叉头状转录因子O1对糖尿病肾病近端小管间质纤维化的影响及机制研究

发布时间：2018-05-13 19:19

本文选题：叉头状转录因子O1 + 糖尿病肾病　；参考：《郑州大学》2017年硕士论文

【摘要】：背景糖尿病肾病(diabetic nephropathy,DN)是终末期肾病最常见的病因。临床肾病最早的表现是出现持续微量白蛋白尿,继而出现持续性蛋白尿,进展为显性糖尿病肾病。随后,肾小球滤过率(GFR)逐渐下降,5年内,50%患者发展为终末期肾脏疾病。虽然过去糖尿病肾病被认为是肾小球疾病为主的病变,现今越来越多的证据表明,肾功能降低程度与肾小管间质病变程度相关性更高。虽然多数糖尿病肾病患者最初是由于肾小球改变导致蛋白尿,肾间质改变在糖尿病肾病长期病变中也发挥了重要作用。随着越来越多研究关注病理性间质改变,主要集中在细胞在纤维化发生中的作用,如近端小管上皮细胞(PTC)或间质细胞。因此,研究高糖环境下PTC功能改变,以及PTC在糖尿病肾病肾间质纤维化中的作用具有重要意义。叉头框转录因子O1(FoxO1)是一类重要的调节因子,主要调控葡萄糖和脂质代谢、氧化应激反应与氧化还原信号、细胞周期和细胞凋亡等方面。本课题组前期研究发现,上调DN大鼠肾皮质中FoxO1,可缓解肾小球病变及肾小管病变,包括系膜细胞、足细胞和肾小管上皮细胞损伤。硫氧还蛋白相互作用蛋白(TXNIP),也被称为维生素D3上调蛋白-1(VDUP-1)或硫氧还蛋白结合蛋白2(TBP-2),是硫氧还蛋白(Trx)的内源抑制剂,在调节血糖和血脂代谢、炎症反应、糖尿病等均有重要作用。Trx-Txnip系统对维持细胞氧化还原状态非常重要。有研究显示,高糖状态下系膜细胞中,VDUP-1的表达迅速升高,IV型胶原α1链(COL4A1)mRNA升高,IV型胶原蛋白积累。基因芯片分析显示,高糖环境下人近端小管细胞系(HK-2细胞),硫氧还蛋白相互作用蛋白(TXNIP)上调明显。因此,本研究推测,在肾小管中,FoxO1可能通过抑制TXNIP,减轻高糖诱导的ROS升高,进而缓解糖尿病肾病肾小管氧化损伤和间质纤维化。目的研究FoxO1/Txnip对糖尿病肾病近端小管氧化损伤和间质纤维化的影响,并对其机制进行探讨。方法体外实验中,应用CRISPR/CAS 9技术构建FoxO1敲除(KO)及过表达(KI)稳转HK-2细胞株,并在FoxO1敲除细胞株中转染Txnip siRNA。分组为正常糖浓度组(NG组)、高糖(25mmol/L)组(HG组),高糖+FoxO1敲除组(KO组)、高糖+FoxO1过表达组(KI组)、高糖+FoxO1敲除+Txnip siRNA组(KO+Txnip siRNA组)以及高糖+FoxO1敲除+阴性对照组(NC组)。实时荧光定量PCR(Real-time PCR)以及Western blotting检测各组FoxO1、pFoxO1、Txnip、Trx、FN、ColⅣ等mRNA和蛋白的表达水平;流式细胞仪检测ROS水平等相关氧化应激指标。体内实验中,应用受精卵显微注射构建肾脏特异性过表达FoxO1转基因小鼠,分组:正常小鼠组(NG组)、糖尿病组(DM组)、转基因正常组(FoxO1-Tg组)、转基因糖尿病组(FoxO1-Tg DM组)。12周末心脏抽血检测血清肌酐、尿素氮;留取24小时尿检测24小时尿总蛋白,HE染色、Masson染色、PAS染色及透射电镜观察肾脏病理学变化情况;取肾组织行实时荧光定量PCR(Real-time PCR)以及Western blotting检测各组FoxO1、pFoxO1、Txnip、Trx、FN、ColⅣ等mRNA和蛋白的表达水平;免疫组化检测FN、ColⅣ等蛋白表达水平。结果1.各组FoxO1表达及活性改变:与NG组相比,HG组Fox O1 mRNA及蛋白水平无明显变化(P0.05),p-FoxO1/FoxO1的比值增高(P0.05),表明高糖对FoxO1转录活性有抑制作用;与HG组相比,KI组FoxO1 mRNA及蛋白水平均升高,p-FoxO1/FoxO1的比值增大(P0.05)。2.各组细胞中FoxO1对Txnip-Trx和ROS的影响:与NG组相比,HG组Txnip mRNA及蛋白表达均增多,Trx表达减少,ROS水平升高(均P0.05)。与HG组相比,KI组Txnip mRNA及蛋白表达均减少,Trx表达增多,ROS水平降低(均P0.05);KO组中Txnip mRNA及蛋白表达明显增多,Trx表达明显减少,ROS明显升高(均P0.05)。而与KO组相比,KO+Txnip siRNA组Txnip mRNA及蛋白表达均降低,Trx表达增多,ROS水平有所降低(均P0.05)。3.FoxO1对肾间质纤维化的影响:HG组与NG组相比,FN及Col mRⅣNA及蛋白表达均升高(均P0.05)。与HG组相比,KI组FN和ColⅣmRNA及蛋白表达降低(均P0.05);KO组FN和ColⅣmRNA及蛋白表达增多(均P0.05)。与KO组相比,KO+Txnip siRNA组FN及ColⅣmRNA和蛋白表达明显降低(均P0.05)。4.各组小鼠肾小管中FoxO1表达情况:与NG组相比,DM组小鼠肾脏Fox O1mRNA和蛋白表达无变化(均P0.05),p-FoxO1/FoxO1比值明显升高(均P0.05)。与DM组相比,FoxO-Tg DM组中p-FoxO1/FoxO1比值明显降低(均P0.05)。与NG和DM组小鼠相比,FoxO1-Tg组和FoxO1-Tg DM组小鼠肾脏FoxO1 mRNA及蛋白表达增多(均P0.05)。5.各组小鼠肾小管中FoxO1抑制TXNIP信号通路的激活:与NG组和FoxO1-Tg组相比,DM组与FoxO1-Tg DM组Txnip mRNA和蛋白水平升高,Trx表达减少(均P0.05)。与DM组相比,FoxO1-Tg DM组Txnip mRNA和蛋白水平相对降低,Trx表达增多(均P0.05)。6.FoxO1缓解糖尿病肾病小鼠肾小管间质纤维化:与NG组相比,DM组FN和Col mRⅣNA及蛋白表达均升高(均P0.05),表明糖尿病肾病时,小鼠肾脏发生间质纤维化。与DM组相比,FoxO1-Tg DM组FN和ColⅣ表达明显降低(均P0.05)。7.FoxO1可以有效缓解糖尿病小鼠肾脏损伤:与NG组相比,DM组体重明显降低,肾重比明显升高,血糖水平明显升高,24h尿蛋白及血肌酐、尿素氮水平均明显升高(均P0.05)。与DM组相比,FoxO1-Tg DM组体重明显增加,肾重比明显降低(均P0.05),血糖未见明显变化(P0.05),24h尿蛋白及血肌酐、尿素氮水平均明显降低(均P0.05)。与正常组相比,DM组小鼠肾小管细胞萎缩,基底膜异常增厚,胶原纤维增多,间质纤维化明显;与DM组相比,FoxO1-Tg DM组病变较轻,基膜均匀,稍增厚,胶原纤维少,间质纤维化程度较轻。结论1.高糖能降低肾小管中FoxO1活性,诱导氧化损伤和间质纤维化。2.过表达FoxO1明显改善糖尿病小鼠蛋白尿和间质纤维化程度,其机制可能与抑制TXNIP/Trx/ROS通路有关。
[Abstract]:Background diabetic nephropathy (diabetic nephropathy, DN) is the most common cause of end-stage renal disease. The earliest manifestation of clinical nephropathy is the emergence of persistent microalbuminuria, followed by persistent proteinuria and progressive diabetic nephropathy. Then, the glomerular filtration rate (GFR) decreases gradually, and within 5 years, 50% patients develop to end-stage renal disease. Although in the past, diabetic nephropathy is considered as a major glomerular disease, there is growing evidence that the degree of