针刺对胰岛素抵抗大鼠氧化应激的影响

发布时间：2018-08-02 21:07

【摘要】：胰岛素抵抗与高血压、糖尿病、血脂异常、心脑血管疾病、肥胖等多种疾病密切相关,是导致这些疾病的“共同土壤”。近年来的研究表明：胰岛素抵抗是心脑血管疾病中众多代谢性异常的始动原因和致病基础,而血管内皮功能障碍是动脉粥样硬化、高血压、心脑血管疾病发生的病理基础和早期阶段。胰岛素抵抗和血管内皮功能障碍二者之间存在相互影响、相互促进的关系,形成恶性循环,在动脉粥样硬化的病理过程中扮演着重要的角色。大量的临床研究报道证实,针刺在这些疾病的治疗中具有不可替代的优势,其作用机理与改善这些疾病中存在的胰岛素抵抗现象密切相关。通过本课题组的前期研究,我们发现针刺能有效改善胰岛素的敏感性,能从受体后水平调整和改善胰岛素抵抗状态。本研究是以胰岛素抵抗与血管内皮功能障碍相互作用为基础,设计实验观察针刺干预对胰岛素抵抗模型大鼠的内皮功能影响；以自发性胰岛素抵抗模型-OLETF大鼠及同系正常大鼠-LETO大鼠为研究对象,用ELISA法、硝酸还原酶法、硫代巴比妥酸等等多种检测方法,检测空腹血糖(FPG)、血清胰岛素(FINS)、C肽、胰岛素敏感指数(ISI)、一氧化氮(NO)、丙二醛(MDA)等血液指标,观察针刺干预对OLETF大鼠胰岛素抵抗及内皮功能障碍共同基础的氧化应激的影响,探讨针刺对胰岛素抵抗及血管内皮功能障碍的作用机制。全文分为文献研究、实验研究及小结三大部分。文献研究：从胰岛素抵抗的概念、胰岛素抵抗的发病机制、血管内皮的生理功能、胰岛素抵抗于内皮功能障碍之间的关系及共同病理基础、中医药防治胰岛素抵抗的研究现状、胰岛素的动物模型及评价等多方面阐述了胰岛素抵抗的最新研究进展。通过对大量与胰岛素抵抗相关的文献进行综合分析,本文提出了今后的研究方向,认为针刺是改善胰岛素抵抗的有效方法之一,对胰岛素抵抗具有一定的逆转效应,应加强对其作用机理的深入研究及探讨。实验研究：目的：观察针刺对自发性胰岛素抵抗模型OLETF大鼠的氧化应激的影响,探讨针刺干预对改善胰岛素抵抗及血管内皮功能障碍的作用机理。方法：实验选用雄性OLETF大鼠16只,正常雄性LETO大鼠8只。将8只雄性LETO大鼠作为空白组,16只雄性OLETF大鼠按体重根据随机数字表分为：模型组和针刺组,每组各8只。空白组和模型组不予针刺干预,针刺组则给予针刺干预。针刺穴位：肾俞、内关、足三里和三阴交穴(均取双侧)。针刺方式选用疏密波的电针(疏波2Hz,密波50Hz,调制频率10次／分),加电穴位选取双侧三阴交和足三里穴,电针刺激强度以大鼠肢体轻微抖动为标准,针刺时间每日1次,每次持续20min,针刺干预持续4周。针刺前4周及针刺4周疗程中,每周称重一次。在治疗结束后,各组大鼠禁食不禁水12h。次日早晨取材。先用10%水合氯醛按体重将大鼠麻醉,采用眼眶静脉窦采血,离心取上清,检测针刺干预后FPG、FINS、C-P、NO、ET、SOD及MDA等指标,用葡萄糖氧化酶法测定FPG；用ELISA法测定FINS、C-P、ET;用硝酸还原酶法测定NO；用WST-1微孔板法测定SOD；硫代巴比妥酸(TBA法)测定丙二醛(MDA)；用1/(CFPG×JFINS)公式计算ISI。取完血液样品后,将大鼠四肢固定于鼠板上,沿腹中线开腹,从下往上分离出主动脉,截取胸主动脉,沿血管腔纵向剪开,置于4%多聚甲醛固定,HE染色。观察针刺作用前后,OLETF大鼠主动脉形态学改变情况。结果：1针刺对胰岛素抵抗模型一般状况及体重的影响在实验过程中,正常对照组大鼠体重增加,精神状态良好,反应灵敏,动作自如,毛发有光泽。针刺前4周开始可见模型组及针刺组大鼠性情变温顺,嗜睡少动,大鼠皮毛蓬乱、无光泽,大便量较正常组偏少,色灰,质较软,腹部肥胖。针刺4周后,可见针刺组大鼠体重仍有增长,表现出精神萎靡,反应迟钝,动作迟缓等较前有所好转。2针刺对大鼠血清FPG、FINS、ISI、C-P的影响模型组的FPG、FINS、C-P相比空白组显著升高(P0.01),ISI较空白组显著降低(P0.01)。针刺组的C-P相比空白组显著降低(P0.05),FPG、FINS和ISI与空白组相比无统计学差异。针刺组的FPG、FINS、C-P较模型组显著降低(P0.01),ISI相比模型组显著升高(P0.01)3针刺对大鼠血清NO与ET-1的影响模型组的血清NO的水平相比空白组显著降低(P0.01),模型组ET-1水平相比空白组显著升高(P0.01)。针刺组的NO、ET-1水平相较空白组无统计学差异(P0.05)。针刺组的血清NO水平相比模型组显著升高(P0.05),ET-1水平与模型组相比明显降低(P0.01)。4针刺对大鼠血清SOD及MDA的影响模型组的血清SOD活力水平相比空白组明显降低(P0.05),模型组MDA水平相比空白组显著升高(P0.01)。针刺组的SOD活力水平、MDA相较空白组无统计学差异(P0.05)。针刺组的血清SOD活力相比模型组显著升高(P0.05),MDA水平与模型组相比明显降低(P0.01)。5针刺对大鼠主动脉的形态学改变各组大鼠主动脉HE染色后于数码成像显微镜下以200倍观察,可见空白对照组大鼠的血管内膜光滑完整、连续,无局部缺损或增厚,弹力纤维、血管平滑肌细胞排列整齐,未见浸润的淋巴细胞等病理改变(见图9)。而模型组大鼠的主动脉内皮层不连续,明显失去内膜的完整性,内皮下层呈疏松或断裂状态,血管平滑肌细胞增生、肥大且排列紊乱(见图10)。当给予一定疗程针刺干预治疗后,针刺组大鼠的主动脉内膜层基本连续完整,未见明显增厚,内皮下间隙稍宽,但程度相比模型组减轻,弹力纤维及平滑肌细胞排列尚整齐,病理损伤均得到不同程度的改善(见图11),提示针刺治疗具有保护主动脉内皮损伤的作用。结论：研究结果表明,针刺可以通过改善大鼠的胰岛素抵抗及氧化应激状态,调整血管内皮功能障碍,逆转血管内皮病理改变。
[Abstract]:Insulin resistance is closely related to many diseases such as hypertension, diabetes, dyslipidemia, cardiovascular and cerebrovascular diseases, obesity and so on. It is a "common soil" that causes these diseases. Recent studies have shown that insulin resistance is the origin and pathogenesis of many metabolic abnormalities in cardiovascular and cerebrovascular diseases, and vascular endothelial dysfunction is a movement. The pathological basis and early stage of atherosclerotic, hypertensive, cardio cerebral vascular diseases. There is a mutual influence between the two cases of insulin resistance and vascular endothelial dysfunction, the relationship of mutual promotion and the formation of a vicious circle, which plays an important role in the pathological process of atherosclerosis. A large number of clinical studies have confirmed that acupuncture has been used. It has an irreplaceable advantage in the treatment of these diseases, and its mechanism is closely related to the improvement of insulin resistance in these diseases. Through the earlier study of this group, we found that acupuncture can effectively improve the sensitivity of insulin and can adjust and improve the state of insulin resistance from the post receptor level. This study is based on this study. On the basis of the interaction of insulin resistance and vascular endothelial dysfunction, the effect of