【作者】 朱威；

【导师】 胡福良；

【作者基本信息】 浙江大学 ， 特种经济动物饲养， 2010， 博士

【摘要】 糖尿病是一种慢性、全身性、代谢性疾病,主要是由于体内胰岛素分泌绝对或相对不足引起,表现为血糖升高,伴以脂肪、蛋白质、水、电解质代谢紊乱。糖尿病发病机制复杂,能引发多种血管并发症。目前市场上的药物不能完全有效地达到治疗目的,且治疗费用昂贵,存在一定的副作用。研究人员试图寻找有效的天然产物以改善糖尿病症状,同时达到减少副作用,降低治疗费用的目的。蜂胶是蜜蜂采集的一种树脂类混合物,其成分复杂,目前已知蜂胶所含成分超过300种,主要包括黄酮类、酚类、芳香族类、有机酸类、酯类、醛类、醇类、烯烃类、萜类和挥发性成分等。蜂胶具有免疫调节、抗氧化、抗炎、护肝、抗肿瘤、抑制病原微生物等多种生物学活性。我们先前的研究也证明蜂胶能有效降低糖尿病模型大鼠血糖,改善氧化应激。蜂胶的生物学活性受植物来源、采集地点、采集蜂种和提取方法等多种因素影响,但是化学成分的差异是造成蜂胶活性变化的主要原因。巴西蜂胶(绿蜂胶)主要胶源植物是Baccharis dracunculifolia DC,其主要的活性成分是异戊二烯-对-香豆酸及其衍生物,而中国蜂胶(杨树型蜂胶)的主要活性成分为黄酮类化合物。巴西蜂胶和中国蜂胶成分的差异可能导致两种蜂胶对糖尿病症状的改善效果存在差异。在本研究中,我们利用链脲霉素构建1型糖尿病(T1DM)大鼠模型,链脲霉素+高脂饲料构建2型糖尿病(T2DM)大鼠模型,通过研究大鼠尿液、血液和肝肾生化指标及肝肾组织病理变化,比较中国蜂胶和巴西蜂胶改善糖尿病大鼠糖代谢、脂质代谢、蛋白质代谢、氧化应激和肝肾功能的效果。同时测定T2DM大鼠肾组织内促炎细胞因子IL-2、IL-6、TNF-α及MCP-1 mRNA表达,测定PKCmRNA表达以及TGF-β1表达,测定大鼠肾脏血流动力学指标,探讨蜂胶改善糖尿病肾病可能的作用机理。实验结果如下：1.蜂胶改善T1DM大鼠的效果以拜糖平为阳性药(1mg/100g,每天两次),观察中国蜂胶和巴西蜂胶(设定高、低剂量组,分别为10mg/100g和5mg/100g,每天两次)改善T1DM模型大鼠的效果。正常组和模型组大鼠给予生理盐水(1mg/100g,每天两次)。实验持续8周。1)体重和糖代谢：中国蜂胶和巴西蜂胶均能明显减缓T1DM大鼠体重下降以及血糖升高,呈现剂量效应。在相同剂量下,中国蜂胶效果略优于巴西蜂胶；中国蜂胶能显著降低T1DM大鼠血清HbAlc水平,呈现剂量效应,巴西蜂胶效果不明显；2)脂质代谢：与正常组大鼠相比,模型组大鼠血清TG和TC含量显著增加。高剂量中国蜂胶能明显降低T1DM大鼠血清TC水平；3)血液氧化应激：与正常组大鼠相比,模型组大鼠血清MDA、NO和NOS含量明显增加,而SOD和CAT含量明显下降。中国蜂胶和巴西蜂胶能改善T1DM大鼠氧化损伤。与模型组相比,高剂量中国蜂胶能显著降低大鼠血清MDA含量,低剂量中国蜂胶能有效提升血清SOD含量；巴西蜂胶能明显增加血清SOD含量,且低剂量巴西蜂胶能显著降低血清MDA含量。巴西蜂胶还能有效降低血清NOS含量；4)肝功能和氧化应激：与正常组相比,模型组大鼠血清ALT和AST含量显著增加,并出现氧化应激现象。中国蜂胶和巴西蜂胶能显著降低血清ALT和AST含量,而阳性药效果不显著。中国蜂胶显著提升肝脏GSH-px,但对肝脏MDA含量影响不显著,而巴西蜂胶能全面恢复肝脏各类抗氧化酶含量,抑制MDA产生。肝脏组织切片HE染色结果表明,与正常组大鼠相比,模型组大鼠肝脏病变程度并不严重,而各给药组大鼠肝组织形态基本维持正常；5)肾功能和氧化应激：与正常组相比,模型组大鼠BUN、尿液总蛋白、尿液白蛋白含量显著增加,而肾脏病理组织HE染色结果显示模型组大鼠肾脏肾小管上皮细胞空泡化,出现肾小管上皮管型,这表示糖尿病大鼠开始出现肾病症状。中国蜂胶和巴西蜂胶均能降低尿液中白蛋白含量,同时巴西蜂胶能抑制BUN和尿液总蛋白升高。观察蜂胶组大鼠肾脏组织切片发现,蜂胶能减少肾小管上皮细胞空泡化现象。对肾脏组织氧化应激水平检测发现,两种蜂胶均能有效提升肾脏CAT,抑制肾脏脂质过氧化并降低GSH-px含量。2.蜂胶改善T2DM大鼠的效果以卡司平为阳性药(1mg/100g,每天两次),观察中国蜂胶和巴西蜂胶(单剂量,5mg/100g大鼠体重,每天两次)改善T2DM模型大鼠的效果。正常组和模型组大鼠给予生理盐水(1mg/100g,每天两次)。实验维持8周。1)体重：中国蜂胶和巴西蜂胶均能减缓T2DM大鼠体重下降,但效果不显著,中国蜂胶在实验第8周能明显抑制实验大鼠体重下降；2)糖代谢和胰岛素：巴西蜂胶能降低糖尿病大鼠血糖含量,但效果不明显；中国蜂胶在实验前4周能显著降低血糖含量,而在实验后4周则不能显著降低血糖含量：阳性药卡司平在第2周能显著降低血糖含量,但在第8周却促进血糖升高。尿糖检测结果发现,蜂胶和阳性药在整个实验周期能有效降低尿糖含量。从HbAlc分析发现,阳性药能降低糖尿病大鼠HbAlc含量,但效果不明显：A组大鼠(饲喂中国蜂胶)和B组大鼠(饲喂巴西蜂胶)HbAlc含量在第4周比模型组大鼠分别低8.6%和10.2%,效果显著；实验第8周A组大鼠(饲喂中国蜂胶)HbAlc含量比模型组大鼠低5.3%,差异显著。T2DM大鼠空腹胰岛素含量与正常组大鼠相比无显著差异,中国蜂胶和巴西蜂胶对T2DM大鼠胰岛素含量无显著影响；3)蛋白质代谢：T2DM大鼠出现蛋白质代谢紊乱现象,中国蜂胶和巴西蜂胶均能改善T2DM大鼠蛋白质代谢紊乱现象,其中中国蜂胶能显著抑制糖尿病大鼠血清总蛋白和白蛋白下降,巴西蜂胶能显著抑制血清总蛋白下降：4)脂质代谢：与正常组大鼠相比,模型组大鼠HDL-C含量变化不显著,但是TC、TG和LDL-C含量则显著增加。中国蜂胶和阳性药对TC和HDL-C含量影响不明显,但在实验前期能有效降低LDL-C和TG含量,而在实验后期效果下降：5)血液氧化应激：中国蜂胶和巴西蜂胶对T2DM大鼠血清NO和NOS含量均无显著影响,但能明显降低MDA含量；中国蜂胶能显著提升CAT含量,巴西蜂胶则能显著提升GSH-px含量；6)肝功能：与正常组大鼠相比,模型组大鼠血清ALT含量显著高于正常大鼠,而AST含量在实验中期明显偏低,中国蜂胶和巴西蜂胶能有效降低糖尿病大鼠血清ALT含量,但对AST含量影响不显著。肝脏病理观察发现,模型组大鼠肝细胞浊肿,且大泡状脂滴空泡化,已有脂肪肝产生,但尚未产生肝纤维化,蜂胶和阳性药能减少糖尿病大鼠肝脏脂肪化程度,且蜂胶效果优于阳性药；7)肾功能和氧化应激：T2DM大鼠BUN、CCR及尿白蛋白排泄率明显高于正常大鼠,观察其肾脏病理发现,模型组大鼠肾脏肾小球体积增大,系膜细胞增生,肾小球基底膜增生严重,部分肾小管上皮细胞空泡化,出现肾小管上皮管型,大量PAS阳性物质染成红色。蜂胶和阳性药能显著降低BUN和尿液白蛋白含量,阳性药卡司平还能明显降低肾脏CCR值。蜂胶和阳性药可减轻T2DM引起的大鼠肾损伤。中国蜂胶能降低肾组织MDA含量并提升CAT含量,巴西蜂胶能降低MDA含量而提升GSH-px含量。