壳寡糖及其配合物对糖尿病的作用研究
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摘要
糖尿病(Diabetes mellitus,DM)是一种常见的内分泌代谢病,具有遗传倾向,是由于胰岛素绝对或相对不足所致的血糖及尿糖增高为主要特征的并导致糖、蛋白和脂肪代谢障碍全身慢性代谢异常综合症,可引发心脑血管、肾、眼及神经等多种并发症,危害性大。据WHO报告,糖尿病已成为世界上继恶性肿瘤、心脑血管病后第3位严重威胁人类健康的慢性非传染性疾病。
壳寡糖(chitooligosaccharides,COS)是壳聚糖的降解产物,分子量小,溶于水,可被人体、动物及植物机体吸收利用。研究发现,壳寡糖和壳聚糖不仅具有一些相似的性质,而且一些生理活性或功能性质更加显著,如作为功能性食品具有提高机体免疫、抗肿瘤、调节血脂、抗感染、防治病原生物感染、促进双歧杆菌生长等功能,尤其是在糖尿病的防治研究方面有潜在的应用价值。
本论文首先在均相条件下,以醋酸酐为乙酰化试剂对壳聚糖〔脱乙酰度90%)进行N-乙酰化反应,制备出脱乙酰度50%和70%左右的壳聚糖。通过壳聚糖酶降解壳聚糖制备出水溶性不同分子量和不同脱乙酰度的系列壳寡糖。并以壳寡糖为原料,制备出了含有铬、钒、硒的壳寡糖配合物,并通过紫外光谱(UV)、红外光谱(FT/IR)、元素分析(EA〕和铬、钒、硒的含量测定分析,确证了壳寡糖与元素的结合形式。
在细胞水平上,通过形态学观察、MTT比色法、放射免疫等方法进行不同分子量、不同脱乙酰度的系列壳寡糖及其配合物对大鼠原代培养的胰岛细胞和胰岛β细胞系NIT-1细胞的增殖及促胰岛素分泌作用的试验,以及对其细胞毒性的影响;通过形态学观察、MTT比色法、DNA凝胶电泳、彗星电泳等方法研究不同分子量、不同脱乙酰度的系列壳寡糖及其配合物对链脲佐菌素损伤胰岛β细胞的保护和抑制细胞凋亡的作用。
实验结果表明,不同分子量以及不同脱乙酰度的系列壳寡糖及其配合物均能明显促进大鼠胰岛β细胞的生长。低分子量高脱乙酰度的壳寡糖在一代生长期内能显著促进胰岛β细胞的生长,在刺激大鼠胰岛β细胞胰岛素的释放方面也效果显著。另外,以STZ处理的NIT-1细胞符合细胞凋亡的形态学特征, DNA发生了断裂,形成凋亡细胞特有的DNA改变;低分子量、高脱乙酰度的壳寡糖可以抑制链脲佐菌素诱导的胰岛β细胞凋亡,对受损的胰岛β细胞有显著的保护作用。细胞毒性实验结果表明,壳寡糖及其配合物是安全可靠的生物制品,为今后壳寡糖及其配合物应用于糖尿病的研究提供了理论依据。
采用一次性腹腔内注射(ip)STZ制备糖尿病大鼠模型,随机分成糖尿病治疗组、糖尿病对照组、正常对照组。治疗组分高、中、低剂量三组分别按每日250、500、1500 mg·kg-1灌胃壳寡糖水溶液,糖尿病对照组、正常组灌胃同体积的水连续60d,每10d测一次血糖值。60d后:①做糖耐量试验,按2.5g·kg-1体重灌胃(ig)葡萄糖水溶液,测定各组大鼠0h、0.5h、1h、2h血糖值;②对DM大鼠血清中抗氧化、血脂、肝功能及肾功能等指标进行测定;③对胰、肝、脾、肾、胸腺等脏器称重,计算器官系数;④对胰腺、肝、肾脏、骨骼肌做病理组织学检查;⑤测定肝、肌糖原含量;⑥对大鼠肠道菌群培养,鉴定,计算B/E值。
实验结果表明,不同剂量的壳寡糖均能不同程度的改善糖尿病大鼠的体重减轻、多饮、多食症状;降低2h血糖值和尿糖;改善葡萄糖耐量。中剂量组餐后2h血糖值降至16.14mmol·L-1,降糖率达到47.48%,糖耐量曲线下面积(AUC)为68.69%与正常组比较有极显著差异(P<0.01)。壳寡糖各剂量组对总抗氧化能力,SOD活力均有显著改善,MDA浓度越少。中剂量组载脂蛋白B质量浓度和载脂A1/载脂B值与阴性对照组比较有显著差异;中、高剂量组可以显著降低血清中尿素氮、谷丙转氨酶、谷草转氨酶、总胆红素、间接胆红素、N-乙酰氨基葡萄糖苷酶及尿液中尿素氮/肌酐、总蛋白/肌酐、N-乙酰氨基葡萄糖苷酶/肌酐的水平,并提高血清中总蛋白、白蛋白、球蛋白、白球比、谷草/谷丙比值;不同剂量的壳寡糖对胰腺、肝、肾脏均有保护作用,可以增加胰腺指数,降低肝、肾脏指数。病理学组织检查表明壳寡糖组均能不同程度地减轻胰岛萎缩、数量减少、胰岛细胞丢失、核固缩等胰岛细胞退行性改变症状。PAS染色显示,壳寡糖各组肝索排列整齐,脂变程度低,骨骼肌和肝组织糖原比模型组明显增多。壳寡糖组可以使大肠肝菌和肠球菌数量下降,乳酸杆菌和双歧杆菌的数量上升, B/E值升高。
结论:壳寡糖能改善糖尿病大鼠的体重减轻、多饮、多食等症状,降低2h血糖值和尿糖,改善葡萄糖耐量;调节糖尿病大鼠血脂和提高其机体抗氧化能力;对胰腺、肝脏、肾脏均有保护作用;对肠道微生态均有调节作用;降低骨骼肌和肝组织的糖原输出性的作用可能是其降血糖机制之一。
The diabetes mellitus (DM) is a kind of endocrine metabolism disease, having the heredity tendency caused by absolute or opposite hyposecretion of insulin. The high plasma glucose and urine glucose are it’s main characteristics, which usually leads to the metabolize obstacle of sugar, protein and fat, also many organ systems have significant long-term complications in such things