小檗碱对糖尿病肾病大鼠的肾脏保护作用以及对大鼠肾脏AGEs-RAGE信号通路的影响
发布时间：2018-07-23 13:22
【摘要】：目的:在大鼠体内实验中,观察小檗碱(Berberine,BBR)对糖尿病肾病(diabetic nephropathy,DN)大鼠肾脏生理病理和肾功能生化指标的影响,明确BBR对DN大鼠的肾功能及组织病变的改善作用;检测大鼠肾脏中部分组织细胞分泌的信号蛋白表达和相应通路的调节变化以及由BBR给药后影响的变化,探究BBR的肾脏保护作用是通过何种机制产生的。在体外细胞实验中,通过模拟DN条件刺激肾脏肾小球系膜细胞(Glomerular mesangial cells,GMCs)和足细胞,进而检测系膜细胞中AGEs-RAGE(晚期糖基化终末产物,advanced glycation end products,AGEs;晚期糖基化终末产物受体,receptor of advanced glycation end products,RAGE)信号通路的异常改变和足细胞与系膜细胞相互影响的信号通路VEGF-VEGFR2(血管内皮细胞生长因子,vascular endothelial growth factor,VEGF;血管内皮细胞生长因子Ⅱ型受体,vascular endothelial growth factor receptor 2,VEGFR2)的调节变化,同时观察BBR给药以及相应刺激剂和阻断剂给予后的影响作用,进一步探讨BBR对DN的肾脏保护作用以及潜在的分子机制和重要的分子靶点;为合理开发利用BBR防治DN提供理论依据,并且为探索新的治疗DN药物开拓新的研究方向和思路。方法:DN大鼠模型的制备,采用长周期(4-6周)高糖高脂饲料喂养并联合低剂量链脲佐菌素(STZ,streptozocin,35mg/kg)腹腔注射诱导。于72h后测量DN大鼠空腹血糖值(FBG,fasting blood-glucose),将FBG≥11.1mmol/L的SD大鼠纳入实验DN模型组。实验动物分组如下:正常组(Normal group),DN模型组(DN model group),BBR低剂量组(50mg/kg),BBR中剂量组(100mg/kg),BBR高剂量组(200mg/kg),二甲双胍组(200mg/kg)和卡托普利组(15mg/kg),每组12只大鼠。正常组大鼠喂以正常饲料,模型组和给药组继续给予高糖高脂饲料喂养,自由饮水。所有给药组按大鼠体重计算给药量,每日一次灌胃给药(Intragastric administration,i.g),正常组与模型组按体重给予相同溶剂羧甲基纤维素钠溶液(Sodium carboxymethylcellulose,CMC-Na)平行对照灌胃。FBG值每俩周检测一次,体重每周检测一次,处死前一天收集24h尿样用以检测尿总蛋白(UTP,total urine protein)和尿肌酐(Ucr,urine creatinine)的含量;麻醉后股动脉取血,分离血清检测血尿素氮(BUN,blood urea nitrogen),血肌酐(Scr,serum creatinine)和AGEs的含量。取同侧肾脏称重,制作组织切片进行肾脏病理学检查,包括HE染色和PAS染色。免疫组化法观察肾脏皮质中AGEs,RAGE,TGF-β1(转化生长因子,transforming growth factor-β1)和PKC-β(蛋白激酶C-β,protein kinase C-β)的分布与表达;Western blot法检测肾皮质中各信号通路蛋白表达水平;酶联免疫吸附法(ELISA,enzyme-linked immuno sorbent assay)法检测各组血清中AGEs的表达水平。体外实验以肾小球系膜细胞和足细胞为研究对象,首先分离大鼠的系膜细胞和足细胞进行原代培养和传代培养,建立DN模型细胞系。利用CCK-8试剂盒检测BBR对系膜细胞和足细胞DN状态下的增殖影响。使用ELISA法检测系膜细胞分泌AGEs的情况。对于AGEs-RAGE通路的蛋白,首先使用western blot法观察正常情况,模型情况和不同浓度BBR给药后以及使用AGEs刺激剂和RAGE阻断剂(Ab-RAGE抗体)后各信号蛋白的表达和变化情况;使用激光共聚焦法观察RAGE蛋白分布,表达类型和变化情况。对于VEGF-VEGFR2信号通路蛋白,首先使用蛋白芯片检测DN状态下足细胞分泌,确定关键因子;western bolt法验证VEGF,激光共聚焦法确定蛋白的分布和表达情况。CCK-8法确定VEGF对系膜细胞的适宜刺激浓度,用western bolt法观察DN状态,高糖,VEGF蛋白,足细胞上清液刺激和给药情况下的VEGFR2的表达和磷酸化的表达;激光共聚焦法检测p-VEGFR2的分布和表达情况。结果:1.BBR对DN大鼠的体重和空腹血糖的影响与正常组相比,造模成功后的DN大鼠的空腹血糖值显著升高;各BBR治疗组在前四周无明显的降血糖效果,而在第6到8周中剂量和高剂量的BBR组与DN模型组相比具有明显的降血糖作用;二甲双胍组显著性降低血糖作用从第四周开始。对于体重,仅高剂量BBR组在第8周具有改善作用。2.BBR对DN大鼠的肾功能及生化指标的影响与正常组相比,DN模型组的肾重/体重比值,尿总蛋白/尿肌酐比值,血清尿素氮(BUN,blood urea nitrogen)和血肌酐(SCr,serum creatinine)含量均明显升高。BBR中剂量组和高剂量组,以及阳性药二甲双胍组和卡托普利组均能显著改善肾功能和降低生化指标。3.BBR对DN大鼠的肾脏组织病理学的影响组织病理学检查结果显示,与正常组相比,模型组大鼠的肾脏肾小球明显肥大,透明样变化,基底膜明显增厚,细胞外基质聚集,出现较为严重的肾小球硬化,纤维化以及伴有大量的炎性细胞浸润。DN大鼠在给予中剂量和高剂量的BBR的治疗后,上述病理现象具有明显改善;阳性药二甲双胍组和卡托普利组也具有明显的减轻病理表现作用。4.BBR对DN大鼠肾脏皮质组织中的AGEs、RAGE、p-PKC-β和TGF-β1蛋白的分布与表达的影响免疫组化法显示蛋白分布:在DN大鼠的肾组织中,AGEs弥散性的分布在整个肾小球及其周围;RAGE主要分布在系膜细胞的胞膜上,少量分散在胞浆中;p-PKC-β在细胞质、细胞核和细胞膜上均有分布,但主要过表达集中于细胞质中;TGF-β1主要位于细胞外液和细胞外基质中。免疫组化法检测蛋白表达:与正常组相比,DN模型组的大鼠肾脏中以上蛋白的表达显著性的升高,而BBR给药组中,低剂量BBR能够有效的降低异常蛋白的表达,中剂量和高剂量的BBR则能够更加明显的改善和降低过高的含量。根据以上结果,使用western bolt法进行验证,发现各剂量的BBR均可以降低以上蛋白的过表达。其中,对于TGF-β1低剂量的BBR虽有降低,但不具有统计学意义;对于p-PKC-β的影响,中剂量的BBR效果最为明显;BBR对于所有蛋白的影响,中剂量和高剂量的BBR效果均强于低剂量的BBR。5.BBR对肾小球系膜细胞和足细胞的生长增殖的影响CCK-8细胞增值实验结果显示,DN状态下系膜细胞过度增殖,而自15μmol/L开始,BBR即具有显著的抑制系膜细胞增殖作用。BBR抑制系膜细胞过度增殖的IC50约为73μmol/L,根据此结果,设定后续系膜细胞的给药浓度为30μmol/L,60μmol/L和90μmol/L。而对于足细胞,DN状态下,足细胞数量减少明显,当BBR的浓度为60μmol/L时,足细胞的吸光度最高,在此浓度及之前,BBR具有显著保护足细胞的作用;此后随浓度增高,吸光度逐渐降低,推测可能是BBR的细胞毒性随浓度增大而逐渐增强,产生对足细胞的损伤。故对于足细胞的后续BBR给药浓度确定为30μmol/L,60μmol/L和90μmol/L。6.VEGF对肾小球系膜细胞生长增殖的影响CCK-8法细胞增殖实验结果显示,高糖和VEGF均能诱导系膜细胞明显过度增殖;当VEGF浓度达到2ng/ml时,系膜细胞的增殖达到最高值,此后随VEGF浓度增高,系膜细胞的增殖出现抑制。7.BBR对大鼠血清和系膜细胞上清液中的AGEs含量的影响ELISA法检测结果显示,DN模型大鼠血清和高糖诱导的系膜细胞上清液中AGEs含量明显高于正常组;各剂量和浓度的BBR均具有显著的抑制作用,其中,中剂量(浓度)和高剂量(浓度)组的BBR效果强于低剂量(浓度)组。8.BBR对系膜细胞中AGEs-RAGE通路的影响Western bolt法结果显示,DN状态下的系膜细胞AGEs、RAGE、p-PKC-β和TGF-β1的表达明显高于正常组;不同浓度的BBR均具有显著的抑制作用,其中,中浓度组的BBR(60μmol/L)抑制效果最强。当使用AGEs刺激系膜细胞后,RAGE、p-PKC-β和TGF-β1的表达显著升高,但BBR此时无抑制作用;而阻断剂Ab-RAGE能显著降低p-PKC-β和TGF-β1的表达。激光共聚焦法结果显示,RAGE在DN状态下的系膜细胞上主要表达为膜性蛋白,此时细胞膜上RAGE表达明显高于正常组,而BBR能够有效的抑制膜性RAGE的过表达。9.BBR对足细胞/系膜细胞之间VEGF-VEGFR2通路的影响蛋白芯片检测足细胞上清液结果显示,VEGF表达水平在DN状态下显著升高。