益气解毒活络法对早期DN大鼠肾组织SREBP影响机制研究

【作者】 金珉廷；

【导师】 李敬林；

【作者基本信息】 辽宁中医药大学 ， 中西医结合临床， 2013， 博士

【摘要】 目的：探讨益气解毒活络中药复方对早期糖尿病肾病(DN)大鼠药效学指标及对肾组织固醇调节元件结合蛋白(SREBP-1c、SREBP-2c)等基因与蛋白表达的影响,观察益气解毒活络中药复方对其防治疗效及其作用机理。材料与方法：SPF级健康雄性Wistar大鼠90只,适应性饲养一周,按体重随机分为2组：正常对照组(10只)、造模组(80只)。正常对照组给予普通饲料喂养,造模组给予高糖高脂饲料喂养。1周后造模组大鼠予腹腔注射STZ42mg/kg,正常对照组予腹腔注射等量枸橼酸缓冲液。72小时后测大鼠空腹血糖,≥16.7mmol/L确定为DM模型成模。再根据大鼠血糖值高低对各造模组进行随机分组,模型组、预防组、高量组、低量组、西药组。益气解毒活络中药复方选用中药免煎颗粒剂溶于蒸馏水中。各治疗组每日用药量按大鼠人体(g/kg)的等效剂量6.3倍计算；中药高剂量治疗组的用药量为中药低剂量治疗组的3倍。正常对照组、模型空白组大鼠给予等量生理盐水灌胃。中药预防组在成DM模型后立即给予灌服益气解毒活络中药复方,每日1次,共计给药10周：中药低剂量治疗组、中药高剂量治疗组及阳性西药对照组均在成DM模型2周后,即成DN模型后灌服中药复方,每日1次,共计给药8周。在灌胃结束后收集大鼠尿液待测尿微量白蛋白；行腹主动脉穿刺取血检测大鼠血糖、糖化血红蛋白、血β2微球蛋白水平、血脂、肾功：肌酐尿素氮；光镜下观察大鼠肾脏组织HE, PAS染色图片改变：用ELISA法检测DN大鼠血清中SREBP-1c、SREBP-2c的含量；观察益气解毒活络中药复方对早期DN大鼠的肾组织中SREBP-1c、SREBP-2c基因及蛋白表达的影响。结果：1.各组大鼠血糖变化：各组大鼠血糖变化结果显示,与正常组比较,模型组大鼠的血糖值明显升高,两组之间有显著性差异(P<0.01)：与模型组比较,药物干预治疗各组大鼠血糖值均下降明显(P<0.01),但均未达到正常血糖值的范围,在使用药物各治疗组中,中药预防组的血糖下降最为明显,实验结果表明：使用药物干预治疗可以显著降低早期DN大鼠的血糖值升高,中药预防组的干预效果最好,西药组的干预效果次之。2.各组大鼠糖化血红蛋白的变化：与正常对照组比较,模型组大鼠的糖化血红蛋白含量明显升高,两组有显著性差异(P<0.01)：与模型组比较,各治疗组大鼠的糖化血红蛋白均有显著降低(P<0.01),但与正常组比较有显著性差异(P<0.01),提示药物干预能够有效降低DN大鼠糖化血红蛋白,但尚未达到正常组的水平；中药预防治疗组的干预效果在各使用药物干预治疗组中最明显。3.各组大鼠尿微量白蛋白变化：与正常组比较,模型组大鼠的尿微量白蛋白明显升高,两组有显著性差异(P<0.01)；与模型组相比,各组大鼠的尿微量白蛋白使用药物干预治疗各组均有显著降低(P<0.01),但与正常组比较有显著性差异(P<0.01),提示药物干预能够有效降低DN大鼠尿微量白蛋白的含量,但尚未达到正常组的水平；中药预防治疗组的干预效果在各使用药物干预治疗组中最明显,实验结果表明,中药预防治疗组对大鼠尿微量白蛋白的含量的影响最明显。4.各组大鼠血p2-微球蛋白的变化：与正常组比较,模型组大鼠的血β2-微球蛋白明显升高,两组有显著性差异(P<0.01)；与模型组相比,各组大鼠的血β2-微球蛋白使用药物干预治疗各组均有显著降低(P<0.01),但与正常组比较有显著性差异(P<0.01),提示药物干预能够有效降低DN大鼠血β2-微球蛋白；各使用药物治疗组间无明显差异(P>0.05)。实验结果表明,中药预防治疗组对β2-微球蛋白含量的影响最明显。5.各组大鼠血清中肌酐的变化：各组大鼠血清中肌酐含量无明显变化,各组间无明显差异(P>0.05)。使用药物干预治疗各组中,中药预防组最高,与西药组比较,它们两组间无明显差异(P>0.05)。6.各组大鼠血清中尿素氮变化：与正常组比较,模型组大鼠的血清中尿素氮明显升高,两组有显著性差异(P<0.01)；与模型组比较,使用药物各组大鼠的血清中尿素氮含量中药预防治疗组的干预效果在各使用药物干预治疗组间中最明显(P<0.01),提示药物干预能够有效降低DN大鼠血清中尿素氮含量,但尚未达到正常组的水平。实验结果表明,中药预防治疗组对血清中尿素氮影响最明显。7.各组大鼠肾脏病理变化HE染色：低倍镜下观察,正常组大鼠肾小球、肾小管及系膜基质未见明显病理变化；高倍镜下可见,模型组肾小球硬化,系膜细胞及系膜基质增生,肾小管上皮细胞空泡变形；各治疗组及中药预防组大鼠肾脏病理变化程度明显轻于模型对照组,中药预防组大鼠肾脏的肾小球及肾小管比正常组大鼠稍微增大；中药高剂量与西药组大鼠肾脏肾小球体积大小相当,但均大于正常组。PAS染色：正常组大鼠肾小球、肾小管及系膜基质未见明显病理变化,肾脏组织PAS染色未见阳性物质；模型组大鼠肾脏组织可见广泛的PAS染色阳性物质,主要位于小叶中央部,肾小球基底膜增厚,系膜基质明显增多,肾小球弥漫性病变,小动脉玻璃样变,肾小球内部偶见圆形、椭圆形或锥形的结节,个别肾小球荒废；而各治疗组及中药预防组大鼠肾脏PAS染色呈弱阳性,西药组可见弥漫肾小球硬化,系膜基质和细胞外基质明显增多,肾小管萎缩；中药预防组可见肾小球系膜基质轻度增多,肾小球基底膜及肾小管无病变；中药高剂量组可见肾小球系膜基质及系膜细胞增多,肾小球基底膜无变化,肾小管无病变；中药低剂量组大鼠可见节断性肾小球硬化,肾小球系膜基质增多,基底膜增厚。8.各组大鼠血清中血脂的变化：(1)甘油三酯实验结果显示,与正常组比较,模型组大鼠的血清中甘油三酯明显升高,两组有显著性差异(P<0.01)；与模型组比较,各药物治疗组大鼠的血清中甘油三酯明显降低有显著性差异(P<0.01),与正常组比较也有显著性差异(P<0.01),但使用中药治疗各组间无明显差异(P>0.05),提示药物干预能够有效降低DN大鼠血清中甘油三酯含量,可以接近正常组的水平。(2)胆固醇实验结果显示,与正常组比较,模型组大鼠的血清中胆固醇明显升高,两组有显著性差异(P<0.01)；与模型组比较,各药物治疗组的血清中胆固醇明显降低有显著性差异(P<0.01),与正常组比较有显著性差异(P<0.01),但使用中药治疗各组无明显差异(P>0.05),提示药物干预能够有效降低DN大鼠血清中胆固醇,可以接近正常组的水平。(3)高密度脂蛋白实验结果显示,各组大鼠血清中高密度脂蛋白含量无明显变化,各组间无显著性差异(P>0.05)。(4)低密度脂蛋白实验结果显示,与正常组比较,模型组大鼠的血清低密度脂蛋白明显升高,两组有显著性差异(P<0.01)；与模型组比较,各药物治疗组大鼠的血清中低密度脂蛋白明显降低有显著性差异(P<0.01),与正常组比较也有显著性差异(P<0.01),提示药物干预能够有效降低DN大鼠血清中低密度脂蛋白,但未达到正常组的水平。9.各组大鼠血清中SREBP-1c、SREBP-2c的含量测定结果(1)与正常组比较,模型组大鼠的血清中SREBP-1c的含量明显升高,两组有显著性差异(P<0.01)；与模型组比较,使用药物各组大鼠的血清中SREBP-1c含量明显降低有显著性差异(P<0.01),但与正常组比较无显著性差异(P>0.05),提示药物干预能够有效降低DN大鼠血清中SREBP-1c的含量,但尚未达到正常组的水平。实验结果表明,中药预防治疗组降低血清中SREBP-1c的含量的影响最明显。(2)与正常组比较,模型组大鼠的血清中SREBP-2c的含量明显升高,两组有显著性差异(P<0.01)；与模型组比较,使用药物各组大鼠的血清中SREBP-2c的含量均下降(P<0.01),在使用药物各治疗组间,使用药物各组大鼠的血清中SREBP-2c的含量中药预防治疗组的降低最明显(P<0.01),提示药物干预能够有效降低DN大鼠血清中SREBP-2c的含量,但尚未达到正常组的水平。