小檗碱对D-半乳糖诱导糖基化模型大鼠脑损害的干预作用

背景:蛋白糖基化反应是糖尿病的发生机制之一,探讨小檗碱对体内蛋白糖基化反应的抑制作用以及对蛋白糖基化造成脑细胞损害的保护作用将有助于糖尿病相关疾病的治疗。目的:观察小檗碱对D-半乳糖诱导的糖基化模型大鼠并发脑损害的干预作用。设计:随机分组设计、对照动物实验。单位:厦门中医院眼科。材料:实验于2005-06/10在暨南大学药学院药理实验室完成。选择6周龄SD大鼠90只。按随机数字表法分为4组,正常对照组、模型组、盐酸氨基胍组和小檗碱高(300mg/kg)、中(150mg/kg)、低(75mg/kg)剂量组,每组15只。采用腹腔注射D-半乳糖诱导糖基化模型。实验用药:小檗碱(广东万基药业有限公司);D-半乳糖(上海源聚生物科技有限公司)。方法:正常对照组腹腔注射生理盐水,持续8周;其他组动物每天腹腔注射5%D-半乳糖(150mg/kg),持续8周。第3周开始,盐酸氨基胍组开始灌胃盐酸氨基胍(150mg/kg),小檗碱组分别灌胃相应剂量的小檗碱,正常对照组和模型组动物均灌胃蒸馏水,连续给药6周。灌胃容积为10mL/kg。第8周末采用考马斯亮蓝法测定红细胞醛糖还原酶活性,采用硫代巴比妥酸比色法测定糖化血红蛋白含量,采用硝基四氮唑蓝比色法测定血清果糖胺。测定血清中晚期糖基化终末产物含量及脑组织中晚期糖基化终末产物、丙二醛含量、超氧化物歧化酶活性及脑神经细胞内钙离子水平,并以透射电镜观察脑内海马神经元线粒体的变化。主要观察指标:①血清中晚期糖基化终末产物、糖化血红蛋白、果糖胺含量,红细胞醛糖还原酶活性。②脑组织中晚期糖基化终末产物含量。③脑神经细胞内钙离子水平。④脑组织中丙二醛含量、超氧化物歧化酶活性。⑤脑内海马神经元线粒体结构变化。结果:纳入动物90只,均进入结果分析。①血清中红细胞醛糖还原酶活性和糖化产物含量:D-半乳糖处理8周后,模型组大鼠红细胞醛糖还原酶活性和果糖胺、糖化血红蛋白、晚期糖基化终末产物水平高于正常对照组(P<0.01);小檗碱高、中剂量组处理6周后,红细胞醛糖还原酶活性和果糖胺、糖化血红蛋白(每10g血红蛋白的吸光度值)、晚期糖基化终末产物水平均低于模型组分别为(1.07±0.39),(1.22±0.47),(1.76±0.30)nkat/g,t=5.052,5.484,P<0.01;(0.740±0.142),(0.862±0.131),(0.958±0.083)mmol/L,t=7.829,P<0.01,t=2.404,P<0.05;58.434±12.135,64.614±13.418,83.747±7.990,t=4.922,6.748,P<0.01;(3.104±0.814),(2.937±0.514),(4.156±0.860)U/mg,t=4.104,3.440,P<0.05];小檗碱低剂量组红细胞醛糖还原酶活性低于模型组(P<0.05),对糖化产物未见明显影响。②脑组织内晚期糖基化终末产物含量:盐酸氨基胍组、小檗碱高、中剂量组大鼠脑组织内晚期糖基化终末产物含量低于模型组分别为(10.52±1.22),(10.95±1.75),(11.95±2.27),(14.26±3.51)U/mg,t=-3.892,-3.263,P<0.01,t=-2.139,P<0.05],小檗碱低剂量处理影响不明显(P>0.05)。③脑神经细胞内钙离子水平:盐酸氨基胍组、小檗碱高剂量组大鼠脑组织内脑神经细胞内钙离子水平低于模型组分别为(271.52±32.71),(293.84±31.58),(337.15±58.49)nmol/L,t=-3.421,P<0.01,t=-2.275,P<0.05],小檗碱低剂量处理影响不明显(P>0.05)。④脑组织内丙二醛含量和超氧化物歧化酶活性:盐酸氨基胍组、小檗碱高、中剂量组脑组织内丙二醛含量显著低于模型组,超氧化物歧化酶活性显著高于模型组分别为(2.09±0.16),(2.12±0.22),(2.41±0.12),(2.54±0.21)μmol/g,t=6.601,5.348,P<0.01,t=2.082,P<0.05;(8.79±1.09),(8.80±1.52),(7.90±1.48),(6.48±1.34)mkat/g,t=4.571,4.254,P<0.01,t=2.226,P<0.05]。⑤脑内海马神经元线粒体结构:透射电镜显示,模型组大鼠脑海马细胞线粒体出现明显肿胀,线粒体嵴断裂,结构紊乱,甚至出现明显的大空泡。盐酸氨基胍组和小檗碱高、中剂量组线粒体肿胀不明显,仅有少量线粒体出现小空泡,小檗碱低剂量组线粒体肿胀明显,嵴断裂,结构紊乱,并有空泡出现。结论:D-半乳糖诱导的糖基化模型大鼠脑线粒体损害可能与脑组织中晚期糖基化终末产物形成、脑细胞内钙稳态失调以及氧化应激有关,小檗碱具有抑制D-半乳糖诱导的蛋白糖基化反应,并对糖基化状态并发的脑神经细胞损害具有保护作用。

