【作者】 王菲；

【导师】 张农；

【作者基本信息】 复旦大学 ， 病理学， 2011， 博士

【摘要】 醛糖还原酶(Aldose Reductase, AR)为NADPH依赖性醛-酮还原酶家族成员之一,是多元醇代谢通路中的限速酶,可催化己糖或戊糖的NADPH依赖性还原反应,使之变成相应的糖醇或多元醇。本课题组前期发现并通过实验鉴定,AR作为转化生长因子-β1(transforming growth factor-β1, TGF-β1)相关反应性基因之一,并在其诱导的肾小球硬化、肾脏纤维化中起着重要作用。近年来,随着对AR研究的逐渐深入,学者们开始认识到,AR在体内的最适底物并非葡萄糖,而是细胞内的一些有毒醛类化合物,例如4-hydroxy-trans-2-nonenal (HNE), AR对于这类底物的结合能力远高于糖类化合物。该类醛化合物最主要的来源为脂质过氧化,特别是ROS介导的脂质过氧化过程。有研究显示,TGF-β1致纤维化的作用机制中与ROS和氧化应激关系密切,ROS可以激活大量加剧组织损伤的信号通路,Nrf2就是其中之一,它的活化可以反过来加速ROS的生成以及ECM的沉积,提示ROS和Nrf2可能参与了TGF-β1上调AR的表达。Nrf2作为一个转录因子,其本身是否受到ROS通路的调节,是否在TGF-β1上调AR的表达中也具有桥梁作用,目前未见国内外报道。本课题从研究HMC表达AR与TGF-β1的相关性研究作为切入点,首次阐明了ROS和Nrf2在其中的作用及其分子机制。采用电子自旋共振(electron spin resonance,ESR)技术、Real-time PCR、Western Blot、报告基因等实验方法,证明了TGF-β1可以诱导HMC表达AR的增加；ROS和Nrf2参与了TGF-β1上调AR的表达。揭示了作为TGF-β1的相关反应性基因,AR的表达受其调控,分别体现在核酸、蛋白水平和活性上。进一步的信号通路研究表明,TGF-β1引起细胞内ROS水平增高,进而激活Nrf2信号通路,从而导致AR的转录水平以及翻译水平上调。对AR基因的启动子研究更加证明了转录因子Nrf2在TGF-β1诱导的AR上调过程中起着重要作用。目的探索ROS-Nrf2信号通路介导TGF-β1诱导HMC高表达AR过程的机制。方法HMC体外培养；Western blot、real-time RT-PCR、荧光法检测TGF-β1因子诱导HMC中AR高表达；ESR技术检测TGF-β1作用HMC后ROS量的变化；瞬时转染Nrf2siRNA和加入抗氧化剂NAC和SOD,对AR高表达的影响；加入外源性H202对Nrf2表达的影响：双荧光素酶报告系统检测Nrf2对含有ARE结合位点AR启动子转录活性的影响。结果1. TGF-β1可以诱导HMC表达AR的增加；随着TGF-β1作用时间的延长和剂量的增大,AR的基因表达水平发生变化,在24h内表达上调明显；AR蛋白表达增高和AR活性水平增强,与作用时间和剂量呈正相关。2. TGF-β1可以诱导HMC产生ROS的增加；随着TGF-β1作用时间的延长和剂量的增大,ROS数量发生变化,在30min内达到最多,与TGF-β1剂量呈正相关；3. TGF-β1可以诱导HMC表达Nrf2的增加,随着TGF-β1作用剂量的增大,Nrf2的蛋白表达水平逐渐增高,与TGF-β1作用剂量呈正相关；加入外源性ROS刺激HMC,Nrf2的蛋白表达水平增高。4.抗氧化剂SOD、NAC能够抑制TGF-β1诱导ROS的生成增多,进而抑制TGF-β1诱导AR基因水平、蛋白表达水平的增高和AR活性的增强。5.抗氧化剂SOD、NAC和Nrf2 siRNA都可以有效干扰Nrf2的表达,下调Nrf2表达能够抑制’TGF-β1诱导的AR基因水平、蛋白表达水平的增高和AR活性的增强。6.双荧光素酶报告基因证实,与转染pGL3-basic的对照组相比,HA-Nrf2可以使AR启动子区含有ARE结合位点的1.06k-AR-luc、ARE质粒转录活性显著增加；突变ARE序列的两个位点(ARE1、ARE2)后,可以显著降低ARE质粒的转录活性。结论1.作为TGF-β1的相关反应性基因,AR的表达受其调控,分别体现在核酸、蛋白水平和活性上。2.进一步的信号通路研究表明,TGF-β1引起细胞内ROS水平增高,并进而激活Nrf2信号通路,从而导致AR的转录水平、翻译水平上调以及酶活性增强。3.对AR基因的启动子研究更加证明了转录因子Nrf2在TGF-β1诱导的AR上调过程中起着重要作用。更多还原

【Abstract】 Aldose reductase (Aldose Reductase, AR) is a member of the NADPH-dependent aldo-keto reductase superfamily, the rate-limiting enzyme in polyol pathway. It can catalyze the NADPH-dependent reduction reaction of hexose and pentose and turn it into the corresponding sugar alcohols or polyols. Our group previously transfer the TGF-β1 gene into rat MC and obtained 28 TGF-β1-responsive gene clones in the screening of positive clones. By sequencing and comparing in Genebank, we found AR, one of TGF-β1-responsive genes.Glomerulosclerosis is a final common outcome of glomerular injury in various types of human glomerular diseases. Under normal circumstances, the normal mesangial cells can express a certain level of TGF-β1, which is necessary for cell physiological activities. It plays an important role in the regulation of extracellular matrix metabolism, involves in inflammation and interstitial fibrosis and is considered to be one of the important cytokines leading to fibrosis. Because TGF-β1 is an important mediator in glomerulonephritis and glomerulosclerosis and closely related to ECM deposition, we takes into account that AR may induce the proliferation of MC and then will participate in the pathogenesis of glomerular sclerosis.It has been reported that many growth factors including TGF-β1 induce fibrosis