实验结果表明,中药预防治疗组大鼠血清中SREBP-2c的含量的最明显。10.各组大鼠的肾组织中SREBP-1c、SREBP-2c mRNA的表达结果(1)与正常组比较,模型组大鼠的肾组织中SREBP-1c的mRNA含量明显升高,两组有显著性差异(P<0.01)；与模型组比较,使用药物各组大鼠的肾组织中SREBP-1c的mRNA含量明显降低(P<0.01),但与正常组比较有显著性差异(P<0.01),与中药预防组比较,使用药物各组有显著性差异(P<0.05)。提示药物干预能够有效降低DN大鼠的肾组织中SREBP-1c的mRNA含量,但尚未达到正常组的水平。(2)与正常组比较,模型组大鼠的肾组织中SREBP-2c的mRNA含量明显升高,两组有显著性差异(P<0.01)；与模型组比较,使用药物各组大鼠的肾组织中SREBP-2c的mRNA含量明显降低(P<0.01),但与正常组比较有显著性差异(P<0.01),与中药预防组比较,使用药物各组有显著性差异(P<0.05),提示药物干预能够有效降低DN大鼠的肾组织中SREBP-2c的mRNA含量,但尚未达到正常组的水平。11.各组大鼠的肾组织中SREBP-lc、SREBP-2c蛋白表达结果(1)与正常组比较,模型组大鼠的肾组织中SREBP-lc的蛋白表达明显升高,两组有显著性差异(P<0.01)；与模型组比较,使用药物各组大鼠的肾组织中SREBP-1c的蛋白表达明显降低(P<0.01),各药物治疗组中药预防组大鼠的肾组织中SREBP-lc的蛋白表达最低(P<0.01),提示药物干预能够有效降低DN大鼠的肾组织中SREBP-1c的蛋白表达水平。(2)与正常组比较,模型组大鼠的肾组织中SREBP-2c的蛋白表达明显升高,两组有显著性差异(P<0.01)；与模型组比较,各药物治疗组的肾组织中SREBP-2c的蛋白表达明显降低(P<0.01),但与正常组比较有显著性差异(P<0.01),各药物治疗组间中药预防组大鼠的肾组织中SREBP-2c的蛋白表达最低(P<0.01),提示药物干预能够有效降低DN大鼠的肾组织中SREBP-2c的蛋白表达水平,但尚未达到正常组的表达水平；中药高剂量组和中药低剂量组高于西药组和中药预防组。结论：1.采用STZ一次腹腔注射法造模诱导的早期DN大鼠,可以成功复制建立早期DN模型。2.益气解毒活络中药复方对早期DN大鼠的药效学指标有改善作用,能够有效控制早期DN大鼠的血糖、糖化、尿素氮、血脂、肾功、血β2、尿蛋白变化、保护受损的肾组织,对早期DN有明显的预防和治疗作用。3.益气解毒活络中药对DN的防治作用机制可能与脂毒和固醇调节元件结合蛋白作用密切相关。更多还原

【Abstract】 Purpose:Investigate the effects of Yiqi Jiedu meridians of Chinese medicine compound on early diabetic nephropathy (DN) rats pharmacodynamic indexes and renal tissue of sterol regulatory element binding protein (SREBP-lc, SREBP-2c) and the effect of gene and protein expression, observation of Yiqi Jiedu adjustable mechanism of traditional Chinese medicine compound on the protective effect and its role.Material and method:The SPF level of90healthy male Wistar rats, fed a week, were randomly divided into2groups:normal control group (10rats), model group (80rats). The control group was given normal diet, model group fed with high fat diet.1weeks Houzao group rats were given intraperitoneal injection of STZ42mg/kg, normal control group, were injected with citrate buffer. After72hours the rats were measured fasting blood glucose, identified as DM models over16.7mmol/L. According to the blood glucose level of each group were randomly divided into two groups, model group, prevention group, high dose group, low dose group, western medicine group. Effect of Yiqi Jiedu meridians of Chinese medicine compound selection of Chinese medicine decoction and granule dissolved in distilled water. The treatment group daily dosage of human in rats (g/kg) equivalent dose6.3times computation; dosage Chinese medicine high dose treatment group was3times of low dosage Chinese medicine treatment groups. The normal control group, model control group rats were given normal saline. Traditional Chinese medicine prevention group were given orally active compound Yiqi Jiedu immediately in the DM model,1times a day, a total of10weeks of administration of traditional Chinese medicine; low dose group, high dose treatment group and positive control group of Western medicine in DM model after2weeks, the DN model after the perfusion of Chinese herbal compound,1times a day, a total of8weeks of administration. Collection of urine of rats in the stomach after urinary albumin measured; abdominal aortic puncture blood blood glucose, glycosylated hemoglobin, serum β2microglobulin levels, blood lipid, renal function:creatinine blood urea nitrogen; HE in renal tissues of rats were observed under the light microscope, PAS staining images change by ELISA