as, cardiovascular, cerebrovascular, retinopathy, nephropathy and neuropathy, result in the very big bane. Diabetes mellitus is a disease with high prevalence all over the world that grows almost at epidemic rates. The long-term tissue complications that affect small and large blood vessels are directly connected with the time that the patients suffer of hyperglycemia. Diabetes has become the third chronic uncontagious disease, which threatens the human health seriously after malignancy, cardiovascular and cerebrovascular problems. Chitooligosaccharides(COS) can be obtained by degradation of chitosan, which has the less molecular weight than a chitosan, it dissolves easily in water so it can be absorbed and utilized by the human body, animals and plant organisms. It has been found through research, that the chitooligosaccharides not only has familiar characteristics with chitosan,but also has the other physiology activities and pharmacological effects, such as improving the organism’s immunity, regulating plasma liquid and the effects of antitumor etc. COS possess various biological activities and have a considerable potential to be utilized in diabetes mellitus applications.
Being treated with acetic anhydride under homogeneous conditions,chitosan(D.D.=90%)can be used to prepare 50%、70% degree of deacetylation chitosan by controlled partial N-acetylationmethod.Then soluble chitooligosaccharides were prepared by enzymatic hydrolysis of chitosan with the chitosanase. one had the different molecular weight, and the other had the same molecular weight but different degree of deacetylation.What’s more the preparations of COS-Cr,COS-VO,COS-Se had been studied. The characterization results of UV ,FT/IR, EA indicated that Cr3+、VO2+、SeO32- are attached to C2 amino and C6 hydroxyl hydroxyl group of chitooligosaccharides.
In vitro,the effect of varying molecular and degree of deacetylation COS and COS-Cr,COS-VO,COS-Se on the proliferation of pancreatic islet cells and the release of insulin were detected with optical microscope、MTT colorimetric assay、radioimmunoassay methods respectively. In addition to, we studied the cytotoxicity of different samples. What’s more ,the protective effect of varying molecular and degree of deacetylation COS and COS-Cr,COS-VO,COS-Se on the DNA damage of pancreasticβcells induced by streptozotocin were detected with optical microscope、MTT colorimetric assay、DNA gel electrophoresis and Comet assay methods respectively.