Western bolt法检测结果显示,模型组足细胞分泌的VEGF蛋白相比正常组显著升高,BBR给药组可以有效降低VEGF的含量;高糖状态下系膜细胞的p-VEGFR2相比正常组表达增多,而VEGF刺激后,系膜细胞的p-VEGFR2的表达升高更加明显,不同浓度的BBR均有不同程度的改善作用。使用足细胞上清液刺激系膜细胞,p-VEGFR2的表达升高显著,中浓度的BBR和VEGF抑制剂均可以有效抑制p-VEGFR2的过表达。激光共聚焦法检测结果显示,足细胞中VEGF弥散性分布,为分泌性因子;系膜细胞中p-VEGFR2主要分布于细胞膜上,而BBR可以显著性的降低VEGFR2的磷酸化。结论:1.BBR可以有效的改善DN大鼠的肾功能,降低血糖,保护肾脏,改善DN大鼠的肾脏组织病理学。2.DN状态下AGEs-RAGE通路在大鼠肾脏中的下游因子包括PKC-β和TGF-β1,且整个AGEs-RAGE-PKC-β-TGF-β1通路在DN中发挥重要作用,而BBR可以显著影响此通路,影响的重要靶点因子为AGEs。3.DN状态下,足细胞通过过度分泌VEGF,激活系膜细胞膜上的VEGFR2,影响系膜细胞的增殖与分泌功能,从而损伤肾脏的结构和作用。BBR能够有效的降低足细胞分泌VEGF同时抑制VEGFR2的过度磷酸化,进而影响整个VEGF-VEGFR2通路,达到保护肾脏的作用。
[Abstract]:Objective: To observe the effects of Berberine (BBR) on the physiological and pathological changes of kidney and renal function in rats with diabetic nephropathy (diabetic nephropathy, DN) in rats, and to clarify the effect of BBR on the renal function and pathological changes of DN rats, and to detect the expression and phase of the signal protein secreted by some tissue cells in the kidney of the rat. The changes in the regulation of the pathway and the changes in the effect of BBR after the administration of the drugs are used to explore the mechanism of the renal protective effect of BBR. In vitro cell experiments, the renal glomerular mesangial cells (Glomerular mesangial cells, GMCs) and podocytes are stimulated by simulated DN conditions to detect AGEs-RAGE (late glycosylation) in mesangial cells. Final products, advanced glycation end products, AGEs, late glycosylation end product receptor, receptor of advanced glycation end products, RAGE) signal pathways and the signal pathway of the interaction between podocytes and mesangial cells (vascular endothelial cell growth factor); The regulatory changes of vascular endothelial growth factor type II receptor, vascular endothelial growth factor receptor 2, VEGFR2), and observe the effects of BBR administration and corresponding stimulants and blockers on the protection of the kidneys and the molecular mechanism and important molecular targets of BBR to DN. BBR provides a theoretical basis for the prevention and treatment of DN, and opens up new research directions and ideas for exploring new therapeutic DN drugs. Methods: preparation of DN rat model, feeding with long period (4-6 weeks) high glucose and high fat diet and combined with low dose streptozotocin (STZ, streptozocin, 35mg/kg) intraperitoneally induced by intraperitoneal injection. The fasting blood glucose in DN rats was measured after 72h. The value (FBG, fasting blood-glucose), the SD rats with FBG > 11.1mmol/L were included in the experimental DN model group. The experimental animals were grouped as follows: the normal group (Normal group), the DN model group (DN model), the low dose group, the dose group, the metformin group and the Kato Pury group, each group of 1 2 rats. The normal rats were fed with normal diet, and the model group and the administration group continued to feed high fat and high fat feed and free drinking water. All the groups were given the dosage of the rats' weight per day (Intragastric administration, i.g). The normal group and the model group were given the same solvent carboxymethyl cellulose sodium solution by the same solvent (Sod Ium carboxymethylcellulose, CMC-Na) the.FBG values in parallel control were measured once every two weeks, and the body weight was detected once a week, and the day before the death of 24h urine samples were collected to detect the content of total urine protein (UTP, total urine protein) and urinary creatinine (Ucr, urine creatinine). Nitrogen), the content of blood creatinine (Scr, serum creatinine) and AGEs. Take the same side of the kidney to weigh and make the tissue section for the pathological examination of kidney, including HE staining and PAS staining. Immunohistochemical method is used to observe AGEs in the renal cortex, RAGE, TGF- beta 1 (TGF, transforming growth) and