【作者】 殷桂香；

【导师】 朱德增；

【作者基本信息】 第二军医大学 ， 中西医结合临床， 2009， 博士

【摘要】 大量的流行病学资料显示,大血管病变是2型糖尿病患者致死和致残的主要原因。在我国,约60%的2型糖尿病患者合并有心血管并发症,80%死于大血管病变。与非糖尿病患者相比,糖尿病患者患心脑血管病的风险高4倍,低位截肢风险高40%。糖尿病大血管病变(即在糖尿病下的血管重构)是一个复杂的多因素疾病,动脉粥样硬化是其主要病理改变之一。研究发现,高血糖与高血脂的毒性作用、蛋白质非酶糖基化、内皮功能障碍等因素共同作用于血管壁,导致平滑肌细胞增殖、胶原过度表达、血管结缔组织基质成分显著改变,与粥样斑块形成、血管壁纤维化、硬化、管腔狭窄密切相关。血管纤维化增加动脉僵硬度,引发血流紊乱,最终造成血流动力学障碍,成为动脉粥样硬化和动脉硬化发生发展的力学基础。糖尿病为全身代谢性疾病,需要终身治疗。而血管病变由于机制复杂,一种药物往往很难控制病情的发生发展。目前共同的观点是,对于糖尿病患者,应进行包括控制血糖、调节血脂、改变生活方式等多因素干预治疗,以改善其预后,特别是降低大血管事件的危险性。但长期、多因素干预治疗在提高疗效的同时大大增加了患者的治疗成本,同时也增加了不良反应的发生率。因此,寻找高效、低毒、多靶点发挥作用的药物非常必要。中医药具有整体调节和多重治疗作用,单味中药就是一个多成份的复合体,中药复方的成分就更复杂了。虽然中药在某单方面的作用并不强,但由于不同的成分各自作用于不同的靶点,从而发挥协同治疗作用。在糖尿病,尤其是血管病变的防治方面显示出了良好的临床应用前景。导师在长期临床实践的基础上,查阅大量古今文献,依据中医“木克土”、“酸克甘”理论,结合现代医学的新认识,提出了用“酸胜甘”法治疗糖尿病的学术观点和治法,并据此而立酸味中药复方。前期我们将该复方与甘味、苦味中药复方作对比,结果表明,该方可明显改善2型糖尿病大鼠糖、脂肪代谢紊乱和胰岛素抵抗状态,其效果优于甘味、苦味中药复方。由于中药具有整体、多靶点发挥作用的特点,我们推测该方可能对2型糖尿病大血管病变也具有防治作用,本实验就是在前期研究的基础上,进一步观察酸味中药复方对2型糖尿病大鼠大血管病变的作用及机制。研究目的1、观察酸味中药复方对2型糖尿病大鼠主动脉病理形态学的影响2、研究酸味中药复方对2型糖尿病大鼠空腹血糖、血脂及胰岛素的影响3、研究酸味中药复方对2型糖尿病大鼠主动脉非酶糖化的影响4、研究酸味中药复方对2型糖尿病大鼠主动脉内皮细胞功能的影响研究方法采用高脂饲料喂养加小剂量STZ腹腔注射的方法建立2型糖尿病动物模型。成模大鼠按血糖值随机分为模型组、中药组、AG组,在药物干预8周和12周两个时间点观察相关指标变化。采用HE染色观察主动脉病理变化,VG以及立春红/维多利亚蓝染色观察血管胶原纤维和弹性纤维变化,Care sense血糖仪测FBG,全自动生化仪测血脂,硝酸还原酶法测血清NO含量、NOS活性,放免法测血浆INS、ET–1水平,荧光法测主动脉AGEs、胶原含量,实时荧光定量PCR法检测主动脉RAGE基因表达量,免疫组化法检测主动脉NF–κB蛋白表达及α–SMA蛋白表达,原位杂交法检测主动脉内皮细胞ICAM–1基因表达。研究结果1、酸味中药复方对2型糖尿病大鼠主动脉病理变化及VSMC表型变化的影响(1)HE染色各时间点正常大鼠主动脉内膜光滑、平坦,内皮细胞扁平,紧贴于平直的内弹力板上,平滑肌细胞呈长梭形,排列整齐,与弹力板近似平行相间排列,细胞核大小均一,细胞膜及核膜清晰、完整,胞浆染色均匀。模型组大鼠在成模8周末,主动脉横断面上有多少不等的内膜增厚病灶,局部主要由排列紊乱的平滑肌细胞和泡沫细胞组成。病灶表面内皮细胞肿胀、肥胖,有的脱落、甚至坏死。内弹力板厚薄不匀或分层,中膜浅层平滑肌细胞增生,中层弹力板与平滑肌细胞间排列紊乱。成模12周末上述病理变化更明显。AG组和中药组在成模8周时主动脉内膜尚整齐,有少许泡沫细胞,平滑肌细胞排列尚整齐。12周时动脉内膜局部轻度增厚,破坏较轻,尚连续。(2)胶原纤维及弹力纤维变化VG染色显示:正常组大鼠主动脉胶原组织分布均匀,相邻细胞的胶原纤维网完好;模型组血管壁胶原纤维明显增多,围绕细胞的胶原纤维网断裂、不完整、排列紊乱;中药组和AG组胶原纤维较模型组减少,排列较规整,胶原纤维少有断裂。立春红/维多利亚蓝染色显示:正常组大鼠主动脉弹力纤维完整,无断裂,排列整齐,模型组弹力纤维断裂、不完整,排列紊乱,含量明显减少,中药组和AG组明显改善。半定量分析结果表明:各时间点糖尿病大鼠主动脉胶原纤维相对含量明显高于同时间正常大鼠(P<0.05),C/E比值增加(P<0.05);中药组和AG组大鼠主动脉胶原纤维相对含量及C/E比值均低于模型组(P<0.05)。(3)主动脉VSMC表型变化药物干预8周和12周后,各组大鼠主动脉均可见α–SMA蛋白阳性表达,与同时间点正常组相比,糖尿病大鼠主动脉α–SMA蛋白阳性表达指数明显降低(P<0.05),说明糖尿病大鼠主动脉VSMC以合成表型为主;酸味中药复方组和AG组α-SMA蛋白阳性表达指数明显高于模型组(P<0.05),合成表型减少,收缩表型增多;在用药8周时中药组α-SMA蛋白阳性表达指数高于AG组(P<0.05),12周时无差异(P>0.05)。2、酸味中药复方对2型糖尿病大鼠体重、FBG、INS及血脂的影响(1)大鼠FBG、体重变化正常组大鼠体重增加,模型组、中药组、AG组糖尿病大鼠体重均有下降,FBG升高,与同时间点正常组相比,差异有统计学意义(P<0.05)。与同时间点模型组和AG组相比,中药组大鼠体重增加、FBG降低(P<0.05)。AG组大鼠体重与同时间点模型组相比,只有12周时升(P<0.05)高,而FBG与同时间点模型组相比,无统计学差异(P>0.05)。(2)大鼠血脂变化在用药8周后,中药组大鼠血清TG、TC含量与同时间点正常组、模型组和AG组相比无统计学差异(P>0.05);HDL含量低于同时间点正常组(P<0.05),但与同时间点模型组和AG组相比,无统计学差异(P>0.05);LDL含量低于同时间点模型组和AG组,高于同时间点正常组(P<0.05)。用药12周后,中药组大鼠血清TG、LDL含量低于同时间点模型组和AG组,高于同时间点正常组(P<0.05);HDL含量高于同时间点模型组(P<0.05),而TC与同时间点模型组相比无差异(P>0.05)。AG组糖尿病大鼠在药物干预8周和12周后,血清TC、TG、LDL、HDL含量高于同时间点正常组,HDL含量低于同时间点正常组(P<0.05),而与同时间点模型组相比,无统计学差异(P>0.05)。(3)大鼠INS变化中药组、模型组、AG组糖尿病大鼠在药物干预8周和12周后,血清INS均低于同时间点正常组大鼠,差异有统计学意义(P<0.05);中药组糖尿病大鼠血清INS与同时间点模型组、AG组相比,无统计学差异(P>0.05)。3、酸味中药复方对2型糖尿病大鼠主动脉非酶糖化的影响(1)主动脉AGEs及胶原含量变化药物干预8周和12周后,模型组、中药组、AG组糖尿病大鼠主动脉AGEs、胶原含量均高于同时间点正常组(P<0.05);与同时间点模型组比较,中药组和AG组大鼠主动脉胶原、AGEs含量明显降低(P<0.05),中药组大鼠主动脉胶原、AGEs含量与同时间点AG组相比,无统计学差异(P>0.05)。(2)主动脉RAGE基因表达变化所有大鼠主动脉均可见RAGE因表达,但正常组RAGE基因表达量较少(P<0.05),与同时间点模型组相比,中药组和AG组RAGE基因表达量均降低(P<0.05);同时间点AG组和中药组相比无统计学差异(P>0.05)。