renal dysfunction is more associated with the degree of renal tubulointerstitial lesions. Although most diabetic nephropathy patients are initially due to glomerular changes in proteinuria, renal interstitial changes are in the long-term pathological changes of diabetic nephropathy. It also plays an important role. As more and more attention is paid to pathological interstitial changes, it is mainly focused on the role of cells in the pathogenesis of fibrosis, such as proximal tubular epithelial cells (PTC) or interstitial cells. Therefore, it is important to study the changes of PTC function in high glucose environment and the role of PTC in the renal interstitial fibrosis of diabetic nephropathy. The forked frame transcription factor O1 (FoxO1) is an important regulation factor, which mainly regulates glucose and lipid metabolism, oxidative stress reaction and redox signal, cell cycle and cell apoptosis. Earlier study in our group found that up regulation of FoxO1 in renal cortex of DN rats could relieve glomerular lesions and renal tubular lesions, including mesangial cells. Thioredoxin protein interaction protein (TXNIP), also known as vitamin D3 up-regulated protein -1 (VDUP-1) or thioredoxin binding protein 2 (TBP-2), is an endogenous inhibitor of thioredoxin (Trx). It has an important role in regulating blood glucose and blood lipid metabolism, inflammatory response, and diabetes, and so on. It is very important to maintain the redox state of cells. Studies have shown that the expression of VDUP-1 in mesangial cells of high glucose state increases rapidly, IV collagen alpha 1 chain (COL4A1) mRNA increases and IV collagen accumulation. Gene chip analysis shows that human proximal tubule cell line (HK-2 cell) and thioredoxin protein interaction protein (TXNIP) in high glucose environment Therefore, this study suggests that in renal tubules, FoxO1 may alleviate the oxidative damage of renal tubules and interstitial fibrosis in diabetic nephropathy by inhibiting TXNIP and alleviating the increase of ROS induced by high glucose. Objective to study the effect of FoxO1/Txnip on the oxidative damage and interstitial fibrosis of proximal tubules in diabetic nephropathy and to explore its mechanism. Methods in vitro, CRISPR/CAS 9 technique was used to construct FoxO1 knockout (KO) and overexpression (KI) to stabilize HK-2 cell lines, and transfected Txnip siRNA. into normal glucose concentration group (NG group), high sugar (25mmol/L) group (HG group), high sugar +FoxO1 knockout group, high sugar overexpression group, high sugar overexpression group, and high glucose knocking in FoxO1 knockout cell lines. SiRNA group (group KO+Txnip siRNA) and high glucose +FoxO1 knockout + negative control group (NC group). Real time fluorescent quantitative PCR (Real-time PCR) and Western blotting were used to detect the expression level of each group and protein; flow cytometry was used to detect the levels of related oxidative stress. In vivo experiments, the fertilized eggs were applied. Renal specific overexpression FoxO1 transgenic mice were microinjected into normal mice (group NG), diabetes group (group DM), transgenic normal group (group FoxO1-Tg), and genetically modified diabetes group (group FoxO1-Tg DM).12 weekend cardiac blood test for serum creatinine and urinary nitrogen; 24 hour urine test was left for 24 hours urinary total protein, HE staining, Masson staining, P. The pathological changes of kidney were observed by AS staining and transmission electron microscopy, and the expression