蓝莓及活性物质对肠道菌群失衡及肠癌作用的初步研究
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摘要
目的:
1.分析盲肠指数、脾脏指数及肠道菌群的变化情况,探讨蓝莓对于小鼠肠道菌群的调节作用。
2.分析蓝莓花青素对肠癌细胞HT29和Caco2生长的影响,探讨蓝莓花青素对于肠癌细胞的抑制作用。
方法:
1.应用抗生素头孢曲松钠灌胃的方法建立小鼠肠道菌群失衡模型。以头孢曲松钠终浓度为8g/kg/d的剂量,连续灌胃8天,监测肠道菌群,建立小鼠菌群失衡模型。
2.利用观察称量等方法观察小鼠临床表征的变化,以及盲肠指数和脾指数的变化。
3.利用活菌计数法分析在正常情况、肠道菌群失衡、自然恢复、回灌蓝莓及回灌大豆低聚糖时双歧杆菌、类杆菌、韦荣球菌、乳酸杆菌、消化球菌、优杆菌、葡萄球菌、肠球菌、大肠杆菌、链球菌、酵母菌等11种肠道优势菌群的变化趋势。
4.用液相色谱法鉴定花青素,用MTT方法检测花青素对HT29和Caco2的生长抑制作用。
结果:
1.通过抗生素头孢曲松钠的诱导,建立稳定的菌群失衡小鼠模型。
2.与正常组小鼠相比,菌群失衡组盲肠指数增高(p<0.05);与失衡组小鼠相比,自然恢复组盲肠指数变化不明显;蓝莓回灌组与大豆低聚糖回灌组盲肠指数均显著下降(p<0.05),呈恢复趋势,且二者作用效果相似。
3.与正常组小鼠相比,菌群失衡组脾脏指数有下降趋势,但无显著差异(p>0.05),与失衡组小鼠相比,自然恢复组脾脏指数无明显变化;蓝莓回灌组与大豆低聚糖回灌组脾脏指数均有所上升,呈恢复趋势,但差异不显著。
4.与正常组相比,菌群失衡组厌氧菌被抑制,需氧菌大量繁殖。与菌群失衡组比较,自然恢复组菌群种类和数量变化不明显;蓝莓回灌组厌氧菌重新出现,尤其是双歧杆菌、优杆菌和类杆菌的出现预示着菌群正在逐渐恢复正常;蓝莓回灌的效果与大豆低聚糖效果相似。
5.花青素对大肠癌HT29细胞有很强的抑制作用,对Caco2的抑制效果稍低。
结论:
1.蓝莓可以促进菌群失衡小鼠盲肠和脾脏大小的恢复。
2.蓝莓可以调节小鼠肠道菌群失衡,效果与大豆低聚糖相似。
3.蓝莓花青素对大肠癌HT29和Caco2细胞具有抑制作用。
Objective:
1.Analyze the variation of the cecum index, spleen index and intestinal flora, and discusses the moderating effect of blueberry on mice intestinal flora.
2.Analyze the effect of blueberry anthocyanin on the growth of colon cancer cells HT-29 and Caco2, and discusses the inhibition of blueberry anthocyanin on colon-cancer cells.
Methods:
1.Set up imbalance of intestinal dysbacteria model by treating with ceftriaxone sodium . Using 8g/kg/d doses of ceftriaxone sodium to continuous gavage for 8 days, to establish mice dysbacteria model with monitoring intestinal microflora.
2.Observe the change of clinical symptoms, cecum index and spleen index by observing and weighing.
3.Analyze the change trend of the 11 species intestinal predominant flora , such as Bifidobacterium、Bacteroides et al, under the normal、intestinal microflora imbalance、natural restoration、blueberry treatment and soybean oligosaccharides treatment.
4.Identify anthocyanin by liquid chromatography and test the inhibition effect of the anthocyanin on HT-29 and Caco2.
Results:
1.To establish stable intestinal dysbacteria model by treating with antibiotics ceftriaxone sodium.
