lncRNA260 siRNA Accelerates M2 Macrophage Polarization and Alleviates Oxidative Stress via Inhibiting IL28RA Gene Alternative Splicing

Abstract

The macrophage transformation of inflammatory M1 to anti-inflammatory M2 could be promoted by activating PI3K/AKT signaling pathway. In our previous study, it was found that downregulation of lncRNA260 could ameliorate hypoxic cardiomyocyte injury by regulating IL28RA through the activation of PI3K/AKT signaling pathways. It was suggested that lncRNA260 siRNA could promote the macrophages toward M2 polarization by regulating IL28RA. In this study, lncRNA260 siRNA was used to observe its effect on the polarization of murine bone marrow-derived macrophages (BMDM) and investigate its related mechanisms. lncRNA 260 specific siRNA were designed and synthesized which were transfected into murine BMDM with liposomes. The experiment was divided into three groups: Hypoxia group, Hypoxia+lncRNA 260-specific siRNA transfection group, and Normoxia group. The CD206-APC/CD11b-FITC or CD206-FITC/CD107b (Mac-3) double positive proportions were used to compare the M2 polarization proportions in the hypoxia process by using the immunofluorescence staining method. The p-AKT, Arg 1, PI3KCG, IL28RAV1, and IL28RAV2 protein expression changes were observed by using the western blot method. Compared with the Normoxia group, the M2 proportions were significantly decreased in the Hypoxia group (P < 0.05). Compared with the hypoxia group, the M2 proportions were significantly increased in the Hypoxia+lncRNA260 siRNA transfection group (P < 0.05). In the Hypoxia group, the ratios of Arg 1/β-Actin, p-AKT/β-Actin, PI3KCG/β-Actin, and IL28RAV1/β-Actin were significantly lower than those in the Normoxia group (P < 0.05). After transfection with lncRNA260 siRNA, the ratios of Arg1/β-Actin, p-AKT/β-Actin, PI3KCG/β-Actin, and IL28RAV1/β-Actin were significantly higher than those in the Hypoxia group (P < 0.05). Compared with the Normoxia group, the IL28RAV2/β-Actin in the Hypoxia group was significantly increased (P < 0.05). After transfection with lncRNA260 siRNA, the ratio of IL28RAV2/β-Actin was significantly decreased than that in the Hypoxia group (P < 0.05). lncRNA260 siRNA could promote the M2 polarization of the hypoxia macrophages by reducing the IL28RAV2 alternative splicing variant, which might be related to the activation of the JAK-STAT and PI3K/AKT signaling pathways. It will provide a new strategy for the anti-inflammation, antioxidative stress therapy, and cardiac remodeling after AMI.

Figure 1

The morphological characteristics of murine BMDM. Under the microscope, the murine BMDM were round or irregular, with pseudofoot protrusion, and the cell size was basically uniform. (a) The normal cell shape under 100x magnification. (b) The normal cell shape under 200x magnification.

Figure 2

The CD11b and CD206 proteins localization and relative levels by an immunostaining method in the murine BMDM hypoxia process. (a) The M2 macrophage changes during the hypoxia process by an immunofluorescence method. In the CD11b-FITC and CD206-APC double staining group, green was positive CD11b-FITC, red was positive CD206-APC, and blue was DAPI staining. The scale in each picture means 100 μm. (b) The M2 macrophage proportion changes during the hypoxia process. ^∗P < 0.05, compared with the Hypoxia group; ^△P < 0.05, compared with the normal control group.

Figure 3

The Mac-3 and CD206 proteins localization and relative levels by immunostaining in the murine BMDM hypoxia process. (a) The M2 macrophage changes during the hypoxia process by an immunofluorescence method. In Mac-3 and CD206-FITC double staining group, green was positive CD206-FITC, red was positive Mac-3, and blue was DAPI staining. The scale in each picture means 100 μm. (b) The M2 macrophage proportion changes during the hypoxia process. ^∗P < 0.05, compared with the Hypoxia group; ^△P < 0.05, compared with the normal control group.

Figure 4

The expression changes of Arg 1, p-AKT, and PI3KCG proteins detected by Western blot in the murine BMDM hypoxia process. (a) The western blot developing figure for the Arg 1, p-AKT, and PI3KCG proteins in the murine BMDM hypoxia process. (b) The ratio changes of Arg 1/β-Actin, p-AKT/β-Actin, and PI3KCG/β-Actin were detected by Western blot in the murine BMDM hypoxia process. ^∗P < 0.05, compared with the Hypoxia group; ^△P < 0.05, compared with the normal control group.

Figure 5

The expression changes of IL28RAV1 and IL28RAV2 proteins detected by Western blot in the murine BMDM hypoxia process. (a) The Western blot developing figure for the IL28RAV1 and IL28RAV2 proteins in the murine BMDM hypoxia process. Figure 5(b). The ratio changes of IL28RAV1/β-Actin and IL28RAV2/β-Actin were detected by Western blot in the murine BMDM hypoxia process. ^∗P < 0.05, compared with the Hypoxia group; ^△P < 0.05, compared with the normal control group.