莱菔子外泌体样纳米颗粒在Caco-2单层细胞中的抗炎活性OA

OBJECTIVE To extract and analyze the active components of Semen Raphani-derived exosome-like nanoparticles(SRENs)and to delve into their biological effects within human colorectal adenocarcinoma(Caco-2).METHODS SRENs were isolated and purified from Semen Raphani via ultracentrifugation and sucrose density gradient centrifugation.The morphology was characterized by transmission electron microscopy imaging,and the average particle size and zeta potential by nanopar-ticle tracking analysis.Liquid chromatography tandem mass spectrometry was employed to analyze the lipid composition and proteomics of SRENs.RNA transcriptome sequencing technology was used to identify the sequences of microRNA(miRNA).The Caco-2 cells were divided into 6 groups:control group(PBS)and SRENs 1,10,20,40,80 mg·L-1 groups.After 24 h of incubation,the cell survival rate was calculated.Caco-2 monolayer intestinal epithelial cells were divided into 7 groups:control group(PBS),lipopolysaccharide(LPS)1 mg·L-1 group,LPS+SRENs 1,10,20,40,80 mg·L-1 groups(SRENs of corresponding concentrations were added after adding LPS 1 mg·L-1).After 24 h incubation,the cell survival rate was calculated.Caco-2 cells were co cultured with PKH67-labeled SRENs 10 mg·L-1,and the uptake of SRENs by the cells was observed under a fluorescence microscope at 0,0.5,1,2,3,4,and 6 hours,respectively.Caco-2 monolayer cells were divided into 5 groups:control group(PBS),LPS 1 mg·L-1 group and LPS+SRENs 1,5,10 mg·L-1 groups(after 24 h of culture with SRENs,LPS 1 mg·L-1 was added and cultured for 24 h).The supernatant was collected.The concentration of nitric oxide(NO)was determined by nitric oxide kit.The levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-6 and interferon-γ(IFN-γ)were detected by ELISA.The base side solution was taken,and the fluorescence intensity was detected by a microplate reader to calculate the apparent permeability coefficient(Papp).The specific activity of intracellular alkaline phosphatase(AKP)was determined by AKP kit.The expression levels of tight junction proteins[occludin,claudin-1,zonula occludens protein 1(ZO-1)]in the cells were determined by Western blotting.Caco-2 cells were divided into 6 groups:control group(PBS),SRENs 5 mg·L-1,LPS 1 mg·L-1,LPS 1 mg·L-1+SRENs 5 mg·L-1,LPS 1 mg·L-1+SRENs 5 mg·L-1+Toll like receptor 4(TLR4)inhibitor resatorvid(TAK-242)1 μmol·L-1,LPS 1 mg·L-1+TAK-242 1 μmol·L-1,In the TAK-242 treatment group,LPS and SRENS were added after one-hour pretreatment with TAK-242,and incubated for 24 h.The expression of TLR4 and the levels of tight junction proteins occludin and IL-6 were measured by Western blotting.RESULTS SRENs were nanoscale cup-shaped vesicles with an average particle size of(112.64±29.52)nm and a zeta potential of(-10.41±0.64)mV,containing 33 types of lipids,330 types of proteins,and 298 types of miRNAs.Among the lipid components,fatty acids had the highest content.SRENs had no significant cytotoxicity and were effec-tively taken up by Caco-2 cells within 6 h.Compared with LPS group,SRENs 1,5,10 mg·L-1 signifi-cantly reduced Papp of cells,lowered the production of NO,increased the activity of AKP.SRENs 5,10 mg·L-1 significantly decreased the levels of IL-6,TNF-α,IL-1β and IFN-γ,and significantly increased the protein levels of Occludin,Claudin-1 and ZO-1.Compared with LPS group,the protein expressions of TLR4 and IL-6 in LPS+TAK-242 group were significantly decreased while that of Occludin was increased.Compared with LPS+TAK-242 group,there was no significant change in the levels of Occludin protein or IL-6 in LPS+SRENs+TAK-242 group.CONCLUSION SRENs have good biocom-patibility and can repair intestinal barrier damage.Its anti-inflammatory activity may be related to the inhi-bition of TLR4 signaling pathway.