Fig. 7
Exosomes release NLRP3 inflammasome complex propagates ZnONP- and UVB-induced keratinocytes inflammation. A Western blot analysis of isolated exosomes. The presence of canonical exosome proteins and the HSP70 decreasing indicate a pure exosome preparation. B TEM imaging of exosomes, arrows indicated the exosomes with cup-shaped morphology. C NTA analysis demonstrated a size distribution of particles that was consistent with the size range of exosomes. D, E Western blot analysis of exosomes isolated from the medium of HaCaT cells treated with ZnONPs and UVB. The NLRP3 inflammasome and pyroptosis proteins NLRP3, pro-caspase-1 and GSDMD were detected, Flotillin-1 was used as a housekeeping protein (loading control). F, G Western blot analysis of NLRP3, caspase-1 and GSDMD expression in HaCaT cells after treatment with PT and exosomes isolated from control cells or cells exposed to ZnONPs and UVB. H, I Western blot analysis of exosomes isolated from HaCaT cells after treatment with rapamycin (autophagy inducer), chloroquine (autophagy inhibitor), ZnONPs and UVB. The NLRP3 inflammasome and pyroptosis proteins NLRP3, caspase-1 and GSDMD were detected, Flotillin-1 was used as a housekeeping protein (loading control). J A Transwell culture system was used to mimic the cellular milieu. HaCaT cells were exposed to foreign DioC18-labeled exosomes from donor HaCaT cells that were exposed to ZnONPs and UVB with or without Rapa or Rapa + CQ treatment. K Histograms represent the relative fluorescence intensity of DioC18 staining. L DioC18-labeled exosomes propagation was measured via flow cytometry. DioC18 positive HaCaT cells (Q2-UR + LR regions) indicate were exposed to foreign DioC18-labeled exosomes from donor HaCaT cells that were exposed to ZnONPs and UVB with or without Rapa or Rapa + CQ treatment. M Histograms represent the DioC18 positive cells. Values are presented as the mean ± SD (n = 3). *p < 0.05, the control group versus the treatment groups. #p < 0.05, the UVB + ZnONPs group versus the UVB + ZnONPs + PT group, Exosome (UVB + ZnONPs) group versus Exosome (UVB + ZnONPs) + PT group, UVB + ZnONPs + Rapa group versus UVB + ZnONPs + CQ group, UVB + ZnONPs + Rapa group versus UVB + ZnONPs + Rapa + CQ group