黄芪甲苷抑制糖尿病肾病肾间质纤维化的作用和机制

发布时间：2018-06-05 22:27

本文选题：黄芪甲苷 + 糖尿病肾病　；参考：《山东大学》2017年博士论文

【摘要】：研究背景糖尿病(diabetes mellitus,DM)是严重影响人类健康的慢性非传染性疾病,随着饮食结构和生活方式的改变,其患病率、发病率和患者数量急剧上升,给家庭及社会带来沉重的经济负担。糖尿病肾病(diabetic kidney disease,DKD)是糖尿病微血管病变的主要并发症之一,即使血压及血糖控制达标,糖尿病患者最终有30%—40%发展至糖尿病肾病。在全球范围内,糖尿病肾病是引起终末期肾病(end-stage renal disease,ESRD)的主要原因。既往认为糖尿病肾病在进展过程中,最重要的病理生理变化为不可逆的肾小球纤维化及瘢痕形成,肾小管损伤只是继发现象。但随着研究的深入人们发现,肾小管病变,尤其是肾小管间质的纤维化(tubulointerstitial fibrosis, TIF)在推动糖尿病肾病进展中起了非常重要的作用,肾小管病变可不依赖于肾小球的病变。因此,探讨延缓肾小管间质纤维化的治疗策略,对于改善患者预后具有十分重要的意义。尽管TIF的发病机理尚未明了,但越来越多的证据指向肾小管上皮细胞凋亡及肾小管上皮细胞-成纤维细胞(间充质细胞)转分化(epithelial-to-mesenchymal transition,EMT)。EMT是糖尿病肾脏疾病小管损伤的经典病理改变,且与肾间质纤维化的程度相平行。越来越多的研究强有力地证实了EMT在不同类型的肾脏疾病中发挥着重要作用,而逆转EMT则能有效改善肾脏纤维化。虽然大量研究揭示了各种EMT的始动因素、调节因子及存在的信号通路,但在所有因素中,TGF-β1是多种组织和器官纤维化的关键调控因子,是糖尿病肾病进展过程中的主要炎性因子,其启动并调节EMT的全过程,其主要通过①诱导细胞凋亡导致细胞丢失②直接作用于成纤维细胞、系膜细胞等上调细胞外基质(extracellular matrix,ECM)合成,抑制ECM降解③促进肾小管上皮细胞EMT参与肾脏纤维化过程。已有研究表明,TGF-β1可诱导肾小管上皮细胞呈现肌成纤维细胞的形态,即极性改变、出现肌动蛋白微丝及致密小体,且胶原蛋白产生增加。尽管TGF-β1可通过RhoA,ERK,P38-MAPK, JNK和Wnt/β-catenin途径介导上述改变,但目前TGF-β/Smad被认为是介导这一过程最主要的信号通路。活化的TGF-β1使得Smad2和Smad3磷酸化,随后,磷酸化的Smad2和Smad3同Smad4结合后形成Smad低聚复合物,并转入细胞核调节靶基因转录。糖尿病时,许多促炎因子能激活Smad信号通路,在糖尿病肾病患者肾小球及间质纤维化区域可见到磷酸化的Smad2及Smad3沉积,证明有TGF-β/Smad信号通路的活化。目前临床上尚无治疗糖尿病肾病的特效药物,只能通过控制饮食、降糖、降压等措施进行治疗,效果有限。中医药治疗历史悠久,中药因其多成分、治疗多靶点的特点,具有良好的应用前景。黄芪为豆科植物蒙古黄芪或膜荚黄芪的干燥根,具有补气固表、利尿托毒、排脓、敛疮生肌的功效。黄芪甲苷(AstragalosideⅣ,ASI)是黄芪皂苷的单体成分,具有抗氧化、减轻缺血再灌注损伤等作用,在防治急性肾损伤、膜性肾病、糖尿病肾病等肾脏疾病中发挥重要作用。目前,对ASI防治糖尿病肾病的报道大多局限于足细胞及系膜细胞,对肾小管间质纤维化研究较少,因此本研究拟通过体内及体外实验探讨ASI抑制糖尿病肾病肾间质纤维化的作用和机制。研究目的观察ASI对2型糖尿病模型KKAy小鼠肾组织TGF-β/Smad信号通路及其对高糖诱导下肾小管上皮细胞NRK-52E的影响,探讨ASI对肾间质纤维化的保护作用及机制,为临床应用ASI提供实验依据。研究方法1.动物分组:购入小鼠适应性喂养2周,KKAy小鼠予以KK饲料(高脂)喂养至14周,随机血糖超过13.9mmol/L提示造模成功;血糖水平相近的KKAy小鼠随机分为模型组(10只)和ASI组(10只),正常饮食的同龄10只雄性C57BL/6J小鼠作为对照组。其中对照组和模型组每日予以生理盐水予以40mg/kg灌胃,ASI组予以ASI 40mg/kg灌胃;2.血糖、血肌酐、尿ACR检测:每周测量小鼠体重,于第16周、20周、24周采取提尾反射法收集随机尿液,ELISA试剂盒检测尿微量白蛋白和尿肌酐,计算尿ACR,尾静脉取血测血糖,内眦取血测血肌酐;3. 24周时断颈处死小鼠,分离肾脏,行HE染色、Masson染色观察各组肾脏病理变化;4.应用免疫组化染色检测各组肾脏TGF-β1、Smad2/3、α-SMA的表达;5. ASI对NRK-52E细胞活力的影响:NRK-52E细胞生长至融合状态时,同步化培养24小时,以不同浓度的ASI (0、10、20、40、80、100μg/ml,依次简写为 0、ASI10、ASI20、ASI40、ASI80、ASI100)刺激 NRK-52E 细胞 24小时,细胞计数试剂盒CCK-8检测细胞活性;6. ASI对高糖诱导下NRK-52E细胞凋亡的影响:①细胞长至融合状态时,同步化培养24小时,设正常对照组、高糖组、ASI各组:高糖DMEM中加入ASI,浓度分别为 20、40、80、100μg/ml (依次简写为 HG+ASI20、HG+ASI40、HG+ASI 80、HG+ASI 100),分别刺激NRK-52E细胞24小时,或②用高糖+ASI 100μg/ml 分别刺激细胞 0、4、8、12、24、48 小时,用 AnnexinV-FITCPI 试剂盒检测细胞凋亡;7. ASI 对高糖诱导的NRK-52E 细胞 Smad2、Smad3、α-SMA、TGF-β1 表达的影响:细胞生长至融合状态时,同步化培养24小时,设正常对照组、高糖组、ASI各组(同前述),分别刺激NRK-52E细胞24小时,以Real-Time PCR检测 Smad2、Smad3、α-SMA、TGF-β1 核酸水平的表达,Western Blot 检测 Smad2、Smad3、p-Smad2、p-Smad3、α-SMA、TGF-β1 蛋白水平的表达;8.统计学方法:计量资料以均数±标准误表示,根据方差齐性检验结果,两组间比较采用独立样本t检验,多组比较采用单因素方差分析,所有数据均由统计软件SPSS 19.0完成,以P0.05为差异有统计学意义,P0.01为差异有显著统计学意义。研究结果1. 一般状态:对照组小鼠精神状态好,反应灵敏,毛色顺滑;模型组小鼠精神萎靡不振,步履迟缓,烦渴多尿,反应迟钝,毛发无光泽,且随着周龄的增加,上述症状更加明显。ASI组小鼠的状态介于上述两组之间。在16、20、24周龄时,模型组及ASI组小鼠体重均比同龄对照组小鼠大(均P0.01);与模型组小鼠相比,ASI组体重增长减缓(均P0.05);2.