The results manifest that varying molecular and degree of deacetylation COS and COS-Cr,COS-VO,COS-Se can stimulate the growth of islet cell which has outstanding molecular-dependence. These different molecular and degree of deacetylation samples all can stimulate the isletβcell release insulin. Our study indicate that lower molecular weight and 90% degree of deacetylation COS ,can improve the overgrowth of pancreatic isletβcells very much ,and which also leads to a increase in insulin secretion of pancreatic cells.The cytotoxicity experiment demonstrates that COS and COS-Cr,COS-VO,COS-Se is security.
The results show that long time exposure to STZ is capable of causing a significant increase in DNA damage levels. Conversely, no induction of DNA damage was observed in lower molecular weight and 90% degree of deacetylation COS treated group(500 mg·L-1) in vitro treatment of NIT-1 cell lines. Similar differences in DNA damage between other COS groups and positive control group were observed after treatments with STZ ,2mM . The results indicate that the lower molecular weight and 90% degree of deacetylation COS has prominent protective effect on the NIT-1 cells induced by STZ and can prohibit the apoptosis of NIT-1 cells.
The diabetic model of Wistar rats was duplicated with STZ once intraperitoneally(65mg·kg-1), then divided into diabetes treating groups ,diabetes control groups and normol groups randomly. The diabetes treating groups were administered (ig) chitooligosaccharides at dose of 250、500、1500 mg·kg-1 daily for successive 60d. Each group measured the plasma glucose every 10 days. after 60 days feeding study:①oral glucose tolerance test was examined;②the effects of COS on regulating plasma lipid and antioxidant ability of DM mouse were measured ,the index about the function of kidney、liver in plasma and urine were evaluated;③weight of pancreas、thymus、spleen、liver、kidney were measured and calculated the coefficient of organs;④pancreas、liver、kidney and muscle were examined by pathlogic methods;⑤The hepatic and muscular glycogen levels were detected by anthrone;⑥the intestinal flora were cultured、calculated and identified,the counts of B/E were also calculated.
All dose of chitooligosaccharides have effective effects on the general clinical symptom of DM rats,decrease the 2hPG and urine glucose, normalize the disorders of glucose tolerance.Chitooligosaccharides of 500 mg·kg-1 daily decrease the plasma glucose to 16.14mmol·L-1,the decrease rate is 47.48%,and the AUC (Areu Under Curve) in oral glucose tolerance is merely 68.69%, compare with DM group have significant difference(P<0.01).All dose of chitooligosaccharides improve the capability of T-AOC and activity of SOD,decrease the content of MDA drastically, and have significant difference(P<0.01)compare with DM group. All dose of chitooligosaccharides decrease the level of TG,ApoB and A/B, have significant difference compare with DM group. High and middle dose of chitooligosaccharides decrease the level of BUN、ALT、AST、TBIL、IBIL、NAG in plasma and BUN/Cr、TP/Cr、NAG/Cr in urine,increase the level of TP、ALB、GLO、A/G、AST/ALT, compare with DM group have significant difference. All dose of chitooligosaccharides have protective effects on the pancreastic islet、kidney、liver,increase the coefficient of pancreas and decrease the coefficient of kidney and liver. Morphological investigation on pancreas show different dose of chitooligo saccharides had different effects on the reduction of islets,loss of pancreastic cells,nuclear pyknosis of pancreastic cells,etc. The normal liver structure and abundant glycogen are observed in the treatment group .There are obvious liver fatty degeneration and little or no glycogen in diabetes group. After administration with chitooligo- saccharides,the numbers of Bifidobacteria and Lactobacillus increase significantly,but the numbers of pathogenetic E.coli and Enterococcus decrease significantly, compare with DM group.The counts of B/E also increase significantly accordingly.