acupuncture intervention on the endothelial function of rats with insulin resistance model was designed by the design experiment. The -OLETF rats and -LETO rats of the same normal rats were used as the research object. The ELISA, nitrate reductase, thiobarbituric acid and so on were used. The method of detecting fasting blood glucose (FPG), serum insulin (FINS), C peptide, insulin sensitivity index (ISI), nitric oxide (NO), malondialdehyde (MDA) and other blood indexes, to observe the effects of acupuncture intervention on the oxidative stress of insulin resistance and endothelial dysfunction in OLETF rats, and to explore the effect of acupuncture on insulin resistance and vascular endothelial function. The full text is divided into literature research, experimental research and summary three parts. Literature study: the concept of insulin resistance, the pathogenesis of insulin resistance, the physiological function of vascular endothelium, the relationship between insulin resistance and endothelial dysfunction and the common pathophysiological basis, the study of the prevention and treatment of insulin resistance in traditional Chinese Medicine The latest research progress in insulin resistance is described in many aspects, such as animal model and evaluation of insulin. Through a comprehensive analysis of a large number of literature related to insulin resistance, this paper puts forward the future research direction. It is considered that acupuncture is one of the effective methods to improve insulin resistance and has a certain reversal effect on insulin resistance. In order to investigate the effect of acupuncture on oxidative stress in spontaneous insulin resistance model OLETF rats and to explore the mechanism of acupuncture intervention on improving insulin resistance and vascular endothelial dysfunction. Methods: 16 male OLETF rats, normal male rats were selected. 8 male LETO rats. 8 male LETO rats were taken as blank group, and 16 male OLETF rats were divided into two groups according to the weight of random numbers: model group and acupuncture group, 8 rats in each group. The blank group and model group were not needled, and acupuncture group was given acupuncture intervention. Acupuncture points: Shenshu, Neiguan, Zusanli and three yin acupoints (both sides). The electroacupuncture (sparse wave 2Hz, dense wave 50Hz, modulation frequency 10 times) and the acupoint of electric acupuncture were selected to select the bilateral and Zusanli points. The stimulation intensity of the electroacupuncture was taken as the standard of the mild limb jitter of the rats. The acupuncture time was 1 times a day, each time 20min was sustained, and the acupuncture intervention lasted for 4 weeks. In the course of 4 weeks before acupuncture and 4 weeks of acupuncture, weight was weighed once a week. After the end of the treatment, the rats in each group were forbidden to feed the water 12h. the following morning. First, the rats were anaesthetized with 10% chloral chloral hydrate and the rats were anaesthetized by the orbital venous sinus and centrifuged to pick up the supernatant. The indexes of FPG, FINS, C-P, NO, ET, SOD and MDA were measured, and FPG was measured by the method of glucosinase. The FINS, C-P, and nitrate were measured by ELISA. NO was determined by enzyme method, SOD was measured with WST-1 microplate method, and malondialdehyde (MDA) was measured by thiobarbituric acid (TBA method). The rat limbs were fixed on the rat plate with 1/ (CFPG x JFINS) formula, and the aorta was separated from the middle line, the aorta was isolated from the lower abdomen, the thoracic aorta was intercepted, and the 4% polymethylene was cut along the blood tube and placed in the polymethyl methylene. The morphological changes in the aorta of OLETF rats before and after acupuncture were observed. Results: 1 the effect of acupuncture