多西环素抑制M2巨噬细胞诱导的角膜基质细胞纤维化

Doxycycline Inhibits M2 Macrophage-Induced Fibrosis of Corneal Stromal Cells

摘要: 目的：探究多西环素(DOXY)对M2巨噬细胞诱导的人角膜基质细胞纤维化的影响及PI3K/Akt信号通路的调控作用。方法：选取THP-1人白血病单核细胞系、人角膜基质细胞为实验材料，诱导THP-1细胞分化为M0、M2型巨噬细胞，构建人角膜基质细胞与巨噬细胞体外共培养模型，分为M0巨噬细胞共培养组、M2巨噬细胞共培养组、M2 + DOXY共培养组；同时构建条件培养基实验分组，开展功能挽救实验。采用RT-qPCR检测角膜基质细胞α-SMA、Col-3、PI3K/Akt相关基因表达，ELISA检测细胞上清羟脯氨酸(HYP)、TGF-β1含量，Western blot法检测p-Akt、Akt蛋白相对表达量。结果：与M0组相比，M2组角膜基质细胞α-SMA、Col-3、PI3K/Akt相关基因表达及细胞上清HYP、TGF-β1含量显著升高(P < 0.05)；与M2组相比，M2 + DOXY组及蛋白表达、细胞因子含量均显著降低(P < 0.05)。功能挽救实验显示，PI3K抑制剂与DOXY显著降低了p-Akt/Akt表达(P < 0.05)，而Akt激动剂可逆转DOXY的作用(P > 0.05)。结论：多西环素抑制了M2巨噬细胞诱导的角膜基质细胞纤维化。

Abstract: Objective: To investigate the effect of doxycycline (DOXY) on M2 macrophage-induced fibrosis of human corneal stromal cells and its regulatory role in the PI3K/Akt signaling pathway. Methods: THP-1 human acute monocytic leukemia cell line and human corneal stromal cells were selected as experimental materials. THP-1 cells were induced to differentiate into M0 and M2 macrophages, and an in vitro co-culture model of human corneal stromal cells and macrophages was established, which was divided into M0 macrophage co-culture group, M2 macrophage co-culture group and M2 + DOXY co-culture group. Meanwhile, experimental groups with conditioned medium were set up to conduct functional rescue experiments. RT-qPCR was used to detect the expression of α-SMA, Col-3 and PI3K/Akt pathway-related genes in corneal stromal cells; ELISA was performed to measure the contents of hydroxyproline (HYP) and TGF-β1 in the cell supernatant; Western blot (WB) was adopted to detect the relative expression levels of p-Akt and Akt proteins. Results: Compared with the M0 group, the expression levels of α-SMA, Col-3, PI3K/Akt-related genes in corneal stromal cells, as well as the contents of HYP and TGF-β1 in cell supernatants, were significantly increased in the M2 group (P < 0.05). In contrast, all the above indices were significantly decreased in the M2 + DOXY group compared with the M2 group (P < 0.05). Functional rescue experiments showed that both PI3K inhibitor and DOXY could reduce the expression ratio of p-Akt/Akt (P < 0.05), while Akt agonist could reverse the inhibitory effect of DOXY (P > 0.05). Conclusion: Doxycycline can inhibit M2 macrophage-induced fibrosis of human corneal stromal cells via regulating the PI3K/Akt signaling pathway.