4、酸味中药复方对2型糖尿病大鼠主动脉内皮细胞功能的影响(1)血清NO、NOS变化模型组、中药组、AG组大鼠血清NO、NOS水平均低于同时间点正常大鼠(P<0.05);与同时间点模型组比较,中药组和AG组大鼠血清NO、NOS含量均明显升高(P<0.05);中药组和AG组相比,无统计学差异(P>0.05)。(2)血浆ET-1变化模型组、中药组、AG组大鼠血浆ET–1水平均高于同时间点正常大鼠(P<0.05);与同时间点模型组比较,中药组和AG组大鼠血浆ET–1含量均明显降低(P<0.05);中药组和AG组相比,无统计学差异(P>0.05)。(3)主动脉ICAM-1基因表达变化原位杂交结果表明,大鼠主动脉均可见ICAM–1基因表达,但正常组ICAM–1基因表达量较少,低于同时间点糖尿病大鼠(P<0.05);与同时间点模型组相比,中药组和AG组大鼠主动脉ICAM–1基因表达量均降低(P<0.05)。结论1、酸味中药复方能够调节2型糖尿病大鼠糖、脂肪代谢;2、酸味中药复方能够减轻2型糖尿病大鼠主动脉内膜损伤,抑制VSMC增殖,降低胶原纤维相对含量及C/E值;3、酸味中药复方可减少2型糖尿病大鼠主动脉AGEs生成,下调RAGE基因表达,具有抑制非酶糖化的作用;4、酸味中药复方可提高2型糖尿病大鼠血清NO含量及NOS活性,降低血浆ET–1含量,抑制内皮细胞ICAM–1基因表达,保护内皮细胞功能。5、酸味中药复方对2型糖尿病大鼠动脉粥样硬化病变具有一定的防治作用。更多还原

【Abstract】 Many epidemiological findings showed that macroangiopathy was the main cause to induce type 2 diabetic patients to die and mutilate .About 60% of the type 2 diabetic patients suffered from coronary heart disease, and 80% patients died of macroangiopathy.The risk of diabetic patients to coronary heart disease and cerebrovascular disease is 4 times and the amputation ratio is higher 40%than that of no diabetic patients. It has been suggested that the pathologic changes of diabetes-induced macroangiopathy are atherosclerosis and angiosclerosis. Many vocative factors such as the toxic effect of hyperglycemia and hyperglycemia, protein glycosylation, endothelial tissue functional impairment may induce to smooth muscle cell proliferation, collagen overexpression, connective tissue matrix alteration. Above-mentioned factors could lead to vascular fibrosis and stiffness.Vasular fibrosis may increase the vascular wall stiffness and turbulent blood flow, and eventually disturb of haemodynamics, which is the mechanics basis of atherosclerosis and angiosclerosis.Now the coincident concept is that doctors should regulate blood glucose and blood fat of type 2 diabetic patients. Meanwhile the patients should change their life styles. These thoracic methods could improve the patients’quality of life. Especially they could down regulate the risk factor for macroangiopathy.But patients may pay much money .So it is necessary to find high performance and economic thoracic methods.Traditional Chinese Medicine (TCM) has effect of multiple regulation and treatment, and it has got well clinical results in diabetes especially its complications. Compounded Sour Medicinal Herbs (CSMH) is organized according to five elements and flavors theory of TCM.We found it could improve glucolipid metabolic disorder and insulin resistance of type 2 diabetic rats. The aim of the present study is to identify the antimacroangiopathic effects of CSMH in vivo. Furthermore, relative mechanisms of antimacroangiopathic effects of CSMH were studied.Objective1. To observe the influence of CSMH on thoracic aortic pathological change in type 2 diabetic rat2. To investigate the influence of CSMH on the changes of FBG、blood fat and INS in diabetic rats 3. To investigate the influence of CSMH on the changes of thoracic aortic no enzymatic in type 2 diabetic rat4. To investigate the influence of CSMH on the changes of thoracic aortic endothelial function in type 2 diabetic ratsMaterials and methodsTwenty animals