【作者】 边云；

【导师】 田风胜；

【作者基本信息】 河北医科大学 ， 中西医结合临床， 2015， 硕士

【摘要】 目的:通过观察益气养阴、活血通络的中药复方制剂—复荣通脉胶囊对糖尿病大鼠e NOS、ADMA、NF-κB表达的影响,进一步阐述其防治糖尿病血管病变的作用机制,为临床应用提供理论依据。方法:60只成年雄性Wistar大鼠随机分为两组,正常对照组10只,造模组50只。造模组大鼠禁食12h后,一次性腹腔注射STZ 60mg/kg(STZ临用前用0.1M柠檬酸钠缓冲液配成浓度为1%、p H=4.2的溶液),正常组腹腔注射等量的柠檬酸钠缓冲液。72h后尾静脉取血,测血糖≥16.7mmol/L,确定为糖尿病大鼠。成模后,随机抽取40只分为糖尿病组(10只)及复荣通脉胶囊低剂量组(10只)、复荣通脉胶囊中剂量组(10只)、复荣通脉胶囊高剂量组(10只)。复荣通脉胶囊中剂量组给予1.4g/(kg体重)灌胃、复荣通脉胶囊低剂量组给予0.7g/(kg体重)灌胃、复荣通脉胶囊高剂量组给予2.8g/(kg体重)灌胃,其余两组每日给予同等剂量生理盐水灌胃。实验期间,各组大鼠自由进食水。连续灌胃8周后,禁食12h以3%戊巴比妥(80mg/kg)腹腔注射麻醉,迅速开腹,分离胸主动脉从胸主动脉采血,取2 m L分离血清测定ADMA。留取胸主动脉时,另留取一段以4%多聚甲醛固定,HE染色观察形态学变化,SP法免疫组化染色观察病理切片组织中阳性染色面积及强度。计量资料均采用X——±S表示,各组自身前后比较采用配对t检验;各组间比较采用单因素方差分析,方差不齐时应用多个独立样本比较的Kruskal-Wallis H检验,所有数据用SPSS 16.0软件统计处理,检验水准α=0.05。结果:1对大鼠一般状态的影响糖尿病组及复荣通脉胶囊低、中、高剂量治疗组大鼠精神状态差,饮水量增多,尿量增多,毛色晦暗无光泽,反应迟钝,抱团少动。2对血糖的影响治疗前,与正常对照组相比,糖尿病组、复荣通脉胶囊低、中、高剂量组血糖明显升高(P<0.05);治疗前,糖尿病组、复荣通脉胶囊低、中、高剂量组间相比血糖无明显差异(P>0.05)。治疗后,与正常对照组相比,糖尿病组、复荣通脉胶囊低、中、高剂量组血糖明显升高(P<0.05);治疗后,糖尿病组、复荣通脉胶囊低、中、高剂量组间相比血糖无明显差异(P>0.05)。各组大鼠治疗前后血糖无明显差异(P>0.05)。3对胆固醇、甘油三酯的影响灌胃治疗8周后,与正常对照组相比,糖尿病组、复荣通脉胶囊低、中、高剂量治疗组胆固醇明显升高(P<0.05);糖尿病组与复荣通脉胶囊低、中、高剂量组组间相比胆固醇无明显差异(P>0.05)。治疗后,与正常对照组相比,糖尿病组、复荣通脉胶囊低、中、高剂量治疗组甘油三酯明显升高(P<0.05);糖尿病组与复荣通脉胶囊低、中、高剂量组组间相比甘油三酯无明显差异(P>0.05)。4对血管组织形态学的影响光镜下观察HE染色后各组大鼠胸主动脉形态学改变:正常对照组血管形态正常,内皮细胞排列紧密,内膜完整,未见炎性细胞及不定形物质沉积;糖尿病组可见动脉内膜增厚,内膜断裂不完整,部分内膜向腔内突起,可见泡沫细胞,中膜可见较多大小不等的裂隙,平滑肌细胞排列紊乱;复荣通脉胶囊低、中、高剂量治疗组胸主动脉形态介于正常对照组与糖尿病组之间,低剂量治疗组光镜下可见动脉内膜增厚,部分内皮细胞脱落消失,偶可见泡沫细胞;复荣通脉胶囊中、高剂量治疗组光镜下无明显差别,内膜较完整,偶见内皮细胞脱落。5对血清ADMA的影响灌胃治疗8周后,与正常对照组相比,糖尿病组及复荣通脉胶囊低、中、高剂量组血清ADMA明显升高(P<0.05);与糖尿病组相比,复荣通脉胶囊低、中、高剂量组血清ADMA明显降低(P<0.05);与复荣通脉胶囊低剂量组相比,复荣通脉胶囊中、高剂量组血清ADMA水平明显降低(P<0.05);复荣通脉胶囊中剂量组与高剂量组间无明显差异(P>0.05)。6对大鼠胸主动脉NF-κB表达的影响灌胃治疗8周后,与正常对照组相比,糖尿病组及复荣通脉胶囊低、中、高剂量组NF-κB表达明显升高(P<0.05);与糖尿病组相比,复荣通脉胶囊低、中、高剂量组NF-κB表达明显降低(P<0.05);与复荣通脉胶囊低剂量组相比,复荣通脉胶囊中、高剂量组NF-κB表达明显降低(P<0.05);复荣通脉胶囊中剂量组与高剂量组相比无明显差异(P>0.05)。7对大鼠胸主动脉e NOS表达的影响灌胃治疗8周后,与正常对照组相比,糖尿病组及复荣通脉胶囊低、中、高剂量组e NOS表达明显降低(P<0.05);与糖尿病组相比,复荣通脉胶囊低、中、高剂量组e NOS表达明显升高(P<0.05);与复荣通脉胶囊低剂量组相比,复荣通脉胶囊中、高剂量组e NOS表达明显升高(P<0.05);复荣通脉胶囊中剂量组、高剂量组相比无明显差异(P>0.05)。结论:1大鼠中NF-κB表达强弱及ADMA水平与胸主动脉病理变化相一致,e NOS表达强弱与胸主动脉病理变化相反,提示糖尿病血管病变的发生发展中,e NOS、ADMA和NF-κB均起重要作用。2 DM大鼠胸主动脉中NF-κB表达增强而e NOS表达降低,血清ADMA水平升高,提示糖尿病血管病变中,NF-κB和ADMA可能存在相互作用,NF-κB和e NOS之间可能存在相互拮抗。3复荣通脉胶囊各治疗组大鼠胸主动脉形态学均较DM组改善,提示复荣通脉胶囊对血管组织具有保护作用,对糖尿病大血管病变有一定治疗作用。4复荣通脉胶囊各治疗组大鼠与DM组比较,血清ADMA水平及NF-κB表达降低,e NOS表达增强,提示复荣通脉胶囊可降低血清ADMA水平,抑制NF-κB、促进e NOS在大鼠胸主动脉的表达。5本实验发现复荣通脉胶囊的治疗效果存在剂量依赖性,但是为非线性关系。复荣通脉胶囊低、中、高剂量治疗组相比,低剂量治疗组效果劣于中、高剂量治疗组,且有统计学差异,中、高剂量治疗组间无明显差别,故推荐临床上以中剂量作为治疗剂量。更多还原

【Abstract】 Objective: To observe the effect of Traditional Chinese medicine compound preparations-Furongtongmai capsule on the expression of e NOS, ADMA and NF-κB on thoracic aorta tissue of diabetic rats, and the protective effects of Furongtongmai capsule on the diabetes vascular lesions were examined in this study. The mechanism of its prevention on diabetes vascular lesions was explored to provide theoretical basis for clinical application.Methods: 60 adult male Wistar rats were randomly divided into two groups: normal control group(NC, n = 10) and diabetes mellitus(DM) group(n = 50). The rats in DM group were injected STZ 60 