method; detection of serum DN SREBP-lc in rats, the content of SREBP-2c; the expression of SREBP-2c gene and protein SREBP-1c, observation of Yiqi Jiedu meridians of Chinese medicine compound on early DN in kidney of rat.Results:1. The changes of blood glucose of rats in each group:rats blood glucose changes showed, compared with the normal group, the model group rats blood glucose increased significantly, there was significant difference between the two groups (P<0.01); compared with the model group, drug intervention treatment of blood glucose of rats in each group were significantly decreased (P<0.01), but did not reach normal glucose range of values, in the use of drugs in the treatment group, Chinese medicine prevention group blood sugar decreased the most obvious, the experimental results show that:the use of drug intervention therapy can significantly reduce early DN rats blood glucose level increases, the best intervention effect of Chinese medicine prevention group, western medicine group and the effect of intervention time.2. Changes in rats of glycated hemoglobin:compared with the normal control group, the content of glycosylated hemoglobin in rats of model group increased obviously, there was significant difference between two groups (P<0.01); compared with the model group, glycosylated hemoglobin of rats in each treatment group decreased significantly (P<0.01), but compared with the normal group there was a significant difference (P<0.01), indicating that the drug intervention can effectively reduce the DN rat glycated hemoglobin, but has not yet reached the normal level; traditional Chinese medicine prevention intervention treatment group was most obvious in the drug intervention group.3. Urine albumin changes of rats in each group:compared with the normal group, urinary albumin in rats of model group increased obviously, there was significant difference between two groups (P<0.01); compared with the model group, albumin in the urine of rats in each group using the drug intervention group were significantly decreased (P<0.01), but with the the normal group were significant difference (P<0.01), indicating that the drug intervention can effectively reduce the content of albumin DN in rat urine, but has not yet reached the normal level; traditional Chinese medicine prevention intervention treatment group was most obvious in the treatment group, the experimental results show that, the most obvious effect of prevention and treatment group content of albumin in rat urine.4. Changes of2-microglobulin β groups of rats:compared with the normal group, the blood β2-microglobulin in the rats of model group increased obviously, there was significant difference between two groups (P<0.01); compared with the model group, the blood β2-microglobulin rats using drug intervention group were significantly decreased (P<0.01), but compared with the normal group, there was significant difference (P<0.01), indicating that the drug intervention can effectively reduce DN rats blood β2-microglobulin; the use of drug treatment groups had no significant difference (P>0.05). The experimental results show that, traditional Chinese medicine prevention treatment group was most obvious influence on the content of2-β2-microglob.5. Changes in serum creatinine of rats in each group:the serum creatinine content in rats of every group had no obvious