without any treatment was randomly choosed from 150 male SD rats after freedom bred, and set up for normal control group with normal diet. To induce T2DM animal models, the others fed with high-fat and high-caloric diets for two months. Then the rats with insulin resistance were injected with streptozotocin(STZ)40mg/kg through peritoneum after.Fourty eight an seventy two hours after injection, the fasting blood glucose (FBG)were tested. The rats which FBG≥16.7mmol/L were as T2DM animal models. Then the T2DM rats were randomly divided into three groups: untreated group,AG treated group and CSMH treated group. At the end of the 8week and 12week, 5-8 rats were random selected from each group. The rats were weight, tested FBG and taken blood to collect serum was before killed. The pathological change of thoracic aorta was observed with optical microscop.The content of collagen fibers and elastic fibers were detected with computer image analysis system. The change of VSMC was detected by immunohistochemical method. NO and NOS in serum were examined by checking their OD values, ET in plasma were detected by radio-immunization. The deposition of AGEs and gene expression of their receptor in aorta tissue of T2DM rats were detected separately by fluorescence and real time fluorescent quantization PCR method. The gene expression of ICAM-1 in the aorta endotheliocyte was detected with real in-situ hybridization method. The activity of NF-κB was detected by mmunohistochemical method.Result1. Pathological changes:(1) The results of HE stainAt the two points, the aortic endothelial cells in group normal rats were thin and flat, adhering to inner elastic plates. The vascular smooth muscle cells (VSMC) in arranged regularly. The per cellular membrane was continuous and integral. The nuclear membrane was smooth and intact. Elastic lamina was uninterrupted and perfect. A few collagenous were scattered in extra cellular matrix. In untreated group rats, the VSMC arranged irregularly. The endothelial cell was swelling, even necrosis. The per cellular membrane was interrupted .The collagenous fibers in extra cellular matrix invaded elastic lamina. In comparison with untreated group, the pathologic changes were improved in CSMH and AG treated group.(2) The collagen elastic fibers changesVG staining slides showed that the collagen fibers in normal group were intact and well-arranged. In untreated group, the collagen fibers were increased. The dicyto-collagen-fibers around cells were split, unintact and arranged irregularly. The collagen fibers were greatly improved in CSMH and AG treated rats contrast to those in untreated rats.Victoria blue staining showed that the elastic fibers were intact and unintrrrupted in group normal rats. But in untreated group, the elastic fibers were deformed and interrupted. These changes in CSMH and AG treated group were markedly amelioration.The contents of collagen fibers in the aortic tissue of T2DM rats were higher and elastic fiber elastic fibers contents were lower than that in normal control rats at the two time points (P<0.05); While the contents of collagen fibers in aortic tissue of CSMH treated rats were decreased and elastic fiber elastic fibers contents were higher than that in untreated group rats (P<0.05). In comparison with untreated group, the collagen/elastin ratio in CSMH treated rats was higher.