beta protein kinase The distribution and expression of C- beta, Western blot method was used to detect the expression level of signal pathway protein in renal cortex, and the expression level of AGEs in each group was detected by enzyme linked immunosorbent assay (ELISA, enzyme-linked immuno sorbent assay). In vitro, glomerular mesangial cells and foot cell were used to separate the mesangial cells of rats. The DN model cell line was established by primary culture and passage culture, and the effect of BBR on the proliferation of DN in mesangial cells and podocytes was detected by CCK-8 kit. ELISA was used to detect the secretion of AGEs in mesangial cells. For the protein of AGEs-RAGE pathway, the normal condition was observed by Western blot method, and the model was not the same. The expression and change of the signal proteins after the same concentration of BBR and the use of AGEs stimulant and RAGE blocker (Ab-RAGE antibody). The distribution, expression types and changes of RAGE protein were observed by laser confocal method. For VEGF-VEGFR2 signaling protein, the protein chip was first used to detect the secretion of podocyte in DN state. Key factors; Western bolt method to verify VEGF, laser confocal method to determine the distribution and expression of protein,.CCK-8 method to determine the appropriate concentration of VEGF on mesangial cells, Western bolt method to observe the state of DN, high sugar, VEGF protein, the expression of VEGFR2 in the supernatant and the expression of phosphorylated VEGFR2 and the expression of phosphorylation; laser confocal method Results: the distribution and expression of p-VEGFR2 were measured. Results: the effect of 1.BBR on the weight and fasting blood glucose of DN rats was significantly higher than that of the normal group. The value of fasting blood glucose in the DN rats was significantly higher than that of the normal group, and the BBR treatment group had no obvious hypoglycemic effect in the first four weeks, and the BBR group in the middle dose and high dose in the sixth to 8 weeks was compared with the DN model group. Significant hypoglycemic effect; metformin group significantly reduced blood sugar from the first four weeks. For body weight, the effect of.2.BBR on the renal function and biochemical indexes of DN rats in the high dose group of BBR was compared with the normal group, the ratio of kidney weight / weight ratio, total urine protein / creatinine ratio, serum urea nitrogen (BUN) in the DN model group. The content of blood urea nitrogen) and blood creatinine (SCr, serum creatinine) increased obviously in the middle dose group and the high dose group of.BBR, and the positive drug metformin group and the Kato Pury group all could significantly improve the renal function and reduce the biochemical indexes. The histopathological findings of the renal histopathology of DN rats were revealed, and the results showed that it was normal and normal. Compared with the group, the glomerular glomeruli of the model rats were obviously hypertrophy, translucent changes, thickening of the basal membrane, aggregation of the extracellular matrix, serious glomerulosclerosis, fibrosis and a large number of inflammatory cells infiltrating.DN rats after the treatment of medium dose and high dose of BBR, the above pathological phenomena were obviously improved. The effects of.4.BBR on the distribution and expression of AGEs, RAGE, p-PKC- beta and TGF- beta 1 protein in the renal cortical tissue of DN rats affected the distribution and expression of protein in the renal cortical tissue of the rats. The distribution of protein in the renal tissue of DN rats: in the renal tissue of DN rats, the dispersion of AGEs was distributed throughout the glomerulus and around the whole glomerulus and around the kidney. GE mainly distributed in the membrane of mesangial cells and scattered in the cytoplasm; p-PKC- beta