on the general condition of insulin resistance model and the influence of weight on the body weight of the insulin resistance model in the normal control group was increased, the mental state was good, the reaction was sensitive, the movement was free and the hair was luster. The visible model of the 1 weeks before the acupuncture was started. The sexual emotion of the rats in the group and the acupuncture group was gentle, sleepy, shaggy, shiny, shiny, less stool than the normal group, gray, soft and abdominal fat. After 4 weeks of acupuncture, the body weight of the rats in the acupuncture group was still growing, showing the mental retardation, the slow reaction, the slow motion and so on,.2 acupuncture on the rat serum FPG, FINS, ISI, C The effect of -P on FPG, FINS, C-P in the blank group was significantly higher than that in the blank group (P0.01), and ISI was significantly lower than that in the blank group (P0.01). The C-P in the acupuncture group was significantly lower than that in the blank group (P0.05). FPG, FINS and ISI were not significantly different from those in the blank group. 3 the effect of acupuncture on serum NO and ET-1 in rats was significantly lower than that in the blank group (P0.01), and the level of ET-1 in the model group was significantly higher than that in the blank group (P0.01). The level of ET-1 in the acupuncture group was not significantly different than that in the blank group (P0.05). The serum NO level of the acupuncture group was significantly higher than that in the model group (P0.05), and the ET-1 level was significantly higher than that in the model group (P0.05). The model group significantly reduced (P0.01).4 acupuncture on the serum SOD and MDA in rats, the serum SOD activity level of the model group was significantly lower than that in the blank group (P0.05). The MDA level in the model group was significantly higher than that in the blank group (P0.01). The SOD activity level of the acupuncture group was not statistically significant (P0.05) compared with the blank group (P0.05). The serum SOD activity phase of the acupuncture group was not significant (P0.05). Compared with the model group (P0.05), the level of MDA was significantly lower than that of the model group (P0.01). The morphological changes of the aorta in the rat aorta were compared with the model group. After HE staining, the aorta of rats was observed 200 times under the digital imaging microscope. It could be seen that the vascular intima of the blank control group was intact, continuous, no local defect or thickening, elastic fiber, The vascular smooth muscle cells were arranged neatly with no infiltrating lymphocytes and other pathological changes (see Figure 9). But the aortic endothelium in the model group was discontinuous, obviously lost the integrity of the intima, the underlayer of the endothelium was loose or fractured, the vascular smooth muscle cells proliferated and hypertrophy and disorder (see Figure 10). When given a certain course of acupuncture intervention treatment After the treatment, the aortic intima of the rats in the acupuncture group was basically complete, no obvious thickening and a slightly wider space under the endothelium, but the extent of the endothelium was lighter than the model group. The arrangement of elastic fibers and smooth muscle cells was neatly arranged, and the pathological damage was improved in different degrees (see Figure 11). The effect of acupuncture treatment on the protection of aortic endothelial injury was suggested. The results show that acupuncture can adjust the dysfunction of vascular endothelium and reverse the pathological changes of vascular endothelium by improving the state of insulin resistance and oxidative stress in rats.
【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R245

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