Fig 1: Regulation role of IL10-PNP on macrophage polarization in an inflammatory environment simulated in vitro(A) Schematic diagram of the coculture system.(B) Flow cytometry detected the polarization of macrophages cultured alone and cocultured with platelets + PBS, platelets + IL10, platelets + IL10-NP and platelets + IL10-PNP for 24 h.(C) M1 macrophages and (D) M2 macrophages ratio in different groups. Levels of TNF-a (E), IL-1ß (F), and IL-6 (G) in the cell supernatant were measured with ELISA. Data are presented as mean ± SD, ns not significant, *p <0 .05, **p <0 .01, ***p < 0.001, n = 3.
Fig 2: Regulation role of IL10-PNP on macrophage polarization in vivo(A) Immunofluorescence images of F4/80 (red) and CD86 (green) double-immunostaining in the sham group and injured carotid artery with different treatments. Nuclei were stained with DAPI (blue).(B) Immunofluorescence images of F4/80 (red) and CD163 (green) double-immunostaining in the sham group and injured carotid artery with different treatments. Nuclei were stained with DAPI (blue).(C) The proportion of macrophages as a percentage of all cells.(D) The proportion of CD86+ macrophages as a percentage of macrophages.(E) The proportion of CD163+ macrophages as a percentage of macrophages. Data are presented as mean ± SD, ns not significant, *p <0 .05, **p <0 .01, ***p < 0.001, n = 6. Scale bar = 20µm.
Fig 3: Regulation role of IL10-PNP on endothelial cells functions in an inflammatory environment simulated by platelets + macrophages coculture system in vitro(A) The impact of IL10-PNP on HUVECs function by tube formation assay.(B) Quantitative analysis of tube formation assay in different groups.(C) The impact of IL10-PNP on HUVECs repair function by wound healing assay.(D) The relative gap area of wound healing assay in different groups, values were normalized based on the value of the blank group. n = 3. Data are presented as mean ± SD, ns not significant, *p <0 .05, **p <0 .01, ***p <0 .001. Scale bar = 50µm.
Fig 4: PNP characterization(A) Transmission electron micrographs of IL10-NP, and (B) IL10-PNP.(C) Coomassie staining of platelet vesicles and empty PNP on SDS-PAGE at equivalent protein concentrations.(D) Western blot analysis of platelet vesicles and PNP for characteristic platelet membrane glycoprotein bound to macrophages.(E) Size of empty PLGA core, IL10-NP, empty PNP, and IL10-PNP.(F) Zeta potential of empty PLGA core, IL10-NP, empty PNP, and IL10-PNP.(G) Diameter of PNP in deionized water on day 0 and day 7.(H) Encapsulation efficiency and (I) loading capacity of IL10 in IL10-NP, IL10-PNP.(J) In vitro cumulative release curve of IL10- NP and IL10-PNP incubated at 37°C in PBS. *p <0 .05, **p <0 .01, ***p < 0.001. n = 3.
Fig 5: Regulation role of IL10-PNP on SMCs in inflammatory environment simulated by platelets + macrophages coculture system in vitro(A) Schematic diagram of the coculture system.(B) Relative expression of osteopontin (OPN) and (C) SM22 in SMCs by RT-qPCR. n = 3.(D and E) Relative expression of a contractile phenotype related protein in SMCs after 24 h coculture. n = 3.(F and G) The impact of IL10-PNP on the proliferative ability of SMCs detected by Edu assay. n = 3.(H) The impact of IL10-PNP on cell viability of SMCs detected by CCK-8 assay. Values were normalized based on the value of the blank group. n = 5.(I and J) The impact of IL10-PNP on migration ability of SMCs detected by transwell assay. n = 3. Data are presented as mean ± SD, ns not significant, *p <0 .05, **p <0 .01, ***p <0 .001. Scale bar = 100µm.
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