Department of Ophthalmology, Fujian Medical University Union Hospital, Fu Zhou, China.
Invest Ophthalmol Vis Sci. 2022 Sep 1;63(10):20. doi: 10.1167/iovs.63.10.20.
Our previous investigations revealed a significant role of methyltransferase-like 3 (METTL3)-mediated N6-methyladenosine (m6A) modification in the development of corneal inflammation in Fusarium infection, but the exact mechanism is unknown. Therefore, this research aimed to explore how METTL3 affects the inflammatory process of fungal keratitis (FK) in mice.
We established in vitro and in vivo models by inoculating mice and primary corneal stromal cells with F. solani. METTL3 expression was confirmed by real-time quantitative polymerase chain reaction, immunofluorescence, and western blotting. After that, siRNAMETTL3 and AAV-sh-METTL3 were transfected into cells and mice to explore the role of METTL3 in the PI3K/AKT signaling pathway and inflammation. PI3K, p-PI3K, AKT, and p-AKT expression was analyzed by western blotting. Viability of corneal stromal cells was measured using a Cell Counting Kit-8 (CCK-8). Additionally, we detected interleukin (IL)-6, IL-1β, and tumor necrosis factor alpha (TNF-α) levels in corneal tissues and analyzed the role of METTL3 in inflammation in FK using slit-lamp biomicroscopy and hematoxylin and eosin staining.
Here, our results show that METTL3 increased in mouse FK, and the expression of p-PI3K and p-AKT decreased when METTL3 was downregulated. We also found that knockdown of METTL3 expression attenuated the inflammatory response and decreased TNF-α, IL-1β, and IL-6 expression in corneal-infected mice. Furthermore, inhibition of the PI3K/AKT pathway attenuated the inflammatory response of FK, decreased the expression of the above inflammatory factors, and enhanced the viability of corneal stromal cells.
Based on the study results, METTL3 downregulation attenuates Fusarium-induced corneal inflammation via the PI3K/AKT signaling pathway.
我们之前的研究表明，甲基转移酶样 3（METTL3）介导的 N6-甲基腺苷（m6A）修饰在镰刀菌感染导致的角膜炎症发展中起重要作用，但具体机制尚不清楚。因此，本研究旨在探讨 METTL3 如何影响真菌性角膜炎（FK）小鼠模型中的炎症过程。
我们通过接种镰刀菌（F. solani）建立了体外和体内模型，并通过实时定量聚合酶链反应、免疫荧光和 Western blot 验证 METTL3 的表达。然后，将 siRNAMETTL3 和 AAV-sh-METTL3 转染到细胞和小鼠中，以研究 METTL3 在 PI3K/AKT 信号通路和炎症中的作用。通过 Western blot 分析 PI3K、p-PI3K、AKT 和 p-AKT 的表达。使用细胞计数试剂盒-8（CCK-8）测量角膜基质细胞的活力。此外，我们通过裂隙灯生物显微镜和苏木精-伊红染色检测角膜组织中白细胞介素（IL）-6、IL-1β 和肿瘤坏死因子α（TNF-α）的水平，并分析 FK 中 METTL3 对炎症的作用。
结果显示，METTL3 在小鼠 FK 中表达增加，下调 METTL3 表达后 p-PI3K 和 p-AKT 的表达降低。我们还发现，下调 METTL3 表达可减轻角膜感染小鼠的炎症反应，降低 TNF-α、IL-1β 和 IL-6 的表达。此外，抑制 PI3K/AKT 通路可减轻 FK 的炎症反应，降低上述炎症因子的表达，并增强角膜基质细胞的活力。
基于研究结果，METTL3 通过 PI3K/AKT 信号通路下调可减轻镰刀菌诱导的角膜炎症。