血糖、血肌酐、尿ACR检测:在各观察时间点,与同龄对照组相比,模型组及ASI组血糖、尿ACR明显升高(均P0.01);与同龄模型组相比,ASI组血糖、尿ACR较低(P0.05, P0.01)。三组小鼠血清肌酐检测结果无统计学差异(P0.05);3.各组小鼠肾组织病理改变:光镜下可观察到对照组肾小球及肾小管结构清晰,系膜细胞数量正常,间质中未见炎细胞浸润及纤维化;模型组肾小球肥大,系膜基质增宽,系膜细胞增多,肾小管上皮细胞胞浆出现空泡、肾小管腔可见透明管型,肾间质炎症细胞增多,充血水肿;ASI组肾小球及系膜区改变介入对照组及模型组之间,肾小管上皮细胞轻度肿胀,胞浆较少,未间明显的间质纤维化;4.免疫组化染色检测各组肾脏TGF-β1、Smad2/3及α-SMA的表达情况:α-SMA、TGF-β1及Smad2/3主要在肾小管-间质表达;对照组少见TGF-β1表达,而模型组及ASI组TGF-β1表达增强(P0.01,P0.05),与模型组相比,ASI组TGF-β1表达减弱(P0.01);对照组α-SMA多数表达于肾血管平滑肌,在肾小管间质偶有表达,但在模型组可见α-SMA在肾小管上皮细胞、肾小管周围均有所表达(P0.01),与模型组相比,ASI组α-SMA表达明显下调(P0.01);对照组肾小管与肾小球细胞核有少量Smad2/3表达,模型组及ASI组表达增加(P0.01,P0.05),但同模型组比较,ASI组Smad2/3表达减少(P0.01);5. ASI对NRK-52E细胞活力的影响:不同浓度ASI分别作用于NRK-52E细胞24小时,各组之间细胞活性差异无统计学意义(P0.05);6. ASI对高糖诱导下NRK-52E细胞凋亡的影响:与对照组相比,高糖组细胞凋亡增加(P0.01),加入 ASI 后,HG+ASI 40、HG+ASI 80、HG+ASI 100组的细胞凋亡均较高糖组减轻(均P0.05 ): HG+ASI 100μg/ml作用于细胞8h后,细胞凋亡显著受抑,且随着时间的延长,此作用有逐渐增强的趋势(与Oh相比,均P0.05);7. ASI 对高糖诱导的 NRK-52E 细胞 Smad2、Smad3、α-SMA、TGF-β1 mRNA的影响:与对照组相比,高糖组(HG)、HG+ASI20组Smad2、TGF-β1mRNA表达上调(均P0.01),Smad3表达上调(均P0.05) , HG、HG+ASI20、HG+ ASI 40 α-SMA 表达上调(均P0.01);与 HG 相比,HG+ASI 80、HG+ASI 100 组 α-SMA、Smad3 mRNA 表达下调(均P0.05),HG+ASI 40、HG+ASI 80、HG+ASI 100 TGF-β1 表达下调(均P0.01),HG+ASI 20、HG+ASI 40、HG+ASI 80、HG+ASI100Smad2 表达下调(P0.05);8. ASI 对高糖诱导的 NRK-52E 细胞 Smad2、Smad3、p-Smad2、p-Smad3、α-SMA、TGF-β1蛋白水平的影响:与对照组相比,高糖组(HG)、HG+ASI20、HG+ASI40组α-SMA、Smad2、Smad3表达上调,差异有统计学意义(均P0.01),p-Smad2、p-Smad3 在 HG、HG+ASI20、HG+ASI40、HG+ASI80 组表达均上调(均P0.01 ),TGF-β1在HG、HG+ ASI 20组表达上调(均P0.01);与 HG 相比,HG+ASI80、HG+ASI 100 组 α-SMA、Smad2、Smad3、p-Smad2、p-Smad3表达下调,差异有统计学意义(均P0.05),TGF-β1在HG+ASI40、HG+ASI80、HG+ASI100 组表达下调(均P0.05)。研究结论1.黄芪甲苷可减轻糖尿病KKAy小鼠的高血糖、肥胖及尿ACR,但不影响血肌酐;2.黄芪甲苷可改善糖尿病KKAy小鼠的肾小管间质纤维化,其作用机制可能与ASI下调TGF-β/Smad信号通路有关;3.黄芪甲苷可呈剂量及时间依赖性抑制高糖诱导的NRK-52E细胞凋亡;4.黄芪甲苷可抑制高糖诱导的NRK-52E细胞α-SMA表达及TGF-β1/Smad信号通路活性,从而减轻EMT,延缓肾间质纤维化。
[Abstract]:Diabetes mellitus (DM) is a chronic noncommunicable disease which seriously affects human health. With the change of diet structure and lifestyle, the prevalence, incidence and number of patients rise sharply and bring heavy economic burden to family and society. Diabetic nephropathy (diabetic kidney disease, DKD) is diabetic Microblood. One of the major complications of vascular disease, even if the blood pressure and blood glucose control standard, diabetes patients eventually have 30% - 40% to develop to diabetic nephropathy. Diabetic nephropathy is the main cause of end-stage renal disease (ESRD) worldwide. The changes are irreversible glomerular fibrosis and scar formation, and renal tubular injury is only secondary. But with the study, renal tubular lesions, especially tubulointerstitial fibrosis (TIF), play a very important role in promoting diabetic nephropathy, and renal tubular lesions can not be found. It is dependent on the glomerular lesion. Therefore, it is of great significance to explore the treatment strategy of postponing renal tubule interstitial fibrosis. Although the