Mechanism Of Action Of Extracellular Vesicles Loaded With Resveratrol On Chronic Sinusitis With Nasal Polyps

ObjectiveIn this study,extracellular vesicles(EVs-Res)loaded with resveratrol that could be taken up by epithelial cells were prepared to explore the role of EVs-Res in repairing oxidative stress damage to epithelial cells in chronic sinusitis with nasal polyps(CRSw NP)and its anti-inflammatory and antibacterial mechanisms.Methods(1)The extracellular vesicles(EVs)in the culture supernatant of macrophages(RAW)were collected by ultracentrifugation,mixed with resveratrol(Res),and resuspended by repeated sonication in an ice bath to obtain EVs-Res.The appearance characteristics and properties of EVs-Res were observed by transmission electron microscopy and zeta potential detection to verify the successful synthesis of EVs-Res.(2)Clinical samples of nasal polyps from 30 patients with CRSw NP were collected for extraction of nasal polyp primary epithelial cells and divided into nasal polyp group(NP group),resveratrol group(Res group),and extracellular vesicle loaded resveratrol group(EVs-Res group).The inferior turbinate tissues of 10 patients with nasal septal deviation with inferior turbinate hypertrophy were collected,and nasal mucosal epithelial cells were extracted as the control group(Control group).(3)The uptake ability of EVs-Res by nasal epithelial cells was observed using fluorescent staining of cells.DCFH-DA fluorescent probe was used to detect the level of ROS in each group of nasal mucosal epithelial cells.The JC-1 probe was used to detect the change of mitochondrial membrane potential in each group of epithelial cells.Transmission electron microscopy was applied to visualize the morphological changes of mitochondria in each group.To investigate the repair effect of EVs-Res on oxidative stress damage in epithelial cells.(4)The changes of mitochondrial autophagy-related protein(LC3B;Parkin)and the expression of its NLRP3 inflammatory vesicles and its downstream inflammatory factor(Cleaved Caspase-1;IL-1β)in the epithelial cells of the nasal mucosa of each group were detected by immunofluorescence and protein blotting to investigate the molecular mechanism of EVs-Res on improving the inflammatory status of epithelial cells.(5)The inhibition of Staphylococcus aureus(S.aureus)growth by Res and EVs-Res was examined by inhibition loop and in vitro inhibition rate studies.The anti-biofilm ability of Res and EVs-Res was assessed by bacterial biofilm formation assay.Exploration of the antibacterial ability of EVs-Res in vitroResults(1)Transmission electron microscopy results showed that the prepared extracellular vesicle encapsulated resveratrol(EVs-Res)had an intact film structure.The particle size and zeta potential difference between it and EVs indicated the successful EVs extraction and synthesis of EVs-Res.Confocal fluorescence staining showed that epithelial cells could effectively take up EVs-Res,providing a prerequisite for its function.(2)The study results showed that EVs-Res effectively cleared ROS levels in nasal mucosal epithelial cells compared with the nasal polyp group and improved the function and structure of mitochondria in epithelial cells.When exploring the molecular mechanism of EVs-Res role,it was seen that EVs-Res treatment increased the autophagy protein LC3 B expression in nasal mucosal epithelial cells,formed more mitochondrial phagosomes,and induced an increase in the expression of pathway protein Parkin.Thus EVs-Res may inhibit oxidative stress by increasing the level of mitochondrial autophagy in nasal polyp tissue.(3)Activation of NLRP3 inflammasome in nasal mucosal epithelial cells was significantly inhibited after EVs-Res treatment.In addition,the changes in the levels of cleaved Caspase-1 and IL-1β were found to be consistent with the changes in NLRP3.It is suggested that EVs-Res may reduce inflammation in nasal polyps by inhibiting the activation of NLRP3.(4)When evaluating the antimicrobial activity,it was found that a visible ring of inhibition was observed after EVs-Res treatment,along with a reduction in the number of colonies.Scanning electron microscopy visualized the complete disruption of bacterial cell walls and membranes after EVs-Res treatment.The corresponding absorbance and solution color of crystalline violet in the EVs-Res group were the least among all treated groups compared to the control group.EVs-Res could effectively damage the bacterial biofilm.Conclusions(1)EVs-Res can be successfully prepared by low-temperature differential centrifugation and ultrasonication,and it could be effectively taken up by epithelial cells.(2)EVs-Res can mediate Parkin-related mitochondrial autophagy to improve the oxidative stress microenvironment of epithelial cells,which can protect the epithelial barrier function of the nasal mucosa.(3)EVs-Res inhibited the activation of inflammatory vesicles NLRP3 and down-regulated the release of downstream inflammatory factor IL-1β,reducing the level of inflammation in epithelial cells.(4)The prepared EVs-Res had superior antibacterial and inhibited bacterial biofilm formation.