| IntroductionDiabetic nephropathy, the severest and commonest long-term microvascular complications in diabetes mellims, is the leading cause of end-stage renal failure and mortality in diabetic patients. Elucidating the molecular mechanisms of diabetic nephropathy attracts attention of basic research of nephrology all along. The unifying mechanism for the pathogenesis of diabetic complications theory presented by Brownlee in 2001 provided a new breakthrough in understanding pathogenesis of diabetic nephropathy. Many factors and pathways related to pathogenesis of diabetic nephropathy such as genetic susceptibility, metabolic and haemodynamatic disturbances, hemodynamic abnormalities and upregulation of various cytokine, vasoactive hormone and chemokine are all involved in a crucial common pathway-oxidative stress. Excess reactive oxygen species(ROS) mediate hyperglycacmia-induced renal damage through activating the polyol way, increasing activity of protein kinase C(PKC)and hexosamine pathway, and promoting advanced glycation end product formation. All these findings provided a breakthrough in DN pathogenesis.NF-E2-related factor 2(Nrf2), is a vital transcription factor against cellular oxidative stress. Nrf2 signaling pathway plays a significant role in defending human cells from oxidative stress in vitro and in vivo, through binding with antioxidant-response element(ARE), upgrading expression of antioxidant protein and phase Ⅱ antioxidant enzymes, and further improving antioxidation. In conclusion, Nrf2 plays an important role in fighting against endo-and exogenous oxidative stress. Recent studies showed that Nrf2 signaling pathway functioned as a protective factor in diseases who shared the same pathogenesis of oxidative stress, including DN. Therefore, researches on Nrf2 signaling pathway are attracting increasing attention in the field of DN protection. As an antioxidant with renoprotective effect, whether Hepatocyte growth factor (HGF) functions by activating Nrf2 signaling pathway and its relevant mechanisms remains unknown. Physiologically, KEAP1 binds to Nrf2 and sequesters it in the cytoplasm. Under basal conditions, KEAP1 mediates ubiquitination and subsequent degradation of Nrf2 by the proteasome. Upon exposure of cells to oxidative stress, Nrf2 dissociates from KEAP1 and translocates into the nucleus to bind to AREs in the genes encoding antioxidant enzymes. Latest research revealed that apart from Keapl, Glycogen synthase kinase-3(GSK-3β) deregulates Nrf2 signaling pathway by mediating translocation of Nrf2 into the cytoplasm. Sulforaphane(SFN), a Nrf2 signaling pathway activator, exhibits antidiabetic properties through Keap1 modification, inhibition of Nrf2 degradation and translocation into the nucleus, and eventually activating Nrf2 signaling pathway. Unfortunately, it is unclear whether SFN activates Nrf2 signaling pathway by moderating GSK-3β and preventing translocation of Nrf2. Elucidating these would provide innovative therapeutic basis for DN treatment.In vitro, we performed many molecular biologic technologies, including RT-PCR, western blot, flowcytometer detection, mmunohistochemistry and immunofluorescence to examine the influence of HGF on Nrf2 activation in high glucose(HG)-induced rat mesangial cells and explore relevant mechanisms. In vivo and in vitro, we observed the effect of SFN on Nrf2 in DN models and discussed correlated mechanisms. The purpose of our research was to determine the antioxidant role of HGF and SFN in diabetic nephropathy and elucidate involved mechanisms, thus bring new insights into the pathogenesis and open novel avenues toward the therapeutic potential of DN.Part I The antioxidant effect of HGF on HG-induced RMsC and relevant mechanismsObjective:To investigate whether HGF activates Nrf2 signaling pathway to perform the protective role against oxidative stress and involved mechanisms.Methods:The influence of HGF on 8-nitro-cGMP in HG-induced RMsC was examined by immunofluorescence. With fluorescence microscope and floweytometer, intracellular ROS was examined qualitatively and quantitatively in RMsC loaded with the fluoroprobe 2’7’-diehlorofluoresein diacetate. The intracellular levels of glutathione(GSH)and malonaldehyde(MDA)as well as nitric oxide synthase (NOS) and NO production were measured by speetrophotometry. qRT-PCR and western blot were used to analyze the effect of HGF on Nrf2 and glutamate-cysteine ligase catalytic subunit (GCLC) mRNA and protein expression in HG-induced RMsC.Results:A noticeable increase of 8-nitro-cGMP caused by HGF in HG-induced RMsC was completely blocked by HGF inhibitor SU11274, NOS inhibitor L-NMMA and sGC inhibitor NS2028. HGF prevented the increase of intracellular ROS and MDA and diminished GSH level. This was totally abolished by SU11274, and partly inhibited by L-NMMA and NS2028. NOS activity and NO content were remained by HGF in HG-treated RMsC. SU11274、L-NMMA completely diminished this effect. mRNA and protein levels of GCLC in HG-induced RMsC increased by HGF were totally abrogated by SU11274 and partially