豆蛋白);组6,过量蛋白日粮组(大豆蛋白)+60mg/kg半胱胺。对照日粮为基础日粮。试验期14天。再取108只小鼠,随机分为9组。第一组为采食前即0h,其余八组为采食后(酪蛋白和大豆蛋白)0.5h、1h、1.5h和2h。通过测定小鼠十二指肠、肝脏和胰腺中氧化和抗氧化水平,器官重及脏器指数,血、空肠、十二指肠、肝脏和胰腺ROS水平,以及大豆蛋白和酪蛋白水解物体外清除DPPH和·OH自由基作用,研究日粮蛋白水平和种类对小鼠消化器官氧化还原状态的影响及过量蛋白日粮对ROS动态变化的影响。结果表明,过量蛋白日粮显著地增加了小鼠十二指肠、肝脏和胰腺中超氧阴离子和MDA含量,且使体内SOD、GSH-Px和CAT和Na~+K~+-ATP酶活性以及GSH含量显著地降低(P＜0.05)。体内试验表明与酪蛋白组相比,高水平大豆蛋白组的氧化水平要低以及抗氧化要高。体外模拟消化道水解过程,发现大豆蛋白酶解产物清除DPPH和羟自由基的作用高于酪蛋白水解产物,高水平大豆蛋白对机体的氧化应激小于酪蛋白。过量蛋白日粮添加半胱胺后,体内氧化应激被减轻,但其氧化水平仍高于对照组。采食过量蛋白日粮后,随时间延长体内ROS水平呈动态变化,且两种蛋白质的整体ROS水平在1.0h时最高。综上所述,过量蛋白能够破坏机体氧化和抗氧化平衡,诱导氧化应激的产生。
3.过量蛋白日粮对小鼠胰腺功能与氧化损伤研究
试验选取36只体重12-13g的C57BL/6J雄性小鼠,随机分成3个处理组,分别饲喂对照日粮、过量蛋白日粮和过量蛋白日粮+60mg/kg半胱胺。试验期14天。通过测定小鼠器官重及脏器指数,胰腺中蛋白质、DNA和RNA浓度,血浆和胰腺中氧化和抗氧化水平、、消化酶活性以及生长抑素和胰岛素水平,研究过量蛋白日粮对胰腺自由基内外分泌功能的影响。结果表明:过量蛋白导致血浆和胰腺中超氧阴离子(225%和481.82%)和MDA(91%和185.19%)显著升高,SOD、GSH-Px活性和T-AOC显著下降(P＜0.05),脏器中胰腺重影响较大,表现为显著增加,蛋白质、DNA和RNA浓度显著提高,消化酶活性被显著降低,腺泡分泌能力下降,而胰岛细胞分泌功能被提高,表现为生长抑素和胰岛素水平显著提高(P＜0.05)。自由基清除剂半胱胺可以改善上述症状。这些结果证明过量蛋白引发胰腺自由基水平增加,引发氧化损伤,影响胰腺内外分泌功能。
4.过量蛋白日粮对小鼠肠基因表达的影响
试验选取24只体重12-13g的C57BL/6J雄性小鼠,随机分成2个处理组,分别饲喂对照日粮和过量蛋白日粮。试验期14天。利用基因芯片研究过量蛋白日粮对小鼠肠基因表达的影响。结果表明,过量蛋白日粮使小鼠肠大量基因表达发生了改变,其中引起上调的基因数为714,下调的基因数为458,而且从生物过程、分子功能和细胞成分三个方面广泛影响基因表达,它们主要与氧化还原、代谢、运输、免疫应答、防御反应和信号传导有关。利用MAPFinder分析GenMAPPs数据库中与过量蛋白响应基因显著相关的MAPP,结果发现Permute P值小于0.05的MAPP有63个,其中Adjusted P小于0.05的MAPP有3个:B细胞受体信号通路、蛋白质组氨酸激酶活性、线粒体肉毒碱棕榈酸酰转移酶。过量蛋白日粮引起Apo E、Gpx2、Cyba、Noxo1、Cat和Sod1等氧和活性氧族代谢相关基因表达上调,其中Cat的FC=2.143547,上调抑制性调节激素-生长抑素SS(FC=1.5157)。引起谷胱甘肽代谢以及谷胱甘肽转移酶相关基因表达,包括Gpx2、Gsta1/Gsta2、Gstm1、Gstm2等。此外,过量蛋白日粮对氨基酸、蛋白质、脂质和糖代谢相关基因的表达产生广泛影响。
5.过量蛋白日粮对小鼠肝脏基因表达的影响
试验选取24只体重12-13g的C57BL/6J雄性小鼠,随机分成2个处理组,分别饲喂对照日粮和过量蛋白日粮。试验期14天。利用基因芯片研究过量蛋白日粮对小鼠肝脏基因表达的影响。结果表明,过量蛋白日粮使小鼠肝脏大量基因表达发生了改变,其中引起上调的基因数为381,下调的基因数为300,而且从生物过程、分子功能和细胞成分三个方面广泛影响基因表达,它们主要与氧化还原、代谢、运输、免疫应答、防御反应和信号传导有关。利用MAPFinder分析GenMAPPs数据库中与过量蛋白响应基因显著相关的MAPP,结果发现Permute P值小于0.05的MAPP有73个,其中Adjusted P小于0.05的MAPP有7个:胆固醇生物合成、固醇生物合成、生物碱生物合成。过量蛋白日粮引起大部分氧和活性氧族代谢相关基因表达上调,而下调的基因只有Scd1、Mt2和Sod1三个,其中Scd1表达量相对较高,FC=-4.92458。过量蛋白影响包括Gpx2、Gpx1、Gpx4、Gstt2、Gstt1、Gstt3、Gsta4、Gstp1等谷胱甘肽代谢以及谷胱甘肽转移酶以及氨基酸、蛋白质、脂质和糖代谢相关基因的表达。
Oxygen free radical is related to many diseases,for example,diabetes,obesity, inflammatory bowel,cardiovascular disease and so on.Oxidatie damage of digestive system is very serious under the condition of chronic stress..Digestive system includes digestive,absorbtive and metabolic organs.Excessive free radicals can induce deseases of digestive system.The aim of our experiment was to study the effect of soybean trypsin inhibitor and the kind and level of dietary protein on digestive system.
1.The effect of raw soybean on redox state of the peptic and gland alveolus function of pancreas in mice
The objective of the this study was to investigate the effect of STI on redox state of the digestive system and gland alveolus function of pancreas in mice.A total of 108 male,KM mice weighing 12-14g,have been used for the experiments.The One hundred and eight were randomly divided into nine groups of 12 each:group 1,basic diet;group 2,basic diet supplemented with 30mg/kg cysteamine;group 3,basic diet supplemented with 60mg/kg cysteamine;group 4,basic diet containing low level of STI;group 5,basic diet containing low level of STI supplemented with 30mg/kg cysteamine;group6,basic diet containing low level of STI supplemented with 60mg/kg;group 