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Objective:To observe the expressions of miR-183 and retinal dehydrogenase 11 (RDH11) in exosomes derived from bone marrow mesenchymal stem cells (BMSC), and to preliminarily explore their targeting relationship and their effects on retinal pigment epithelial (RPE) cells.Methods:BMSC from C57BL/6 (C57) mice were isolated and cultured, and BMSC-derived exosomes were identified. BMSC were divided into blank group, simulation blank control group (mimic-NC group), miR-183 simulation group (miR-183-mimic group). C57 mice and retinal degeneration 10 (rd10) mouse RPE cells were cultured with reference to literature methods. RPE cells from rd10 mice were transfected with BMSC exosomes and co-cultured and divided into control group, exosome group, mimic-NC-exosome group (mimic-NC-exo group), miR-183-mimic-exosome group (miR-183-mimic-exo group). The relative expression levels of miR-183, RDH11 mRNA and protein in C57 mice, rd10 mice and RPE cells in each group were detected by real-time quantitative polymerase chain reaction and western blotting. The targeting relationship between miR-183 and RDH11 was analyzed by bioinformatics website and dual luciferase reporter. Cell counting kit 8 was used to detect the effect of miR-183 on BMSC exosomes on RPE cell proliferation; in situ labeling end labeling method was used to detect RPE cells apoptosis. One-way ANOVA was used to compare multiple groups.Results:Compared with C57 mouse RPE cells, the relative expression of miR-183 in rd10 mouse RPE cells was down-regulated, and the relative expression of RDH11 mRNA was up-regulated, and the differences were statistically significant ( t=5.230, 8.548; P=0.006, 0.001). Compared with the blank group and the mimic-NC group, the relative expression of miR-183 mRNA in the exosomes of the miR-183-mimics group was significantly increased ( F=60.130, P <0.05 ). After 24 h of co-culture, exosomes entered RPE cells. Compared with the mimic-NC-exo group, the relative expression of miR-183 mRNA in RPE cells in the miR-183-mimic-exo group was significantly increased, the proliferation ability was enhanced ( t=7.311, P=0.002), and the number of apoptotic cells was decreased ( F=10.949, P=0.012), and the differences were statistically significant ( t=4.571, P=0.002). Bioinformatics website and dual-luciferase report confirmed that miR-183 has a targeting relationship with RDH11. Compared with the mimic-NC group, the relative expression of RDH11 mRNA and protein in the exosomes of the miR-183-mimic group was decreased, and the difference was statistically significant ( t=5.361, 6.591; P=0.006, 0.003). After co-culture, compared with the control group, there was no significant difference in the relative expression of RDH11 mRNA and protein in RPE cells in the exosome group ( t=0.169, 1.134; P=0.874, 0.320); The relative expressions of RDH11 mRNA and protein in RPE cells in -183-mimic-exo group were decreased, and the difference was statistically significant ( t=5.554, 5.546; P=0.005, 0.005). Conclusion:Up-regulation of BMSC-derived exosomal miR-183 promote the proliferation of RPE cells in vitro by targeting the expression of RDH11 and reduce the number of apoptosis.
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OBJECTIVE: Retinal dehydrogenase 1 (RALDH1) is a cytosolic enzyme which acts both as a source of retinoic acid (RA) and as a detoxification enzyme. RALDH1 has key functions in the midbrain dopaminergic system, which influences motivation, cognition, and social behavior. Since dopamine has been increasingly linked to autism spectrum disorder (ASD), we asked whether RALDH1 could contribute to the autistic phenotype. Therefore, we investigated for the first time the levels of RALDH1 in autistic patients. To further assess the detoxification function of RALDH1, we also explored 4-hydroxynonenal protein adducts (4-HNE PAs) and reduced glutathione (GSH) levels. Moreover, considering the effect of testosterone on RALDH1 expression, we measured the second to fourth digit ratio (2D:4D ratio) for both hands, which reflects exposure to prenatal testosterone. METHODS: Male patients with ASD (n=18; age, 62.9±4.3 months) and healthy controls (n=13; age, 78.1±4.9 months) were examined. Erythrocyte RALDH1, serum 4-HNE PAs and erythrocyte GSH levels were measured using colorimetric assays, and digit lengths were measured using digital calipers. RESULTS: We found significantly lower (−42.9%) RALDH1 levels in autistic patients as compared to controls (p=0.032). However, there was no difference in 4-HNE PAs levels (p=0.368), GSH levels (p=0.586), or 2D:4D ratios (p=0.246 in the left hand, p=0.584 in the right hand) between healthy controls and autistic subjects. CONCLUSION: We concluded that a subset of autistic patients had a low RALDH1 level. These results suggest that low RALDH1 levels could contribute to the autistic phenotype by reflecting a dopaminergic dysfunction.