2.Compared with the normal group, the cecum index of intestinal dysbacteria group increased significantly(p<0.05); Compared with imbalance group, the cecum index of the natural restoration group did not change remarkably; the cecum index of both blueberry recirculation group and soybean oligosaccharides group decreased remarkably(p<0.05), presenting a recovery trend, and the effects in two groups were similar.
3.Compared with the normal group, the spleen index of the flora imbalance groups showed a downtrending but no distinguishing differences; Compared with imbalance group, the spleen index of the natural restoration group did not change remarkably; the spleen index of both blueberry recirculation group and soybean oligosaccharides group increased, presenting a recovery trend, but no distinguishing differences.
4.Compared with the normal group, the anaerobic bacteria in dysbacteria group were restrained, and aerobes were bred in great amount. Compared with the dysbacteria group, the types and quantities of the natural restoration group did not change remarkably; the anaerobic bacteria in blueberry treated reappeared, especially the appearances of the Bifidobacteria and Bacteroides predicted that the flora were recovering to normal, the effect of blueberry treatment were similar with that of soybean oligosaccharides.
5.Anthocyanin has strong inhibition effect on colon-cancer cell HT-29, and less inhibition on Caco2.
Conclusion:
1.Blueberry can help the intestinal dysbacteria mice to recover their cecum and spleen.
2.Blueberry can regulate the intestinal dysbacteria, its effect is similar with that of soybean oligosaccharides.
3.Blueberry anthocyanin has inhibition on colon-cancer cells HT-29 and Caco2.
1.分析盲肠指数、脾脏指数及肠道菌群的变化情况,探讨蓝莓对于小鼠肠道菌群的调节作用。
2.分析蓝莓花青素对肠癌细胞HT29和Caco2生长的影响,探讨蓝莓花青素对于肠癌细胞的抑制作用。
方法:
1.应用抗生素头孢曲松钠灌胃的方法建立小鼠肠道菌群失衡模型。以头孢曲松钠终浓度为8g/kg/d的剂量,连续灌胃8天,监测肠道菌群,建立小鼠菌群失衡模型。
2.利用观察称量等方法观察小鼠临床表征的变化,以及盲肠指数和脾指数的变化。
3.利用活菌计数法分析在正常情况、肠道菌群失衡、自然恢复、回灌蓝莓及回灌大豆低聚糖时双歧杆菌、类杆菌、韦荣球菌、乳酸杆菌、消化球菌、优杆菌、葡萄球菌、肠球菌、大肠杆菌、链球菌、酵母菌等11种肠道优势菌群的变化趋势。
4.用液相色谱法鉴定花青素,用MTT方法检测花青素对HT29和Caco2的生长抑制作用。
结果:
1.通过抗生素头孢曲松钠的诱导,建立稳定的菌群失衡小鼠模型。
2.与正常组小鼠相比,菌群失衡组盲肠指数增高(p<0.05);与失衡组小鼠相比,自然恢复组盲肠指数变化不明显;蓝莓回灌组与大豆低聚糖回灌组盲肠指数均显著下降(p<0.05),呈恢复趋势,且二者作用效果相似。
3.与正常组小鼠相比,菌群失衡组脾脏指数有下降趋势,但无显著差异(p>0.05),与失衡组小鼠相比,自然恢复组脾脏指数无明显变化;蓝莓回灌组与大豆低聚糖回灌组脾脏指数均有所上升,呈恢复趋势,但差异不显著。
4.与正常组相比,菌群失衡组厌氧菌被抑制,需氧菌大量繁殖。与菌群失衡组比较,自然恢复组菌群种类和数量变化不明显;蓝莓回灌组厌氧菌重新出现,尤其是双歧杆菌、优杆菌和类杆菌的出现预示着菌群正在逐渐恢复正常;蓝莓回灌的效果与大豆低聚糖效果相似。
5.花青素对大肠癌HT29细胞有很强的抑制作用,对Caco2的抑制效果稍低。
结论:
1.蓝莓可以促进菌群失衡小鼠盲肠和脾脏大小的恢复。
2.蓝莓可以调节小鼠肠道菌群失衡,效果与大豆低聚糖相似。
3.蓝莓花青素对大肠癌HT29和Caco2细胞具有抑制作用。
Objective:
1.Analyze the variation of the cecum index, spleen index and intestinal flora, and discusses the moderating effect of blueberry on mice intestinal flora.
