靶向Bcl2L12调控实验性自身免疫性葡萄膜炎来源抗原特异性Th17细胞功能的体外研究OA

Objective To investigate the effect of Bcl2-like protein 12(Bcl2L12)on interphotoreceptor retinoid-binding protein 1-20(IRBP1-20)-specific T helper 17(Th17)cells in mice with experimental autoimmune uveitis(EAU).Methods Twelve female C57BL/6J mice(8-10 weeks old)were divided into an EAU group and a control group,with 6 mice in each group.Mice in the EAU group received an intraperitoneal injection of pertussis toxin,followed by subcutaneous injections of IRBP1-20 and complete Freund's adjuvant emulsion into the footpads and back to establish the model,while mice in the control group received no immunization.At day 13 post-immunization,model establishment was evaluated.T cells were isolated from the spleen and lymph nodes of EAU mice,and Bcl2L12 mRNA expression level was detected by quantita-tive real-time polymerase chain reaction(qRT-PCR).Effective small interfering RNAs targeting Bcl2L12 were screened out using the mouse T lymphoma cell line(EL4)cells.The selected siBcl2L12 or negative control(si-NC)was transfected into CD4+T cells in EAU mice,which were then co-cultured with γ-irradiated antigen-presenting cells.Interleukin(IL)-23 was added to induce Th17 polarization.The mRNA expression levels of Th17-associated genes[IL-17A,retinoid-related orphan receptor γt(RORγt),IL-1 receptor(IL-1R),IL-23 receptor(IL-23R),and granulocyte-macrophage colony-stimulating factor(GM-CSF)]were detected by qRT-PCR,IL-17 levels in the cell supernatant were measured by ELISA,and the proportion of Th17 cells was analyzed by flow cytometry.Results At day 13 post-immunization,EAU mice exhibited inflammatory changes in the fundus,confirming successful model establishment.Compared with the control group,Bcl2L12 mRNA expres-sion was significantly upregulated in CD4+T cells in EAU mice(P<0.01).EL4 cell validation results showed that siBcl2L12-2 exhibited the highest knockdown efficiency(P<0.01)and was used for subsequent experiments.In the co-cul-ture system,compared with the Blank group and si-NC group,the si-Bcl2L12 group showed a significantly reduced propor-tion of Th17 cells(P<0.05),significantly decreased IL-17 levels in the supernatant(P<0.05),and markedly downregulat-ed mRNA expression levels of IL-17A,ROR-γt,IL-1R,IL-23R,and GM-CSF(all P<0.01).Conclusion Knockdown of Bcl2L12 in T cells inhibits the polarization of IRBP1-20-specific Th17 cells and the expression of related inflammatory factors in EAU mice,suggesting that Bcl2L12 positively regulates IRBP1-20-specific Th17 cell responses.