change, no significant differences among the groups (P>0.05). The use of drug intervention groups, traditional Chinese medicine prevention group was the highest, compared with the western medicine group, they are no significant difference between the two groups (P>0.05).6. Changes of serum urea nitrogen of rats in each group:compared with the normal group, serum urea nitrogen in the rats of model group were significantly elevated, there was significant difference between two groups (P<0.01); compared with the model group, the serum drug use rats urea nitrogen content in Chinese medicine prevention intervention treatment group was most obvious in the use of drug intervention group (P<0.01), in the prompt intervention can effectively reduce the blood urea nitrogen content in serum of DN rats, but has not yet reached the normal level. The experimental results show that, traditional Chinese medicine prevention treatment group was most obvious effect on serum urea nitrogen.7. The pathological changes of kidney of ratsHE staining:observation at low magnification, rats in normal control group, renal tubular and glomerular mesangial matrix without obvious pathological changes; high magnification, model group, glomerulosclerosis, mesangial cells and mesangial matrix proliferation, vacuoles in renal tubular epithelial cell deformation; each treatment group and traditional Chinese medicine prevention group change degree renal pathology was obviously lighter than the model control group, traditional Chinese medicine prevention group rat kidney glomerular and tubular than normal rats increased slightly; very high dose and the western medicine group rat kidney glomerular volume size, but were higher than normal group.PAS staining:normal rats, renal tubular and glomerular mesangial matrix without obvious pathological changes of renal tissue, PAS staining was positive substance; kidney tissue of rats in model group showed extensive PAS staining positive material, mainly located in the central part of the lobule, glomerular basement membrane thickening, mesangial matrix increased, glomerular diffuse lesions, hyaline arteriolosclerosis, glomerular internal circular, elliptical or conical occasionally tuberous, individual glomerular obsolescence; and each treatment group and traditional Chinese medicine prevention of kidney of rats in the PAS group was weakly positive, western medicine group showed diffuse glomerular sclerosis, mesangial matrix and extracellular matrix increased, tubular atrophy; traditional Chinese medicine prevention group mesangial matrix increased slightly, glomerular basement membrane and renal tubular no lesion; high dose group showed mesangial matrix and mesangial cells increased, no change in the glomerular basement membrane, tubular no lesion; low dose traditional Chinese medicine group rats showed segmental glomerular sclerosis, mesangial matrix increased, basement membrane thickening.8. The changes of blood lipid in serum of rats in each group:(1)triglycerideExperimental results show that, compared with the normal group, model group rats serum triglyceride was significantly increased, there was significant difference between two groups (P<0.01); compared with the model group, the serum drug treatment group rats triglyceride decreased significantly (P<0.01), compared with the normal group also there was a significant difference (P<0.01), but the use of Chinese medicine treatment was no significant difference between the groups (P>0.05), indicating that the drug intervention can effectively reduce the content of triglyceride in serum of DN rats, can be close to normal level.