Purpose: This study aimed to explore the role of the protein kinase A (PKA) pathway in proliferative vitreoretinopathy (PVR) and the effect of the PKA inhibitor H89 on experimental PVR. Methods: Epiretinal membranes (ERMs) were acquired from PVR patients and analyzed by frozen-section immunofluorescence. An in vivo model was developed by intravitreal injecting rat eyes with ARPE-19 cells and platelet-rich plasma, and changes in eye structures and vision function were observed. An in vitro epithelial-mesenchymal transition (EMT) cell model was established by stimulating ARPE-19 cells with transforming growth factor (TGF)-β. Alterations in EMT-related genes and cell function were detected. Mechanistically, PKA activation and activity were explored to assess the relationship between TGF-β1 stimulation and the PKA pathway. The effect of H89 on the TGF-β-Smad2/3 pathway was detected. RNA sequencing was used to analyze gene expression profile changes after H89 treatment. Results: PKA was activated in human PVR membranes. In vivo, H89 treatment protected against structural changes in the retina and prevented decreases in electroretinogram b-wave amplitudes. In vitro, H89 treatment inhibited EMT-related gene alterations and partially reversed the functions of the cells. TGF-β-induced PKA activation was blocked by H89 pretreatment. H89 did not affect the phosphorylation or nuclear translocation of regulatory Smad2/3 but increased the expression of inhibitory Smad6. Conclusions: PKA pathway activation is involved in PVR pathogenesis, and the PKA inhibitor H89 can effectively inhibit PVR, both in vivo and in vitro. Furthermore, the protective effect of H89 is related to an increase in inhibitory Smad6.
Epithelial-mesenchymal transition (EMT) of the retinal pigment epithelial (RPE) cells is a critical step in the pathogenesis of proliferative vitreoretinopathy (PVR). Some microRNAs (miRNAs) participate in regulating RPE cell EMT as post-transcriptional regulators. However, the function of miR-194 in RPE cell EMT remains elusive. Here, the role of miR-194 in PVR was investigated.Retinal layers were obtained using laser capture microdissection (LCM). Gene expression at the mRNA and protein level in the tissues and cells was examined using quantitative reverse transcription (RT)-polymerase chain reaction and Western blotting, respectively. The related protein expression was observed by immunostaining. The effect of miR-194 on RPE cell EMT was examined by gel contraction, wound healing, and cell migration assays. RNAseq was performed in ARPE-19 with transfection of pSuper-scramble and pSuper-miR-194. The target gene of miR-194 was identified and confirmed via bioinformatics analysis and dual-luciferase reporter assay. ARPE-19 (adult retinal pigment epithelium-19) cells were treated with transforming growth factor (TGF)-β1 in the same fashion as the in vitro RPE cell EMT model. A PVR rat model was prepared by intravitreous injection of ARPE-19 cells with plasma-rich platelets.miR-194 was preferentially expressed in the RPE cell layer compared with the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer in rat retina. RNAseq analysis indicated that miR-194 overexpression was involved in RPE cell processes, including phagocytosis, ECM-receptor interaction, cell adhesion molecules, and focal adhesion. miR-194 overexpression significantly inhibited the TGF-β1-induced EMT phenotype of RPE cells in vitro. Zinc finger E-box binding homeobox 1 (ZEB1), a key transcription factor in EMT, was confirmed as the direct functional target of miR-194. Knockdown of ZEB1 attenuated TGF-β1-induced α-smooth muscle actin expression in ARPE-19 cells, and overexpression of miR-194 could significantly reduce the expression of some genes which were up-regulated by ZEB1. Exogenous miR-194 administration in vivo effectively suppressed PVR in the rat model, both functionally and structurally.Our findings demonstrate for the first time that miR-194 suppresses RPE cell EMT by functionally targeting ZEB1. The clinical application of miR-194 in patients with PVR merits further investigation.
Objective
To study the expression of SGK1 in T lymphocytes from pediatric asthma, and the effect of SGK1 on the differentiation of T cells, also to explore the function of SGK1 regulating the differentiation of T subset in pediatric asthma.
Methods
Twenty-eight children with asthma were recruited in Xi′an children′s hospital and divided into moderate group and severe group according to diagnostic guideline of asthma.The serum levels of IL-4, IL-13 and IL-17A were analyzed by ELISA.The CD4+ T cells from PBMC and naive T cells were selected using magnetic beads.Naive T cells were differentiated in vitro under cytokines.SGK1 expression were analyzed with Real-time PCR.The ability of Th2 and Th17 on secreting IL-4 and IL-17A were detected after SGK1 was inhibited by siRNA.In vivo, shRNA-SGK1 Naive T cells were transferred into the mice asthma models by intravenous injection.The airway inflammation were observed in shRNA-SGK1 Naive T models.
Results
Compared with healthy children, the serum levels of IL-4、IL-13 and IL-17A increased significantly in the children with asthma.Importantly, the levels of these three cytokines were much higher with the development of asthma.SGK1 were up-regulated remarkably in CD4+ T cells from the children with asthma and were positively correlated with IL-13 and IL-17A.Besides, SGK1 expression increased in the differentiated Th2 and Th17 in vitro, but had no change in the differentiated Th1.The levels of IL-4 and IL-17A associated with Th2 and Th17 decreased after SGK1 was inhibited by siRNA.Similarly, In vivo, the serum levels of IL-13 and IL-17A and airway inflammation were reduced in shRNA-SGK1 Naive T models.
Conclusion
The over-expression of SGK1 in pediatric asthma enhances the asthma progress by promoting the differentiation of T subsets.
Key words:
SGK1; T lymphocyte; Differentiation; Cytokine; Asthma
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