of FoxO1, pFoxO1, Txnip, Trx, Trx, FN, Col IV and other proteins were detected by real-time fluorescence quantitative PCR (Real-time PCR) and Western blotting in the renal tissue. Compared with group NG, the level of Fox O1 mRNA and protein in group HG had no obvious changes (P0.05), and the ratio of p-FoxO1/FoxO1 increased (P0.05), indicating that high sugar had a inhibitory effect on the transcriptional activity of FoxO1. Compared with the HG group, the expression of Txnip mRNA and protein increased, the expression of Trx decreased, and the level of ROS increased (P0.05). Compared with the HG group, the Txnip mRNA and protein expression in KI group decreased, Trx expression increased and ROS level decreased. The expression of Txnip mRNA and protein in group O+Txnip siRNA decreased, the expression of Trx increased, and the level of ROS decreased (P0.05).3.FoxO1's effect on renal interstitial fibrosis: HG group and NG group were higher than NG group. The expression of protein was increased (all P0.05). Compared with the KO group, the expression of FN and Col IV mRNA and protein in the KO+Txnip siRNA group decreased significantly (all P0.05) in the renal tubules of each group of.4. mice, and the expression of the kidneys and proteins in the mice of the DM group was significantly higher than that in the NG group. The p-FoxO1/FoxO1 ratio in the DM group was significantly lower (P0.05). Compared with the NG and DM mice, the FoxO1 mRNA and protein expression in the FoxO1-Tg and FoxO1-Tg DM mice increased (P0.05) in the renal tubules of the mice. Trx expression decreased (P0.05). Compared with group DM, Txnip mRNA and protein level in FoxO1-Tg DM group decreased and Trx expression increased (all P0.05).6.FoxO1 alleviated renal tubulointerstitial fibrosis in diabetic nephropathy mice. Interstitial fibrosis. Compared with the DM group, the expression of FN and Col IV in the FoxO1-Tg DM group decreased significantly (all P0.05).7.FoxO1 could effectively alleviate the renal injury in diabetic mice. Compared with the NG group, the weight of the DM group was significantly reduced, the renal weight ratio was significantly elevated, the blood glucose level was significantly elevated, the 24h urine protein and serum creatinine, and the urea nitrogen water increased significantly (P0.05). And DM group. The weight of FoxO1-Tg DM group was significantly increased, the renal weight ratio was significantly decreased (P0.05), blood sugar was not significantly changed (P0.05), 24h urine protein and blood creatinine, and urea nitrogen water decreased significantly (P0.05). Compared with the normal group, the renal tubule cells in the DM group were atrophied, the basement membrane was thickened, the collagen fibers increased and the interstitial fibrosis was obvious; compared with the DM group, it was compared with the DM group. The pathological changes of FoxO1-Tg DM group are lighter, the basement membrane is homogeneous, the thickness of the collagen fiber is slightly thicker, the collagen fiber is less and the degree of interstitial fibrosis is lighter. Conclusion 1. high glucose can reduce the activity of FoxO1 in the renal tubules. The induced oxidative damage and the overexpression of.2. over the interstitial fibrosis can obviously improve the proteinuria and the degree of interstitial fibrosis in diabetic mice. The mechanism may be related to the inhibition of the TXNIP/Trx/ROS pathway. Of

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

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