was distributed in cytoplasm, nucleus and cell membrane, but the main overexpression was concentrated in the cytoplasm; TGF- beta 1 was mainly located in extracellular fluid and extracellular matrix. Immunohistochemistry was used to detect protein expression: the rat kidney of the DN model group compared with the normal group. In the BBR administration group, low dose BBR could effectively reduce the expression of abnormal protein in the BBR administration group, while the medium dose and high dose of BBR could improve and reduce the high content. Based on the above results, the Western bolt method was used to verify that all the doses of BBR could reduce the above eggs. White overexpression. Among them, the low dose BBR of TGF- beta 1 decreased, but did not have statistical significance; for the effect of p-PKC- beta, the effect of medium dose BBR was most obvious; the effect of BBR on all proteins, the medium dose and high dose of BBR were stronger than that of the low dose BBR.5.BBR on the growth and proliferation of glomerular mesangial cells and podocytes. The results of the CCK-8 cell increment experiment showed that the mesangial cells were overproliferated in the DN state, and from 15 mu mol/L, BBR had a significant inhibition of mesangial cell proliferation, and the IC50 of.BBR inhibited mesangial cells overproliferation was about 73 mu mol/L. According to this result, the dosage of subsequent mesangial cells was set to 30 u mol/L, 60 u mol/L and 90 micron mol/L.. For the podocyte, the number of podocytes decreased obviously in DN state. When the concentration of BBR was 60 u mol/L, the absorbance of podocytes was the highest. Before this concentration and the concentration, BBR had a significant protective effect on the podocyte. After that, the absorbance decreased gradually with the increase of concentration. It is presumed that the cytotoxicity of BBR may be increased with the increase of concentration. The damage of podoni cells. Therefore, the effect of the following BBR dosage on the podocyte was determined to be 30 mu mol/L, 60 mol/L and 90 mu mol/L.6.VEGF on the proliferation of mesangial cells. The results of CCK-8 cell proliferation experiment showed that high glucose and VEGF could induce the hyperproliferation of mesangial cells. When VEGF concentration reached 2ng/ml, the proliferation of mesangial cells The proliferation of mesangial cells inhibited the AGEs content in the supernatant of rat sera and mesangial cells with the increase of the concentration of VEGF. The results showed that the AGEs content in the supernatant of the mesangial cells induced by DN model rats and high glucose was higher than that of the normal group, and the BBR of each dose and concentration of BBR had a higher level of AGEs than that of the normal group. The effect of BBR on the AGEs-RAGE pathway in mesangial cells was stronger than that in the low dose (concentration) group. The effect of.8.BBR on the AGEs-RAGE pathway in mesangial cells by Western bolt method showed that the expression of AGEs, RAGE, p-PKC- beta and TGF- beta in the mesangial cells in DN state was obviously higher than that in the normal group; the BBR of different concentrations were all obvious. The inhibitory effect of BBR (60 mu mol/L) in the medium concentration group was the strongest. When AGEs was used to stimulate mesangial cells, the expression of RAGE, p-PKC- beta and TGF- beta 1 increased significantly, but BBR at this time did not inhibit the expression, but the blocker Ab-RAGE could significantly reduce the expression of p-PKC- beta and TGF- beta 1. Membrane cells were mainly expressed as membranous proteins, and the expression of RAGE on the membrane was significantly higher than that of the normal group, while BBR could effectively inhibit the overexpression of membranous RAGE and the effect of.9.BBR on the VEGF-VEGFR2 pathway between podocytes