(3) Expression ofα-SMA protein (immunohistochemistry)There were high expression ofα-SMA in the aortic tissue of T2DM rats at 8th and 12th week after the models induced (P<0.05). the expression density of NF-κB p65 in CSMH treated group rats were lower than that in untreated model group (P<0.05).2. The changes of weight,FBG,blood fat and INS in rat(1) The changes of FBG and weightIn comparison with normal group rats, body weight of diabetic rats were decreased, while FBG were higher (P<0.05). At the two time points, body weight in CSMH treated group rats were higher and FBG lower than those in untreated group (P<0.05). There was no significant difference in AG treated and untreated group (P>0.05). (2) The changes of blood fatAt the end of 8week, TC and LDL in CSMH treated group were lower than those of untreated group while HDL were higher (P<0.05); while HDL in CSMH group comparable with those AG treated and untreated group (P>0.05). In comparison with untreated group,FBG, TG, TC and LDL were lower(P<0.05).At the end of 12week, TG and LDL in CSMH treated group were lower than those of untreated group while HDL were higher(P<0.05).There was no significant difference of index of AG treated group in comparison with untreated group(P>0.05).(3) The changes of plasma INSIn comparison with control group, INS in untread, CSMH and AG treated groups were decreased (P<0.05), while there was no significant in the three groups (P>0.05). .3. The influence of CSMH on the changes of thoracic aortic noenzymatic in type 2 diabetic ratThe changes of AGEs and RAGE gene expressionThe deposition of AGEs and RAGE gene expression in the aorta tissues of control group were lower than that of T2DM rats (P<0.05). The deposition of AGEs and gene expression of their receptor in aorta tissues in CSMH treated group rats were decreased as compared with that of untreated group rats(P<0.05).4. The influence of CSMH on the changes of thoracic aortic endothelial function in type 2 diabetic rat(1) In comparison with the control group, The quantity of NO and NOS in blood serum were lower an ET-1 were higher of T2DM rats (P<0.05).The quantity of NO and NOS of CSMH treated group were higher and quantity of ET-1 in plasma were lower than those of untreated group (P<0.05).(2) The gene expression of ICAM-1 in the aorta tissues of control group were lower than that of T2DM rats (P<0.05). gene expression of ICAM-1in the aorta tissues of CSMH treated group rats were decreased as compared with that of untreated group rats(P<0.05).Conclusion1. CSMH could improve aorta injury; inhibit VSMC generation, decrease collagen fibers and C/E.2. CSMH could regulate diabetic rats’blood glucose and lipid metabolism.3. CSMH could inhibit the deposition of AGEs in aorta and down regulate its receptor gene expression.4. CSMH could obviously elevate the levels of NO and NOS in serum, decrease content of ET-1 in plasma and inhibit the gene expression of ICAM-1. It could improve the function of endothelial cell.更多还原