pathogenesis of TIF is not clear, more and more evidence points to the apoptosis of renal tubular epithelial cells and the transdifferentiation of renal tubular fibroblasts (mesenchymal cells). (epithelial-to-mesenchymal transition, EMT).EMT is a classic pathological change of tubular injury in diabetic nephropathy and is parallel to the degree of renal interstitial fibrosis. More and more studies have strongly confirmed that EMT plays an important role in different types of renal diseases, while reversing EMT can effectively improve renal fibrosis. A large number of studies have revealed the initiating factors, regulatory factors and existing signaling pathways of various EMT, but in all factors, TGF- beta 1 is a key regulator of fibrosis in various tissues and organs. It is the main inflammatory factor in the progression of diabetic nephropathy. It initiates and regulates the whole process of EMT. It mainly induces apoptosis by inducing cell apoptosis. Cell loss (2) directly acts on fibroblasts, mesangial cells and so on, up the synthesis of extracellular matrix (extracellular matrix, ECM), inhibiting ECM degradation and promoting renal tubular epithelial cell EMT to participate in the process of renal fibrosis. It has been shown that TGF- beta 1 can induce the morphology of myofibroblast in renal tubular epithelial cells, that is, polarity change, Actin microfilaments and compact bodies appear and collagen production increases. Although TGF- beta 1 can mediate these changes through RhoA, ERK, P38-MAPK, JNK and Wnt/ beta -catenin pathways, TGF- beta /Smad is currently considered as the most important signaling pathway to mediate this process. Activated TGF- beta 1 makes Smad2 and Smad3 phosphorylation, followed by phosphorylation The Smad oligomer complex is formed with the combination of Smad3 with Smad4 and transferred into the nuclear regulation target gene transcription. In diabetes, many proinflammatory factors can activate the Smad signaling pathway. In the glomeruli and interstitial fibrosis areas of diabetic nephropathy patients, phosphorylated Smad2 and Smad3 deposition can be seen, proving the activation of TGF- beta /Smad signaling pathway. There is no special drug to treat diabetic nephropathy in bed. It can only be treated by controlling diet, reducing sugar and lowering blood pressure, and the effect is limited. Traditional Chinese medicine has a long history. The traditional Chinese medicine has a good application prospect because of its multi-component and multi target characteristics. The radix astragali is the dry root of Mongolia Astragalus or membranous membranous membranous Astragalus membranaceus. The effects of Astragalus glycoside (Astragaloside IV, ASI) are the monomers of Astragalus saponins, which have the effect of antioxidation and ischemia reperfusion injury, and play an important role in the prevention and treatment of renal diseases such