ABSTRACT
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:To observe the expression of miR-142-5p and forkhead transcription protein O subgroup 3 (FOXO3) in CD4 + T cells of experimental autoimmune uveitis (EAU) model rats, and preliminarily explore the targeting relationship between the two and the effect on EAU impact. Methods:Ten Lewis rats were randomly divided into model group and control group. Rats in the model group wree induced an EAU animal model by adoptive immunization. Twenty days after immunization, CD4 + T cells were extracted from the eyeballs and draining lymph nodes of rats in the control group and model group, and divided into control group, model group, mimic-negative control (NC) group, miR-142-5p-mimic group, and small interference (si)-NC group, si-FOXO3 group for in vitro experiments. The miR-142-5p-mimic group and si-FOXO3 group were transfected with miR-142-5p-mimic and si-FOXO3, respectively. Twenty-five Lewis rats were randomly divided into model group, mimic-NC transfected group, miR-142-5p-mimic transfected group, si-NC transfected group, and si-FOXO3 transfected group. The above-mentioned in vitro experimental groups were injected with cells respectively. Slit lamp microscopy and EAU score were performed on 4, 8, 12, 16, 20 days after immunization; on 20 days after immunization, hematoxylin-eosin staining was performed for histopathological grading. Real-time fluorescence quantitative polymerase chain reaction was used to detect the relative expression of miR-142- 5p and FOXO3 mRNA in CD4 + T cells and eye tissues of rats in each group, and helper T cell 17 (Th17) marker interleukin (IL)-17, IL-22, retinoic acid-related orphan receptor gamma (ROR gamma) relative expression level in the supernatant. Bioinformatics website and dual luciferase was used to predict the targeting relationship between miR-142-5p and FOXO3. One-way analysis of variance or t test was used for comparison between groups. Results:All rats in the model group showed symptoms of EAU to varying degrees, and the symptoms became worse with time. Compared with the control group, the relative expression of miR-142-5p mRNA in CD4 + T cells of the model group increased, and the relative expression of FOXO3 mRNA decreased. The differences were statistically significant ( t=7.374, 10.423; P=0.002, 0.001). Compared with the mimic-NC group, the relative expression of miR-142-5p mRNA in the CD4 + T cells of the miR-142-5p-mimic group increased, and the difference was statistically significant ( t=6.540, P=0.003). Compared with the model group, mimic-NC group, and si-NC group, the relative expression of IL-17, IL-22, and RORγ mRNA in CD4 + T cells in the miR- 142-5p-mimic group and si-FOCO3 group increased significantly. The difference was statistically significant ( F=26.110, 6.292, 5.269, 55.660, 10.490, 11.430; P<0.05). Compared with the mimic-NC transfected group, the relative expression of miR-142-5p mRNA in the ocular tissues of the miR-142-5p-mimic transfected rats increased significantly, and the difference was statistically significant ( t=6.690, P<0.05). Compared with the transfected si-NC group, the relative expression of FOXO3 mRNA in the eye tissue of the transfected si-FOXO3 group was significantly decreased, and the difference was statistically significant ( t=17.751, P<0.05). Rats in the mimic-NC transfected group, miR-142-5p-mimic transfected group, si-NC transfected group, and si-FOXO3 transfected group prolonged with time after immunization, and the EAU scores showed an upward trend. The EAU score and histopathological grade of rats in the miR-142-5p-mimic transfected group were higher than those in the mimic-NC transfected group, and the difference was statistically significant ( t=5.633, 6.286; P<0.05). The EAU score and histopathological grade of the rats in the transfected si-FOXO3 group were higher than those in the transfected si-NC group, and the difference was statistically significant ( t=6.852, 6.635; P<0.05). FOXO3 has a targeting relationship with miR-142-5p. Conclusions:In EAU rat CD4 + T cells, the expression of miR-142-5p is up-regulated, while the expression of FOXO3 is down-regulated. miR-142-5p targets the expression of FOXO3 to promote the development of Th17 cell-related inflammatory factors.
ABSTRACT
Objective To explore the clinical efficacy and safety of cyclosporin A combined with glucocorti-costeroid in the treatment of juvenile uveitis.Methods 66 juvenile uveitis patients(93 eyes)were selected.Accord-ing to the conditions,the patients were treated by oral administration of cyclosporin A,2-5mg·kg -1 ·d -1 ,twice per day,combined with prednisone,0.5-1mg·kg -1 ·d -1 ,taken in the morning.After 4-6 months treatment,the inflam-mation control and visual acuity improvement were observed,and before and after treatment liver and kidney function, blood glucose,blood pressure,white blood cell counts were checked.Adverse reactions in patients were recorded with regular follow-up.Results After 4-6 months treatment,heal inflammation 78 eyes,accounted for 83.87%;improved 9 eyes,accounted for 9.68%;no respond in 6 eyes,accounted for 6.45%;the total effective rate was 93.55%.Visual acuity was obviously improved in 50 eyes,improved in 27 eyes and no response in 16 eyes;the total effective rate was 82.79%;56 eyes were removed from residual standard.In the stage of therapy,6 patients had liver and kidney dys-function,6 patients had blood abnormalities,and 3 patients had low blood sugar,all were controlled by symptomatic treatment in the normal range,and no systemic progression.Conclusion Cyclosporin A combined with glucocortico-steroid applied in juvenile uveitis improved the results with obvious clinical effect and fewer systemic side effect.