mg/kg one-time into enterocoelia(STZ before use with 0.1 M with the concentration of 1% sodium citrate buffer solution, p H = 4.2) and the rats in normal control group were injected same volume of sodium citrate buffer. After 72 hours, the rats whose blood glucose≥16.7mmol/L had chosen as diabetic rats. 40 of 50 diabetes rats were randomly divided into the follow group: DM, FRL, FRM and FRH group(n=10). The rats in FRL, FRM and FRH group was given the 0.7 g, 1.4 g and 2.8 g/(kg body weight) Furongtongmai medicine with intragastric administration. The rats in NC and DM groups have given the same volume of saline. All of rats drink and eat freely in the experiment. After 8 weeks, The pentobarbital anesthesia with 80 mg/kg used in rats to harvest blood and thoracic aorta tissue. 2 m L blood was taken from thoracic aortic for serum ADMA. The thoracic aorta fixed with 4% paraformaldehyde for HE staining to known the pathological changes. The expression of NF-κB and e NOS on thoracic aortic tissue were examined with immunohistochemical staining. The positive area and integrated optical density(IOD) was measured by colour image analytic system. The difference of measurement data was detected by analysis of variance. The one-way ANOVA used for test of each group. Kruskal-Wallis H test is used when variance is not neat. All the data were processed with SPSS 16.0 statistical processing software and inspection level: α=0.05.Results:1 The influence of the general condition of rats The rats of DM group and FRL, FRM, FRH groups compared with the normal group: mental state is poor, water quantity increased, urine volume increased, the colour dark matt, unresponsive, means less still.2 The effect on blood sugar Before the treatment, DM group and FRL, FRM, FRH groups were higer than normal control group(P<0.05); Before the treatment, DM group and FRL, FRM, FRH groups compared with each other has no obvious difference(P<0.05). After the treatment, DM group and FRL, FRM, FRH groups were higer than normal control group(P < 0.05); After the treatment, DM group and FRL, FRM, FRH groups compared with each other has no obvious difference(P<0.05). No difference between the groups of rats, blood glucose before and after the treatment(P<0.05).3 The effect on cholesterol and triglyceride After lavage treatment for 8 weeks, compared with normal control group, DM group and FRL, FRM, FRH groups cholesterol significantly increased(P<0.05); DM group and FRL, FRM, FRH groups compared with each other has no obvious difference(P<0.05). After the treatment, compared with the normal control group, DM group and FRL, FRM, FRH groups triglyceride significantly increased(P<0.05); DM group and FRL, FRM, FRH groups compared with each other triglycerides has no obvious difference(P<0.05).4 The impact on the vascular tissue morphology Observe each rat thoracic aorta morphological changes after HE staining: The blood vessels form of NC group is normal, endothelial cells arranged closely, membrane is integrated, no inflammatory cells and amorphous material deposition; The intimal artery of DM group is visible incrassated and incomplete, part of