by L-NMMA and NS2028. HGF upregulated Nrf2 protein expression both in the cytoplasm and the nucleus, whereas gene level of Nrf2 did not change appreciably. This was wholly inhibited by SU11274、L-NMMA and NS2028.Conclusions:HGF protected RMsC against oxidant stress by activating Nrf2 signaling pathway and upregulating GCLC-downstream gene of ARE. The antioxidant properties of HGF in HG-treated RMsC were exerted partly through upregulating intracellular 8-nitro-cGMP and thus activating Nrf2 signaling pathway.Part Ⅱ The protective role of SFN against oxidant stress in STZ-induced diabetic nephropathy rat model and relevant mechanismsObjective:To elucidate whether SFN exert antioxidant function in STZ-induced DN rat model via Nrf2 signaling pathway and explore its mechanism.Methods:Blood glucose, urine glucose and creatinine and ratio of urine albumin/urine creatinine(UACR) were tested in STZ-induced DN rat model. Tissue sections from paraffin-embedded kidneys were stained with hematoxylin-eosin (H&E), periodic acid Schiff (PAS) and Masson’s trichrome to observe morphology changes. Immunohistochemistry analysis was performed to evaluate 8-oxo-dG, Transforming Growth Factor-β1 (TGF-β1), Fibronectin (FN), Type IV collagen (COL IV), Nrf2, Heme Oxygenasel (Ho1), Quinine Qxidoreductase 1(NQO1), Keap1, GSK-3β, p-GSK-3β(Ser9) and Fyn expression. With qRT-PCR, mRNA levels of TGF-β1, FN, COL IV, Nrf2, Nqo1, Ho1, GSK-3β and Fyn were determined.Results:Compared with STZ group, STZ+SFN decreased urine creatinine and ratio of urine albumin/urine creatinine, reduced Extracellular Matrix(ECM) deposition, glomerular fibrosis and sclerosis, lessened 8-oxo-dG production and TGF-β1、FN、 COL IV at mRNA and protein levels, increased expression of Nrf2 and its downstream genes Nqoland Ho1 at protein levels. mRNA expression of Nqoland Ho1 were also increased. However, mRNA level of Nrf2 and Keapland mRNA expression of the latter showed no significant difference. Comparing with the Control group, there was noticeable rise in GSK-3β and Fyn protein levels in STZ-induced models, but not in mRNA levels. SFN increased phosphorylation of Ser9, an activity inhibition point of GSK-3β, but had no noticeable influence on GSK-3β and Fyn mRNA and protein expression.Conclusions:SFN performed antioxidant properties by diminishing 8-oxo-dG, TGF-β1 expression and ECM deposition. SFN activated NRF2 signaling pathway in STZ-induced DM rat models. GSK-3β/Fyn signaling pathway was inhibited by SFN through phosphor-Ser9 in experimental rats.Part III The defending influence of SFN on RMsC and involved mechanismsObjective:To observe whether SFN activates Nrf2 signaling pathway in HG-induced RMsC to function as a protective factor and relevant mechanisms.Methods:Transfection was used to reconstruct RMsC with high expression of GSK-3β. With fluorescence microscope, intracellular ROS was examined qualitatively and quantitatively in RMsC loaded with the fluoroprobe 2’7’-diehlorofluoresein diacetate. Western blot analysis was utilized to examine protein expression of TGF-β1、 FN、COL IV、Nrf2、Nqo1、Ho1、Keap1、GSK-3β、p-GSK-3β(Ser9) and Fyn. mRNA levels of TGF-β1、FN、COL Ⅳ、Nrf2、Nqo1、Ho1、GSK-3β and Fyn were measured by qRT-PCR.Results:The PcDNA3.0GSK-3β transfected RMsC showed noticeable increase in GSK-3β mRNA and protein levels. When comparing with HG group, RMsC treated with HG+SFN had a dramatic drop in ROS as well as TGF-β1, FN, COL IV at protein and mRNA levels. There was considerable rise in Nqoland Ho1 at both levels andNrf2 expression in the nucleus. No significant difference was found in protein and mRNA expression of Keapl,Nrf2 expression in the cytoplasm, and mRNA level of Nrf2. In contrast to the NG cohort, RMsC in HG group showed significant increase in GSK-3β and Fyn nuclear protein expression, decrease in Fyn cytoplasmic protein expression, while mRNA levels of GSK-3β and Fyn did not change obviously. In HG-induced RMsC, SFN upregulated phosphorylation of GSK-30 activity inhibition point Ser9 as well as Fyn expression in the cytoplasm, while deregulated Fyn expression at nuclear protein level. Compared with HG+SFN group, HG+SFN+GSK-3β revealed dramatic increase in ROS level, mRNA and protein expression of GSK-3β. mRNA and protein levels of TGF-β1, FN and COL IV were highly increased, while those of Nqol and Ho1 were decreased severely. Simultaneously, there was considerable drop in nuclear expression of Nrf2 and cytoplasmic expression of Fyn. Nuclear protein of Fyn increased. No significant difference was noticed in mRNA level of Fyn. All these effects of GSK-3β were abrogated by its micromolecular inhibitor LiCL.Conclusions:Through decreasing ROS production, TGF-β1, FN and COL Ⅳ at both protein and mRNA levels, SFN acted as an antioxidant in HG-induced RMsC. In experimental rats, SFN upregulated phosphorylation of an active point (Ser9) in GSK-β to inhibit GSK-3β/Fyn pathway, therefore activated Nrf2 signaling pathway. This process is involved in increasing mRNA and nulear protein levels Fyn. |
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