as acute renal injury, membranous nephropathy, diabetic nephropathy and so on. At present, the prevention and treatment of diabetic nephropathy by ASI is reported. Most of the channels are limited to podocyte and mesangial cells, and there are few studies on renal tubulointerstitial fibrosis. Therefore, this study intends to explore the role and mechanism of ASI inhibition of renal interstitial fibrosis in diabetic nephropathy in vivo and in vitro. The purpose of this study is to observe the TGF- beta /Smad signaling pathway and the high glucose induced by ASI in the renal tissue of type 2 diabetes model KKAy mice. The effect of NRK-52E on renal tubular epithelial cells was guided to explore the protective effect and mechanism of ASI on renal interstitial fibrosis, and to provide experimental basis for clinical application of ASI. Methods 1. animals were divided into groups: 2 weeks of adaptive feeding in mice, KKAy mice were fed with KK feed (high fat) for 14 weeks, and the blood glucose was more than 13.9mmol/L. The KKAy mice were randomly divided into model group (10 rats) and group ASI (10 rats), and 10 male C57BL/6J mice of the same age of normal diet were used as control group. The control group and model group were given 40mg/kg gavage with normal saline daily, ASI group was given ASI 40mg/kg, 2. blood glucose, blood creatinine, and urine ACR test: the weight of mice was measured every week, sixteenth weeks, 20 weeks, 24 weeks to take the tail reflection method to collect random urine, ELISA kit to detect urine microalbumin and urine creatinine, calculate urine ACR, take blood from the tail vein to measure blood sugar, and take blood to measure creatinine in the inner canthus; 3.24 weeks when the neck was killed, the kidneys were separated, HE staining was performed, and Masson staining was used to observe the pathological changes of kidney in each group; 4. the immunohistochemical staining was used to detect each group. The expression of TGF- beta 1, Smad2/3, and alpha -SMA, and the effect of 5. ASI on the vitality of NRK-52E cells: synchronization culture for 24 hours when NRK-52E cells grew to fusion state, and stimulated the cells for 24 hours at different concentrations of ASI (0,10,20,40,80100 mu g/ml, ASI10, ASI20, ASI40, etc.) for 24 hours, cell count kit inspection The effect of 6. ASI on the apoptosis of NRK-52E cells induced by high glucose: (1) when the cells grew to the fusion state, the cell culture was synchronized for 24 hours, and the normal control group, the high sugar group, and the ASI groups were added to the high sugar DMEM, and the concentration was 20,40,80100 g/ml (HG +ASI20, HG+ASI40, HG+ASI 80, HG+ASI 100) respectively. Cells were stimulated for 0,4,8,12,24,48 hours with high glucose +ASI 100 g/ml for 24 hours, and cell apoptosis was detected by AnnexinV-FITCPI kits, and 7. ASI on the expression of Smad2, Smad3, alpha -SMA, TGF- beta 1 