Adipose-derived Stem Cell-derived Exosomes Immunomodulate Allergic Rhinitis Through TLR4/NF-κB Pathway

ObjectiveBy co-cultivating adipose stem cell-derived exosomes(ADSC-Exos)with the human nasal mucosal epithelial cells to explore the influence of adipose-derived stem cell-derived exosomes on the TLR4/NF-κB inflammatory signal pathway,and seek new ideas for the treatment of allergic rhinitis.Materials and Methods1.Obtaining and culture of adipose stem cells and nasal mucosal epitThe adult primary adipose stem cell line and the nasal mucosal epithelial cell line were purchased from Guangzhou Saiye and Guangzhou Jinio Biotechnology Co.,Ltd.respectively.After 48h～72h of resuscitation,change the medium according to the cell status.When the cell growth status is good and the cell fusion degree is about 80%,trypsinization and passage.2.Extraction and identification of adipose stem cell-derived exosomesWhen the cultured human adipose stem cells reach 70%-80%fusion rate,ADSC were cultured to the 4～5 generation,the original medium was removed,exosomal-free serum medium was replaced,then continue culturing for 48 hours and collecting the supernatant.The supernatant was processed by ultracentrifugation to extract exosomes derived from adipose stem cells.Then the exosomes were fixed,negatively stained and dried,and the morphology of ADSC-Exos was observed by transmission electron microscope;Then,exosomes were diluted to a certain ratio,and the diameter range of ADSC-Exos was analyzed by nanoparticle tracking analysis.Western Blot identified the expression of ADSC-Exos surface specific membrane proteins CD9,CD63 and membrane-bound protein TSG101.3.ADSC-Exos was co-cultured with nasal mucosal epithelial cells,then detect the expression levels of related inflammatory factors and proteinsThe experiment was divided into four groups,which were co-cultured with nasal mucosa epithelial cells.Group 1:blank control group(no treatment group);Group 2:LPS(1μg/mL)group;Group 3:ADSC-Exos(20μg/mL)group;Group 3:LPS(1μg/mL)+ADSC-Exos(20μg/mL)group.To determine the effect of human adipose stem cell exosomes on damaged human nasal mucosal epithelial cells,nasal epithelial cells were treated with adipose stem cell-derived exosomes and cocultured for 24 h,the cytokines of IL-6,IFN-γ,IL-1βand TNF-α in the supernatant of cells were detected by ELISA test kit;The expressions of TLR4,P65 and p-p65 proteins were detected;Extract cellular RNA,the mRNA levels of IL-6,IFN-γ,IL-1β and TNF-α were detected by qRT-PCR system,finally,inoculate the cells in a 6-well plate,process the cells and observe the transportation to the nucleus of P65 under an inverted fluorescence microscope.Results1.Culture of human adipose stem cells and nasal mucosa epithelial cellsAfter 24h of adipose stem cells resuscitation,a large number of adipose stem cells were observed to adhere to the wall under microscope,the cell shape is mostly polygonal or short spindle-like shape,after 72 hours,the cells were observed to be long spindle-shaped,chrysanthemum-like radial and spiral arrangement,and passaged 1:3,the cells grew faster after passage.24h after the recovery of human nasal mucosal epithelial cells,the cells grew evenly adherent to the wall,they are short and spindle-shaped with clear membrane,the epithelial cells of the nasal mucosa were fused into slices at 48h～72h,showing a typical paving stone pattern,and the bottom of the culture bottle was filled with 4～5 days.2.Extraction and identification of ADSC-ExosExosomes were extracted by overspeed centrifugal method.Under transmission electron microscope,ADSC-Exos had clear edges,and most of them were round or elliptical in shape,with a double membrane capsule structure,most exosomes are 20～300nm in diameter and have a typical cup-shaped morphology.The particle size distribution of ADSC-Exos was detected by using a nanoparticle tracker,the results indicate that the measured average particle size is 143.5nm,and the exosomes are basically in the distribution range of 30-200nm.Western Blot analysis indicated that adipose stem cell-derived exosomes were positive for specific membrane proteins CD9,CD63 and TSG101.3.The expression of TLR4,NF-κB p65 and Phospho-NF-κB p65 proteins and related inflammatory cytokines in the control group and the experimental groupAccording to experimental groups,LPS(1μg/mL)and ADSC-Exos(20μg/mL)were added according to the predesigned experimental scheme,and the test was conducted 24 hours later.TLR4,NF-κB p65,and Phospho-NF-κB p65 were upregulated in the LPS group compared with the control group and the Exos group,the mRNA levels of IL-6,IFN-γ,TNF-α,IL-1β and inflammatory cytokines associated with cell supernatant in nasal mucosa epithelial cells were also higher than those in control group and Exos.The difference between the control group and the Exos group is not statistically significant.Intervention with ADSC-Exos in the LPS+ADSC-Exos group,Western Blot test showed that the expressions of the three proteins were down-regulated compared with those in the LPS group,The mRNA levels of IL-6,INF-γ,TNF-α,IL-1β and inflammatory cytokines related to cell supernatant in nasal mucosa epithelial cells were also significantly decreased.4.The transportation to the nucleus of P65 was observed under an inverted fluorescence microscopeIntervention was carried out on cells according to experimental groups,two hours later,all P65 in the LPS group and the LPS+ADSC-Exos group can quickly enter the nucleus from the cytoplasm under inverted fluorescence microscope.Conclusion1.After the primary adipose stem cells were cultured to the 4-5 generation,exosomes could be successfully separated and extracted by hypervelocity centrifugation technology,moreover,The exosomes have a particle size range of 30-200 nm,and with a double-layer membrane vesicle structure.The expression of CD9,CD63 and TSG101 protein markers was positive.2.LPS can successfully activate the TLR4/NF-κB inflammatory pathway leading to up-regulation of TLR4,NF-κB p65 and phospho-NF-κB p65 protein expression in nasal mucosal epithelial cells.The expression level of IL-6,IFN-γ,IL-1β,TNF-α was increased.3.ADSC-Exos can inhibit the production of inflammatory factors(IL-6,IFN-γ,IL-1 β,TNF-α)in nasal mucosa epithelial cells by regulating TLR4/NF-κB signaling pathway and affecting the expression of TLR4,NF-κB protein and downstream genes.