under the participation of oxidative stress. Some scholars believe that renal fibrosis and ROS are also related to oxidative stress. ROS played an important role in the course of a variety of fibrotic diseases and ROS can activate a large number of signaling pathways, thus to exacerbate the tissue injury. Nrf2 is one of the activated pathways and its activation would accelerate the generation of ROS and ECM deposition. Nrf2 is an important transcription factor, it enters into the nucleus, binding to specific DNA sequences and inducing the expression of downstream genes.It is not sufficient to simply explain TGF-β1 induced renal fibrosis, TGF-β1 induced high expression of AR, TGF-β1 induced metabolic changes with Smad pathway and MAPK pathway. ROS is both free radical oxidative stress factors and a growth factor signaling molecules. Here, we infer that, ROS are involved in TGF-β1 fibrosis, Nrf2 is also a bridge in the development process of renal fibrosis. Nrf2’s research in this area is still rare. Objective To explore the pathway mechanism that TGF-β1 induced high expression of AR.Methods Cultured HMC; Western blot, real-time RT-PCR, fluorescence detection of TGF-β1 cytokine-induced AR expression in HMC; ESR detection of the ROS volume in TGF-β1 treated HMC; Transient transfection of Nrf2 siRNA and the addition of anti oxidants NAC and SOD, the impact to high expression of AR; the addition of exogenous H2O2 on the expression of Nrf2; dual luciferase reporter system detects the Nrf2 impact to transcriptional activity of AR promoter containing ARE binding sites.Results Treated by 4ng/ml and 10ng/ml TGF-β1 factor, AR mRNA levels, protein expression and activity in HMC were increased, positively correlated to the acting time and dosage of TGF-β1, compared with the control group. Respectively, after the effect of 4ng/ ml and 10ng/ml TGF-β1, AR mRNA expression levels began to increase with the increased affecting time or concentration, leading to a peak at 24h. Compared with the control group, it rose 9.78 times and 11.53 times higher (P＜0.01), mRNA levels then appeared to reduce.AR protein expression and activity increased with increasing time and concentration of TGF-β1(P<0.05), reached the peak at 48h, compared with the control group, increased by 24.19 and 27.44 times(P＜0.01);at the 6h TGF-β1 effect time point, Nrf2 protein expression increased, respectively,4.27 times and 4.75 times, compared with control group(P<0.05); at TGF-β1 concentration of 10 ng/ml point, Nrf2 protein expression reach its peak,4.85 times than control group, and the difference was statistically significant (P＜0.01). With the increased TGF-β1 dose, Nrf2 protein expression increased with TGF-β1 effect dose and they are positively correlated;Nrf2 siRNA interference, transfection of Nrf2 siRNA (10 nM) 72h, real-time RT-PCR, Western blot and fluorescence methods were used to detect AR protein expression and AR activity, the results show that with comparisons to negative control group, AR mRNA levels decreased by 48.7%(P＜0.01), AR protein levels decreased 53.1%(P＜0.01), AR activity level decreased 34.6%, (P＜0.01); After 72h Nrf2 siRNA,36h TGF-β1 stimulation, compared with Nrf2 siRNA group, expression of AR protein levels and AR