从MicroRNAs调控M1/M2巨噬细胞极化探讨角膜移植排斥的发生机制和治疗方式

Corneal graft rejection（CGR) is a leading cause for the surgery failure of corneal transplantation. Currently, it is still a therapeutic challenge in ophthalmology. Macrophages (Mф) play key roles in CGR. Our preliminary studies show that macrophages (Mф) are the main inflammatory cells in the rejected tissues, with two subtypes of M1Mф and M2Mф; M1Mф promoted CGR, whereas M2Mф inhibited CGR. After searching in grafted corneas, we found that 18 microRNAs expression are changed (> 5 fold) in allograft group, compared with isograft group. We further found that mir-155 promoted M1Mф polarization, whereas mir-let-7c elicited M2Mф polarization. Furthermore, in vitro, we found that recombinant HMGB-1 increased the expression of TLR-4, Akt2 and mir-155, and decreased mir-let-7c expression. Using public website, we searched predicted targets of let-7c that could participate in this process and found that the transcriptional factor C-EBP-β and C-EBP-δ are among mir-155 and mir-let-7c targets, respectively. Thus, we speculate that HMGB-1→TLR-4→Akt2→mir-155/let-7c→C-EBP-β/δ pathway may elicit M1Mф polarization, and play crucial roles for CGR initiation and development, and targeting regulation of HMGB-1→TLR-4→Akt2→mir-155/let-7c→C-EBP-β/δ pathway may inhibit CGR. It is expected that this project further elucidates the molecular mechanism of CGR, and therefore develop a new and effective clinical approach for CGR treatment.

角膜移植排斥（CGR）是移植失败的主要原因，治疗棘手。前期研究发现：CGR 中巨噬细胞（Mф）为主要参与细胞，存在M1Mф和M2Mф两种亚型, M1Mф促进CGR，而M2Mф抑制CGR；芯片筛选发现，CGR中18种MicroRNAs的表达变化5倍以上，进一步体外筛选发现mir-155促进M1Mф极化，mir-let-7c促进M2Mф极化；体外应用HMGB-1可上调TLR-4，Akt2，mir-155，下调mir-let-7c的表达。专业网站预测C-EBPβ和C-EBPδ分别是mir-155和mir-let-7c的靶基因。推测CGR中HMGB-1→TLR-4→Akt2→mir-155/let-7c→C-EBPβ/δ通路诱导M1Mф极化，促进CGR。本课题拟通过选择性阻断、激活和基因敲除等靶向调控方法，验证我们的推测，进一步阐明CGR 发生机制，探索调控MicroRNAs治疗CGR的可行性。

角膜移植排斥是移植失败的主要原因，治疗困难。本项目揭示了角膜移植排斥的发生发展的新机制，明确了HMGB-1→TLR-4→Akt2→mir-155/let-7c→C-EBPβ/δ信号通路调控巨噬细胞极化，启动和促进移植排斥反应的发生。此外，HMGB1/TLR4/Caspase-8/NLRP3 inflammasome/HMGB1正反馈通路也被证明可以促进Caspase-8介导的巨噬细胞激活和募集，在角膜移植排斥中也起重要作用。应用HMGB1 抑制剂（甘草酸）、Caspase-8抑制剂，及临床药物富马酸二甲酯和氯化血红素能够治疗角膜移植排斥。本项目的成果为角膜移植排斥靶向治疗药物的研发提供了新的选择。