泄浊解毒、活血通络中药对糖尿病肾病大鼠肾组织中血管生成素1表达的影响
发布时间：2018-08-24 07:36
【摘要】：目的：本研究通过高糖高脂饲料饲养加腹腔注射小剂量链脲佐菌素(streptozotocin, STZ)建立DM大鼠模型，应用泄浊解毒、活血通络中药、厄贝沙坦以及两者联合应用对其进行干预，通过观察实验动物各生化指标，肾脏病理形态学变化以及肾皮质组织中血管生成素1(Angiopoietin-1，Ang-1)的表达水平，来探讨Ang-1与DN的关系，以及泄浊解毒、活血通络中药对DN大鼠肾脏的保护作用和可能机制，为临床治疗DN提供实验依据。 方法：雄性Sprague-Dauley大鼠85只，体重170±10g，适应性饲养一周，测定血糖和尿蛋白均为阴性后，随机选取15只为正常组（NG)，给予普通饲料喂养。其余大鼠给予高糖高脂饲料喂养，12周后给予一次性腹腔注射STZ35mg/kg，正常组大鼠给予一次性腹腔注射相当剂量的0.1mol/L枸橼酸缓冲液。72小时后尾静脉采血测随机血糖≥16.7mmol/L为糖尿病造模成功，造模过程中死亡两只，另有一只大鼠血糖不达标，均予以剔除。将成模大鼠随机分为4组：模型组(MG)、中药组(ZG)、西药组(XG)、中药加西药组(JG)，中药加西药组16只，剩余各组17只。成模后开始给药，每日一次，治疗组每只大鼠给予4ml/d的药物溶液，正常组与模型组均灌服等体积的生理盐水，共给药16周。自由饮水、进食，室温22-25℃，相对湿度40%-60%，12小时交替照明。在给药期间的第4周、8周、12周各监测体重、血糖及尿蛋白。于给药第16周末，禁食12小时，不禁水，留取各组大鼠24小时尿液，测定24小时尿蛋白定量，称量体重，10%水合氯醛麻醉动物，腹主动脉取血，摘取右侧肾脏并称重，计算肥大指数，留取肾脏标本，光镜、电镜下观察肾脏病理形态学变化，提取各组肾皮质RNA，用实时荧光定量PCR方法检测Ang-1mRNA的含量，同时免疫组化染色观察Ang-1的表达情况。 结果： 1各组大鼠体重、肾重、肥大指数（肾重/体重）与正常组比较，各治疗组及模型组体重明显减轻（P0.05）；各治疗组及模型组之间体重无显著差异（P0.05）。与正常组比较，各治疗组及模型组肥大指数明显升高（P0.05)；各治疗组及模型组之间比较无明显差别（P0.05)。各组右肾重比较差异均无统计学意义（P0.05）。 2各组大鼠24小时尿蛋白定量与正常组比较，各治疗组及模型组24小时尿蛋白定量（24-hour urinary totalprotein,24hUTP）均升高(P0.05)；与模型组比较，各治疗组UTP下降(P0.05)；与中药组及西药组比较，中药加西药组UTP下降(P0.05)；中药组UTP与西药组相比无明显差异(P0.05)。 3各组大鼠血清总蛋白、白蛋白、血脂及肾功能的比较与正常组相比，模型组及各治疗组血清总蛋白（TP）及白蛋白（ALB)均降低（P0.05)；与模型组比较，各治疗组TP及ALB均升高（P0.05)；与中药组及西药组比较，中药加西药组TP及ALB升高（P0.05)；中药组与西药组相比无明显差异（P0.05）。与正常组相比，模型组及各治疗组总胆固醇(TC)及甘油三脂(TG)均明显升高（P0.05）；与模型组比较，，各治疗组TC及TG降低（P0.05）；与中药组及西药组比较，中药加西药组TC及TG降低（P0.05)；中药组与西药组相比无明显差异(P0.05)。正常组、模型组、各治疗组血肌酐及尿素氮比较均无统计学意义(P0.05)。 4各组大鼠糖化血红蛋白（HbA1c)的比较与正常组比较，模型组及各治疗组HbA1c均明显升高（P0.05）；与模型组及西药组相比，中药加西药组及中药组HbA1c均明显降低（P0.05）；西药组HbA1c比模型组降低但无统计学意义(P0.05)；中药加西药组HbA1c比中药组降低但无统计学意义(P0.05)。 5肾脏病理形态学观察 5.1光镜观察正常组大鼠肾脏肾小球正常大小，结构清晰，未见肾小球肥大或萎缩，基底膜无增厚，系膜基质分布面积正常。模型组大鼠肾脏肾小球明显肥大，基底膜增厚，系膜区显著增宽。各治疗组以上病理改变较模型组为轻，各治疗组间无明显差异。 5.2电镜观察正常组肾小球内皮细胞未见异常，基底膜厚度均匀，结构完整，足突无融合、缺如等异常改变；模型组肾小球内皮细胞明显增生、突出，基底膜明显增厚，伴有足细胞足突广泛融合；各治疗组也均有不同程度的病理改变，但较模型组轻，各组间差异不明显。 6各组大鼠肾皮质Ang-1的表达 6.1Real-Time PCR检测肾组织Ang-1mRNA的表达与正常组相比，模型组及各治疗组Ang-1mRNA表达量均明显升高（P0.05）；与模型组比较，各治疗组Ang-1mRNA表达量均明显升高(P0.05)；与西药组和中药组比较，中药加西药组Ang-1mRNA表达量明显升高(P0.05)；西药组与中药组相比差异无统计学意义(P0.05)。 6.2免疫组织化学法检测肾组织Ang-1的表达正常组Ang-1在肾小球足细胞少量表达；与正常组相比，各造模组大鼠肾组织中Ang-1表达明显增加(P0.05)，突出表达于足细胞；与模型组比较，各治疗组Ang-1表达明显增加(P0.05)；各治疗组之间比较，中药加西药组肾组织Ang-1表达最强，与中药组和西药组相比有明显差异(P0.05)；中药组与西药组相比Ang-1的表达无明显差别(P0.05)。 结论： 1泄浊解毒、活血通络中药可减少尿蛋白的排泄，升高血清总蛋白、白蛋白，降低血糖、血脂，延缓DN的进展。 2泄浊解毒、活血通络中药可以有效抑制系膜基质的增生、基底膜的增厚及内皮细胞的增生和足细胞的融合，延缓肾脏病理进展。 3泄浊解毒、活血通络中药能够上调DN大鼠肾组织Ang-1的表达，提示该方可以减轻血管的渗漏并促进血管的成熟和稳定，减少尿蛋白的排泄，延缓DN进展从而达到对DN大鼠肾脏保护作用。 4泄浊解毒、活血通络中药和厄贝沙坦联合应用在减少蛋白尿，升高血清蛋白，降低血脂，调节Ang-1表达等方面比单纯用中药或西药作用均强。