the lining to the cavity, foam cells and cranny is visible, smooth muscle cells are disordered; FRL, FRM, FRH groups of thoracic aorta shape between the NC group and DM group; The intimal artery of FRL group is visible thickening, part of the endothelial cells disappeared, foam cells can be seen occasionally; There is no significant differences between and FRM and FRH group, intimal artery is integrated, the endothelial cell loss occasional.5 Impact on serum ADMA After lavage treatment for 8 weeks, compared with the normal control group, DM group and FRL, FRM, FRH groups serum ADMA significantly increased(P<0.05); Compared with DM group, FRL, FRM, FRH groups significantly lower(P<0.05); Compared with FRL group, the levels of ADMA of FRM, FRH groups significantly decreased(P<0.05); FRM and FRH group was no significant difference(P<0.05).6 The expression of NF-κB After lavage treatment for 8 weeks, compared with normal control group, DM group and FRL, FRM, FRH groups significantly increased(P<0.05); Compared with DM group, FRL, FRM, FRH groups significantly decreased(P<0.05); Compared with FRL group, FRM and FRH groups significantly decreased(P<0.05); FRM and FRH group was no significant difference(P<0.05).7 The influence of the expression of e NOS After lavage treatment for 8 weeks, compared with normal control group, DM group and FRL, FRM, FRH groups e NOS expression significantly decreased(P<0.05); Compared with DM group, FRL, FRM, FRH groups significantly increased(P<0.05); Compared with FRL group, FRM and FRH groups significantly increased(P<0.05); FRM and FRH group was no significant difference(P<0.05).Conclusion:1 The expression of NF-κB and the level of serum ADMA are consistent with the pathological changes of thoracic aortic, on the contrary is the expression of e NOS, suggesting that e NOS, ADMA and NF-κB might play important roles in the development of diabetes vascular lesions.2 The expression of NF-κB increased and e NOS decreased on thoracic aortic in diabetic rats, and the level of serum ADMA increased indicating that there might exist interaction between NF-κB and ADMA. NF-κB and e NOS might play opposite roles on the diabetes vascular lesions.3 The pathology of thoracic aortic in FRL, FRM, FRH rats is better than that in DM group, suggesting that Furongtongmai capsule plays a protective role on blood vessels. So it has a certain therapeutic effect on diabetic vascular lesions.4 Compared with DM group, the expression of NF-κB on thoracic aortic and serum ADMA in FRL, FRM, FRH group rats decreased, the expression of e NOS increased, indicating that Furongtongmai capsule can reduce serum ADMA level. Beside it can inhibit the NF-κB and promote the expression of e NOS in thoracic aorta in rats.5 The experiments show that the clinical application of Furongtongmai capsule are dose dependent, but for nonlinear relationship. Compared with Furongtongmai capsule middle dose in the treatment group, the inefficiency of lowe dose treatment groups, statistically significant, high dose treatment group with no significant difference, Middle dose is enough when we treat diabetes vascular lesions.更多还原