| Objective:To investigate the correlation between miR-30b-5p,the Notch signaling pathway activation,CD4~+T cell differentiation balance,and the progression of experimental autoimmune uveitis(EAU),and to explore the mechanism of intravitreal injection of miR-30b-5p lentivirus to orchestrate the Notch signaling activation,thereby inhibiting Th1 and Th17 differentiation and promoting Th1/Th2 and Th17/Treg ratio balance in the treatment of experimental autoimmune uveitis.In addition,we further explored the molecular mechanism of prednisone acetate(PA)in the treatment of uveitis through inhibiting the activation of Notch signal pathway and regulating the differentiation of Th1 and Th17 cells to promote the balance of Th1/Th2 and Th17/Treg differentiation in EAU rats.Totally,our study provides a theoretical basis for the clinical application using small RNA to treat uveitis.Methods:The current study consists of two parts.Part I:Intravitreal injection of miR-30b-5p modulates the balance of CD4~+T cell differentiation in experimental autoimmune uveitis via orchestrating the Notch signaling pathway.First,a dual luciferase assay was constructed to verify the regulatory effect of miR-30b-5p on Notch1 and Dll4gene expression.Lewis rats were randomly divided into normal control(NC)group,model control(EAU)group,miR-30b-5p-carrying lentivirus injection group(miR-30b-5p group),DAPT inhibitor group and miR-30b-5p vector-carrying lentivirus injection group(miR-30b-5p-N group.Except for the normal control group,all the rats were induced uveitis.At 0 d after immunization,8μL of miR-30b-5p lentivirus and miR-30b-5p vector-carrying lentivirus were injected into the vitreous cavity of both eyes in the miR-30b-5p and miR-30b-5p-N groups,respectively;meanwhile,50μL of DAPT(0.016μg/μL)was injected intraperitoneally once daily in the DAPT inhibitor group.On day 12post-immunization,the inflammatory manifestations of the anterior segment in each group were observed by the Genesis-D fundus camera and retinal imaging system,and clinical scores were recorded.The changes in organelles such as mitochondria and inflammatory cell infiltration in the ciliary muscle and retina of each group were observed by transmission electron micrographs.Then,the levels of Notch1 and Dll4 proteins in the iris,ciliary muscle and retina of each group were detected by immuno-fluorescence assay(IFA)and immunohistochemistry(IHC).In addition,the protein expression levels of Notch1 and Dll4 in the spleen,lymph nodes,and eyes in each group were determined using Western blot.The frequencies of Th1,Th2,Th17 and Treg cells were detected by flow cytometry and variations in the ratios of Th1/Th2 and Th17/Treg were assessed.The protein expression levels of IFN-γ,IL-4,IL-10 and IL-17 were determined by ELISA.Part Ⅱ:The impact of PA on Th1/Th2 and Th17/Treg homeostasis in EAU rats through the Notch signaling pathway.Lewis rats were randomly divided into normal control(NC)group,a model control(EAU)group,and PA intervention group.After modeling in the EAU and PA groups,PA gavage was given daily in the PA group and the EAU model group was given the same amount of normal saline gavage.On days 9,12,and15 after immunization,the inflammatory manifestations of the anterior segment were monitored daily with the Genesis-D fundus camera,and the clinical scores were recorded;On day 12 post-immunization,histopathological changes in the iris and ciliary muscle were observed.The expression levels of Notch1,Notch2,Dll3,Dll4,and Rbpj m RNAs were detected by real-time fluorescence quantitative PCR(Q-PCR)after isolation of spleen,lymph nodes,and eye tissues of the three groups,and ELISA was performed to detect Notch1,Notch2,Dll3,Dll4,Rbpj,IFN-γ,IL-4,IL-17,and IL-10 proteins,and the frequencies of Th1,Th2,Th17,and Treg cells in each tissue were detected by flow cytometry,and the variations in the ratios of Th1/Th2 and Th17/Treg were assessed.In addition,we have also carried out molecular docking research on molecules from