[Abstract]:Objective: To establish DM rat model by feeding high-sugar and high-fat diet and intraperitoneal injection of low-dose streptozotocin (STZ), and to observe the biochemical indexes, pathological morphological changes of kidney and kidney of experimental animals. The expression of Angiopoietin-1 (Ang-1) in cortical tissue was studied to explore the relationship between Ang-1 and DN, and the protective effect and possible mechanism of Chinese herbs on kidney of DN rats.
Methods: Eighty-five Sprague-Dauley male rats weighing 170 + 10g were fed adaptively for one week. After the blood glucose and urinary protein were tested negative, 15 rats were randomly selected as normal group (NG) and fed with normal diet. The other rats were fed with high glucose and high fat diet, and the other rats were injected with STZ 35mg/kg by abdominal cavity for 12 weeks. The rats were randomly divided into four groups: model group (MG), Chinese medicine group (ZG), western medicine group (XG), Chinese medicine plus Western medicine (TCM) and Chinese medicine plus Western medicine (WM). Group JG, Chinese medicine plus Western medicine group 16, the remaining 17 in each group. After modeling, the rats in the treatment group were given 4 ml/d of the drug solution once a day. The normal group and the model group were given the same volume of physiological saline, a total of 16 weeks. Free drinking water, eating, room temperature 22-25 C, relative humidity 40% - 60%, 12 hours alternate lighting. At the end of the sixteenth week, fasting for twelve hours, water was not forbidden. 24-hour urine was taken from rats in each group. 24-hour urine protein was measured. Weight was weighed, 10% chloral hydrate was anesthetized, abdominal aorta was taken for blood, right kidney was taken for weighing, hypertrophy index was calculated, kidney samples were taken, light microscope and electricity were used. The pathological morphological changes of kidney were observed under microscope. RNA was extracted from renal cortex of each group. The content of Ang-1 mRNA was detected by real-time fluorescence quantitative PCR and the expression of Ang-1 was observed by immunohistochemical staining.
Result:
1 Compared with the normal group, the body weight, kidney weight and hypertrophy index (kidney weight/body weight) of rats in each treatment group and model group decreased significantly (P 0.05). There was no significant difference in body weight between each treatment group and model group (P 0.05). Compared with the normal group, the hypertrophy index of each treatment group and model group increased significantly (P 0.05). There was no significant difference (P0.05). There was no significant difference in right kidney weight between each group (P0.05).