induced by high glucose induced NRK-52E cells: cell growth to fusion state, synchronized culture for 24 hours, normal control group, high sugar Group (ASI), the NRK-52E cells were stimulated for 24 hours, respectively, and the expression of Smad2, Smad3, alpha -SMA, TGF- beta 1 nucleic acid level was detected by Real-Time PCR, Western Blot was detected in Smad2, Smad3, alpha, alpha, and beta 1 protein level; 8. statistical formula: measurement data were mistaken for mean number, and according to the homogeneity test of variance Results, the two groups were compared with the independent sample t test, and the multiple groups were compared with single factor analysis of variance. All the data were completed by the statistical software SPSS 19. The difference was statistically significant with the difference of P0.05. The difference of P0.01 was statistically significant. The results of the study were 1. general states: the control group had good mental state, sensitive reaction, smooth hair color slippery; The mice in the type group were depressed, slow, thirsty, polyuria, slow reaction and glossy hair, and with the increase of age, the above symptoms were more obvious between the two groups of.ASI mice. At the age of 16,20,24, the weight of the model group and the ASI group was larger than the same age group (P0.01); compared with the model mice, ASI group weight growth slowed (P0.05); 2. blood sugar, blood creatinine, urine ACR detection: compared with the same age control group, the blood glucose of the model group and the ASI group increased significantly (P0.01) at the observation time point. Compared with the same age model group, the blood glucose of the ASI group was lower (P0.05, P0.01). The serum creatinine detection results of the three groups were not statistically significant (P0.05); 3. Pathological changes of renal tissue in each group: the glomerular and tubular structure of the control group could be observed clearly, the number of mesangial cells was normal, the number of mesangial cells was normal, there was no inflammatory cell infiltration and fibrosis in the interstitium; the glomerular hypertrophy, the mesangial matrix increased, the mesangial cells increased, the cytosolic vacuoles appeared in the renal tubules, and the small tubule of the renal tubules could be seen in the renal tubule. The renal interstitial inflammatory cells increased and hyperemia and edema, and the glomerular and mesangial areas of the ASI group changed between the interventional control group and the model group. The renal tubular epithelial cells were slightly swollen, with less cytoplasm and no obvious interstitial fibrosis. 