2.Analyze the effect of blueberry anthocyanin on the growth of colon cancer cells HT-29 and Caco2, and discusses the inhibition of blueberry anthocyanin on colon-cancer cells.
Methods:
1.Set up imbalance of intestinal dysbacteria model by treating with ceftriaxone sodium . Using 8g/kg/d doses of ceftriaxone sodium to continuous gavage for 8 days, to establish mice dysbacteria model with monitoring intestinal microflora.
2.Observe the change of clinical symptoms, cecum index and spleen index by observing and weighing.
3.Analyze the change trend of the 11 species intestinal predominant flora , such as Bifidobacterium、Bacteroides et al, under the normal、intestinal microflora imbalance、natural restoration、blueberry treatment and soybean oligosaccharides treatment.
4.Identify anthocyanin by liquid chromatography and test the inhibition effect of the anthocyanin on HT-29 and Caco2.
Results:
1.To establish stable intestinal dysbacteria model by treating with antibiotics ceftriaxone sodium.
2.Compared with the normal group, the cecum index of intestinal dysbacteria group increased significantly(p<0.05); Compared with imbalance group, the cecum index of the natural restoration group did not change remarkably; the cecum index of both blueberry recirculation group and soybean oligosaccharides group decreased remarkably(p<0.05), presenting a recovery trend, and the effects in two groups were similar.
3.Compared with the normal group, the spleen index of the flora imbalance groups showed a downtrending but no distinguishing differences; Compared with imbalance group, the spleen index of the natural restoration group did not change remarkably; the spleen index of both blueberry recirculation group and soybean oligosaccharides group increased, presenting a recovery trend, but no distinguishing differences.
4.Compared with the normal group, the anaerobic bacteria in dysbacteria group were restrained, and aerobes were bred in great amount. Compared with the dysbacteria group, the types and quantities of the natural restoration group did not change remarkably; the anaerobic bacteria in blueberry treated reappeared, especially the appearances of the Bifidobacteria and Bacteroides predicted that the flora were recovering to normal, the effect of blueberry treatment were similar with that of soybean oligosaccharides.
5.Anthocyanin has strong inhibition effect on colon-cancer cell HT-29, and less inhibition on Caco2.
Conclusion:
1.Blueberry can help the intestinal dysbacteria mice to recover their cecum and spleen.
2.Blueberry can regulate the intestinal dysbacteria, its effect is similar with that of soybean oligosaccharides.
3.Blueberry anthocyanin has inhibition on colon-cancer cells HT-29 and Caco2.
引文
1.李祯祯等.人体肠道益生菌的作用及其应用.江苏调味副食品.2007;24:1-3.
2.Davis CD, Milner JA. Gastrointestinal microflora, food components and colon cancer prevention. J Nutr Biochem. 2009, 20(10):743-52.
3.Le Leu RK, Hu Y, Brown IL, Woodman RJ, Young GP. Synbiotic intervention of Bifidobacterium lactis and resistant starch protects against colorectal cancer development in rats. Carcinogenesis. 2010;31(2):246-51.
4.Lopez M, Li N, Kataria J, Russell M. Live and ultraviolet-inactivated Lactobacillus rhamnosus GG decrease flagellin-induced interleukin-8 production in Caco-2 cells. Neu J.J Nutr. 2008 Nov;138(11):2264-8.