Compared with the normal group, the 24-hour urinary total protein (24-hour UTP) in the treatment group and the model group increased (P 0.05); compared with the model group, the UTP in the treatment group decreased (P 0.05); compared with the traditional Chinese medicine group and the western medicine group, the UTP in the traditional Chinese medicine plus Western medicine group decreased (P 0.05); There was no significant difference between the two groups (P0.05).
Compared with the normal group, the serum total protein (TP) and albumin (ALB) in the model group and the treatment group decreased (P 0.05); compared with the model group, TP and ALB in the treatment group increased (P 0.05); compared with the traditional Chinese medicine group and the western medicine group, TP and ALB in the traditional Chinese medicine plus Western medicine group increased (P 0.05); Compared with the normal group, the total cholesterol (TC) and triglyceride (TG) in the model group and each treatment group were significantly higher (P 0.05); compared with the model group, TC and TG in each treatment group were lower (P 0.05); compared with the traditional Chinese medicine group and the western medicine group, TC and TG in the traditional Chinese medicine plus Western Medicine group were lower (P 0.05); compared with the traditional Chinese medicine group and the western medicine group, TC and TG were lower (P 0.05). Compared with no significant difference (P 0.05). Normal group, model group, the treatment group serum creatinine and urea nitrogen were not statistically significant (P 0.05).
Compared with normal group, HbA1c in model group and treatment group increased significantly (P 0.05); compared with model group and Western medicine group, HbA1c in traditional Chinese medicine plus Western medicine group and traditional Chinese medicine group decreased significantly (P 0.05); HbA1c in western medicine group was lower than that in model group, but there was no statistical significance (P 0.05); HbA1c in traditional Chinese medicine plus Western medicine group was lower than that in model group. Compared with the Chinese medicine group, there was no statistical significance (P0.05).
5 pathological observation of kidney
5.1 The glomeruli of normal rats were normal size, clear structure, no glomerular hypertrophy or atrophy, no basement membrane thickening and normal distribution area of mesangial matrix were observed under light microscope. There was no significant difference.
5.2 Electron microscopic observation showed that there were no abnormal changes in the glomerular endothelial cells in the normal group, such as the thickness of basement membrane was uniform, the structure was intact, the foot process did not fuse, and the absence of abnormal changes; the glomerular endothelial cells in the model group were obviously hyperplasia, prominent, the basement membrane was obviously thickened, and the foot process of podocytes fused extensively; the treatment groups also had different degrees of Patholog There was no significant difference between groups.
6 the expression of Ang-1 in renal cortex of rats in each group.
Compared with the normal group, the expression of Ang-1 mRNA in the model group and each treatment group was significantly increased (P 0.05); compared with the model group, the expression of Ang-1 mRNA in each treatment group was significantly increased (P 0.05); compared with the western medicine group and the traditional Chinese medicine group, the expression of Ang-1 mRNA in the traditional Chinese medicine plus Western medicine group was significantly increased (P 0.05); There was no significant difference between western medicine group and traditional Chinese medicine group (P0.05).
6.2 Immunohistochemical method was used to detect Ang-1 expression in podocytes of the normal group; compared with the normal group, the expression of Ang-1 in renal tissue of rats in each model group was significantly increased (P 0.05), especially in podocytes; compared with the model group, the expression of Ang-1 in each treatment group was significantly increased (P 0.05); The expression of Ang-1 was strongest in the kidney tissue of Chinese medicine plus Western medicine group, which was significantly different from that of Chinese medicine group and Western medicine group (P 0.05).
Conclusion:
1. Dispelling turbidity and detoxification, promoting blood circulation and dredging collaterals can reduce urinary protein excretion, increase serum total protein, albumin, reduce blood sugar and lipid, and delay the progress of DN.
2. The Chinese herbal medicine of relieving turbidity and detoxification, promoting blood circulation and dredging collaterals can effectively inhibit the proliferation of mesangial matrix, thickening of basement membrane, proliferation of endothelial cells and fusion of podocytes, and delay the progress of renal pathology.
3. The Chinese herbal medicine of eliminating turbidity and detoxification, promoting blood circulation and dredging collaterals can up-regulate the expression of Ang-1 in kidney tissue of DN rats, suggesting that this prescription can reduce the leakage of blood vessels, promote the maturation and stability of blood vessels, reduce the excretion of urinary protein, delay the progress of DN, and thus achieve the renal protection of DN rats.