7,basic diet containing high level of STI;group 8,basic diet containing low level of STI supplemented with 30mg/kg cysteamine;group 9,basic diet containing low level of STI supplemented with 60mg/kg cysteamine.Mice were fed for two weeks.The effect of STI on the redox state and gland alveolus of pancreas in were evaluated by determination of oxidative and antioxidative indexes in plasma and the digestive system,and activities of amylase,lipase and trypsin.The results show higher content of oxidative index STI and lower level of antioxidant index were found in plasma and duodenum,liver and pancreas in the basic diet containing STI(P＜0.05);STI caused pancreas pancrease hypertrophy,and significantly lowed activities of amylase,lipase and trypsin in plasma,duodenum and pancreas.These result was dependent-level of STI.A balance between oxidatant and antioxidant was improved and lesion of pancreas was lessened by cysteamine.These results strongly suggest that damage of STI on pancreas was related to production of free radicals.
2.The effect of dietary protein origin and level on the redox status in peptic of mice
To study the effects of dietary protein kind(soybean protein or casein) and level on a balance between free radicals production and antioxidant level in digestive organs of mice and the dynamic change of ROS level.A total of 72 male,C57BL/6J mice weighing 12-13g,have been used for the experiments.The seventy-two were randomly divided into six groups of 12 each:group 1,control diet containing casein; group 2,excessive protein diet containing casein;group 3,excessive protein diet containing casein supplemented with 60mg/kg cysteamine;group 4,control diet containing soybean;group 5,excessive protein diet containing soybean;group 6, excessive protein diet containing soybean supplemented with 60mg/kg cysteamine. Mice were fed for two weeks.In addition,the one hundred and eight mice were randomly divided into nine groups of 12 each:group 1,before intake,that is,0h;other eight groups were 0.5h,1.0h 1.5h and 2.0h after intake(casein and soybean protein). Oxidative and antioxidative levels in digestive system,organs weight and viscera indexes in mice,ROS level in blood,jejunum,duodenum,liver and pancreas,and the scavenging effect of soybean and casein hydrolysate on DPPH and·OH in vitro were determined.The results show that ingestion of excessive protein markedly increased contents of superoxide anion and MDA and decreased activities of SOD,GSH-Px, CAT and Na~+K~+-ATPase,and content of GSH in duodenum,liver and pancreas of mice(P＜0.05).Levels of oxidative parameters were lower and antioxidant capacity of both enzyme and non-enzyme was higher in mice fed with soybean protein than those fed with casein in vivo.The scavenging capacity of soybean hydrolysate on DPPH and·OH was stronger than that of casein hydrolysate in vitro.In groups fed excessive protein diets supplemented with cysteamine,oxidative stress was mitigated;however, oxidative parameters levels were still higher than those of control diet-fed groups. ROS level showed the dynamic change with time after intake,and the whole level of ROS was highest at 1.0h.Based on above results,it was concluded that a balance between oxidant and antioxidant was destroyed,furthermore,oxidative stress was induced by excessive protein diet.