5.Li N, Russell WM, Douglas-escobar M, Hauser N, Lopez M, Neu J. Live and heat-killed Lactobacillus rhamnosus GG: effects on proinflammatory and anti-inflammatory cytokines/chemokines in gastrostomy-fed infant rats. Pediatr Res. 2009 Aug;66(2):203-7.3.
6.汪洪涛.益生元极其应用研究进展.食品科技,2007,10:5-7.
7.Wolfe KL, Liu RH.Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements. J Agric Food Chem. 2007,55(22):8896-8907.
8.Wolfe KL, Kang X, He X, Dong M, Zhang Q, Liu RH.Cellular antioxidant activity of common fruits. J Agric Food Chem. 2008 Sep 24;56(18):8418-8426.
9.Ahmet I, Spangler E, Shukitt-Hale B, Juhaszova M, Sollott SJ, Joseph JA, Ingram DK, Talan M. Blueberry-enriched diet protects rat heart from ischemic damage. PLoS One. 2009 Jun 18;4(6):e5954.
10.Joseph JA, Shukitt-Hale B, Willis LM. Grape juice, berries, and walnuts affect brain aging and behavior. J Nutr. 2009, 139(9): 1813S-1817S.
11.T Vuong, A Benhaddou-Andaloussi, A Brault, D Harbilas, L C Martineau, D Vallerand, C Ramassamy, C Matar and P S Haddad. Antiobesity and antidiabetic effects of biotransformed blueberry juice in KKA(y) mice. International Journal of Obesity (2009) 33, 1166–1173; 2009.149.
12.Seeram NP, Adams LS, Zhang Y, Lee R, Sand D, Scheuller HS, Heber D. Blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts inhibit growth and stimulate apoptosis of human cancer cells in vitro.J Agric Food Chem. 2006 Dec 13;54(25):9329-39.
13.McDougall GJ, Ross HA, Ikeji M, Stewart D. Berry extracts exert different antiproliferative effects against cervical and colon cancer cells grown in vitro.J Agric Food Chem.2008, 56(9):3016-23.
14.Savage DC,Dubos R.Alteration in the mouse cecum and its flora produced by antibacterial drugs [J].J.Exp.Med.1968(128):97-110.
15.朱志中等.针刺结合康复训练对免疫抑制大鼠脾脏指数及血清IL一2的影响[J].中医药学报,2010,38(3):93-94.
16.MacDonald TT, Monteleone G. Immunity, Inflammation,and Allergy in the gut[M].Science Magezine.2005,307(5717):1920-1925.
17.Shu Y T. Effect of High content Isomaltooligosaccharides on the Growth of Bitidobacteria[J] . Thesis for Master of Science Department of Bioengineering, Tatun University,2004.
18.Somer SM, Johannot L. Dietary flavonoid sources in Australian adults[J]. Nutr Cancer, 2008, 60:442-449.
19.JW Yun, WS Lee, MJ Kim, JN Lu, MH Kang HG Kim, D C Kim, E J Choi, J Y Choi, H G Kim, Y K Leef, C H Ryu, G S Kim, Y H Choi, O J Parkand S Cl Shin. Characterization of a profile of the anthocyanins isolated from Vitis coignetiae Pulliat and their anti-invasive activity on HT-29 human colon cancer cells. Food and Chemical Toxicology. 2010, 48(3):903-909.
20.Murillo G, Nagpal V, Tiwari N, etal. Actions of vitamin D mediated by TLR4 pathway in inflammation-induced colon cancer[J].Steroid biochemistry and Molecular Biology,2010,121 (1-2):403-407.
21.Francis C. Lau, James A, Joseph, etal. Attenuation of iNOS and COX2 by blueberry polypherols is mediated through the suppression of NF-Kb activation[J]. Functional foods, 2009,1(3):274-283.
1.Sonnenburg JL,Angenent LT, Gordon JI. Getting a grip on things: how do communities of bacterial symbionts become established in our intestine[J]. Nat Immunol, 2004, 5(6): 569-573.