The combination of Chinese herbal medicine and irbesartan in reducing albuminuria, elevating serum protein, lowering blood lipid and regulating Ang-1 expression is more effective than Chinese herbal medicine or western medicine.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R259;R277.5
本文编号：2200086
[Abstract]:Objective: To establish DM rat model by feeding high-sugar and high-fat diet and intraperitoneal injection of low-dose streptozotocin (STZ), and to observe the biochemical indexes, pathological morphological changes of kidney and kidney of experimental animals. The expression of Angiopoietin-1 (Ang-1) in cortical tissue was studied to explore the relationship between Ang-1 and DN, and the protective effect and possible mechanism of Chinese herbs on kidney of DN rats.
Methods: Eighty-five Sprague-Dauley male rats weighing 170 + 10g were fed adaptively for one week. After the blood glucose and urinary protein were tested negative, 15 rats were randomly selected as normal group (NG) and fed with normal diet. The other rats were fed with high glucose and high fat diet, and the other rats were injected with STZ 35mg/kg by abdominal cavity for 12 weeks. The rats were randomly divided into four groups: model group (MG), Chinese medicine group (ZG), western medicine group (XG), Chinese medicine plus Western medicine (TCM) and Chinese medicine plus Western medicine (WM). Group JG, Chinese medicine plus Western medicine group 16, the remaining 17 in each group. After modeling, the rats in the treatment group were given 4 ml/d of the drug solution once a day. The normal group and the model group were given the same volume of physiological saline, a total of 16 weeks. Free drinking water, eating, room temperature 22-25 C, relative humidity 40% - 60%, 12 hours alternate lighting. At the end of the sixteenth week, fasting for twelve hours, water was not forbidden. 24-hour urine was taken from rats in each group. 24-hour urine protein was measured. Weight was weighed, 10% chloral hydrate was anesthetized, abdominal aorta was taken for blood, right kidney was taken for weighing, hypertrophy index was calculated, kidney samples were taken, light microscope and electricity were used. The pathological morphological changes of kidney were observed under microscope. RNA was extracted from renal cortex of each group. The content of Ang-1 mRNA was detected by real-time fluorescence quantitative PCR and the expression of Ang-1 was observed by immunohistochemical staining.
Result:
1 Compared with the normal group, the body weight, kidney weight and hypertrophy index (kidney weight/body weight) of rats in each treatment group and model group decreased significantly (P 0.05). There was no significant difference in body weight between each treatment group and model group (P 0.05). Compared with the normal group, the hypertrophy index of each treatment group and model group increased significantly (P 0.05). There was no significant difference (P0.05). There was no significant difference in right kidney weight between each group (P0.05).
Compared with the normal group, the 24-hour urinary total protein (24-hour UTP) in the treatment group and the model group increased (P 0.05); compared with the model group, the UTP in the treatment group decreased (P 0.05); compared with the traditional Chinese medicine group and the western medicine group, the UTP in the traditional Chinese medicine plus Western medicine group decreased (P 0.05); There was no significant difference between the two groups (P0.05).
Compared with the normal group, the serum total protein (TP) and albumin (ALB) in the model group and the treatment group decreased (P 0.05); compared with the model group, TP and ALB in the treatment group increased (P 0.05); compared with the traditional Chinese medicine group and the western medicine group, TP and ALB in the traditional Chinese medicine plus Western medicine group increased (P 0.05); Compared with the normal group, the total cholesterol (TC) and triglyceride (TG) in the model group and each treatment group were significantly higher (P 0.05); compared with the model group, TC and TG in each treatment group were lower (P 0.05); compared with the traditional Chinese medicine group and the western medicine group, TC and TG in the traditional Chinese medicine plus Western Medicine group were lower (P 0.05); compared with the traditional Chinese medicine group and the western medicine group, TC and TG were lower (P 0.05). Compared with no significant difference (P 0.05). Normal group, model group, the treatment group serum creatinine and urea nitrogen were not statistically significant (P 0.05).
Compared with normal group, HbA1c in model group and treatment group increased significantly (P 0.05); compared with model group and Western medicine group, HbA1c in traditional Chinese medicine plus Western medicine group and traditional Chinese medicine group decreased significantly (P 0.05); HbA1c in western medicine group was lower than that in model group, but there was no statistical significance (P 0.05); HbA1c in traditional Chinese medicine plus Western medicine group was lower than that in model group. Compared with the Chinese medicine group, there was no statistical significance (P0.05).
5 pathological observation of kidney
5.1 The glomeruli of normal rats were normal size, clear structure, no glomerular hypertrophy or atrophy, no basement membrane thickening and normal distribution area of mesangial matrix were observed under light microscope. There was no significant difference.