Subject(s)
Humans , Male , Autism Spectrum Disorder , Autistic Disorder , Cognition , Cytosol , Dopamine , Erythrocytes , Glutathione , Hand , Mesencephalon , Motivation , Phenotype , Retinal Dehydrogenase , Retinaldehyde , Social Behavior , Testosterone , TretinoinABSTRACT
Objective To investigate the expression of retinoic acid receptors (RARs) subtypes RAR-α, RAR-β, RAR-γ and retinal dehydrogenases 1(Ra/dh1), Raldh2 in the mouse embryonic hematopoiesis, so as to discuss the role of different RAR subtypes in embryonic hematopoiesis. Methods The yolk sac (YS) at embryonic day 9. 5 (E9. 5), aortagonad- mesonephros (AGM) region at E10. 5 and E11. 5, fetal liver (FL) at E13. 5, E14. 5 and E17. 5 were harvested from the pregnant mice. The bonemarrow (BM) was also harvested from adult mice. The mRNA levels of RAR-α, RAR-β, RAR-γ, Raldh1 and Raldh2 at the above four different hematopoietic sites were assessed by quantitative reverse transcription polymerase chain reaction (RT PCR). Results The mRNA levels of RAR-α, RAR-β, RAR-γ, RaldhX and Raldh2 at E9. 5 YS, E11. 5 AGM regions and BM were significantly higher than those in other relevant hematopoietic tissues (P<0. 01). Compared with the E10. 5 AGM region, E11. 5 AGM region had significantly increasedmRNA levels of RAR-α, RAR-β, RAR-γ, Raldh1 and Raldh2 (P<0. 01), but the mRNA levels of these genes in FL at E13. 5-E17. 5 were not significantly different. Conclusion The expressions of RAR-α, RAR-β, RAR-γ, Raldh1 and Raldh2 in YS, AGM regions and BM are consistent with the situation of colony forming unit erythroid (CFU-E), suggesting that retinoic acid is closely related to the formation of hematopoiesis in the YS, AGM region and BM in mouse, but has less correlation with the FL in mouse.
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Objective To study the effect of retinal dehydrogenase type 2 inhibitor (4-diethylaminobenzaldehyde,DEAB) on embryonic CSrdiac develclpment of zebrafish model with retinoic acid(RA)deficiency. Methods Zebrafish embryos were treated with DEAB at various concentrations including 1×10~(-6),5×10~(-6),10×10~(-6),25×10~(-6)mol/L at 5,8 and 10.3 hours post fertilization,respectively.The effects of DEAB on the embryonic development were assessed under microscope.1×10~(-9)mol/L exogenous RA was then added to detect the antagonistic effect against DEAB.The abnormal cardiac phenotype,heart rate and ventricular systolic fraction were observed and analyzed between wild type and DEAB treated groups.The expression of specific cardiac gene, natriuretic peptide precursor A,was monitored by whole-mount in situ hybridization to demonstrate the effect of RA signaling on early cardiac development. Results The survival rate of zebrafish embryos declined with the increase of DEAB concentration at different developmental stage.The percentage of abnormal embryos reached 100% when DEAB over 5×10~(-6)mol/L.1×10~(-9) mol/L exogenous RA could eliminate the teratogenic effect of DEAB(≥5×10~(-6)mol/L).DEAB treated embryos presented abnormal cardiac phenotype,including tubular heart,incomplete D-loop,abnormal atrioventricular development,regurgitation,slow blood flow and weak heart beat.The difference of heart rate and ventrieular systolic fraction between wild type and RA deficiency embryos was of statistical significance(P<0.05).The natriuretic peptide precursor A expression remained in the ventricle,but reduced obviously in the atrium with RA signaling deficiency. Conclusions The effects of DEAB on the embryonic development are dose-dependent and time-dependent,and could be rescued by exogenous RA.RA signaling plays a critical role in several key stages of early cardiac development and natriuretie peptide precursor A expression.