2.Gibson,GR. Roberfroid, M.B.Dietary modulation of the Human colonic microbito:introducing the concept of prebiotics [J]. Journal of Nutrition, 1995(12):15-16.
3.朱路甲等.功能糖的性能和保健作用中国食品添加剂2010, 6(6): 1-2
4.黄德娟等.功能性低聚糖[J].生物学通报, 2005, 40(12):19-20.
5.Gluan Y, Xue L, Ye C, et al. The trans-membrane signal trans-duct ion in HEp-2 cells induces by bacterial adherence[J]. chin Med Sci J, 2000, 15(1): 20-23.
6.Kenneth J, Ryan C, Ray G. sherries. Medical microbiology[M]. 4th ed. New York: McGraw Hill Medical Pub Division, 2004: 141-149.
7.Brooks GF, Butel JS,Morse SA.Medical microbiology[M]. 23th ed.New York:McGraw Hill Medical Pub divisi0n. 2004:196-201.
8.Shrivastava, S, Goyal, GK. Therapeutic benefits of pro-and prebiotics: A review[J]. Indian Food Industry, 2007, 26(2): 41-49.
9.Scholtens Pamj, Alliet P, Raes M, et al. Fecal secretory immunoglobulin A is increased in healthy infants who receive aformula with short-chain galactooligosaccharides and long-chain fructooligosaccharides[J].J Nutr, 2008: 138.
10.Ishikawa T Nanjo F. Dietary Cycloinuloo-ligosaccharides Enhance Intestinal Immu-noglobulin A Production in Mice[J]. Biosci Biotechnol Biochem, 2009, 73(3):677-682.
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2.Davis CD, Milner JA. Gastrointestinal microflora, food components and colon cancer prevention. J Nutr Biochem. 2009, 20(10):743-52.
3.Le Leu RK, Hu Y, Brown IL, Woodman RJ, Young GP. Synbiotic intervention of Bifidobacterium lactis and resistant starch protects against colorectal cancer development in rats. Carcinogenesis. 2010;31(2):246-51.
4.Lopez M, Li N, Kataria J, Russell M. Live and ultraviolet-inactivated Lactobacillus rhamnosus GG decrease flagellin-induced interleukin-8 production in Caco-2 cells. Neu J.J Nutr. 2008 Nov;138(11):2264-8.
5.Li N, Russell WM, Douglas-escobar M, Hauser N, Lopez M, Neu J. Live and heat-killed Lactobacillus rhamnosus GG: effects on proinflammatory and anti-inflammatory cytokines/chemokines in gastrostomy-fed infant rats. Pediatr Res. 2009 Aug;66(2):203-7.3.
6.汪洪涛.益生元极其应用研究进展.食品科技,2007,10:5-7.
7.Wolfe KL, Liu RH.Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements. J Agric Food Chem. 2007,55(22):8896-8907.
8.Wolfe KL, Kang X, He X, Dong M, Zhang Q, Liu RH.Cellular antioxidant activity of common fruits. J Agric Food Chem. 2008 Sep 24;56(18):8418-8426.
9.Ahmet I, Spangler E, Shukitt-Hale B, Juhaszova M, Sollott SJ, Joseph JA, Ingram DK, Talan M. Blueberry-enriched diet protects rat heart from ischemic damage. PLoS One. 2009 Jun 18;4(6):e5954.
10.Joseph JA, Shukitt-Hale B, Willis LM. Grape juice, berries, and walnuts affect brain aging and behavior. J Nutr. 2009, 139(9): 1813S-1817S.
11.T Vuong, A Benhaddou-Andaloussi, A Brault, D Harbilas, L C Martineau, D Vallerand, C Ramassamy, C Matar and P S Haddad. Antiobesity and antidiabetic effects of biotransformed blueberry juice in KKA(y) mice. International Journal of Obesity (2009) 33, 1166–1173; 2009.149.
12.Seeram NP, Adams LS, Zhang Y, Lee R, Sand D, Scheuller HS, Heber D. Blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts inhibit growth and stimulate apoptosis of human cancer cells in vitro.J Agric Food Chem. 2006 Dec 13;54(25):9329-39.