the Notch signaling pathway such as Notch1,Notch2,Dll4,and Rbpj on PA to analyze the interaction between PA and Notch signaling pathway-related molecules to predict their binding mode and affinity.Results:1.Notch1 and Dll4 were identified as miR-30b-5p-regulated target genes according to dual luciferase assay.On day 12 post-immunization,the ocular inflammation was severe and clinical scores were markedly increased in the EAU group compared to the NC group.Compared to the NC group,the retinal ultrastructure of the EAU group showed fewer mitochondria,fewer vacuoles,broken membranes,and more nuclei with deformation.In contrast,Compared to the EAU group,the miR-30b-5p,and DAPT inhibitor groups did not show significant vacuolation,membrane breakage,or nuclear consolidation.In addition,the ciliary muscle in the EAU group showed a large number of mixed neutrophil(NEU)and macrophage(M(?))inflammatory cell infiltration compared to the NC group,and the inflammatory cell infiltration was markedly reduced in the miR-30b-5p and DAPT inhibitor groups compared to the EAU group.Immunofluorescence and immunohistochemistry results showed that the protein levels of Notch1 and Dll4 were markedly increased in the iris,ciliary muscle and retina in the EAU group compared to the NC group,and that the miR-30b-5p and DAPT inhibitor groups markedly reduced the protein levels of Notch1 and Dll4 compared to the EAU group(both P<0.05).WB results showed that the protein levels of Notch1 and Dll4 were significantly increased in the spleen,lymph node,and eyes compared to the NC group,while Notch1and Dll4 proteins in the miR-30b-5p and DAPT inhibitor groups were markedly decreased compared to the EAU group(all P<0.05).Flow cytometry analysis showed that Th1 and Th17 were elevated and Th1/Th2 and Th17/Treg ratios were markedly higher(in disequilibrium)in the EAU group compared to the NC group(both P<0.05).Compared to the EAU group,the levels of Th1 and Th17 cells were significantly decreased in the miR-30b-5p and DAPT groups,while the levels of Th2 and Treg cells were significantly increased,and the Th1/Th2 and Th17/Treg cell ratios were balanced(both P<0.05).ELISA results showed that IFN-γand IL-17 protein levels were elevated in spleen,lymph node and eyes in the miR-30b-5p and DAPT groups compared with the NC group,and IL-10and IL-4 protein levels were remarkably increased compared with the EAU group(all P<0.05).In this study,there was no remarkably difference between the data of both the EAU group and the miR-30b-5p-N group(P>0.05).Conclusions:1.Activation of the Notch signaling pathway by elevated Notch1 and Dll4 levels regulates the differentiation of CD4~+T cells to Th1 and Th17 cells,leading to a severe imbalance of Th1/Th2 and Th17/Treg ratios,which in turn promotes the development of uveitis.miR-30b-5p negatively regulates the expression of Notch1 and Dll4 in EAU rats,significantly inhibiting the activation of the Notch signaling pathway and reducing the differentiation of Th cells to Th1 and Th17 cells.Meanwhile,miR-30b-5p significantly inhibited the activation of the Notch signaling pathway,thereby reducing the differentiation of Th cells to Th1 and Th17 cells,increasing the levels of Th2 and Treg,promoting the balance of Th cell ratio and exerting therapeutic effects on EAU.2.PA can restore the balance of Th1/Th2 and Th17/Treg ratios in EAU rats by down-regulating the expression levels of Notch 1,Notch 2,Dll4,and Rbpj molecules in the spleen,lymph nodes,and eye tissues,up-regulating the expression levels of Dll3molecules,inhibiting the activation of the Notch signaling pathway,decreasing the differentiation of Th1 and Th17,and restoring the balance of Th1/Th2 and Th17/Treg ratios in EAU rats,and significantly increasing the levels of anti-inflammatory molecules IL-4 and IL-10 to achieve the restoration of the balance between ocular and systemic immune microenvironment to achieve the goal of treating uveitis. |
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