In conclusion, chitooligosaccharides has effective effects on the general clinical symptom, 2hPG and urine glucose, disorders of glucose tolerance, regulating plasma lipid and improving antioxidant ability , normalize the index about the function of kidney and liver in plasma and urine, protecting the pancreastic islet、kidney and liver of DM rats. The effects of increase the glycogen storage of liver and muscle, maybe one of the mechanism about chitooligosaccharides’effects on decreasing the plasma glucose.
壳寡糖(chitooligosaccharides,COS)是壳聚糖的降解产物,分子量小,溶于水,可被人体、动物及植物机体吸收利用。研究发现,壳寡糖和壳聚糖不仅具有一些相似的性质,而且一些生理活性或功能性质更加显著,如作为功能性食品具有提高机体免疫、抗肿瘤、调节血脂、抗感染、防治病原生物感染、促进双歧杆菌生长等功能,尤其是在糖尿病的防治研究方面有潜在的应用价值。
本论文首先在均相条件下,以醋酸酐为乙酰化试剂对壳聚糖〔脱乙酰度90%)进行N-乙酰化反应,制备出脱乙酰度50%和70%左右的壳聚糖。通过壳聚糖酶降解壳聚糖制备出水溶性不同分子量和不同脱乙酰度的系列壳寡糖。并以壳寡糖为原料,制备出了含有铬、钒、硒的壳寡糖配合物,并通过紫外光谱(UV)、红外光谱(FT/IR)、元素分析(EA〕和铬、钒、硒的含量测定分析,确证了壳寡糖与元素的结合形式。
在细胞水平上,通过形态学观察、MTT比色法、放射免疫等方法进行不同分子量、不同脱乙酰度的系列壳寡糖及其配合物对大鼠原代培养的胰岛细胞和胰岛β细胞系NIT-1细胞的增殖及促胰岛素分泌作用的试验,以及对其细胞毒性的影响;通过形态学观察、MTT比色法、DNA凝胶电泳、彗星电泳等方法研究不同分子量、不同脱乙酰度的系列壳寡糖及其配合物对链脲佐菌素损伤胰岛β细胞的保护和抑制细胞凋亡的作用。
实验结果表明,不同分子量以及不同脱乙酰度的系列壳寡糖及其配合物均能明显促进大鼠胰岛β细胞的生长。低分子量高脱乙酰度的壳寡糖在一代生长期内能显著促进胰岛β细胞的生长,在刺激大鼠胰岛β细胞胰岛素的释放方面也效果显著。另外,以STZ处理的NIT-1细胞符合细胞凋亡的形态学特征, DNA发生了断裂,形成凋亡细胞特有的DNA改变;低分子量、高脱乙酰度的壳寡糖可以抑制链脲佐菌素诱导的胰岛β细胞凋亡,对受损的胰岛β细胞有显著的保护作用。细胞毒性实验结果表明,壳寡糖及其配合物是安全可靠的生物制品,为今后壳寡糖及其配合物应用于糖尿病的研究提供了理论依据。
采用一次性腹腔内注射(ip)STZ制备糖尿病大鼠模型,随机分成糖尿病治疗组、糖尿病对照组、正常对照组。治疗组分高、中、低剂量三组分别按每日250、500、1500 mg·kg-1灌胃壳寡糖水溶液,糖尿病对照组、正常组灌胃同体积的水连续60d,每10d测一次血糖值。60d后:①做糖耐量试验,按2.5g·kg-1体重灌胃(ig)葡萄糖水溶液,测定各组大鼠0h、0.5h、1h、2h血糖值;②对DM大鼠血清中抗氧化、血脂、肝功能及肾功能等指标进行测定;③对胰、肝、脾、肾、胸腺等脏器称重,计算器官系数;④对胰腺、肝、肾脏、骨骼肌做病理组织学检查;⑤测定肝、肌糖原含量;⑥对大鼠肠道菌群培养,鉴定,计算B/E值。
实验结果表明,不同剂量的壳寡糖均能不同程度的改善糖尿病大鼠的体重减轻、多饮、多食症状;降低2h血糖值和尿糖;改善葡萄糖耐量。中剂量组餐后2h血糖值降至16.14mmol·L-1,降糖率达到47.48%,糖耐量曲线下面积(AUC)为68.69%与正常组比较有极显著差异(P<0.01)。壳寡糖各剂量组对总抗氧化能力,SOD活力均有显著改善,MDA浓度越少。中剂量组载脂蛋白B质量浓度和载脂A1/载脂B值与阴性对照组比较有显著差异;中、高剂量组可以显著降低血清中尿素氮、谷丙转氨酶、谷草转氨酶、总胆红素、间接胆红素、N-乙酰氨基葡萄糖苷酶及尿液中尿素氮/肌酐、总蛋白/肌酐、N-乙酰氨基葡萄糖苷酶/肌酐的水平,并提高血清中总蛋白、白蛋白、球蛋白、白球比、谷草/谷丙比值;不同剂量的壳寡糖对胰腺、肝、肾脏均有保护作用,可以增加胰腺指数,降低肝、肾脏指数。病理学组织检查表明壳寡糖组均能不同程度地减轻胰岛萎缩、数量减少、胰岛细胞丢失、核固缩等胰岛细胞退行性改变症状。PAS染色显示,壳寡糖各组肝索排列整齐,脂变程度低,骨骼肌和肝组织糖原比模型组明显增多。壳寡糖组可以使大肠肝菌和肠球菌数量下降,乳酸杆菌和双歧杆菌的数量上升, B/E值升高。
结论:壳寡糖能改善糖尿病大鼠的体重减轻、多饮、多食等症状,降低2h血糖值和尿糖,改善葡萄糖耐量;调节糖尿病大鼠血脂和提高其机体抗氧化能力;对胰腺、肝脏、肾脏均有保护作用;对肠道微生态均有调节作用;降低骨骼肌和肝组织的糖原输出性的作用可能是其降血糖机制之一。