此外,蜂胶和阳性药均能显著减少肾脏NO和NOS含量的增加。3.蜂胶保护T2DM大鼠肾脏的作用机理在实验第8周用激光多普勒血流仪测定T2DM模型大鼠肾脏血流动力学指标,实验结束后取T2DM模型大鼠肾脏组织,测定相关指标。1)炎症反应：中国蜂胶和巴西蜂胶均能降低T2DM大鼠血清CRP含量,巴西蜂胶效果显著,这表明蜂胶能改善T2DM大鼠炎症现象；中国蜂胶和巴西蜂胶均能减轻T2DM大鼠肾脏炎症程度,其中中国蜂胶能有效降低糖尿病大鼠肾脏中IL-2、IL-6和TNF-α含量,巴西蜂胶能有效抑制糖尿病大鼠肾脏中TNF-α含量的增加及MCP-1 mRNA表达；2)肾脏血流动力学：中国蜂胶能明显改善T2DM大鼠肾脏血流动力学异常现象,升高肾脏血流量,并降低肾脏血管阻力值和过滤分数,巴西蜂胶则效果不明显；3)PKC表达：与正常组相比,T2DM大鼠肾脏PKC mRNA表达明显增加。中国蜂胶和巴西蜂胶能明显抑制T2DM大鼠肾脏PKC mRNA表达；4)TGF-β1表达：免疫组化染色和图像分析结果显示,模型组大鼠肾脏TGF-β1表达明显高于正常组。蜂胶和阳性药能降低大鼠肾脏TGF-β1表达的总光密度、平均光密度和阳性指数,其中巴西蜂胶和阳性药效果显著,而中国蜂胶效果不显著,巴西蜂胶效果优于中国蜂胶而与与阳性药相当。综上所述,中国蜂胶和巴西蜂胶能有效改善T1DM和T2DM对大鼠造成的损伤。两种蜂胶均能抑制糖尿病大鼠体重下降,改善大鼠血液糖代谢、脂质代谢、蛋白代谢以及血液、肝、肾氧化应激。通过测定肝肾功能相关指标及组织切片观察结果表明,蜂胶能改善糖尿病大鼠肝肾功能。比较中国蜂胶和巴西蜂胶的效果发现,中国蜂胶对血糖的控制效果和肝脏的保护效果略优于巴西蜂胶,而巴西蜂胶改善氧化应激的效果要略优于中国蜂胶。同时,通过测定肾脏组织炎症反应、血流动力学、PKC和TGF-β1,初步探明了蜂胶改善糖尿病肾病的作用机理。更多还原

【Abstract】 Diabetes is a chronic disease that occurs when the pancreas does not produce enough insulin, or alternatively, when the body cannot effectively use the insulin it produces. Hyperglycaemia is a common effect of uncontrolled diabetes and over time leads to serious damage to many of the body’s systems, which results in various vascular complications such as retinopathy, neuropathy, kidney failure, heart disease and stroke. Due to the huge medical expenditure and complicated pathobiology of diabetes, also adverse effect of diabetes drug, the research has directed to herb medicine to improve glucose control and lower the risk of its complications.Propolis, a resinous substance collected from the buds of certain trees by bees, is a traditional herb medicine in many countries. More than 300 components have been found in propolis, mainly composed of phenolic compounds, terpenes and essential oil. Propolis has been proved various bioactivities of antibacterial, antivirus, antifungal, immunoloregulatory, anti-oxidative, anti-inflammatory,hepatoprotective and anti-tumor, Our previous studies have shown that Chinese propolis helps to reduce fast blood glucose, improve oxidative stress and lipid metablisom in alloxan-induced diabetic rats. In addition, propolis intake by diabetic patients are proved to reduce blood glucose and glycosylated hemoglobin (HbAlc) level.Some reports suggested the chemistry of propolis varied with botonical source, geographical origins and collecting bees. The botanical difference is a key factor for chemical variability. Chinese propolis contains many kinds of flavonoids and phenolic acid esters.The major components in of Brazilian green propolis are terpenoids and prenylated derivatives of pcoumaric.To guarantees the quality, safety and efficacy of propolis, we compared the antidiabetic effect of Chinese propolis and Brazilian green propolis in this experiment. We induced type 1 diabetes mellitus (T1DM) rat model by injection of streptozotcin, and type 2 diabetes mellitus (T2DM) rat model by injection of streptozotcin plus high lipid diet feeding. By determination of