Effect of berberine on the brain damage of glycated rats induced by D-galactose

BACKGROUND: As the damage caused by protein glycation is one of the mechanisms of diabetes, it is helpful to treat diabetes related diseases with the understanding of the inhibition of berberine on protein glycation and the protection to the brain damage caused by protein glycation.OBJECTIVE: To observe the effects of berberine on glycated brain damages induced by D-galactose in model rats.DESIGN: Randomly grouping paralleled control study.SETTING: Department of Ophthalmology, Xiamen Traditional Chinese Medicine Hospital.MATERIALS: The experiment was conducted in the Department of Pharmacology, Pharmacy College of Jinan University from June to October 2005. Ninety SD rats (6 weeks old) were selected and divided into 6 groups: control group, model group, hydrochloride aminoguanidine group and high (300 mg/kg), middle (150 mg/kg) and low (75 mg/kg) doses berberine groups with 15 rats in each group. The glycated models were established by intraperitoneal injection of D-galactose. The main drugs:berberine was from Guangzhou Wanji Drugs Limited Company; D-galactose was from Shanghai Yuanju Bioscience Technology Limited Company.METHODS: The rats in the control group were intraperitoneally injected the normal saline for 8 weeks; rats in other groups were injected 5%D-galactose (150 mg/kg) for 8 weeks. From the 3rd week, the hydrochloride aminoguanidine group was infused hydrochloride aminoganidine (150 mg/kg); the three doses berberine groups were given corresponding doses berberine; the control group and model group were given distilled water for 6 weeks with the volume of 10 mL/kg. At the end of the 8th week, the erythrocyte aldose reductase activity was determined by coomassie brilliant blue method; the level of plasma glycohemoglobin was measured by thio-barbituric acid colorimetry and the fructosamine in serum was measured by nitroblue tetrazolium colorimetry. The quantity of advanced glycation end products (AGEs) in serum, and AGEs, malondialdehyde (MDA), and activity of superoxide edismutase (SOD) in brain tissue and calcium ion in neurons were also dertermined. Moreover, the changes of mitochondria in brain hippocampus cells were observed under electronic microscope.MAIN OUTCOME MEASURES: ① The AGEs, plasma glycohemoglobin, serum fructosamine and aldose reductase activity. ②AGEs in brain tissues. ③Calcium level in brain. ④MDA content and SOD activity in brain tissues. ⑤Changes of mitochondria in hippocampus neurons.RESULTS: All 90 animals were involved in the result analysis. ①Aldose reductase activity and glycated product content in serum: After the rats were treated with D-galactose for 8 weeks, the aldose reductase activity in red blood cells and the content of fructosamine in serum, glycohemoglobin,AGEs in the model group were significantly higher than those in the normal control group (P ＜ 0.01); After treated by high and middle doses berberine for 6 weeks, the activity of aldose reductase and content of fructosamine in serum (absorbancevalue of hemoglobin every 10 g), glycohemoglobin, and AGEs were obviously lower than those in the control group (1.07±0.39), (1.22±0.47), (1.76±0.30) nkat/g, t=5.052, 5.484, P ＜ 0.01;(0.740±0.142), (0.862±0.131), (0.958±0.083) mmol/L, t=7.829, P ＜ 0.01,t=2.404, P ＜ 0.05; 58.434±12.135, 64.614±13.418, 83.747±7.990,t=4.922, 6.748, P ＜ 0.01; (3.104±0.814), (2.937±0.514), (4.156±0.860) U/mg,t=4.104, 3.440, P ＜ 0.05]; the aldose reductase activity of the low dose berberine group was lower than the model group (P ＜ 0.05), which had no obvious effect on glycated products. ②AGEs in brain tissues: The contents in the hydrochloride aminoganidine group, high and middle doses berberine groups were lower than the model group (10.52±1.22), (10.95±1.75),(11.95±2.27), (14.26±3.51) U/mg, t=-3.892, -3.263, P ＜ 0.01, t=-2.139,P ＜ 0.05], and the low dose berberine had little effect (P ＞ 0.05). ③Calcium level in neurons: The levels in the hydrochloride aminoganidine group,and high dose berberine groups were lower than the model group.(271.52±32.71), (293.84±31.58), (337.15±58.49) nmol/L, t=-3.421, P＜ 0.01, t=-2.275, P ＜ 0.05], the low dose berberine group had no obvious effect (P ＞ 0.05). ④MDA content and SOD activity in brain tissues: MDA contents in the hydrochloride aminoganidine group, high and middle doses berberine groups were lower than the model group, and the SOD activity was markedly higher than the model group (2.09±0.16), (2.12±0.22),(2.41±0.12), (2.54±0.21) μmol/g, t=6.601, 5.348, P ＜ 0.01, t=2.082, P＜ 0.05; (8.79±1.09), (8.80±1.52), (7.90±1.48), (6.48±1.34) mkat/g, t=4.571,4.254, P ＜ 0.01, t=2.226, P ＜ 0.05]. ⑤Mitochondria structure in brain hippocampus cells: Under the electronic microscope, mitochondria in brain hippocampus cells of the model group appeared obvious swelling with broken crests and disorganized structure, even obvious big vacuoles were observed. In the hydrochloride aminoganidine, and high and middle doses berberine groups, no obvious swelling was observed with vacuoles only in a few mitochondria. Nevertheless, obvious swelling appeared in mitochondria of low dose berberine group with broken crest and disorganized structure,and vacuoles were observed.CONCLUSION: D-galactose-induced damage in mitochondria may be related to AGEs formation in brain tissue, maladjustment of calcium ions in neurons and oxidative stress in rat models. Berberine can inhibit glycation induced by D-galactose and protect rat brain tissues from glycated damage.