| 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-?1 gene into rat MC and obtained 28 TGF-?1-responsive gene clones in the screening of positive clones. By sequencing and comparing in Genebank, we found AR, one of TGF-?1-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-?1, 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-?1 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-?1 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-?1 induced renal fibrosis, TGF-?1 induced high expression of AR, TGF-?1 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-?1 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-?1 induced high expression of AR.Methods Cultured HMC; Western blot, real-time RT-PCR, fluorescence detection of TGF-?1 cytokine-induced AR expression in HMC; ESR detection of the ROS volume in TGF-?1 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-?1 factor, AR mRNA levels, protein expression and activity in HMC were increased, positively correlated to the acting time and dosage of TGF-?1, compared with the control group. Respectively, after the effect of 4ng/ ml and 10ng/ml TGF-?1, 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-?1(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-?1 effect time point, Nrf2 protein expression increased, respectively,4.27 times and 4.75 times, compared with control group(P<0.05); at TGF-?1 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-?1 dose, Nrf2 protein expression increased with TGF-?1 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-?1 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-?1 stimulation group (P?0.01). H2O2 and TGF-?1 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-?1 stimulation, Western blot results showed that, Nrf2 protein levels were significantly lower than that H2O2 and TGF-?1 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-?1 factor acting on the HMC, ESR detection of the amount of ROS, as the concentration of TGF-?1, the amount of ROS generation increased.When the concentration of TGF-?1 was 10 ng/ml, ROS generate the most, is 17.35-fold of the control group (P<0.01), positively correlated with TGF-?1 dose, negatively correlated with effect time. After antioxidant NAC, SOD treatment, and then 4 ng/ml TGF-?1 stimulation, ESR signal levels stimulated by TGF-?1 are significantly higher(P?0.01); By adding antioxidant NAC, SOD, stimulating by TGF-?1, ESR signals are significantly decreased, compared to stimulation of TGF-?1 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-?1 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-?1; With the increase of TGF-?1 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-?1 can successfully induce ROS expression increase in HMC; With the increase of TGF-?1 effect time and dose, ROS levels increased significantly in 30min to the top, positively related to TGF-?1 dose; antioxidants SOD, NAC can inhibit TGF-?1-induced ROS generation and inhibit TGF-?1-induced increase of AR expression and AR activity. ROS mediate TGF-?1 induced increase of AR expression in HMC.3. TGF-?1 can successfully induce the expression of Nrf2 in HMC; Nrf2 siRNA can interfere with the expression of Nrf2, and inhibit TGF-?1-induced AR expression increase and AR activity enhancement. Nrf2 mediates TGF-?1 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-?1 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. |
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