urine, blood and hepatorenal biochemical indicators and biopsy, we analyzed the effect of propolis on glycometabolism, lipid metabolism, protein metabolism, oxidative stress and hepatorenal function in T1DM and T2DM rats. To reveal the possible protection mechanism of propolis on diabetic kidney injury in T2DM rats, we measured the expression of pro inflammatory cytokines IL-2, IL-6 and TNF-a protein by ELISA. The TGF-β1 expressions were measured using immunohistochemical staining and the MCP-1 mRNA and protein kinase C (PKC) mRNA was measured using RT-PCR. The results are as follows:1. Effects of propolis on TIDM ratsGrouping and Administration:TIDM rats were randomly divided into groups of model, A1, A2, B1, B2 and positive groups, eight rats each group. Additional 8 rats without STZ induction were selected as normal group. Each rat of Al group was received a dose of 10 mg Chinese propolis per 100 g body weight; Each rat of A2 group was received a dose of 5 mg Chinese propolis per 100 g body weight; Each rat of B1 group was received a dose of 10 mg Brazilian propolis per 100 g body; Each rat of B2 group was received a dose of 5 mg Brazilian propolis per 100 g body; Each rat of positive group was received a dose of 1 mg glucobay perl00 g body weight; Each rat of model group and normal group was received a dose of 1 ml physiological saline per100 g body weight. Administration last continuously eight weeks intragastrically twice daily.Results:1) Body weight and glycometabolism:Chinese propolis and Brazilian green propolis obviously inhibited weight loss and blood glucose increment in TIDM rats in a dose-dependent effect, and Chinese propolis had a slightly better effect than Brazilian green propolis; China Propolis can significantly lower HbAlc levels in a dose-dependent effect; 2) Lipid metabolism:Compared with normal rats, model rats had an obviously elevated serum TC and TG level. High dose Chinese propolis obviously reduced serum TC levels in T1DM rats; 3) Serum oxidative stress: Compared with normal rats, model rats had an obviously elevated MDA, NO and NOS level and reduced SOD and CAT level. Chinese propolis and Brazilian green propolis improved serum oxidative damage in TIDM rats by a dose-independent effect. High dose Chinese propolis significantly reduced serum MDA level, whereas low dose Chinese propolis markedly raise serum SOD level. Brazilian propolis increased serum SOD level significantly. In addition, Brazilian green propolis can apparently reduce serum NOS level; 4) Hepatic function and histological injury: The content of AST and ALT in model rats increased significantly compared with normal rats. Chinese propolis and Brazilian green propolis can significantly reduced AST and ALT level. As to hepatic oxidative damage, Chinese propolis suppressed the hepatic MDA level and showed no effect on antioxidant enzyme, whereas Brazilian green propolis inhibited lipid peroxidation and improve the activity of