The diabetes mellitus (DM) is a kind of endocrine metabolism disease, having the heredity tendency caused by absolute or opposite hyposecretion of insulin. The high plasma glucose and urine glucose are it’s main characteristics, which usually leads to the metabolize obstacle of sugar, protein and fat, also many organ systems have significant long-term complications in such things as, cardiovascular, cerebrovascular, retinopathy, nephropathy and neuropathy, result in the very big bane. Diabetes mellitus is a disease with high prevalence all over the world that grows almost at epidemic rates. The long-term tissue complications that affect small and large blood vessels are directly connected with the time that the patients suffer of hyperglycemia. Diabetes has become the third chronic uncontagious disease, which threatens the human health seriously after malignancy, cardiovascular and cerebrovascular problems. Chitooligosaccharides(COS) can be obtained by degradation of chitosan, which has the less molecular weight than a chitosan, it dissolves easily in water so it can be absorbed and utilized by the human body, animals and plant organisms. It has been found through research, that the chitooligosaccharides not only has familiar characteristics with chitosan,but also has the other physiology activities and pharmacological effects, such as improving the organism’s immunity, regulating plasma liquid and the effects of antitumor etc. COS possess various biological activities and have a considerable potential to be utilized in diabetes mellitus applications.
Being treated with acetic anhydride under homogeneous conditions,chitosan(D.D.=90%)can be used to prepare 50%、70% degree of deacetylation chitosan by controlled partial N-acetylationmethod.Then soluble chitooligosaccharides were prepared by enzymatic hydrolysis of chitosan with the chitosanase. one had the different molecular weight, and the other had the same molecular weight but different degree of deacetylation.What’s more the preparations of COS-Cr,COS-VO,COS-Se had been studied. The characterization results of UV ,FT/IR, EA indicated that Cr3+、VO2+、SeO32- are attached to C2 amino and C6 hydroxyl hydroxyl group of chitooligosaccharides.
In vitro,the effect of varying molecular and degree of deacetylation COS and COS-Cr,COS-VO,COS-Se on the proliferation of pancreatic islet cells and the release of insulin were detected with optical microscope、MTT colorimetric assay、radioimmunoassay methods respectively. In addition to, we studied the cytotoxicity of different samples. What’s more ,the protective effect of varying molecular and degree of deacetylation COS and COS-Cr,COS-VO,COS-Se on the DNA damage of pancreasticβcells induced by streptozotocin were detected with optical microscope、MTT colorimetric assay、DNA gel electrophoresis and Comet assay methods respectively.