(2) cholesterolExperimental results show that, compared with the normal group, serum cholesterol of rats in model group were significantly elevated, there was significant difference between two groups (P<0.01); compared with the model group, serum cholesterol in the drug treatment group decreased significantly (P<0.01), there was significant difference compared with the normal group (P<0.01), but the use of Chinese medicine was not significantly different between treatment groups (P>0.05), indicating that the drug intervention can effectively reduce serum cholesterol in DN rats, can be close to normal level.(3) and high density lipoproteinExperimental results show that, the content of high density lipoprotein in serum of rats in each group had no obvious change, there was no significant difference among the three groups (P>0.05).(4) and low density lipoproteinExperimental results show that, compared with the normal group, the serum low density lipoprotein in rats of model group increased obviously, there was significant difference between two groups (P<0.01);compared with the model group, the treatment group rats serum low density lipoprotein decreased significantly (P<0.01), and compared with normal group had significant difference (P<0.01), indicating that the drug intervention can effectively reduce the serum DN of rats with low density lipoprotein, but did not reach the normal level.9. Results the content of serum of rats in SREBP-lc, SREBP-2c(1) compared with the normal group, the content of SREBP-lc in serum of model group rats in the elevated significantly, there was significant difference between two groups (P<0.01); compared with the model group, the content of SREBP-lc in serum of drug use of rats in each group decreased significantly (P<0.01), but with normal group there is no significant difference (P>0.05), indicating that the drug intervention can effectively reduce the contents of SREBP-lc in serum of DN rats, but has not yet reached the normal level. Experimental results show that the effect of traditional Chinese medicine, preventive treatment group decreased the content of SREBP-lc in serum is the most obvious.(2) compared with the normal group, the content of SREBP-2c in serum of model group rats in the elevated significantly, there was significant difference between two groups (P<0.01); compared with the model group, the content of SREBP-2c in serum of rats in each group of drug use were decreased (P<0.01), in the use of drugs to the treatment group the content of serum SREBP-2c, traditional Chinese medicine drug use rats in the prevention and treatment group was most obvious decreased (P<0.01), indicating that the drug intervention can effectively reduce the contents of SREBP-2c in serum of DN rats, but has not yet reached the normal level. Experimental results show that the content of serum SREBP-2c, traditional Chinese medicine prevention treatment group rats in the most obvious.10. Results expression of rats in the renal tissue of SREBP-lc, SREBP-2c mRNA(1) compared with the normal group, the content of mRNA in SREBP-lc model group rats kidney tissue