13.McDougall GJ, Ross HA, Ikeji M, Stewart D. Berry extracts exert different antiproliferative effects against cervical and colon cancer cells grown in vitro.J Agric Food Chem.2008, 56(9):3016-23.
14.Savage DC,Dubos R.Alteration in the mouse cecum and its flora produced by antibacterial drugs [J].J.Exp.Med.1968(128):97-110.
15.朱志中等.针刺结合康复训练对免疫抑制大鼠脾脏指数及血清IL一2的影响[J].中医药学报,2010,38(3):93-94.
16.MacDonald TT, Monteleone G. Immunity, Inflammation,and Allergy in the gut[M].Science Magezine.2005,307(5717):1920-1925.
17.Shu Y T. Effect of High content Isomaltooligosaccharides on the Growth of Bitidobacteria[J] . Thesis for Master of Science Department of Bioengineering, Tatun University,2004.
18.Somer SM, Johannot L. Dietary flavonoid sources in Australian adults[J]. Nutr Cancer, 2008, 60:442-449.
19.JW Yun, WS Lee, MJ Kim, JN Lu, MH Kang HG Kim, D C Kim, E J Choi, J Y Choi, H G Kim, Y K Leef, C H Ryu, G S Kim, Y H Choi, O J Parkand S Cl Shin. Characterization of a profile of the anthocyanins isolated from Vitis coignetiae Pulliat and their anti-invasive activity on HT-29 human colon cancer cells. Food and Chemical Toxicology. 2010, 48(3):903-909.
20.Murillo G, Nagpal V, Tiwari N, etal. Actions of vitamin D mediated by TLR4 pathway in inflammation-induced colon cancer[J].Steroid biochemistry and Molecular Biology,2010,121 (1-2):403-407.
21.Francis C. Lau, James A, Joseph, etal. Attenuation of iNOS and COX2 by blueberry polypherols is mediated through the suppression of NF-Kb activation[J]. Functional foods, 2009,1(3):274-283.
1.Sonnenburg JL,Angenent LT, Gordon JI. Getting a grip on things: how do communities of bacterial symbionts become established in our intestine[J]. Nat Immunol, 2004, 5(6): 569-573.
2.Gibson,GR. Roberfroid, M.B.Dietary modulation of the Human colonic microbito:introducing the concept of prebiotics [J]. Journal of Nutrition, 1995(12):15-16.
3.朱路甲等.功能糖的性能和保健作用中国食品添加剂2010, 6(6): 1-2
4.黄德娟等.功能性低聚糖[J].生物学通报, 2005, 40(12):19-20.
5.Gluan Y, Xue L, Ye C, et al. The trans-membrane signal trans-duct ion in HEp-2 cells induces by bacterial adherence[J]. chin Med Sci J, 2000, 15(1): 20-23.
6.Kenneth J, Ryan C, Ray G. sherries. Medical microbiology[M]. 4th ed. New York: McGraw Hill Medical Pub Division, 2004: 141-149.
7.Brooks GF, Butel JS,Morse SA.Medical microbiology[M]. 23th ed.New York:McGraw Hill Medical Pub divisi0n. 2004:196-201.
8.Shrivastava, S, Goyal, GK. Therapeutic benefits of pro-and prebiotics: A review[J]. Indian Food Industry, 2007, 26(2): 41-49.
9.Scholtens Pamj, Alliet P, Raes M, et al. Fecal secretory immunoglobulin A is increased in healthy infants who receive aformula with short-chain galactooligosaccharides and long-chain fructooligosaccharides[J].J Nutr, 2008: 138.
10.Ishikawa T Nanjo F. Dietary Cycloinuloo-ligosaccharides Enhance Intestinal Immu-noglobulin A Production in Mice[J]. Biosci Biotechnol Biochem, 2009, 73(3):677-682.
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