activity were increased by 2.66 times and 2.73 times (P＜0.01), but lower than TGF-β1 stimulation group (P＜0.01). H2O2 and TGF-β1 was respectively added in HMC, Western blot results showed that, Nrf2 protein levels were significantly higher (increased by 3.80 times and 3.79 times) (P＜0.01); were added SOD and free radical scavengers NAC, then add H2O2 and TGF-β1 stimulation, Western blot results showed that, Nrf2 protein levels were significantly lower than that H2O2 and TGF-β1 stimulation group (P＜0.01), but higher than the normal control group, and statistically differences (P<0.01).4ng/ml and 10ng/ml of TGF-β1 factor acting on the HMC, ESR detection of the amount of ROS, as the concentration of TGF-β1, the amount of ROS generation increased.When the concentration of TGF-β1 was 10 ng/ml, ROS generate the most, is 17.35-fold of the control group (P<0.01), positively correlated with TGF-β1 dose, negatively correlated with effect time. After antioxidant NAC, SOD treatment, and then 4 ng/ml TGF-β1 stimulation, ESR signal levels stimulated by TGF-β1 are significantly higher(P＜0.01); By adding antioxidant NAC, SOD, stimulating by TGF-β1, ESR signals are significantly decreased, compared to stimulation of TGF-β1 alone group, by 42% and 44%(P＜0.01). AR mRNA levels, protein expression and activity were reduced by 57.2%,61%,51%; ESR detection of ROS quantity, when the concentration of TGF-β1 was10 ng/ml, ROS is 17.35 times of the control group at the peak, and differences were statistically significant (P＜0.01).Dual-luciferase reporter system detected, compared with transfected pGL3-basic control group, HA-Nrf2 increased transcriptional activity of binding sites containing ARE,1.06-AR-luc and ARE plasmid, in AR promoter region by 52.33 times and 49.67 times (P＜0.01); mutant ARE sequences of two loci ARE1, ARE2, compared to unmutant ARE plasmid, its transcriptional activity decreased by 93% and 92.7%(P＜0.01).Conclusion1. Expression of AR in HMC can be successfully induced by TGF-β1; With the increase of TGF-β1 effect time and dose, AR gene expression levels increased significantly in the 24h; AR expression and AR activity levels increased, positively correlated to effect time and dose.2. TGF-β1 can successfully induce ROS expression increase in HMC; With the increase of TGF-β1 effect time and dose, ROS levels increased significantly in 30min to the top, positively related to TGF-β1 dose; antioxidants SOD, NAC can inhibit TGF-β1-induced ROS generation and inhibit TGF-β1-induced increase of AR expression and AR activity. ROS mediate TGF-β1 induced increase of AR expression in HMC.3. TGF-β1 can successfully induce the expression of Nrf2 in HMC; Nrf2 siRNA can interfere with the expression of Nrf2, and inhibit TGF-β1-induced AR expression increase and AR activity enhancement. Nrf2 mediates TGF-β1 induced increase of AR expression in MC.4. Exogenous ROS can successfully induce the increase of Nrf2 expression in HMC. Antioxidants can interfere with the high expression of Nrf2, ROS mediated TGF-β1 induced high expression of Nrf2.5. Nrf2 can increase the expression of AR protein. Nrf2, through the regulation of ARE sequence in AR promoter region plays its roles. AR is likely to be one of Nrf2 downstream molecules.更多还原