【摘要】：目的:在大鼠體內(nèi)實驗中,觀察小檗堿(Berberine,BBR)對糖尿病腎病(diabetic nephropathy,DN)大鼠腎臟生理病理和腎功能生化指標的影響,明確BBR對DN大鼠的腎功能及組織病變的改善作用;檢測大鼠腎臟中部分組織細胞分泌的信號蛋白表達和相應通路的調(diào)節(jié)變化以及由BBR給藥后影響的變化,探究BBR的腎臟保護作用是通過何種機制產(chǎn)生的。在體外細胞實驗中,通過模擬DN條件刺激腎臟腎小球系膜細胞(Glomerular mesangial cells,GMCs)和足細胞,進而檢測系膜細胞中AGEs-RAGE(晚期糖基化終末產(chǎn)物,advanced glycation end products,AGEs;晚期糖基化終末產(chǎn)物受體,receptor of advanced glycation end products,RAGE)信號通路的異常改變和足細胞與系膜細胞相互影響的信號通路VEGF-VEGFR2(血管內(nèi)皮細胞生長因子,vascular endothelial growth factor,VEGF;血管內(nèi)皮細胞生長因子Ⅱ型受體,vascular endothelial growth factor receptor 2,VEGFR2)的調(diào)節(jié)變化,同時觀察BBR給藥以及相應刺激劑和阻斷劑給予后的影響作用,進一步探討B(tài)BR對DN的腎臟保護作用以及潛在的分子機制和重要的分子靶點;為合理開發(fā)利用BBR防治DN提供理論依據(jù),并且為探索新的治療DN藥物開拓新的研究方向和思路。方法:DN大鼠模型的制備,采用長周期(4-6周)高糖高脂飼料喂養(yǎng)并聯(lián)合低劑量鏈脲佐菌素(STZ,streptozocin,35mg/kg)腹腔注射誘導。于72h后測量DN大鼠空腹血糖值(FBG,fasting blood-glucose),將FBG≥11.1mmol/L的SD大鼠納入實驗DN模型組。實驗動物分組如下:正常組(Normal group),DN模型組(DN model group),BBR低劑量組(50mg/kg),BBR中劑量組(100mg/kg),BBR高劑量組(200mg/kg),二甲雙胍組(200mg/kg)和卡托普利組(15mg/kg),每組12只大鼠。正常組大鼠喂以正常飼料,模型組和給藥組繼續(xù)給予高糖高脂飼料喂養(yǎng),自由飲水。所有給藥組按大鼠體重計算給藥量,每日一次灌胃給藥(Intragastric administration,i.g),正常組與模型組按體重給予相同溶劑羧甲基纖維素鈉溶液(Sodium carboxymethylcellulose,CMC-Na)平行對照灌胃。FBG值每倆周檢測一次,體重每周檢測一次,處死前一天收集24h尿樣用以檢測尿總蛋白(UTP,total urine protein)和尿肌酐(Ucr,urine creatinine)的含量;麻醉后股動脈取血,分離血清檢測血尿素氮(BUN,blood urea nitrogen),血肌酐(Scr,serum creatinine)和AGEs的含量。取同側腎臟稱重,制作組織切片進行腎臟病理學檢查,包括HE染色和PAS染色。免疫組化法觀察腎臟皮質中AGEs,RAGE,TGF-β1(轉化生長因子,transforming growth factor-β1)和PKC-β(蛋白激酶C-β,protein kinase C-β)的分布與表達;Western blot法檢測腎皮質中各信號通路蛋白表達水平;酶聯(lián)免疫吸附法(ELISA,enzyme-linked immuno sorbent assay)法檢測各組血清中AGEs的表達水平。