The results manifest that varying molecular and degree of deacetylation COS and COS-Cr,COS-VO,COS-Se can stimulate the growth of islet cell which has outstanding molecular-dependence. These different molecular and degree of deacetylation samples all can stimulate the isletβcell release insulin. Our study indicate that lower molecular weight and 90% degree of deacetylation COS ,can improve the overgrowth of pancreatic isletβcells very much ,and which also leads to a increase in insulin secretion of pancreatic cells.The cytotoxicity experiment demonstrates that COS and COS-Cr,COS-VO,COS-Se is security.
The results show that long time exposure to STZ is capable of causing a significant increase in DNA damage levels. Conversely, no induction of DNA damage was observed in lower molecular weight and 90% degree of deacetylation COS treated group(500 mg·L-1) in vitro treatment of NIT-1 cell lines. Similar differences in DNA damage between other COS groups and positive control group were observed after treatments with STZ ,2mM . The results indicate that the lower molecular weight and 90% degree of deacetylation COS has prominent protective effect on the NIT-1 cells induced by STZ and can prohibit the apoptosis of NIT-1 cells.
The diabetic model of Wistar rats was duplicated with STZ once intraperitoneally(65mg·kg-1), then divided into diabetes treating groups ,diabetes control groups and normol groups randomly. The diabetes treating groups were administered (ig) chitooligosaccharides at dose of 250、500、1500 mg·kg-1 daily for successive 60d. Each group measured the plasma glucose every 10 days. after 60 days feeding study:①oral glucose tolerance test was examined;②the effects of COS on regulating plasma lipid and antioxidant ability of DM mouse were measured ,the index about the function of kidney、liver in plasma and urine were evaluated;③weight of pancreas、thymus、spleen、liver、kidney were measured and calculated the coefficient of organs;④pancreas、liver、kidney and muscle were examined by pathlogic methods;⑤The hepatic and muscular glycogen levels were detected by anthrone;⑥the intestinal flora were cultured、calculated and identified,the counts of B/E were also calculated.
All dose of chitooligosaccharides have effective effects on the general clinical symptom of DM rats,decrease the 2hPG and urine glucose, normalize the disorders of glucose tolerance.Chitooligosaccharides of 500 mg·kg-1 daily decrease the plasma glucose to 16.14mmol·L-1,the decrease rate is 47.48%,and the AUC (Areu Under Curve) in oral glucose tolerance is merely 68.69%, compare with DM group have significant difference(P<0.01).All dose of chitooligosaccharides improve the capability of T-AOC and activity of SOD,decrease the content of MDA drastically, and have significant difference(P<0.01)compare with DM group. All dose of chitooligosaccharides decrease the level of TG,ApoB and A/B, have significant difference compare with DM group. High and middle dose of chitooligosaccharides decrease the level of BUN、ALT、AST、TBIL、IBIL、NAG in plasma and BUN/Cr、TP/Cr、NAG/Cr in urine,increase the level of TP、ALB、GLO、A/G、AST/ALT, compare with DM group have significant difference. All dose of chitooligosaccharides have protective effects on the pancreastic islet、kidney、liver,increase the coefficient of pancreas and decrease the coefficient of kidney and liver. Morphological investigation on pancreas show different dose of chitooligo saccharides had different effects on the reduction of islets,loss of pancreastic cells,nuclear pyknosis of pancreastic cells,etc. The normal liver structure and abundant glycogen are observed in the treatment group .There are obvious liver fatty degeneration and little or no glycogen in diabetes group. After administration with chitooligo- saccharides,the numbers of Bifidobacteria and Lactobacillus increase significantly,but the numbers of pathogenetic E.coli and Enterococcus decrease significantly, compare with DM group.The counts of B/E also increase significantly accordingly.
In conclusion, chitooligosaccharides has effective effects on the general clinical symptom, 2hPG and urine glucose, disorders of glucose tolerance, regulating plasma lipid and improving antioxidant ability , normalize the index about the function of kidney and liver in plasma and urine, protecting the pancreastic islet、kidney and liver of DM rats. The effects of increase the glycogen storage of liver and muscle, maybe one of the mechanism about chitooligosaccharides’effects on decreasing the plasma glucose.
引文
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