and mesangial cells. The results of the protein chip detection in the supernatant showed that the VEGF expression level increased significantly in the DN state. The results of RN bolt assay showed that the VEGF protein secreted by the model group was significantly higher than that in the normal group, and the content of VEGF in the BBR administration group could be effectively reduced. The expression of p-VEGFR2 in the mesangial cells in the high glucose state increased, and the expression of p-VEGFR2 in the mesangial cells increased more obviously after VEGF stimulation, and the BBR of different concentrations had no effect. The expression of p-VEGFR2 increased significantly with the use of podocyte supernatant to stimulate mesangial cells. The concentration of BBR and VEGF inhibitors could effectively inhibit the overexpression of p-VEGFR2. The results of laser confocal detection showed that VEGF was distributed in the podocytes, and the p-VEGFR2 was mainly distributed in the mesangial cells. On the cell membrane, BBR can significantly reduce the phosphorylation of VEGFR2. Conclusion: 1.BBR can effectively improve the renal function of DN rats, reduce blood sugar, protect the kidney, and improve the renal histopathology of DN rats, the downstream factors of AGEs-RAGE pathway in the rat kidney include PKC- beta and TGF- beta 1, and the whole AGEs-RAGE-PKC- beta -TGF- beta 1 The pathway plays an important role in DN, and BBR can significantly affect the pathway. The important target factor of the impact is AGEs.3.DN, the hypersecretion of VEGF, the activation of VEGFR2 on the membrane of the mesangial cells, the proliferation and secretion of mesangial cells, and the damage of the structure and action of the kidneys by.BBR can effectively reduce the podocyte fraction. Secreting VEGF also inhibited the excessive phosphorylation of VEGFR2, thereby affecting the entire VEGF-VEGFR2 pathway and protecting the kidneys.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5
本文编号：2139539
[Abstract]:Objective: To observe the effects of Berberine (BBR) on the physiological and pathological changes of kidney and renal function in rats with diabetic nephropathy (diabetic nephropathy, DN) in rats, and to clarify the effect of BBR on the renal function and pathological changes of DN rats, and to detect the expression and phase of the signal protein secreted by some tissue cells in the kidney of the rat. The changes in the regulation of the pathway and the changes in the effect of BBR after the administration of the drugs are used to explore the mechanism of the renal protective effect of BBR. In vitro cell experiments, the renal glomerular mesangial cells (Glomerular mesangial cells, GMCs) and podocytes are stimulated by simulated DN conditions to detect AGEs-RAGE (late glycosylation) in mesangial cells. Final products, advanced glycation end products, AGEs, late glycosylation end product receptor, receptor of advanced glycation end products, RAGE) signal pathways and the signal pathway of the interaction between podocytes and mesangial cells (vascular endothelial cell growth factor); The regulatory changes of vascular endothelial growth factor type II receptor, vascular endothelial growth factor receptor 2, VEGFR2), and observe the effects of BBR administration and corresponding stimulants and blockers on the protection of the kidneys and the molecular mechanism and important molecular targets of BBR to DN. BBR provides a theoretical basis for the prevention and treatment of DN, and opens up new research directions and ideas for exploring new therapeutic DN drugs. Methods: preparation of DN rat model, feeding with long period (4-6 weeks) high glucose and high fat diet and combined with low dose streptozotocin (STZ, streptozocin, 35mg/kg) intraperitoneally induced by intraperitoneal injection. The fasting blood glucose in DN rats was measured after 