4. immunohistochemical staining was used to detect the expression of TGF- beta 1, Smad2/ 3 and alpha -SMA in each group: alpha -SMA, TGF- beta 1 and Smad2/3 were mainly in the group. In the control group, the expression of TGF- beta 1 was rare in the control group, while the expression of TGF- beta 1 in the model group and ASI group was enhanced (P0.01, P0.05). Compared with the model group, the expression of TGF- beta 1 was weakened (P0.01), and the alpha -SMA majority of the control group was expressed in the renal vascular smooth muscle and the tubulointerstitium was occasionally expressed, but the alpha -SMA in the renal tubular epithelial cells and renal tubules was found in the model group. Compared with the model group, the expression of alpha -SMA in the ASI group was significantly down (P0.01), and in the control group, the renal tubules and the glomerular nuclei had a small amount of Smad2/3 expression, the expression of the model group and the ASI group increased (P0.01, P0.05), but the Smad2/3 expression of the ASI group decreased (P0.01) compared with the model group, and the effect of 5. ASI on the vitality of the 5. ASI cells: different concentrations The effect of I on NRK-52E cells was 24 hours respectively, and there was no significant difference in cell activity between each group (P0.05); the effect of 6. ASI on the apoptosis of NRK-52E cells induced by high glucose: compared with the control group, the apoptosis of the high glucose group increased (P0.01). After adding ASI, the apoptosis of HG+ASI 40, HG+ASI 80, HG+ASI 100 was lower than that of the high sugar group (P0.05): H After the action of G+ASI 100 mu g/ml on cell 8h, apoptosis was significantly inhibited, and with the prolongation of time, the effect was gradually enhanced (compared with Oh, P0.05); 7. ASI to high glucose induced NRK-52E cell Smad2, Smad3, alpha -SMA, TGF- beta 1 mRNA. All P0.01), Smad3 up regulation (P0.05), HG, HG+ASI20, HG+ ASI 40 alpha -SMA expression up-regulated (P0.01), HG+ASI 80 compared with HG, HG+ASI 100, 40, 80, 100, 1, 20, 40, 80, downregulation The effect of 8. ASI on the level of Smad2, Smad3, p-Smad2, p-Smad3, alpha -SMA, TGF- beta 1 protein in high glucose induced NRK-52E cells: compared with the control group, the high glucose group (HG), HG+ASI20, HG+ASI40 group was up regulated. .01), TGF- beta 1 was up-regulated in HG, HG+ ASI 20 groups (P0.01); compared with HG, HG+ASI80, HG+ASI 100 groups of -SMA, Smad2, Smad3, and decreased expression was statistically significant. Blood glucose, obesity and urine ACR, but do not affect the blood creatinine; 2. astragaloside can improve the renal tubulointerstitial fibrosis in diabetic KKAy mice, its mechanism may be related to the downregulation of TGF- beta /Smad signaling pathway in ASI; 3. astragaloside can inhibit high glucose induced apoptosis in a dose and time dependent manner; 4. astragaloside can inhibit high glucose induction The expression of NRK-52E -SMA and TGF- beta 1/Smad signaling pathway can alleviate EMT and delay renal interstitial fibrosis.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

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