5.2 Electron microscopic observation showed that there were no abnormal changes in the glomerular endothelial cells in the normal group, such as the thickness of basement membrane was uniform, the structure was intact, the foot process did not fuse, and the absence of abnormal changes; the glomerular endothelial cells in the model group were obviously hyperplasia, prominent, the basement membrane was obviously thickened, and the foot process of podocytes fused extensively; the treatment groups also had different degrees of Patholog There was no significant difference between groups.
6 the expression of Ang-1 in renal cortex of rats in each group.
Compared with the normal group, the expression of Ang-1 mRNA in the model group and each treatment group was significantly increased (P 0.05); compared with the model group, the expression of Ang-1 mRNA in each treatment group was significantly increased (P 0.05); compared with the western medicine group and the traditional Chinese medicine group, the expression of Ang-1 mRNA in the traditional Chinese medicine plus Western medicine group was significantly increased (P 0.05); There was no significant difference between western medicine group and traditional Chinese medicine group (P0.05).
6.2 Immunohistochemical method was used to detect Ang-1 expression in podocytes of the normal group; compared with the normal group, the expression of Ang-1 in renal tissue of rats in each model group was significantly increased (P 0.05), especially in podocytes; compared with the model group, the expression of Ang-1 in each treatment group was significantly increased (P 0.05); The expression of Ang-1 was strongest in the kidney tissue of Chinese medicine plus Western medicine group, which was significantly different from that of Chinese medicine group and Western medicine group (P 0.05).
Conclusion:
1. Dispelling turbidity and detoxification, promoting blood circulation and dredging collaterals can reduce urinary protein excretion, increase serum total protein, albumin, reduce blood sugar and lipid, and delay the progress of DN.
2. The Chinese herbal medicine of relieving turbidity and detoxification, promoting blood circulation and dredging collaterals can effectively inhibit the proliferation of mesangial matrix, thickening of basement membrane, proliferation of endothelial cells and fusion of podocytes, and delay the progress of renal pathology.
3. The Chinese herbal medicine of eliminating turbidity and detoxification, promoting blood circulation and dredging collaterals can up-regulate the expression of Ang-1 in kidney tissue of DN rats, suggesting that this prescription can reduce the leakage of blood vessels, promote the maturation and stability of blood vessels, reduce the excretion of urinary protein, delay the progress of DN, and thus achieve the renal protection of DN rats.
The combination of Chinese herbal medicine and irbesartan in reducing albuminuria, elevating serum protein, lowering blood lipid and regulating Ang-1 expression is more effective than Chinese herbal medicine or western medicine.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R259;R277.5
【参考文献】
相关期刊论文 前10条
1 陈璐;张洪;;黄连素治疗2型糖尿病机制研究进展[J];辽宁中医药大学学报;2013年11期
2 熊雄;王懿萍;王岳峰;吴玉娟;;黄连多糖的降血糖活性研究[J];时珍国医国药;2013年10期
3 周凤新;南征;;南征教授治疗糖尿病肾病新路径——调散膏,达膜原[J];天津中医药;2012年06期
4 李文;吴晨光;王丽;陈宇宁;陈艳;徐志刚;;积雪草酸对高糖培养大鼠肾小球系膜细胞氧化应激及细胞外基质分泌的影响[J];江苏医药;2011年21期
5 梁正宇;苏文弟;闫威;张柏林;;张柏林治疗糖尿病肾病常用对药介绍[J];天津中医药;2011年05期
6 王晓娟;王旭;;中医药治疗糖尿病肾病研究的最新综述[J];中医临床研究;2011年11期
7 郑承红;孔彩霞;彭聪;柯淑红;;积雪草对2型糖尿病大鼠胰岛素抵抗影响的研究[J];内科急危重症杂志;2011年02期
8 刘亚明;郭继龙;刘必旺;刘丙辰;张慧忠;周保腾;张芳;梁红娟;;中药地龙的活性成分及药理作用研究进展[J];山西中医;2011年03期
9 侯丽然;孙丽娜;侯巍;滕杨;高金波;;蒲公英多糖的提取及降糖作用的研究[J];黑龙江医药科学;2010年06期
10 戈娜;李顺民;孙惠力;邵牧民;程小燕;熊国良;易无庸;郑义侯;;地龙对糖尿病肾病大鼠肾脏保护作用的研究[J];上海中医药杂志;2010年06期
本文编号：2200086
本文链接：https://www.wllwen.com/yixuelunwen/mazuiyixuelunwen/2200086.html
最近更新
教材专著