體外實驗以腎小球系膜細胞和足細胞為研究對象,首先分離大鼠的系膜細胞和足細胞進行原代培養(yǎng)和傳代培養(yǎng),建立DN模型細胞系。利用CCK-8試劑盒檢測BBR對系膜細胞和足細胞DN狀態(tài)下的增殖影響。使用ELISA法檢測系膜細胞分泌AGEs的情況。對于AGEs-RAGE通路的蛋白,首先使用western blot法觀察正常情況,模型情況和不同濃度BBR給藥后以及使用AGEs刺激劑和RAGE阻斷劑(Ab-RAGE抗體)后各信號蛋白的表達和變化情況;使用激光共聚焦法觀察RAGE蛋白分布,表達類型和變化情況。對于VEGF-VEGFR2信號通路蛋白,首先使用蛋白芯片檢測DN狀態(tài)下足細胞分泌,確定關鍵因子;western bolt法驗證VEGF,激光共聚焦法確定蛋白的分布和表達情況。CCK-8法確定VEGF對系膜細胞的適宜刺激濃度,用western bolt法觀察DN狀態(tài),高糖,VEGF蛋白,足細胞上清液刺激和給藥情況下的VEGFR2的表達和磷酸化的表達;激光共聚焦法檢測p-VEGFR2的分布和表達情況。結果:1.BBR對DN大鼠的體重和空腹血糖的影響與正常組相比,造模成功后的DN大鼠的空腹血糖值顯著升高;各BBR治療組在前四周無明顯的降血糖效果,而在第6到8周中劑量和高劑量的BBR組與DN模型組相比具有明顯的降血糖作用;二甲雙胍組顯著性降低血糖作用從第四周開始。對于體重,僅高劑量BBR組在第8周具有改善作用。2.BBR對DN大鼠的腎功能及生化指標的影響與正常組相比,DN模型組的腎重/體重比值,尿總蛋白/尿肌酐比值,血清尿素氮(BUN,blood urea nitrogen)和血肌酐(SCr,serum creatinine)含量均明顯升高。BBR中劑量組和高劑量組,以及陽性藥二甲雙胍組和卡托普利組均能顯著改善腎功能和降低生化指標。3.BBR對DN大鼠的腎臟組織病理學的影響組織病理學檢查結果顯示,與正常組相比,模型組大鼠的腎臟腎小球明顯肥大,透明樣變化,基底膜明顯增厚,細胞外基質聚集,出現(xiàn)較為嚴重的腎小球硬化,纖維化以及伴有大量的炎性細胞浸潤。DN大鼠在給予中劑量和高劑量的BBR的治療后,上述病理現(xiàn)象具有明顯改善;陽性藥二甲雙胍組和卡托普利組也具有明顯的減輕病理表現(xiàn)作用。4.BBR對DN大鼠腎臟皮質組織中的AGEs、RAGE、p-PKC-β和TGF-β1蛋白的分布與表達的影響免疫組化法顯示蛋白分布:在DN大鼠的腎組織中,AGEs彌散性的分布在整個腎小球及其周圍;RAGE主要分布在系膜細胞的胞膜上,少量分散在胞漿中;p-PKC-β在細胞質、細胞核和細胞膜上均有分布,但主要過表達集中于細胞質中;TGF-β1主要位于細胞外液和細胞外基質中。免疫組化法檢測蛋白表達:與正常組相比,DN模型組的大鼠腎臟中以上蛋白的表達顯著性的升高,而BBR給藥組中,低劑量BBR能夠有效的降低異常蛋白的表達,中劑量和高劑量的BBR則能夠更加明顯的改善和降低過高的含量。根據(jù)以上結果,使用western bolt法進行驗證,發(fā)現(xiàn)各劑量的BBR均可以降低以上蛋白的過表達。其中,對于TGF-β1低劑量的BBR雖有降低,但不具有統(tǒng)計學意義;對于p-PKC-β的影響,中劑量的BBR效果最為明顯;BBR對于所有蛋白的影響,中劑量和高劑量的BBR效果均強于低劑量的BBR。5.BBR對腎小球系膜細胞和足細胞的生長增殖的影響CCK-8細胞增值實驗結果顯示,DN狀態(tài)下系膜細胞過度增殖,而自15μmol/L開始,BBR即具有顯著的抑制系膜細胞增殖作用。BBR抑制系膜細胞過度增殖的IC50約為73μmol/L,根據(jù)此結果,設定后續(xù)系膜細胞的給藥濃度為30μmol/L,60μmol/L和90μmol/L。