antioxidant enzyme. By histological observation, model rats showed mild hepatic injury, and both Propolis and glucobay helped to recover hepatic injury.5) Renal function andhistological injury:The level of BUN, urine albumin and urine protein in model group were significantly higher than those in normal group. Treated with Chinese propolis and Brazilian propolis reduced urine albumin. Brazilian green propolis also reduced serum BUN and urine protein level significantly. As to renal oxidative stress, propolis can reduce renal MDA and GSH-px level and raise CAT markedly. By histological observation, model rats showed moderate renal injury, and both Propolis and acarbose helped to recover hepatic injury.2. Effects of propolis on T2DM ratsGrouping and Administration:T2DM rats were randomly divided into groups of model, A, B and positive groups, eight rats each group. Additional 8 rats without STZ induction were selected as normal group. Each rat of A group was received a dose of 5 mg Chinese propolis per 100 g body weight; Each rat of B group was received a dose of 5 mg Brazilian propolis per 100 g body; Each rat of positive was received a dose of 1 mg pioglitazonehcl per100 g body weight; Each rat of model group and normal group was received a dose of 1 ml physiological saline per100 g body weight. Administration last continuously eight weeks intragastrically twice daily.Results:1) Body weight:Chinese propolis and Brazilian green propolis had an indistinctive inhibition on weight loss of T2DM rats. A group rats (treated with Chinese propolis) is obviously heavier than model rats at Week 8; 2) Glycometabolism:Chinese propolis lowered FBG of T2DM significantly during the previous four weeks and showed no marked effect during the following four weeks, whereas Brazilian green propolis had no obvious effect. China Propolis can significantly lower HbAlc levels at Week 4 and Week 8, whereas Brazilian green propolis reduced HbAlc levels at Week 4; 3) Lipid metabolism:Compared with normal rats, model rats had an obviously elevated serum TC, LDL-C and TG level, Chinese propolis obviously reduced serum TG and LDL-C levels in early phase of T2DM rats; 4) Serum oxidative stress:Compared with normal rats, model rats had an obviously elevated serum MDA level and reduced GSH-px level. Chinese propolis increased serum GSH-px level and reduced serum MDA level significantly, whereas Brazilian green propolis improved serum CAT level and reduced serum MDA level significantly; 5) Insulin and protein metabolism:Compared with normal rats, T2DM rats had a normal insulin level and reduced serum protein and albumin level. Propolis showed no effect on insulin. Chinese propolis can raise serum protein and albumin level, whereas Brazilian propolis can raise serum albumin level; 6)Hepaticfunction and histological injury:Obviously reduced serum AST level and increased ALT level of model rats meant damaged hepatic function, propolis-treated rats