increased obviously, there was significant difference between two groups (P<0.01); compared with the model group, the content of mRNA SREBP-1c the use of drugs in the renal tissue of rats in each group were significantly lower (P<0.01), but with normal group had significant differences (P<0.01), and the traditional Chinese medicine prevention group, medication group had significant difference (P<0.05). Prompt medical intervention can effectively reduce the content of mRNA SREBP-lc DN in rat renal tissue, but has not yet reached the normal level.(2) compared with the normal group, the content of mRNA in SREBP-2c model group rats kidney tissue increased obviously, there was significant difference between two groups (P<0.01); compared with the model group, the content of mRNA SREBP-2c the use of drugs in the renal tissue of rats in each group were significantly lower (P<0.01), but with normal group had significant difference (P<0.01), and the traditional Chinese medicine prevention group, medication group had significant difference (P<0.05), indicating that the drug intervention can effectively reduce the content of mRNA SREBP-2c DN in rat renal tissue, but has not yet reached the normal level.11. Results the expression of SREBP-lc, SREBP-2c protein in renal tissue of rats in each group.(1) compared with the normal group, SREBP-lc model group rat kidney tissue protein expression was significantly increased, there was significant difference between two groups (P<0.01); compared with the model group, the expression of SREBP-lc rats using drugs in renal tissue protein decreased significantly (P<0.01), SREBP-lc prevention group rats treated with each drug group of traditional Chinese medicine in renal tissue protein expression in the lowest (P<0.01), indicating that the drug intervention can effectively reduce the SREBP-lc DN in rat renal tissue expression. (2) compared with the normal group, SREBP-2c model group rat kidney tissue protein expression was significantly increased, there was significant difference between two groups (P<0.01); compared with the model group, the expression of SREBP-2c in renal tissue of each drug group in the protein decreased significantly (P<0.01), but with normal group there was significant difference (P<0.01), SREBP-2c in renal tissue of each drug group Chinese medicine prevention group rats expressed proteins in the lowest (P<0.01), indicating that the (drug intervention can effectively reduce the SREBP-2c DN in rat renal tissue expression, expression but have not yet reached the normal group; high dose group and low dosage Chinese medicine group was higher than that of Western medicine group and traditional Chinese medicine prevention group.Conclusion:1. Using STZ once intraperitoneal injection molding induced early DN rats, can be replicated successfully establishing early DN model.2. Effect of Yiqi Jiedu active compound traditional Chinese medicine on early DN rats index improved, can effectively control the early DN rats blood glucose, glycosylated, urea nitrogen, serum lipid, renal function, urinary protein, serum β2change, protect the injured renal tissue, has obvious preventing and treating effect on early DN3. Possible mechanism of Yiqi Jiedu active preventive and therapeutic effects of Chinese medicine on DN and lipid toxin and steroid binding protein is closely related to regulatory element.更多还原