而對于足細胞,DN狀態(tài)下,足細胞數(shù)量減少明顯,當BBR的濃度為60μmol/L時,足細胞的吸光度最高,在此濃度及之前,BBR具有顯著保護足細胞的作用;此后隨濃度增高,吸光度逐漸降低,推測可能是BBR的細胞毒性隨濃度增大而逐漸增強,產(chǎn)生對足細胞的損傷。故對于足細胞的后續(xù)BBR給藥濃度確定為30μmol/L,60μmol/L和90μmol/L。6.VEGF對腎小球系膜細胞生長增殖的影響CCK-8法細胞增殖實驗結果顯示,高糖和VEGF均能誘導系膜細胞明顯過度增殖;當VEGF濃度達到2ng/ml時,系膜細胞的增殖達到最高值,此后隨VEGF濃度增高,系膜細胞的增殖出現(xiàn)抑制。7.BBR對大鼠血清和系膜細胞上清液中的AGEs含量的影響ELISA法檢測結果顯示,DN模型大鼠血清和高糖誘導的系膜細胞上清液中AGEs含量明顯高于正常組;各劑量和濃度的BBR均具有顯著的抑制作用,其中,中劑量(濃度)和高劑量(濃度)組的BBR效果強于低劑量(濃度)組。8.BBR對系膜細胞中AGEs-RAGE通路的影響Western bolt法結果顯示,DN狀態(tài)下的系膜細胞AGEs、RAGE、p-PKC-β和TGF-β1的表達明顯高于正常組;不同濃度的BBR均具有顯著的抑制作用,其中,中濃度組的BBR(60μmol/L)抑制效果最強。當使用AGEs刺激系膜細胞后,RAGE、p-PKC-β和TGF-β1的表達顯著升高,但BBR此時無抑制作用;而阻斷劑Ab-RAGE能顯著降低p-PKC-β和TGF-β1的表達。激光共聚焦法結果顯示,RAGE在DN狀態(tài)下的系膜細胞上主要表達為膜性蛋白,此時細胞膜上RAGE表達明顯高于正常組,而BBR能夠有效的抑制膜性RAGE的過表達。9.BBR對足細胞/系膜細胞之間VEGF-VEGFR2通路的影響蛋白芯片檢測足細胞上清液結果顯示,VEGF表達水平在DN狀態(tài)下顯著升高。Western bolt法檢測結果顯示,模型組足細胞分泌的VEGF蛋白相比正常組顯著升高,BBR給藥組可以有效降低VEGF的含量;高糖狀態(tài)下系膜細胞的p-VEGFR2相比正常組表達增多,而VEGF刺激后,系膜細胞的p-VEGFR2的表達升高更加明顯,不同濃度的BBR均有不同程度的改善作用。使用足細胞上清液刺激系膜細胞,p-VEGFR2的表達升高顯著,中濃度的BBR和VEGF抑制劑均可以有效抑制p-VEGFR2的過表達。激光共聚焦法檢測結果顯示,足細胞中VEGF彌散性分布,為分泌性因子;系膜細胞中p-VEGFR2主要分布于細胞膜上,而BBR可以顯著性的降低VEGFR2的磷酸化。結論:1.BBR可以有效的改善DN大鼠的腎功能,降低血糖,保護腎臟,改善DN大鼠的腎臟組織病理學。2.DN狀態(tài)下AGEs-RAGE通路在大鼠腎臟中的下游因子包括PKC-β和TGF-β1,且整個AGEs-RAGE-PKC-β-TGF-β1通路在DN中發(fā)揮重要作用,而BBR可以顯著影響此通路,影響的重要靶點因子為AGEs。3.DN狀態(tài)下,足細胞通過過度分泌VEGF,激活系膜細胞膜上的VEGFR2,影響系膜細胞的增殖與分泌功能,從而損傷腎臟的結構和作用。BBR能夠有效的降低足細胞分泌VEGF同時抑制VEGFR2的過度磷酸化,進而影響整個VEGF-VEGFR2通路,達到保護腎臟的作用。
[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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R285.5

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