| BackgroundProliferative vitreoretinopathy(PVR) is not only the main reason of complications after reattachment surgery of rhegmatogenous retinal detachment(RRD), but also the main reason for the PVR surgical failure. PVR series of pathological processes and process-related cellular activities, including cell depolarization, migration, adhesion, proliferation-related, in addition to the secretion of collagen and other extracellular matrix(extracellular matrix, ECM) close links; After the above process, front and back surfaces of the retina and vitreous in proliferative form a film having the ability to shrink, resulting in traction retinal detachment(tractional retinal detachment, TRD). In recent decades the study, the occurrence and development mechanism PVR has been the focus and the content of one of the difficulties fundus study. Retinal pigment epithelium(retinal pigment epithelial, RPE) cells in peripheral retinal photoreceptor cell layer, a layer structure consisting of rows of a single layer of hexagonal cells. The current study has confirmed that, RPE cells is essential for the occurrence and development of PVR. Epithelial- mesenchymal transition(Epithelial-mesenchymal transition, EMT, EMT) for the development and progression of PVR’s an important part, refers to an epithelioid phenotype cells lose polarity and enhanced activity in the intercellular substance between free to move and exhibit transformation process fiber-like phenotype. EMT is usually divided into three subtypes, one is to make gastrulation, epithelial cells of the body in the original action by EMT involved in a variety of cell biological embryonic development and organ formation; the other is a second endothelial or epithelial transition for the organization of fibroblasts, which play an important role in the body’s wound healing and organ fibrosis; the third is the loss of polarity of epithelial-derived cells, which translates to having the ability to invade cells.Transforming growth factor β activated kinase-1(transforming growth factor-βactivated kinase-1, TAK1) as an important member of MAP3 K important family belonging tryptophan / threonine protein kinases, whose activity is regulated by TGF-β and BMP’s. In living organisms, TAK1 activation via P38, JNK and NF-κB pathway regulatory role in a series of cell-specific transcription factor, which affects cell survival, differentiation and other physiological and pathological processes, while also having a regulatory role for inflammation. Since TAK1 functional diversity, so TAK1 in inflammatory diseases has become a major target kinases. In addition, TAK1 also involved in activation MKK3 / 4-P38 / JNK-AP-1 and IKK-NF-κB and other important signal transduction pathways, cell survival, differentiation and inflammatory response has an important regulatory effect. Transforming growth factor-β(Transforming growth factor-β, TGF-β) is a newly discovered class of cell growth and differentiation has processes play a regulatory role multifunctional cytokine, there are a large number of studies have confirmed, TGF-β is now it is known to have the most important cytokines promote tissue fibrosis progression, which is highly expressed in the blood like too renal fibrosis, liver fibrosis and lung fibrosis. TGF-β can be derived mesenchymal cells play a stimulating role, including connective tissue cells, endothelial cells and smooth muscle cells, while still in development and progression of PVR plays an important role.ObjectiveOur aim is to clear qualitative effect between TAK1 in human RPE cells to TGF-β1-induced epithelial cells through experimental studies while exploring the effects between TAK1 inhibitor LYTAK1 on RPE cell proliferation and its impact on quality of human retinal pigment epithelial cells of the mechanism of action is intended to provide an experimental basis for the treatment of PVR.MethodsWe were detected by ELISA in June 2014- December 2104 period at the First People hospital of Yunnan Province,PVR 30 cases of patients, 20 cases of IMH patients and the same period in 15 normal volunteers or vitreous humor peripheral blood expression levels of TGF-β1 and TAK1. All patients were aged between 45 to 65 years old. Wherein, PVR patients in the male 18, female 12 cases; IMH patients, 8 cases of male, female 12 cases; 9 healthy male volunteers, female 6 cases.We use 0.01 ng / mL TGF-β1 effects on human RPE cell line ARPE-19, for 24 h after fluorescence quantitative PCR method to detect cells TAK1 mRNA expression using; were treated with different concentrations of LYTAK1(0 μM, 1μM, 10 μM, 25 μM, 50 μM) acting on the 0.01 ng / mL TGF-β1-induced post-ARPE-19 cells, western blot assay cells expressing TAK1 protein; invasion of Transwell cell assay cells; flow cytometry( Annexin V-FITC / PI double staining) to detect cell apoptosis; cell scratch migration assay cells.We used quantitative PCR were detected in the control group, the expression of TGF-β1 + DMSO group and TGF-β1 + LYTAK1 group ARPE-19 cells, a-SMA and fibronectin mRNA’s. Using western blot were detected in the control group, TGF-β1 + DMSO group and TGF-β1 + LYTAK1 various concentrations(0 μM, 1μM, 10 μM, 25 μM, 50 μM) ARPE-19 cells, a-SMA, fibronectin, p-Smad2, p-Smad3, IKKα, NF-κBp65 protein.ResultsThere was a significant difference(t = 3.114, P = 0.035 <0.05) concentrations PVR and IMH TAK1 between the two groups, namely, the concentration of PVR vitreous TAK1 patients was significantly higher than IMH group; on the PVR between the two groups and the normal group TAK1 whether there are differences between the concentration of a comparative analysis, the results showed that there was a significant difference(t = 3.156, P = 0.034 <0.05), that is, the concentration of serum PVR TAK1 IMH group was significantly higher than the concentration between the two groups of TAK1. These results indicate that patients with PVR vitreous TAK1 and serum levels were significantly higher in patients with IMH and healthy people, suggesting that it might happen and development of PVR has a role in promoting. PVR patients, IMH patients and healthy volunteers vitreous humor and / or the presence of concentrations of TGF-β1 in serum significant difference(t = 2.319, P = 0.033 <0.05), that is, the concentration of PVR in patients with vitreous TGF-β1 was significantly higher than IMH group. PVR and normal for both groups between TGF-β1 concentration of whether there are differences between using the t test was used for comparative analysis showed that TGF-β1 present in a concentration of significant differences between the two groups(t = 3.312, P = 0.037 <0.05), that is, PVR serum concentration of TGF-β1 was significantly higher than IMH group. These results indicate that patients with PVR vitreous and TGF-β1 in serum were significantly higher in patients with IMH and healthy people, suggesting that the occurrence and development of PVR related.Fluorescence quantitative PCR results show that, TGF-β1-treated group ARPE-19 cells TAK1 mRNA expression was significantly higher, TGF-β1 can significantly increase the expression of TAK1 protein, and when LYTAK1 with different concentrations of cells, TAK1 protein expression levels in different degrees of reduced(P <0.05), which, when LYTAK1 concentration of 25 μM when the expression of TAK1 protein minimum. CCK-8 assay showed that when LYTAK1 role during 24 h, cell proliferation activity of each concentration group showed no significant change(P> 0.05); and when LYTAK1 for 48 h and after 72 h, with the effect of increasing concentrations of LYTAK1, when the concentration of 50 μM LYTAK1 effect on proliferation of ARPE-19 cells inhibition rate highest. ARPE-19 cell proliferation activity decreased, indicating LYTAK1 of ARPE-19 cells in a dose-dependent activity. Test results Transwell cell method shows that, LYTAK1 each concentration group and control group compared to the View average invasive cell number was significantly decreased, and with LYTAK1 concentration increased its number of invasive cells gradually decreased, suggesting LYTAK1 invasiveness ARPE-19 cells inhibit effect and dose-dependent manner. Flow cytometry results showed that with LYTAK1 effect concentration increased apoptosis rate ARPE-19 cells along with the rise, suggesting LYTAK1 ARPE-19 cells induced apoptosis in a dose-dependent manner. Experimental results show that cell scratch, 48 h after the migration distance of each concentration LYTAK1 action group ARPE-19 cells was significantly shorter than the control group(P <0.05); and with the increase of LYTAK1 concentration, cell migration distance is gradually decreased, suggesting LYTAK1 ARPE-19 inhibition of cell migration in a dose-dependent manner.Compared with the control group, TGF-β1 + DMSO group expression and TGF-β1 + LYTAK1 group a-SMA and fibronectin mRNA were significantly increased; and TGF-β1 + DMSO group compared to, TGF-β1 + LYTAK1 group a- SMA and fibronectin mRNA expression was significantly reduced. Western blot analysis showed that the detection result, TGF-β1 + DMSO group cells p-Smad2 p-Smad3 IKKα expression of a-SMA, fibronectin,,,, NF-κBp65 protein were significantly higher. TGF-β1 + LYTAK1 each concentration of cells in a-SMA, p-Smad2 IKKα expression of fibronectin,,, NF-κBp65 protein with increasing LYTAK1 concentration decreased in a dose-dependent manner, while LYTAK1 expression of p-Smad3 protein no significant effect.ConclusionsSerum and PVR vitreous TAK1 expression and TGF-β1 was significantly higherTAK1 and TGF-β1 may be associated with the occurrence and development related to PVR;TGF-β1 can significantly increase ARPE-19 cells, the expression of TAK1;LYTAK1 significantly inhibited in ARPE-19 cells expressing TAK1, and dose-dependent manner;TGF-β1 able to upregulate the expression of ARPE-19 cells TAK1, a-SMA, fibronectin, p-Smad2, p-Smad3, IKKα, NF-κBp65 to promote the development and progression of EMT;TAK1 by inhibiting TAK1, the expression of a-SMA, fibronectin, p-Smad2, IKKα, NF-κBp65 play inhibition of EMT and Development;LYTAK1 capable of proliferation of ARPE-19 cells, apoptosis, invasion and migration was significantly inhibited in a dose-dependent manner. |
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