72h. The value (FBG, fasting blood-glucose), the SD rats with FBG > 11.1mmol/L were included in the experimental DN model group. The experimental animals were grouped as follows: the normal group (Normal group), the DN model group (DN model), the low dose group, the dose group, the metformin group and the Kato Pury group, each group of 1 2 rats. The normal rats were fed with normal diet, and the model group and the administration group continued to feed high fat and high fat feed and free drinking water. All the groups were given the dosage of the rats' weight per day (Intragastric administration, i.g). The normal group and the model group were given the same solvent carboxymethyl cellulose sodium solution by the same solvent (Sod Ium carboxymethylcellulose, CMC-Na) the.FBG values in parallel control were measured once every two weeks, and the body weight was detected once a week, and the day before the death of 24h urine samples were collected to detect the content of total urine protein (UTP, total urine protein) and urinary creatinine (Ucr, urine creatinine). Nitrogen), the content of blood creatinine (Scr, serum creatinine) and AGEs. Take the same side of the kidney to weigh and make the tissue section for the pathological examination of kidney, including HE staining and PAS staining. Immunohistochemical method is used to observe AGEs in the renal cortex, RAGE, TGF- beta 1 (TGF, transforming growth) and beta protein kinase The distribution and expression of C- beta, Western blot method was used to detect the expression level of signal pathway protein in renal cortex, and the expression level of AGEs in each group was detected by enzyme linked immunosorbent assay (ELISA, enzyme-linked immuno sorbent assay). In vitro, glomerular mesangial cells and foot cell were used to separate the mesangial cells of rats. The DN model cell line was established by primary culture and passage culture, and the effect of BBR on the proliferation of DN in mesangial cells and podocytes was detected by CCK-8 kit. ELISA was used to detect the secretion of AGEs in mesangial cells. For the protein of AGEs-RAGE pathway, the normal condition was observed by Western blot method, and the model was not the same. The expression and change of the signal proteins after the same concentration of BBR and the use of AGEs stimulant and RAGE blocker (Ab-RAGE antibody). The distribution, expression types and changes of RAGE protein were observed by laser confocal method. For VEGF-VEGFR2 signaling protein, the protein chip was first used to detect the secretion of podocyte in DN state. Key factors; Western bolt method to verify VEGF, laser confocal method to determine the distribution and expression of protein,.CCK-8 method to determine the appropriate concentration of VEGF on mesangial cells, Western bolt method to observe the state of DN, high sugar, VEGF protein, the expression of VEGFR2 in the supernatant and the expression of phosphorylated VEGFR2 and the expression of phosphorylation; laser confocal method Results: the distribution and expression of p-VEGFR2 were measured. Results: the effect of 1.BBR on the weight and fasting blood glucose of DN rats was significantly higher than that of the normal group. The value of fasting blood glucose in the DN rats was significantly higher than that of the normal group, and the BBR treatment group had no obvious hypoglycemic effect in the first four weeks, and the BBR group in the middle dose and high dose in the sixth to 8 weeks was compared with the DN model group. Significant hypoglycemic effect; metformin group significantly reduced blood sugar from the first four weeks. For body weight, the effect of.2.BBR on the renal function and biochemical indexes of DN rats in the high dose group of BBR was compared with the normal group, the ratio of kidney weight / weight ratio, total urine protein / creatinine ratio, serum urea nitrogen (BUN) in the DN model group. The content of blood urea nitrogen) and blood creatinine (SCr, serum creatinine) increased obviously in the middle dose group and the high dose group of.BBR, and the positive drug metformin group and the Kato Pury group all could significantly improve the renal function and reduce the biochemical indexes. The histopathological findings of the renal histopathology of DN rats were revealed, and the results showed that it was normal and normal. Compared with the group, the glomerular glomeruli of the model rats were obviously hypertrophy, translucent changes, thickening of the basal membrane, aggregation of the extracellular matrix, serious glomerulosclerosis, fibrosis and a large number of inflammatory cells infiltrating.DN rats after the treatment of medium dose and high dose of BBR, the above pathological phenomena were obviously improved. The effects of.4.BBR on the distribution and expression of AGEs, RAGE, p-PKC- beta and TGF- beta 1 protein in the renal cortical tissue of DN rats affected the distribution and expression of protein in the renal cortical tissue of the rats. The distribution of protein in the renal tissue of DN rats: in the renal tissue of DN rats, the dispersion of AGEs was distributed throughout the glomerulus and around the whole glomerulus and around the kidney. GE mainly distributed in the membrane of mesangial cells and scattered in the cytoplasm; p-PKC- beta was distributed in cytoplasm, nucleus and cell membrane, but the main overexpression was concentrated in the cytoplasm; TGF- beta 1 was mainly located in extracellular fluid and extracellular matrix. Immunohistochemistry was used to detect protein expression: the rat kidney of the DN model group compared with the normal group. In the BBR administration group, low dose BBR could effectively reduce the expression of abnormal protein in the BBR administration group, while the medium dose and high dose of BBR could improve and reduce the high content. Based on the above results, the Western bolt method was used to verify that all the doses of BBR could reduce the above eggs. White overexpression. Among them, the low dose BBR of TGF- beta 1 decreased, but did not have statistical significance; for the effect of p-PKC- beta, the effect of medium dose BBR was most obvious; the effect of BBR on all proteins, the medium dose and high dose of BBR were stronger than that of the low dose BBR.5.BBR on the growth and proliferation of glomerular mesangial cells and podocytes. The results of the CCK-8 cell increment experiment showed that the mesangial cells were overproliferated in the DN state, and from 15 mu mol/L, BBR had a significant inhibition of mesangial cell proliferation, and the IC50 of.BBR inhibited mesangial cells overproliferation was about 73 mu mol/L. According to this result, the dosage of subsequent mesangial cells was set to 30 u mol/L, 60 u mol/L and 90 micron mol/L.. For the podocyte, the number of podocytes decreased obviously in DN state. When the concentration of BBR was 60 u mol/L, the absorbance of podocytes was the highest. Before this concentration and the concentration, BBR had a significant protective effect on the podocyte. After that, the absorbance decreased gradually with the increase of concentration. It is presumed that the cytotoxicity of BBR may be increased with the increase of concentration. The damage of podoni cells. Therefore, the effect of the following BBR dosage on the podocyte was determined to be 30 mu mol/L, 60 mol/L and 90 mu mol/L.6.VEGF on the proliferation of mesangial cells. The results of CCK-8 cell proliferation experiment showed that high glucose and VEGF could induce the hyperproliferation of mesangial cells. When VEGF concentration reached 2ng/ml, the proliferation of mesangial cells The proliferation of mesangial cells inhibited the AGEs content in the supernatant of rat sera and mesangial