3.The study of excessive protein diet on pancreas function and oxidative damage
To estimate the effect of excessive protein diet on free radicals and the pancreas function in mice,thirty-six mice with an average weight of 12-13g were randomly divided into three groups of twelve each.The mice received control diet,excessive protein diet and excessive protein diet supplemented with 60mg/kg cysteamine, respectively,for 14 days.Organs weight and viscera indexes,protein,DNA and RNA concentration in pancreas,oxidative and antioxidative levels,digestive enzyme activity,and level of somatostatin and insulin in plasma and pancreas in mice were determined.The results show that ingestion of excessive protein markedly increased contents of superoxide anion(225%and 481.82%) and MDA(91%and 185.19%) and decreased activities of SOD,GSH-Px,and T-AOC(P＜0.05).Pancreas weight and concentration of protein,DNA and RNA were significantly increased.But capacity of islet cell secretion was enhanced,that is,levels of somatostatin and insulin were enhanced(P＜0.05).Cysteamine improved the above symptom.These results show excessive protein diet might cause the increase of free radicals levels and oxidative damage,and influence pancreas function.
4.The effect of excessive protein diet on gene expression of intestine in mice
A total of 24 C57BL/6J male mice of an average body weight of 12-13g were randomly allocated to two dietary treatments of twelve each:control diet and excessive protein diet.These animals were fed for two weeks.The effect of excessive protein diet on gene expression of intestine in mice was estimated by gene chip.The results show excessive protein diet made genes of 714 up-regulated and genes of 458 down-regulated,and influenced gene expresstion of intestine at biological process,molecule function and cell component,mainly including oxidoreduction,metabolism,transport,immune response,defense reaction and signal conduction.We found that there were 63 Permute P less than 0.05 and 3(B cell receptor signaling pathway,Protein histidine kinase activity, Mitochondrial-Carnitine-Palmitoyl transferase-CPT-System-BioCarta) Adjusted P less than 0.05 by analyzing GenMAPPs datacase.Excessive protein diet induced up-regulation of Apoe,Gpx2,Cyba,Noxo1,Cat and Scd1 and so on related oxygen and reactive oxygen species metabolism(Cat,FC=2.143547),and genes expression of glutathione metabolism and glutathione transferase including Gpx2、Gsta1/Gsta2、Gstm1、Gstm2 and so on.In addition,excessive protein diet had wide effect on gene expression of metabolism of amino acid,protein,lipid and glucose.Gene of somatostatin was up-regulated and FC of it was 1.515717.
5.The effect of excessive protein diet on gene expression of liver in mice
A total of 24 C57BL/6J male mice of an average body weight of 12-13g were randomly allocated to two dietary treatments of twelve each:control diet and excessive protein diet.These animals were fed for two weeks.The effect of excessive protein diet on gene expression of liver in mice was estimated by gene chip. The results show excessive protein diet made genes of 381 up-regulated and genes of 300 down-regulated,and influenced gene expresstion of intestine at biological process, molecule function and cell component,mainly including oxidoreduction,metabolism, transport,immune response,defense reaction and signal conduction.We found that there were 73 Permute P less than 0.05 and 7(Cholesterol Biosynthetic Process, Sterol Biosynthesis,Alkaloid BiosynthesisⅡ) Adjusted P less than 0.05 by analyzing GenMAPPs datacase.Excessive protein diet induced up-regulation of major of genes related oxygen and reactive oxygen species metabolism and down-regulation of three genes including Scd1(FC,-4.92458),Mt2,Sod1,and genes expression of glutathione metabolism and glutathione transferase including Gpx2、Gpx1、Gpx4、Gstt2、Gstt1、Gstt3、Gsta4、Gstp1 and so on.In addition,excessive protein diet had wide effect on gene expression of metabolism of amino acid,protein,lipid and glucose.
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