had a significantly reduced ALT level. By histological observation, model rats liver showed severe adipose infiltration. Propolis and positive drug helped to recover hepatic injury.7) Renal function and histological injury:The level of serum BUN, CCR and urine albumin in model group were significantly higher than those in normal group. Propolis treating can reduce serum BUN and urine albumin. As to renal oxidative stress, Chinese propolis can reduce renal MDA and increase CAT level significantly, whereas Brazilian propolis can reduce renal MDA and increase GSH-px level. By histological observation, model rats showed moderate renal injury and propolis and acarbose helped to recover hepatic injury.3. Protection mechanism of propolis on T2DM rat kidneyAt Week 8 we measured kidney hemodynamics of T2DM rats by laser Doppler flowmetery. Once rats were sacrificed, kidney was removed to determine relevant indicators. 1) Inflammation:Compared with normal rats, model rats had an obviously increased serum CRP level and renal proinflammatory cytokines. Brazilian green propolis can reduce serum CRP levels significantly. As to renal inflammation, Chinese propolis can markedly reduce renal IL-2, IL-6 and TNF-a levels, whereas the Brazilian green propolis can effectively inhibit the expression of TNF-a protein and MCP-1 mRNA; 2) Renal hemodynamics:Diabetes/high glucose can change rats renal hemodynamics. Chinese propolis can significantly improve the abnormal renal hemodynamics in T2DM rats by increasing renal blood flow and reducing renal vascular resistance and filtration fraction, whereas Brazilian propolis showed little effect; 3) Expression of renal PKC mRNA:Both Chinese propolis and Brazilian green propolis inhibit the PKC mRNA expression in T2DM rat kidney; 4) Expression of renal TGF-β1:Immunohistochemical staining and image analysis showed that model rats had a significantly higher renal TGF-β1 expression compared with normal group. Brazilian green propolis and positive drug can improve total optical density, taverage optical density and positive index in rat kidneys, whereas Chinese propolis showed an inapparent improvement.In summary, Chinese propolis and Brazilian green propolis can effectively alleviate the diabetes-induced metabolism disorders in rats. Propolis can improve weight loss, glycometabolism, lipid metabolism, protein metabolism and serum, liver and kidney oxidative stress in diabetic rats. Determination of relevant indicators and histological observation suggested the protection effect of propolis on hepatorenal function in diabetic rats. In comparison, Chinese propolis showed a better hypoglycemic and hepatic-protection effect than Brazilian propolis, whereas Brazilian green propolis had a more comprehensive antioxidant effect. By measuring the renal proinflammatory cytokines, hemodynamics, PKC mRNA, and TGF-β1 protein, we analyzed a possible protection mechanism of propolis on diabetic nephropathy.更多还原