cells with the increase of the concentration of VEGF. The results showed that the AGEs content in the supernatant of the mesangial cells induced by DN model rats and high glucose was higher than that of the normal group, and the BBR of each dose and concentration of BBR had a higher level of AGEs than that of the normal group. The effect of BBR on the AGEs-RAGE pathway in mesangial cells was stronger than that in the low dose (concentration) group. The effect of.8.BBR on the AGEs-RAGE pathway in mesangial cells by Western bolt method showed that the expression of AGEs, RAGE, p-PKC- beta and TGF- beta in the mesangial cells in DN state was obviously higher than that in the normal group; the BBR of different concentrations were all obvious. The inhibitory effect of BBR (60 mu mol/L) in the medium concentration group was the strongest. When AGEs was used to stimulate mesangial cells, the expression of RAGE, p-PKC- beta and TGF- beta 1 increased significantly, but BBR at this time did not inhibit the expression, but the blocker Ab-RAGE could significantly reduce the expression of p-PKC- beta and TGF- beta 1. Membrane cells were mainly expressed as membranous proteins, and the expression of RAGE on the membrane was significantly higher than that of the normal group, while BBR could effectively inhibit the overexpression of membranous RAGE and the effect of.9.BBR on the VEGF-VEGFR2 pathway between podocytes and mesangial cells. The results of the protein chip detection in the supernatant showed that the VEGF expression level increased significantly in the DN state. The results of RN bolt assay showed that the VEGF protein secreted by the model group was significantly higher than that in the normal group, and the content of VEGF in the BBR administration group could be effectively reduced. The expression of p-VEGFR2 in the mesangial cells in the high glucose state increased, and the expression of p-VEGFR2 in the mesangial cells increased more obviously after VEGF stimulation, and the BBR of different concentrations had no effect. The expression of p-VEGFR2 increased significantly with the use of podocyte supernatant to stimulate mesangial cells. The concentration of BBR and VEGF inhibitors could effectively inhibit the overexpression of p-VEGFR2. The results of laser confocal detection showed that VEGF was distributed in the podocytes, and the p-VEGFR2 was mainly distributed in the mesangial cells. On the cell membrane, BBR can significantly reduce the phosphorylation of VEGFR2. Conclusion: 1.BBR can effectively improve the renal function of DN rats, reduce blood sugar, protect the kidney, and improve the renal histopathology of DN rats, the downstream factors of AGEs-RAGE pathway in the rat kidney include PKC- beta and TGF- beta 1, and the whole AGEs-RAGE-PKC- beta -TGF- beta 1 The pathway plays an important role in DN, and BBR can significantly affect the pathway. The important target factor of the impact is AGEs.3.DN, the hypersecretion of VEGF, the activation of VEGFR2 on the membrane of the mesangial cells, the proliferation and secretion of mesangial cells, and the damage of the structure and action of the kidneys by.BBR can effectively reduce the podocyte fraction. Secreting VEGF also inhibited the excessive phosphorylation of VEGFR2, thereby affecting the entire VEGF-VEGFR2 pathway and protecting the kidneys.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5
【参考文献】
相关期刊论文 前4条
1 陈好利;万毅刚;赵青;黄燕如;史喜苗;孟宪杰;姚建;;糖尿病肾病肾组织炎症信号通路p38MAPK的调节机制及中药的干预作用[J];中国中药杂志;2013年14期
2 赵青;万毅刚;王朝俊;魏晴雪;陈好利;孟宪杰;姚建;;慢性肾脏病肾组织炎症信号通路p38MAPK的调节机制及中药的干预作用[J];中国中药杂志;2012年12期
3 ;Therapeutic effects of berberine in impaired glucose tolerance rats and its influence on insulin secretion[J];Acta Pharmacologica Sinica;2004年04期
4 潘国宇,王广基,孙建国,黄志江,赵小辰,顾轶,刘晓东;小檗碱对葡萄糖吸收的抑制作用[J];药学学报;2003年12期
，本文编号：2139539
本文链接：https://www.wllwen.com/shoufeilunwen/mpalunwen/2139539.html
最近更新
教材专著
